U.S. patent application number 09/888358 was filed with the patent office on 2002-08-29 for cgi-69 compositions and methods of use.
Invention is credited to Adams, Sean H., Lewin, David, Yu, Xing Xian.
Application Number | 20020119137 09/888358 |
Document ID | / |
Family ID | 22794566 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119137 |
Kind Code |
A1 |
Adams, Sean H. ; et
al. |
August 29, 2002 |
CGI-69 compositions and methods of use
Abstract
An isolated polypeptide comprising an amino acid sequence having
at least 80% sequence identity to the sequence SEQ ID NO:1,
polynucleotides encoding these peptides, and antibodies to the
polypeptides are useful in treating metabolic disorders or
disorders associated with changes in adipose tissue physiological
function or mass.
Inventors: |
Adams, Sean H.; (Randolph
Township, NJ) ; Lewin, David; (New Haven, CT)
; Yu, Xing Xian; (San Mateo, CA) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Family ID: |
22794566 |
Appl. No.: |
09/888358 |
Filed: |
June 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60213307 |
Jun 22, 2000 |
|
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Current U.S.
Class: |
424/94.4 ;
435/189; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 3/04 20180101; C12Q
1/6883 20130101; G01N 2800/52 20130101; A61K 38/00 20130101; C07K
14/47 20130101; A61K 48/00 20130101; G01N 33/5035 20130101; C07K
2319/43 20130101; C12Q 1/6809 20130101; C07K 2319/00 20130101; G01N
33/502 20130101; G01N 33/5079 20130101; A01K 2217/05 20130101; C12Q
2600/158 20130101; A61P 3/00 20180101; G01N 33/5008 20130101 |
Class at
Publication: |
424/94.4 ;
435/69.1; 435/189; 435/325; 435/320.1; 536/23.2 |
International
Class: |
A61K 038/44; C07H
021/04; C12N 009/02; C12P 021/02; C12N 005/06 |
Claims
1. An isolated CGI-69 nucleic acid comprising: (a) a nucleic acid
sequence comprising at least 80% identity with SEQ ID NO:1,
excluding the CGI-69 nucleic acid having the sequence of SEQ ID
NO:2; or (b) a complement of the nucleic acid sequence of (a).
2. The isolated CGI-69 nucleic acid of claim 1 comprising: (a) a
nucleic acid sequence comprising at least 80% identity with a
fragment of SEQ ID NO:1 consisting of nucleotide 14 to nucleotide
1093, excluding the CGI-69 nucleic acid having the sequence of SEQ
ID NO:2 consisting of nucleotide 118 to nucleotide 1173; or (b) a
complement of the nucleic acid sequence of (a).
3. The isolated CGI-69 nucleic acid of claim 1, wherein the nucleic
acid sequence encodes a polypeptide comprising at least 80%
identity with the polypeptide of SEQ ID NO:3, excluding the
polypeptide of SEQ ID NO:4, wherein the polypeptide comprises at
least one biological activity of the polypeptide of SEQ ID
NO:3.
4. The isolated CGI-69 nucleic acid of claim 3, wherein biological
activity comprises mitochondrial localization.
5. A vector comprising the isolated CGI-69 nucleic acid of claim
1.
6. The vector of claim 5, wherein the vector is an expression
vector comprising the CGI-69 nucleic acid of claim 1 operably
linked to a promoter.
7. The vector of claim 6, wherein the promoter is recognized by a
mammalian cell transformed with the vector.
8. A host cell comprising the vector of claim 5.
9. The host cell of claim 8, wherein the cell is a 293 cell.
10. An isolated CGI-69 polypeptide comprising an amino acid
sequence having at least 80% sequence identity to the sequence of
SEQ ID NO:3, excluding the CGI-69 polypeptide having the sequence
of SEQ ID NO:4.
11. The isolated CGI-69 polypeptide of claim 10, wherein said
polypeptide is a biologically active CGI-69 polypeptide.
12. The isolated CGI-69 polypeptide of claim 10, wherein said amino
acid sequence has at least 90% sequence identity to the sequence of
SEQ ID NOS:3.
13. A CGI-69 fusion protein comprising a polypeptide fused to the
carboxy-terminus of a polypeptide comprising an amino acid sequence
having at least 80% sequence identity to the sequence of SEQ ID
NO:3.
14. The CGI-69 fusion polypeptide of claim 13, wherein the fusion
protein acts as an uncoupling protein.
15. The CGI-69 fusion polypeptide of claim 14, wherein the
polypeptide fused to the carboxy-terminus is negatively
charged.
16. The CGI-69 fusion polypeptide of claim 14, wherein the
polypeptide fused to the carboxy-terminus comprises the sequence of
SEQ ID NO:17.
17. An antibody that specifically binds to the polypeptide of claim
10.
18. A method of treating a metabolic disorder comprising modulating
the activity of CGI-69.
19. The method of claim 18, wherein said modulating activity of
CGI-69 comprises decreasing the activity of CGI-69.
20. The method of claim 19, wherein said decreasing activity
comprises decreasing the expression of CGI-69.
21. The method of claim 20, wherein said metabolic disorder is
selected from the group consisting of cachexia, tumors, cancers,
viral infections and bacterial infections.
22. The method of claim 18, wherein said modulating activity of
CGI-69 comprises increasing the activity of CGI-69.
23. The method of claim 22, wherein said increasing activity
comprises increasing the expression of CGI-69.
24. The method of claim 22, wherein said metabolic disorder is
selected from the group consisting of obesity, tumors, cancers,
viral infections and bacterial infections.
25. