U.S. patent application number 10/800865 was filed with the patent office on 2004-07-29 for isolated human ras-like proteins, nucleic acid molecules encoding human ras-like proteins, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Ketchum, Karen A., Ye, Jane.
Application Number | 20040146978 10/800865 |
Document ID | / |
Family ID | 25145152 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146978 |
Kind Code |
A1 |
Ye, Jane ; et al. |
July 29, 2004 |
Isolated human Ras-like proteins, nucleic acid molecules encoding
human Ras-like proteins, and uses thereof
Abstract
The present invention provides amino acid sequences of
polypeptides that are encoded by genes within the human genome, the
Ras-like protein polypeptides of the present invention. The present
invention specifically provides isolated polypeptide and nucleic
acid molecules, methods of identifying orthologs and paralogs of
the Ras-like protein polypeptides, and methods of identifying
modulators of the Ras-like protein polypeptides.
Inventors: |
Ye, Jane; (Boyds, MD)
; Ketchum, Karen A.; (Germantown, MD) ; Di
Francesco, Valentina; (Rockville, MD) ; Beasley,
Ellen M.; (Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
|
Family ID: |
25145152 |
Appl. No.: |
10/800865 |
Filed: |
March 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10800865 |
Mar 16, 2004 |
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09788654 |
Feb 21, 2001 |
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6733992 |
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Current U.S.
Class: |
435/69.1 ;
435/193; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
C07K 14/82 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/193; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12N 009/10; C07H
021/04 |
Claims
That which is claimed is:
1. An isolated polypeptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a polypeptide of
claim 2.
4. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. A method for producing any of the polypeptides of claim 1
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the polypeptides are expressed
from the nucleotide sequence.
11. A method for producing any of the polypeptides of claim 2
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the polypeptides are expressed
from the nucleotide sequence.
12. A method for detecting the presence of any of the polypeptides
of claim 2 in a sample, said method comprising contacting said
sample with a detection agent that specifically allows detection of
the presence of the polypeptide in the sample and then detecting
the presence of the polypeptide.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. A method for identifying a modulator of a polypeptide of claim
2, said method comprising contacting said polypeptide with an agent
and determining if said agent has modulated the function or
activity of said polypeptide.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
polypeptide.
16. A method for identifying an agent that binds to any of the
polypeptides of claim 2, said method comprising contacting the
polypeptide with an agent and assaying the contacted mixture to
determine whether a complex is formed with the agent bound to the
polypeptide.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human Ras-like protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 16.
19. A method for identifying a modulator of the expression of a
polypeptide of claim 2, said method comprising contacting a cell
expressing said polypeptide with an agent, and determining if said
agent has modulated the expression of said polypeptide.
20. An isolated human Ras-like protein polypeptide having an amino
acid sequence that shares at least 70% homology with an amino acid
sequence shown in SEQ ID NO:2.
21. A polypeptide according to claim 20 that shares at least 90
percent homology with an amino acid sequence shown in SEQ ID
NO:2.
22. An isolated nucleic acid molecule encoding a human Ras-like
protein polypeptide, said nucleic acid molecule sharing at least 80
percent homology with a nucleic acid molecule shown in SEQ ID NOS:1
or 3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS:1 or 3.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of Ras-like proteins
that are related to the RRP22 subfamily, recombinant DNA molecules
and protein production. The present invention specifically provides
novel Ras-like protein polypeptides and proteins and nucleic acid
molecules encoding such peptide and protein molecules, all of which
are useful in the development of human therapeutics and diagnostic
compositions and methods.
BACKGROUND OF THE INVENTION
[0002] The novel human protein, and encoding gene, provided by the
present invention is related to the Ras-like protein family. The
protein of the present invention shows the highest degree of
similarity to the RRP22 gene, which is found on chromosome 22.
RRP22 defines a subgroup within the Ras-like protein family
(Zucman-Rossi et al., Genomics 38 (3), 247-254 (1996)). The gene
encoding the Ras-like protein of the present invention is found on
chromosome 17. It has been suggested that growth-arrest-specific
and Ras-related genes may be involved in tumorigenic processes
(Zucman-Rossi et al., Genomics 38 (3), 247-254 (1996)).
[0003] Ras-like proteins, particularly members of the RRP22
subfamilies, 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 members
of these subfamily of Ras-like proteins. The present invention
advances the state of the art by providing a previously
unidentified human Ras-like proteins that have homology to members
of the RRP22 subfamilies.
[0004] Ras Protein
[0005] Ras proteins are small regulatory GTP-binding proteins, or
small G proteins, which belong to the Ras protein superfamily. They
are monomeric GTPases, but their GTPase activity is very slow (less
than one GTP molecule per minute).
[0006] Ras proteins are key relays in the signal transducing
cascade induced by the binding of a ligand to specific receptors
such as receptor tyrosine kinases (RTKs), since they trigger the
MAP kinase cascade. The ligand can be a growth factor (epidermal
growth factor (EGF), platelet-derived growth factor (PDGF) . . . ),
insulin, an interleukin (IL), granulocyte colony-stimulating factor
(G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF).
. . .
[0007] Ras proteins contain sequences highly conserved during
evolution. Their tertiary structure includes ten loops connecting
six strands of beta-sheet and five alpha helices.
[0008] In mammalians, there are four Ras proteins, which are
encoded by Ha-ras, N-ras, Ki-rasA and Ki-rasB genes. They are
composed of about 170 residues and have a relative molecular mass
of 21 kD. Ras proteins contain covalently attached modified lipids
allowing these proteins to bind to the plasma membrane. Ha-Ras has
a C-terminal farnesyl group, a C-terminal palmitoyl group and a
N-terminal myristoyl group. In Ki-Ras(B), a C-terminal polylysine
domain replaces the palmitoyl group.
[0009] Ras proteins alternate between an inactive form bound to GDP
and an active form bound to GTP. Their activation results from
reactions induced by a guanine nucleotide-exchange factor (GEF).
Their inactivation results from reactions catalyzed by a
GTPase-activating protein (GAP).
[0010] When a Ras protein is activated by a GEF such as a Sos
protein, the N-terminal region of a serine/threonine kinase, called
"Raf protein", can bind to Ras protein. The C-terminal region of
the activated Raf thus formed binds to another protein, MEK, and
phosphorylates it on both specific tyrosine and serine residues.
Active MEK phosphorylates and activates, in turn, a MAP kinase
(ERK1 or ERK2), which is also a serine/threonine kinase. This
phosphorylation occurs on both specific tyrosine and threonine
residues of MAP kinase.
[0011] MAP kinase phosphorylates many different proteins,
especially nuclear transcription factors (TFs) which regulate
expression of many genes during cell proliferation and
differentiation.
[0012] Recent researches suggest that, in mammalians, phosphatidyl
inositol 3'-kinase (PI3-kinase) might be a target of Ras protein,
instead of Raf protein. In certain mutations, the translation of
ras genes may produce oncogenic Ras proteins.
[0013] Ras-Like Protein
[0014] Guanine nucleotide-binding proteins (GTP-binding proteins,
or G proteins) participate in a wide range of regulatory functions
including metabolism, growth, differentiation, signal transduction,
cytoskeletal organization, and intracellular vesicle transport and
secretion. These proteins control diverse sets of regulatory
pathways in response to hormones, growth factors, neuromodulators,
or other signaling molecules. When these molecules bind to
transmembrane receptors, signals are propagated to effector
molecules by intracellular signal transducing proteins. Many of
these signal transducing proteins are members of the Ras
superfamily.
[0015] The Ras superfamily is a class of low molecular weight (LMW)
GTP-binding proteins which consist of 21-30 kDa polypeptides. These
proteins regulate cell growth, cell cycle control, protein
secretion, and intracellular vesicle interaction. In particular,
the LMW GTP-binding proteins activate cellular proteins by
transducing mitogenic signals involved in various cell functions in
response to extracellular signals from receptors (Tavitian, A.
(1995) C. R. Seances Soc. Biol. Fil. 189:7-12). During this
process, the hydrolysis of GTP acts as an energy source as well as
an on-off switch for the GTPase activity of the LMW GTP-binding
proteins.
[0016] The Ras superfamily is comprised of five subfamilies: Ras,
Rho, Ran, Rab, and ADP-ribosylation factor (ARF). Specifically, Ras
genes are essential in the control of cell proliferation. Mutations
in Ras genes have been associated with cancer. Rho proteins control
signal transduction in the process of linking receptors of growth
factors to actin polymerization which is necessary for cell
division. Rab proteins control the translocation of vesicles to and
from membranes for protein localization, protein processing, and
secretion. Ran proteins are localized to the cell nucleus and play
a key role in nuclear protein import, control of DNA synthesis, and
cell-cycle progression. ARF and ARF-like proteins participate in a
wide variety of cellular functions including vesicle trafficking,
exocrine secretion, regulation of phospholipase activity, and
endocytosis.
[0017] Despite their sequence variations, all five subfamilies of
the Ras superfamily share conserved structural features. Four
conserved sequence regions (motifs I-IV) have been studied in the
LMW GTP-binding proteins. Motif I is the most variable but has the
conserved sequence, GXXXXGK. The lysine residue is essential in
interacting with the .beta.- and .gamma.-phosphates of GTP. Motif
II, III, and IV contain highly conserved sequences of DTAGQ, NKXD,
and EXSAX, respectively. Specifically, Motif II regulates the
binding of gamma-phosphate of GTP; Motif II regulates the binding
of GTP; and Motif IV regulates the guanine base of GTP. Most of the
membrane-bound LMW GTP-binding proteins generally require a carboxy
terminal isoprenyl group for membrane association and biological
activity. The isoprenyl group is added posttranslationally through
recognition of a terminal cysteine residue alone or a terminal
cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid
(CAAX) motif. Additional membrane-binding energy is often provided
by either internal palmitoylation or a carboxy terminal cluster of
basic amino acids. The LMW GTP-binding proteins also have a
variable effector region, located between motifs I and II, which is
characterized as the interaction site for guanine nucleotide
exchange factors (GEFs) or GTPase-activating proteins (GAPs). GEFs
induce the release of GDP from the active form of the G protein,
whereas GAPs interact with the inactive form by stimulating the
GTPase activity of the G protein.
[0018] The ARF subfamily has at least 15 distinct members
encompassing both ARF and ARF-like proteins. ARF proteins
identified to date exhibit high structural similarity and
ADP-ribosylation enhancing activity. In contrast, several ARF-like
proteins lack ADP-ribosylation enhancing activity and bind GTP
differently. An example of ARF-like proteins is a rat protein,
ARL184. ARL184 has been shown to have a molecular weight of 22 kDa
and four functional GTP-binding sites (Icard-Liepkalns, C. et al.
(1997) Eur. J. Biochem. 246: 388-393). ARL184 is active in both the
cytosol and the Golgi apparatus and is closely associated with
acetylcholine release, suggesting that ARL184 is a potential
regulatory protein associated with Ca.sup.2+-dependent release of
acetylcholine.
[0019] A number of Rho GTP-binding proteins have been identified in
plasma membrane and cytoplasm. These include RhoA, B and C, and D,
rhoG, rac 1 and 2, G25K-A and B, and TC10 (Hall, A. et al. (1993)
Philos. Trans. R. Soc. Lond. (Biol.) 340:267-271). All Rho proteins
have a CAAX motif which binds a prenyl group and either a
palmitoylation site or a basic amino acid-rich region, suggesting
their role in membrane-associated functions. In particular, RhoD is
a protein which functions in early endosome motility and
distribution by inducing rearrangement of actin cytoskeleton and
cell surface (Murphy, C. et al. (1996) Nature 384:427-432). During
cell adhesion, the Rho proteins are essential for triggering focal
complex assembly and integrin-dependent signal transduction
(Hotchin, N. A. and Hall, A. (1995) J. Cell Biol.
131:1857-1865).
[0020] The Ras subfamily proteins already indicated supra are
essential in transducing signals from receptor tyrosine kinases
(RTKs) to a series of serine/threonine kinases which control cell
growth and differentiation. Mutant Ras proteins, which bind but
cannot hydrolyze GTP, are permanently activated and cause
continuous cell proliferation or cancer. TC21, a Ras-like protein,
is found to be highly expressed in a human teratocarcinoma cell
line (Drivas, G. T. et al. (1990) Mol. Cell. Biol. 10: 1793-1798).
Rin and Rit are characterized as membrane-binding, Ras-like
proteins without the lipid-binding CAAX motif and carboxy terminal
cysteine (Lee, C.-H. J. et al. (1996) J. Neurosci. 16: 6784-6794).
Further, Rin is shown to localize in neurons and have
calcium-dependant calmodulin-binding activity.
[0021] The discovery of new human Ras-like proteins and the
polynucleotides that encode them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of inflammation and disorders associated
with cell proliferation and apoptosis.
SUMMARY OF THE INVENTION
[0022] The present invention is based in part on the identification
of amino acid sequences of human Ras-like protein polypeptides and
proteins that are related to the RRP22 Ras-like protein subfamily,
as well as allelic variants and other mammalian orthologs thereof.
These unique peptide sequences, and nucleic acid sequences that
encode these peptides, can be used as models for the development of
human therapeutic targets, aid in the identification of therapeutic
proteins, and serve as targets for the development of human
therapeutic agents that modulate Ras-like protein activity in cells
and tissues that express the Ras-like protein. Experimental data as
provided in FIG. 1 indicates expression in humans in lung small
cell carcinomas and in the brain.
DESCRIPTION OF THE FIGURE SHEETS
[0023] FIG. 1 provides the nucleotide sequence of a cDNA molecule
that encodes the Ras-like protein of the present invention. (SEQ ID
NO:1) In addition, structure and functional information is
provided, such as ATG start, stop and tissue distribution, where
available, that allows one to readily determine specific uses of
inventions based on this molecular sequence. Experimental data as
provided in FIG. 1 indicates expression in humans in lung small
cell carcinomas and in the brain.
[0024] FIG. 2 provides the predicted amino acid sequence of the
Ras-like protein of the present invention. (SEQ ID NO:2) In
addition structure and functional information such as protein
family, function, and modification sites is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence.
[0025] FIG. 3 provides genomic sequences that span the gene
encoding the Ras-like protein of the present invention. (SEQ ID
NO:3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, the following SNPs were identified: A2455C, A2785G, T3482A,
A6189G, T6491C, A7353T, A8688G, G10789C, G11079A, and A12087G.
DETAILED DESCRIPTION OF THE INVENTION
[0026] General Description
[0027] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a Ras-like protein or part of a Ras-like protein and are
related to the RRP22 subfamily. Utilizing these sequences,
additional genomic sequences were assembled and transcript and/or
cDNA sequences were isolated and characterized. Based on this
analysis, the present invention provides amino acid sequences of
human Ras-like protein polypeptides that are related to the RRP22
subfamily, nucleic acid sequences in the form of transcript
sequences, cDNA sequences and/or genomic sequences that encode
these Ras-like protein polypeptide, nucleic acid variation (allelic
information), tissue distribution of expression, and information
about the closest art known protein/peptide/domain that has
structural or sequence homology to the Ras-like protein of the
present invention.
[0028] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
Ras-like proteins of the RRP22 subfamily and the expression pattern
observed. Experimental data as provided in FIG. 1 indicates
expression in humans in lung small cell carcinomas and in the
brain. The art has clearly established the commercial importance of
members of this family of proteins and proteins that have
expression patterns similar to that of the present gene. Some of
the more specific features of the peptides of the present
invention, and the uses thereof, are described herein, particularly
in the Background of the Invention and in the annotation provided
in the Figures, and/or are known within the art for each of the
known RRP22 family or subfamily of Ras-like proteins.
[0029] Specific Embodiments
[0030] Peptide Molecules
[0031] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the Ras-like protein family and are related to the RRP22
subfamily (protein sequences are provided in FIG. 2,
transcript/cDNA sequences are provided in FIG. 1 and genomic
sequences are provided in FIG. 3). The peptide sequences provided
in FIG. 2, as well as the obvious variants described herein,
particularly allelic variants as identified herein and using the
information in FIG. 3, will be referred herein as the Ras-like
proteins or peptides of the present invention, Ras-like proteins or
peptides, or peptides/proteins of the present invention.
[0032] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the Ras-like protein polypeptide disclosed
in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG.
1, transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0033] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components.
[0034] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0035] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the Ras-like protein polypeptide having
less than about 30% (by dry weight) chemical precursors or other
chemicals, less than about 20% chemical precursors or other
chemicals, less than about 10% chemical precursors or other
chemicals, or less than about 5% chemical precursors or other
chemicals.
[0036] The isolated Ras-like protein polypeptide can be purified
from cells that naturally express it, purified from cells that have
been altered to express it (recombinant), or synthesized using
known protein synthesis methods. Experimental data as provided in
FIG. 1 indicates expression in humans in lung small cell carcinomas
and in the brain. For example, a nucleic acid molecule encoding the
Ras-like protein polypeptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Many of these techniques are
described in detail below.
[0037] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence
of such a protein is provided in FIG. 2. A protein consists of an
amino acid sequence when the amino acid sequence is the final amino
acid sequence of the protein.
[0038] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues,
for example from about 1 to about 100 or so additional residues,
typically from 1 to about 20 additional residues in the final
protein.
[0039] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2),
for example, proteins encoded by the transcript/cDNA nucleic acid
sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences
provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid
sequence when the amino acid sequence is at least part of the final
amino acid sequence of the protein. In such a fashion, the protein
can be only the peptide or have additional amino acid molecules,
such as amino acid residues (contiguous encoded sequence) that are
naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few
additional amino acid residues or can comprise several hundred or
more additional amino acids. The preferred classes of proteins that
are comprised of the Ras-like protein polypeptide of the present
invention are the naturally occurring mature proteins. A brief
description of how various types of these proteins can be
made/isolated is provided below.
[0040] The Ras-like protein polypeptides of the present invention
can be attached to heterologous sequences to form chimeric or
fusion proteins. Such chimeric and fusion proteins comprise a
Ras-like protein polypeptide operatively linked to a heterologous
protein having an amino acid sequence not substantially homologous
to the Ras-like protein polypeptide. "Operatively linked" indicates
that the Ras-like protein polypeptide and the heterologous protein
are fused in-frame. The heterologous protein can be fused to the
N-terminus or C-terminus of the Ras-like protein polypeptide.
[0041] In some uses, the fusion protein does not affect the
activity of the Ras-like protein polypeptide per se. For example,
the fusion protein can include, but is not limited to, enzymatic
fusion proteins, for example beta-galactosidase fusions, yeast
two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and
Ig fusions. Such fusion proteins, particularly poly-His fusions,
can facilitate the purification of recombinant Ras-like protein
polypeptide. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a protein can be increased by using
a heterologous signal sequence.
[0042] A chimeric or fusion protein can be 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. 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 Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A Ras-like protein
polypeptide-encoding nucleic acid can be cloned into such an
expression vector such that the fusion moiety is linked in-frame to
the Ras-like protein polypeptide.
[0043] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the peptides
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art know techniques in the fields of
recombinant nucleic acid technology and protein biochemistry. It is
understood, however, that variants exclude any amino acid sequences
disclosed prior to the invention.
[0044] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the Ras-like
protein polypeptides of the present invention. The degree of
homology/identity present will be based primarily on whether the
peptide is a functional variant or non-functional variant, the
amount of divergence present in the paralog family, and the
evolutionary distance between the orthologs.
[0045] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment,
the length of a reference sequence aligned for comparison purposes
is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the
length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0046] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0047] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, word length=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, word length=3, to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
[0048] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the Ras-like protein polypeptides of the present
invention as well as being encoded by the same genetic locus as the
Ras-like protein polypeptide provided herein. The gene encoding the
novel Ras-like protein of the present invention is located on a
genome component that has been mapped to human chromosome 17 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0049] Allelic variants of a Ras-like protein polypeptide can
readily be identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the Ras-like protein polypeptide as well as being encoded by the
same genetic locus as the Ras-like protein polypeptide provided
herein. Genetic locus can readily be determined based on the
genomic information provided in FIG. 3, such as the genomic
sequence mapped to the reference human. The gene encoding the novel
Ras-like protein of the present invention is located on a genome
component that has been mapped to human chromosome 17 (as indicated
in FIG. 3), which is supported by multiple lines of evidence, such
as STS and BAC map data. As used herein, two proteins (or a region
of the proteins) have significant homology when the amino acid
sequences are typically at least about 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous. A significantly
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence that will hybridize to a
Ras-like protein polypeptide encoding nucleic acid molecule under
stringent conditions as more fully described below.
[0050] FIG. 3 provides information on SNPs that have been found in
the gene encoding the Ras-like protein of the present invention.
The following variations were seen: A2455C, A2785G, T3482A, A6189G,
T6491C, A7353T, A8688G, G10789C, G11079A, and A12087G. Some of
these SNPs, particularly the SNPs 5' of the ORF and in the first
intron, may affect control/regulatory elements.
[0051] Paralogs of a Ras-like protein polypeptide can readily be
identified as having some degree of significant sequence
homology/identity to at least a portion of the Ras-like protein
polypeptide, as being encoded by a gene from humans, and as having
similar activity or function. Two proteins will typically be
considered paralogs when the amino acid sequences are typically at
least about 40-50%, 50-60%, and more typically at least about
60-70% or more homologous through a given region or domain. Such
paralogs will be encoded by a nucleic acid sequence that will
hybridize to a Ras-like protein polypeptide encoding nucleic acid
molecule under moderate to stringent conditions as more fully
described below.
[0052] Orthologs of a Ras-like protein polypeptide can readily be
identified as having some degree of significant sequence
homology/identity to at least a portion of the Ras-like protein
polypeptide as well as being encoded by a gene from another
organism. Preferred orthologs will be isolated from mammals,
preferably primates, for the development of human therapeutic
targets and agents. Such orthologs will be encoded by a nucleic
acid sequence that will hybridize to a Ras-like protein polypeptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins.
[0053] Non-naturally occurring variants of the Ras-like protein
polypeptides of the present invention can readily be generated
using recombinant techniques. Such variants include, but are not
limited to deletions, additions and substitutions in the amino acid
sequence of the Ras-like protein polypeptide. For example, one
class of substitutions is conserved amino acid substitutions. Such
substitutions are those that substitute a given amino acid in a
Ras-like protein polypeptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the replacements, one for another, among the aliphatic amino acids
Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr, exchange of the acidic residues Asp and Glu, substitution
between the amide residues Asn and Gln, exchange of the basic
residues Lys and Arg, replacements among the aromatic residues Phe,
Tyr, and the like. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
[0054] Variant Ras-like protein polypeptides can be fully
functional or can lack function in one or more activities. Fully
functional variants typically contain only conservative variations
or variations in non-critical residues or in non-critical regions.
Functional variants can also contain substitution of similar amino
acids that result in no change or an insignificant change in
function. Alternatively, such substitutions may positively or
negatively affect function to some degree.
[0055] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0056] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity
such as receptor binding or in vitro proliferative activity. Sites
that are critical for ligand-receptor binding can also be
determined by structural analysis such as crystallography, nuclear
magnetic resonance, or photoaffinity labeling (Smith et al., J.
Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312
(1992)).
[0057] The present invention further provides fragments of the
Ras-like protein polypeptides, in addition to proteins and peptides
that comprise and consist of such fragments. Particularly those
comprising the residues identified in FIG. 2. The fragments to
which the invention pertains, however, are not to be construed as
encompassing fragments that have been disclosed publicly prior to
the present invention.
[0058] As used herein, a fragment comprises at least 8, 10, 12, 14,
16 or more contiguous amino acid residues from a Ras-like protein
polypeptide. Such fragments can be chosen based on the ability to
retain one or more of the biological activities of the Ras-like
protein polypeptide, or can be chosen for the ability to perform a
function, e.g., act as an immunogen. Particularly important
fragments are biologically active fragments, peptides that are, for
example about 8 or more amino acids in length. Such fragments will
typically comprise a domain or motif of the Ras-like protein
polypeptide, e.g., active site. Further, possible fragments
include, but are not limited to, domain or motif containing
fragments, soluble peptide fragments, and fragments containing
immunogenic structures. Predicted domains and functional sites are
readily identifiable by computer programs well known and readily
available to those of skill in the art (e.g., PROSITE, HMMer,
eMOTIF, etc.). The results of one such analysis are provided in
FIG. 2.
[0059] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in Ras-like protein polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the art
(some of these features are identified in FIG. 2).
[0060] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0061] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Postranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0062] Accordingly, the Ras-like protein polypeptides of the
present invention also encompass derivatives or analogs in which a
substituted amino acid residue is not one encoded by the genetic
code, in which a substituent group is included, in which the mature
Ras-like protein polypeptide is fused with another compound, such
as a compound to increase the half-life of the Ras-like protein
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature Ras-like protein
polypeptide, such as a leader or secretory sequence or a sequence
for purification of the mature Ras-like protein polypeptide, or a
pro-protein sequence.
[0063] Protein/Peptide Uses
[0064] The proteins of the present invention can be used in assays
to determine the biological activity of the protein, including in a
panel of multiple proteins for high-throughput screening; to raise
antibodies or to elicit another immune response; as a reagent
(including the labeled reagent) in assays designed to
quantitatively determine levels of the protein (or its ligand or
receptor) in biological fluids; and as markers for tissues in which
the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state). Where the protein binds or
potentially binds to another protein (such as, for example, in a
receptor-ligand interaction), the protein can be used to identify
the binding partner so as to develop a system to identify
inhibitors of the binding interaction. Any or all of these research
utilities are capable of being developed into reagent grade or kit
format for commercialization as research products.
[0065] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0066] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, Ras-like proteins
isolated from humans and their human/mammalian orthologs serve as
targets for identifying agents for use in mammalian therapeutic
applications, e.g. a human drug, particularly in modulating a
biological or pathological response in a cell or tissue that
expresses the Ras-like protein. Experimental data as provided in
FIG. 1 indicates that the Ras-like proteins of the present
invention are expressed in humans in lung small cell carcinomas (as
indicated by virtual northern blot analysis) and in the brain (as
indicated by PCR-based tissue screening panels). A large percentage
of pharmaceutical agents are being developed that modulate the
activity of Ras-like proteins, particularly members of the RRP22
subfamily (see Background of the Invention). The structural and
functional information provided in the Background and Figures
provide specific and substantial uses for the molecules of the
present invention, particularly in combination with the expression
information provided in FIG. 1. Experimental data as provided in
FIG. 1 indicates expression in humans in lung small cell carcinomas
and in the brain. Such uses can readily be determined using the
information provided herein, that which is known in the art, and
routine experimentation.
[0067] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to Ras-like
proteins that are related to members of the RRP22 subfamily. Such
assays involve any of the known Ras-like protein functions or
activities or properties useful for diagnosis and treatment of
Ras-like protein-related conditions that are specific for the
subfamily of Ras-like proteins that the one of the present
invention belongs to, particularly in cells and tissues that
express the Ras-like protein. Experimental data as provided in FIG.
1 indicates that the Ras-like proteins of the present invention are
expressed in humans in lung small cell carcinomas (as indicated by
virtual northern blot analysis) and in the brain (as indicated by
PCR-based tissue screening panels).
[0068] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the Ras-like protein, as a biopsy or expanded in cell culture.
Experimental data as provided in FIG. 1 indicates expression in
humans in lung small cell carcinomas and in the brain. In an
alternate embodiment, cell-based assays involve recombinant host
cells expressing the Ras-like protein.
[0069] The polypeptides can be used to identify compounds that
modulate Ras-like protein activity. Both the Ras-like protein of
the present invention and appropriate variants and fragments can be
used in high-throughput screens to assay candidate compounds for
the ability to bind to the Ras-like protein. These compounds can be
further screened against a functional Ras-like protein to determine
the effect of the compound on the Ras-like protein activity.
Further, these compounds can be tested in animal or invertebrate
systems to determine activity/effectiveness. Compounds can be
identified that activate (agonist) or inactivate (antagonist) the
Ras-like protein to a desired degree.
[0070] Therefore, in one embodiment, RRP22 or a fragment or
derivative thereof may be administered to a subject to prevent or
treat a disorder associated with an increase in apoptosis. Such
disorders include, but are not limited to, AIDS and other
infectious or genetic immunodeficiencies, neurodegenerative
diseases such as Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebellar
degeneration, myelodysplastic syndromes such as aplastic anemia,
ischemic injuries such as myocardial infarction, stroke, and
reperfusion injury, toxin-induced diseases such as alcohol-induced
liver damage, cirrhosis, and lathyrism, wasting diseases such as
cachexia, viral infections such as those caused by hepatitis B and
C, and osteoporosis.
[0071] In another embodiment, a pharmaceutical composition
comprising RRP22 may be administered to a subject to prevent or
treat a disorder associated with increased apoptosis including, but
not limited to, those listed above.
[0072] In still another embodiment, an agonist which is specific
for RRP22 may be administered to prevent or treat a disorder
associated with increased apoptosis including, but not limited to,
those listed above.
[0073] In a further embodiment, a vector capable of expressing
RRP22, or a fragment or a derivative thereof, may be used to
prevent or treat a disorder associated with increased apoptosis
including, but not limited to, those listed above.
[0074] In cancer, where RRP22 promotes cell proliferation, it is
desirable to decrease its activity. Therefore, in one embodiment,
an antagonist of RRP22 may be administered to a subject to prevent
or treat cancer including, but not limited to, adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, and
teratocarcinoma, and, in particular, cancers of the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary
glands, skin, spleen, testis, thymus, thyroid, and uterus. In one
aspect, an antibody specific for RRP22 may be used directly as an
antagonist, or indirectly as a targeting or delivery mechanism for
bringing a pharmaceutical agent to cells or tissue which express
RRP22.
[0075] In another embodiment, a vector expressing the complement of
the polynucleotide encoding RRP22 may be administered to a subject
to prevent or treat a cancer including, but not limited to, the
types of cancer listed above.
[0076] In inflammation, where RRP22 promotes cell proliferation, it
is desirable to decrease its activity. Therefore, in one
embodiment, an antagonist of RRP22 may be administered to a subject
to prevent or treat an inflammation. Disorders associated with
inflammation include, but are not limited to, Addison's disease,
adult respiratory distress syndrome, allergies, anemia, asthma,
atherosclerosis, bronchitis, cholecystitis, Crohn's disease,
ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout,
Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, rheumatoid arthritis, scleroderma,
Sjogren's syndrome, and autoimmune thyroiditis; complications of
cancer, hemodialysis, extracorporeal circulation; viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections and trauma.
In one aspect, an antibody specific for RRP22 may be used directly
as an antagonist, or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express RRP22.
[0077] Further, the Ras-like protein polypeptides can be used to
screen a compound for the ability to stimulate or inhibit
interaction between the Ras-like protein and a molecule that
normally interacts with the Ras-like protein, e.g. a ligand or a
component of the signal pathway that the Ras-like protein normally
interacts. Such assays typically include the steps of combining the
Ras-like protein with a candidate compound under conditions that
allow the Ras-like protein, or fragment, to interact with the
target molecule, and to detect the formation of a complex between
the protein and the target or to detect the biochemical consequence
of the interaction with the Ras-like protein and the target, such
as any of the associated effects of signal transduction.
[0078] Candidate 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 et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) 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 et al., Cell 72:767-778 (1993)); 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). (Hodgson, Bio/technology, 1992,
Sep. 10(9);973-80).
[0079] One candidate compound is a soluble fragment of the Ras-like
protein that competes for ligand binding. Other candidate compounds
include mutant Ras-like proteins or appropriate fragments
containing mutations that affect Ras-like protein function and thus
compete for ligand. Accordingly, a fragment that competes for
ligand, for example with a higher affinity, or a fragment that
binds ligand but does not allow release, is within the scope of the
invention.
[0080] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) Ras-like
protein activity. The assays typically involve an assay of events
in the Ras-like protein mediated signal transduction pathway that
indicate Ras-like protein activity. Thus, the phosphorylation of a
protein/ligand target, the expression of genes that are up- or
down-regulated in response to the Ras-like protein dependent signal
cascade can be assayed. In one embodiment, the regulatory region of
such genes can be operably linked to a marker that is easily
detectable, such as luciferase. Alternatively, phosphorylation of
the Ras-like protein, or a Ras-like protein target, could also be
measured.
[0081] Any of the biological or biochemical functions mediated by
the Ras-like protein can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[0082] Binding and/or activating compounds can also be screened by
using chimeric Ras-like proteins in which any of the protein's
domains, or parts thereof, can be replaced by heterologous domains
or subregions. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the Ras-like protein is derived.
[0083] The Ras-like protein polypeptide of the present invention is
also useful in competition binding assays in methods designed to
discover compounds that interact with the Ras-like protein. Thus, a
compound is exposed to a Ras-like protein polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble Ras-like protein polypeptide is also
added to the mixture. If the test compound interacts with the
soluble Ras-like protein polypeptide, it decreases the amount of
complex formed or activity from the Ras-like protein target. This
type of assay is particularly useful in cases in which compounds
are sought that interact with specific regions of the Ras-like
protein. Thus, the soluble polypeptide that competes with the
target Ras-like protein region is designed to contain peptide
sequences corresponding to the region of interest.
[0084] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the Ras-like protein, or fragment,
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.
[0085] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. 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/15625
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 Ras-like protein-binding protein found
in the bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin with techniques well known in the art.
Alternatively, antibodies reactive with the 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 the protein
trapped in the wells by antibody conjugation. Preparations of a
Ras-like protein-binding protein and a candidate compound are
incubated in the Ras-like protein-presenting wells 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 Ras-like protein target
molecule, or which are reactive with Ras-like 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.
[0086] Agents that modulate one of the Ras-like proteins of the
present invention can be identified using one or more of the above
assays, alone or in combination. It is generally preferable to use
a cell-based or cell free system first and then confirm activity in
an animal/insect model system. Such model systems are well known in
the art and can readily be employed in this context.
[0087] Modulators of Ras-like protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the Ras-like protein associated pathway, by
treating cells that express the Ras-like protein. Experimental data
as provided in FIG. 1 indicates expression in humans in lung small
cell carcinomas and in the brain. These methods of treatment
include the steps of administering the modulators of protein
activity in a pharmaceutical composition as described herein, to a
subject in need of such treatment.
[0088] In yet another aspect of the invention, the Ras-like
proteins can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al., Cell 72:223-232 (1993); Madura et al., J. Biol. Chem.
268:12046-12054 (1993); Bartel et al., Biotechniques 14:920-924
(1993); Iwabuchi et al., Oncogene 8:1693-1696 (1993); and Brent
WO94/10300), to identify other proteins that bind to or interact
with the Ras-like protein and are involved in Ras-like protein
activity. Such Ras-like protein-binding proteins are also likely to
be involved in the propagation of signals by the Ras-like proteins
or Ras-like protein targets as, for example, downstream elements of
a Ras-like protein-mediated signaling pathway, e.g., a pain
signaling pathway. Alternatively, such Ras-like protein-binding
proteins are likely to be Ras-like protein inhibitors.
[0089] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a Ras-like
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GALA). 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 a Ras-like 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 Ras-like protein.
[0090] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a Ras-like protein
modulating agent, an antisense Ras-like protein nucleic acid
molecule, a Ras-like protein-specific antibody, or a Ras-like
protein-binding partner) can be used in an animal or insect model
to determine the efficacy, toxicity, or side effects of treatment
with such an agent. Alternatively, an agent identified as described
herein can be used in an animal or insect model to determine the
mechanism of action of such an agent. Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatments as described herein.
[0091] The Ras-like proteins of the present invention are also
useful to provide a target for diagnosing a disease or
predisposition to a disease mediated by the peptide, Accordingly,
the invention provides methods for detecting the presence, or
levels of, the protein (or encoding mRNA) in a cell, tissue, or
organism. Experimental data as provided in FIG. 1 indicates
expression in humans in lung small cell carcinomas and in the
brain. The method involves contacting a biological sample with a
compound capable of interacting with the receptor protein such that
the interaction can be detected. Such an assay can be provided in a
single detection format or a multi-detection format such as an
antibody chip array.
[0092] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells, and fluids present within a
subject.
[0093] The peptides also are useful to provide a target for
diagnosing a disease or predisposition to a disease mediated by the
peptide, Accordingly, the invention provides methods for detecting
the presence, or levels of, the protein in a cell, tissue, or
organism. The method involves contacting a biological sample with a
compound capable of interacting with the receptor protein such that
the interaction can be detected.
[0094] The peptides of the present invention also provide targets
for diagnosing active disease, or predisposition to a disease, in a
patient having a variant peptide. Thus, the peptide can be isolated
from a biological sample and assayed for the presence of a genetic
mutation that results in translation of an aberrant peptide. This
includes amino acid substitution, deletion, insertion,
rearrangement, (as the result of aberrant splicing events), and
inappropriate post-translational modification. Analytic methods
include altered electrophoretic mobility, altered tryptic peptide
digest, altered receptor activity in cell-based or cell-free assay,
alteration in ligand or antibody-binding pattern, altered
isoelectric point, direct amino acid sequencing, and any other of
the known assay techniques useful for detecting mutations in a
protein. Such an assay can be provided in a single detection format
or a multi-detection format such as an antibody chip array.
[0095] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence using a detection
reagents, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide 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 that
detect the allelic variant of a peptide expressed in a subject and
methods which detect fragments of a peptide in a sample.
[0096] The peptides are also useful in pharmacogenomic analysis.
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.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
receptor protein in which one or more of the receptor functions in
one population is different from those in another population. The
peptides thus allow a target to ascertain a genetic predisposition
that can affect treatment modality. Thus, in a ligand-based
treatment, polymorphism may give rise to amino terminal
extracellular domains and/or other ligand-binding regions that are
more or less active in ligand binding, and receptor activation.
Accordingly, ligand dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing a polymorphism. As an alternative to genotyping,
specific polymorphic peptides could be identified.
[0097] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in FIG. 1
indicates expression in humans in lung small cell carcinomas and in
the brain. Accordingly, methods for treatment include the use of
the Ras-like protein or fragments.
[0098] Antibodies
[0099] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0100] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0101] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0102] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0103] Antibodies are preferably prepared from regions or discrete
fragments of the Ras-like proteins. Antibodies can be prepared from
any region of the peptide as described herein. However, preferred
regions will include those involved in function/activity and/or
receptor/binding partner interaction. FIG. 2 can be used to
identify particularly important regions while sequence alignment
can be used to identify conserved and unique sequence
fragments.
[0104] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0105] Detection of an antibody of the present invention 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.133I, .sup.35S, or .sup.3H.
[0106] Antibody Uses
[0107] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in FIG. 1 indicates that the Ras-like
proteins of the present invention are expressed in humans in lung
small cell carcinomas (as indicated by virtual northern blot
analysis) and in the brain (as indicated by PCR-based tissue
screening panels). Further, such antibodies can be used to detect
protein in situ, in vitro, or in a cell lysate or supernatant in
order to evaluate the abundance and pattern of expression. Also,
such antibodies can be used to assess abnormal tissue distribution
or abnormal expression during development. Antibody detection of
circulating fragments of the full-length protein can be used to
identify turnover.
[0108] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in FIG. 1 indicates expression in humans in lung small cell
carcinomas and in the brain. If a disorder is characterized by a
specific mutation in the protein, antibodies specific for this
mutant protein can be used to assay for the presence of the
specific mutant protein.
[0109] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in FIG. 1 indicates
expression in humans in lung small cell carcinomas and in the
brain. The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting expression level
or the presence of aberrant sequence and aberrant tissue
distribution or developmental expression, antibodies directed
against the or relevant fragments can be used to monitor
therapeutic efficacy.
[0110] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0111] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in lung small cell carcinomas and in the brain. Thus, where
a specific protein has been correlated with expression in a
specific tissue, antibodies that are specific for this protein can
be used to identify a tissue type.
[0112] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the Ras-like protein
to a binding partner such as a substrate. These uses can also be
applied in a therapeutic context in which treatment involves
inhibiting the protein's function. An antibody can be used, for
example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane. See FIG. 2 for structural information relating to the
proteins of the present invention.
[0113] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use.
[0114] Nucleic Acid Molecules
[0115] The present invention further provides isolated nucleic acid
molecules that encode a Ras-like protein polypeptide of the present
invention. Such nucleic acid molecules will consist of, consist
essentially of, or comprise a nucleotide sequence that encodes one
of the Ras-like protein polypeptides of the present invention, an
allelic variant thereof, or an ortholog or paralog thereof.
[0116] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid 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.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB, particularly contiguous peptide encoding
sequences and peptide encoding sequences within the same gene but
separated by introns in the genomic sequence. The important point
is that the nucleic acid is isolated from remote and unimportant
flanking sequences such that it can be subjected to the specific
manipulations described herein such as recombinant expression,
preparation of probes and primers, and other uses specific to the
nucleic acid sequences.
[0117] 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 nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0118] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0119] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule. The
present invention further provides nucleic acid molecules that
consist essentially of the nucleotide sequence shown in FIG. 1 or 3
(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
essentially of a nucleotide sequence when such a nucleotide
sequence is present with only a few additional nucleic acid
residues in the final nucleic acid molecule.
[0120] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0121] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0122] Full-length genes may be cloned from known sequence using
any one of a number of methods known in the art. For example, a
method which employs XL-PCR (Perkin-Elmer, Foster City, Calif.) to
amplify long pieces of DNA may be used. Other methods for obtaining
full-length sequences are well known in the art.
[0123] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life, or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0124] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the Ras-like
protein polypeptide alone, the sequence encoding the mature peptide
and additional coding sequences, such as a leader or secretory
sequence (e.g., a pre-pro or pro-protein sequence), the sequence
encoding the mature peptide, with or without the additional coding
sequences, plus additional non-coding sequences, for example
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding, and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0125] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form of DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0126] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention and that
encode obvious variants of the Ras-like proteins of the present
invention that are described above. Such nucleic acid molecules may
be naturally occurring, such as allelic variants (same locus),
paralogs (different locus), and orthologs (different organism), or
may be constructed by recombinant DNA methods or by chemical
synthesis. Such non-naturally occurring variants may be made by
mutagenesis techniques, including those applied to nucleic acid
molecules, cells, or whole organisms. Accordingly, as discussed
above, the variants can contain nucleotide substitutions, deletions
inversions, and/or insertions. Variation can occur in either or
both the coding and non-coding regions. The variations can produce
both conservative and non-conservative amino acid
substitutions.
[0127] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in the FIGS. 1 and 3.
Preferred non-coding fragments include, but are not limited to,
promoter sequences, enhancer sequences, gene modulating sequences,
and gene termination sequences. Such fragments are useful in
controlling heterologous gene expression and in developing screens
to identify gene-modulating agents.
[0128] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could be
at least 30, 40, 50, 100 250, or 500 nucleotides in length. The
length of the fragment will be based on its intended use. For
example, the fragment can encode epitope-bearing regions of the
peptide, or can be useful as DNA probes and primers. Such fragments
can be isolated using the known nucleotide sequence to synthesize
an oligonucleotide probe. A labeled probe can then be used to
screen a cDNA library, genomic DNA library, or mRNA to isolate
nucleic acid corresponding to the coding region. Further, primers
can be used in PCR reactions to clone specific regions of gene.
[0129] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50, or
more consecutive nucleotides.
[0130] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. The gene encoding the novel Ras-like
protein of the present invention is located on a genome component
that has been mapped to human chromosome 17 (as indicated in FIG.
3), which is supported by multiple lines of evidence, such as STS
and BAC map data.
[0131] FIG. 3 provides information on SNPs that have been found in
the gene encoding the Ras-like protein of the present invention.
The following variations were seen: A2455C, A2785G, T3482A, A6189G,
T6491C, A7353T, A8688G, G10789C, G11079A, and A12087G. Some of
these SNPs, particularly the SNPs 5' of the ORF and in the first
intron, may affect control/regulatory elements.
[0132] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% 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. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45C, followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 50-65.degree. C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0133] Nucleic Acid Molecule Uses
[0134] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, the following SNPs were identified: A2455C, A2785G, T3482A,
A6189G, T6491C, A7353T, A8688G, G10789C, G11079A, and A12087G.
[0135] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as those, which may encompass
fragments disclosed prior to the present invention.
[0136] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0137] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via:
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0138] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0139] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. The gene encoding the
novel Ras-like protein of the present invention is located on a
genome component that has been mapped to human chromosome 17 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0140] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0141] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0142] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides. Moreover, the nucleic acid molecules are useful for
constructing transgenic animals wherein a homolog of the nucleic
acid molecule has been "knocked-out" of the animal's genome.
[0143] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0144] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0145] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form, and distribution
of nucleic acid expression. Experimental data as provided in FIG. 1
indicates that the Ras-like proteins of the present invention are
expressed in humans in lung small cell carcinomas (as indicated by
virtual northern blot analysis) and in the brain (as indicated by
PCR-based tissue screening panels). Accordingly, the probes can be
used to detect the presence of, or to determine levels of, a
specific nucleic acid molecule in cells, tissues, and in organisms.
The nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the peptides described herein
can be used to assess expression and/or gene copy number in a given
cell, tissue, or organism. These uses are relevant for diagnosis of
disorders involving an increase or decrease in Ras-like protein
expression relative to normal results.
[0146] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA include Southern hybridizations and in situ
hybridization.
[0147] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a Ras-like protein, such
as by measuring a level of a receptor-encoding nucleic acid in a
sample of cells from a subject e.g., mRNA or genomic DNA, or
determining if a receptor gene has been mutated. Experimental data
as provided in FIG. 1 indicates that the Ras-like proteins of the
present invention are expressed in humans in lung small cell
carcinomas (as indicated by virtual northern blot analysis) and in
the brain (as indicated by PCR-based tissue screening panels).
[0148] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate Ras-like protein nucleic acid
expression.
[0149] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the Ras-like protein gene, particularly
biological and pathological processes that are mediated by the
Ras-like protein in cells and tissues that express it. Experimental
data as provided in FIG. 1 indicates expression in humans in lung
small cell carcinomas and in the brain. The method typically
includes assaying the ability of the compound to modulate the
expression of the Ras-like protein nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired Ras-like protein nucleic acid
expression. The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
Ras-like protein nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0150] The assay for Ras-like protein nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the signal pathway. Further,
the expression of genes that are up- or down-regulated in response
to the Ras-like protein signal pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0151] Thus, modulators of Ras-like protein gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of Ras-like protein mRNA in the presence of the
candidate compound is compared to the level of expression of
Ras-like protein mRNA in the absence of the candidate compound. The
candidate compound can then be identified as a modulator of nucleic
acid expression based on this comparison and be used, for example
to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[0152] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate Ras-like protein
nucleic acid expression in cells and tissues that express the
Ras-like protein. Experimental data as provided in FIG. 1 indicates
that the Ras-like proteins of the present invention are expressed
in humans in lung small cell carcinomas (as indicated by virtual
northern blot analysis) and in the brain (as indicated by PCR-based
tissue screening panels). Modulation includes both up-regulation
(i.e. activation or agonization) or down-regulation (suppression or
antagonization) of nucleic acid expression.
[0153] Alternatively, a modulator for Ras-like protein nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the Ras-like protein nucleic acid expression in
the cells and tissues that express the protein. Experimental data
as provided in FIG. 1 indicates expression in humans in lung small
cell carcinomas and in the brain.
[0154] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the Ras-like protein gene in clinical trials or in a
treatment regimen. Thus, the gene expression pattern can serve as a
barometer for the continuing effectiveness of treatment with the
compound, particularly with compounds to which a patient can
develop resistance. The gene expression pattern can also serve as a
marker indicative of a physiological response of the affected cells
to the compound. Accordingly, such monitoring would allow either
increased administration of the compound or the administration of
alternative compounds to which the patient has not become
resistant. Similarly, if the level of nucleic acid expression falls
below a desirable level, administration of the compound could be
commensurately decreased.
[0155] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in Ras-like protein nucleic acid,
and particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in Ras-like
protein genes and gene expression products such as mRNA. The
nucleic acid molecules can be used as hybridization probes to
detect naturally occurring genetic mutations in the Ras-like
protein gene and thereby to determine whether a subject with the
mutation is at risk for a disorder caused by the mutation.
Mutations include deletion, addition, or substitution of one or
more nucleotides in the gene, chromosomal rearrangement, such as
inversion or transposition, modification of genomic DNA, such as
aberrant methylation patterns, or changes in gene copy number, such
as amplification. Detection of a mutated form of the Ras-like
protein gene associated with a dysfunction provides a diagnostic
tool for an active disease or susceptibility to disease when the
disease results from overexpression, underexpression, or altered
expression of a Ras-like protein.
[0156] Individuals carrying mutations in the Ras-like protein gene
can be detected at the nucleic acid level by a variety of
techniques. FIG. 3 provides information on SNPs that have been
found in the gene encoding the Ras-like protein of the present
invention. The following variations were seen: A2455C, A2785G,
T3482A, A6189G, T6491C, A7353T, A8688G, G10789C, G11079A, and
A12087G. Some of these SNPs, particularly the SNPs 5' of the ORF
and in the first intron, may affect control/regulatory elements.
The gene encoding the novel Ras-like protein of the present
invention is located on a genome component that has been mapped to
human chromosome 17 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data. Genomic
DNA can be analyzed directly or can be amplified by using PCR prior
to analysis. RNA or cDNA can be used in the same way. In some uses,
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.,
Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364
(1994)), the latter of which can be particularly useful for
detecting point mutations in the gene (see Abravaya et al., Nucleic
Acids Res. 23:675-682 (1995)). 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 a gene under conditions such that
hybridization and amplification of the 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. Deletions and insertions can be
detected by a change in size of the amplified product compared to
the normal genotype. Point mutations can be identified by
hybridizing amplified DNA to normal RNA or antisense DNA
sequences.
[0157] Alternatively, mutations in a Ras-like protein gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0158] Further, sequence-specific ribozymes (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.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0159] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant Ras-like protein gene and a wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing the
diagnostic assays (Naeve, C. W., Biotechniques 19:448 (1995)),
including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.
Biotechnol. 38:147-159 (1993)).
[0160] Other methods for detecting mutations in the 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.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), 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., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include, selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0161] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the Ras-like protein gene in
an individual in order to select an appropriate compound or dosage
regimen for treatment. FIG. 3 provides information on SNPs that
have been found in the gene encoding the Ras-like protein of the
present invention. The following variations were seen: A2455C,
A2785G, T3482A, A6189G, T6491C, A7353T, A8688G, G10789C, G11079A,
and A12087G. Some of these SNPs, particularly the SNPs 5' of the
ORF and in the first intron, may affect control/regulatory
elements.
[0162] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0163] The nucleic acid molecules are thus useful as antisense
constructs to control Ras-like protein gene expression in cells,
tissues, and organisms. A DNA antisense nucleic acid molecule is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
Ras-like protein. An antisense RNA or DNA nucleic acid molecule
would hybridize to the mRNA and thus block translation of mRNA into
Ras-like protein.
[0164] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of Ras-like protein
nucleic acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired Ras-like protein nucleic
acid expression. This technique involves cleavage by means of
ribozymes containing nucleotide sequences complementary to one or
more regions in the mRNA that attenuate the ability of the mRNA to
be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the Ras-like protein, such as ligand
binding.
[0165] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in Ras-like
protein gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired Ras-like protein to treat the individual.
[0166] The invention also encompasses kits for detecting the
presence of a Ras-like protein nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that the Ras-like
proteins of the present invention are expressed in humans in lung
small cell carcinomas (as indicated by virtual northern blot
analysis) and in the brain (as indicated by PCR-based tissue
screening panels). For example, the kit can comprise reagents such
as a labeled or labelable nucleic acid or agent capable of
detecting Ras-like protein nucleic acid in a biological sample;
means for determining the amount of Ras-like protein nucleic acid
in the sample; and means for comparing the amount of Ras-like
protein nucleic acid 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 Ras-like protein
mRNA or DNA.
[0167] Nucleic Acid Arrays
[0168] The present invention further provides arrays or microarrays
of nucleic acid molecules that are based on the sequence
information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).
[0169] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et. al., U.S.
Pat. No. 5,807,522.
[0170] The microarray is preferably composed of a large number of
unique, single-stranded nucleic acid sequences, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides are preferably about 6-60
nucleotides in length, more preferably 15-30 nucleotides in length,
and most preferably about 20-25 nucleotides in length. For a
certain type of microarray, it may be preferable to use
oligonucleotides that are only 7-20 nucleotides in length. The
microarray may contain oligonucleotides that cover the known 5', or
3', sequence, sequential oligonucleotides that cover the
full-length sequence; or unique oligonucleotides selected from
particular areas along the length of the sequence. Polynucleotides
used in the microarray may be oligonucleotides that are specific to
a gene or genes of interest.
[0171] In order to produce oligonucleotides to a known sequence for
a microarray, the gene(s) of interest (or an ORF identified from
the contigs of the present invention) is typically examined using a
computer algorithm that starts at the 5' or at the 3' end of the
nucleotide sequence. Typical algorithms will then identify
oligomers of defined length that are unique to the gene, have a GC
content within a range suitable for hybridization, and lack
predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray. The "pairs" will be
identical, except for one nucleotide that preferably is located in
the center of the sequence. The second oligonucleotide in the pair
(mismatched by one) serves as a control. The number of
oligonucleotide pairs may range from two to one million. The
oligomers are synthesized at designated areas on a substrate using
a light-directed chemical process. The substrate may be paper,
nylon or other type of membrane, filter, chip, glass slide or any
other suitable solid support.
[0172] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0173] In order to conduct sample analysis using a microarray, the
RNA or DNA from a biological sample is made into hybridization
probes. The mRNA is isolated, and cDNA is produced and used as a
template to make antisense RNA (aRNA). The aRNA is amplified in the
presence of fluorescent nucleotides, and labeled probes are
incubated with the microarray so that the probe sequences hybridize
to complementary oligonucleotides of the microarray. Incubation
conditions are adjusted so that hybridization occurs with precise
complementary matches or with various degrees of less
complementarity. After removal of nonhybridized probes, a scanner
is used to determine the levels and patterns of fluorescence. The
scanned images are examined to determine degree of complementarity
and the relative abundance of each oligonucleotide sequence on the
microarray. The biological samples may be obtained from any bodily
fluids (such as blood, urine, saliva, phlegm, gastric juices,
etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be used to measure the absence, presence, and
amount of hybridization for all of the distinct sequences
simultaneously. This data may be used for large-scale correlation
studies on the sequences, expression patterns, mutations, variants,
or polymorphisms among samples.
[0174] Using such arrays, the present invention provides methods to
identify the expression of one or more of the proteins/peptides of
the present invention. In detail, such methods comprise incubating
a test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention. FIG. 3 provides information on SNPs that have been found
in the gene encoding the Ras-like protein of the present invention.
The following variations were seen: A2455C, A2785G, T3482A, A6189G,
T6491C, A7353T, A8688G, G10789C, G11079A, and A12087G. Some of
these SNPs, particularly the SNPs 5' of the ORF and in the first
intron, may affect control/regulatory elements.
[0175] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0176] The test samples of the present invention include cells,
protein or membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0177] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0178] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid. Preferred kits will include chips
that are capable of detecting the expression of 10 or more, 100 or
more, or 500 or more, 1000 or more, or all of the genes expressed
in Human.
[0179] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified Ras-like protein genes of the present invention can be
routinely identified using the sequence information disclosed
herein can be readily incorporated into one of the established kit
formats which are well known in the art, particularly expression
arrays.
[0180] Vectors/Host Cells
[0181] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0182] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0183] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in procaryotic or
eukaryotic cells or in both (shuttle vectors).
[0184] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0185] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0186] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0187] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0188] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0189] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0190] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0191] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0192] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enteroRas-like protein. Typical fusion expression
vectors include pGEX (Smith et al., Gene 67:3140 (1988)), 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. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0193] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has 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).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:2111-2118 (1992)).
[0194] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0195] The nucleic acid molecules can also 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., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0196] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0197] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance,
propagation, or expression of the nucleic acid molecules described
herein. These are found for example 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.
[0198] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0199] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0200] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those 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).
[0201] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced, or joined to the nucleic acid
molecule vector.
[0202] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0203] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0204] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0205] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as kinases, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0206] Where the peptide is not secreted into the medium, which is
typically the case with kinases, the protein can be isolated from
the host cell by standard disruption procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0207] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0208] Uses of Vectors and Host Cells
[0209] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a Ras-like protein polypeptide that can be further
purified to produce desired amounts of Ras-like protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0210] Host cells are also useful for conducting cell-based assays
involving the Ras-like protein or Ras-like protein fragments. Thus,
a recombinant host cell expressing a native Ras-like protein is
useful for assaying compounds that stimulate or inhibit Ras-like
protein function.
[0211] Host cells are also useful for identifying Ras-like protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant Ras-like protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native Ras-like protein.
[0212] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. 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 in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a Ras-like protein and
identifying and evaluating modulators of Ras-like protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0213] A transgenic animal can be produced by introducing 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. Any of the
Ras-like protein nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0214] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
Ras-like protein to particular cells.
[0215] 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 transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0216] 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. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). 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 is
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.
[0217] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) 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 born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0218] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect ligand binding, Ras-like protein activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo Ras-like protein function,
including ligand interaction, the effect of specific mutant
Ras-like proteins on Ras-like protein function and ligand
interaction, and the effect of chimeric Ras-like proteins. It is
also possible to assess the effect of null mutations, which is
mutations that substantially or completely eliminate one or more
Ras-like protein functions.
[0219] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention, which are obvious to those skilled in the field of
molecular biology or related fields, are intended to be within the
scope of the following claims.
* * * * *
References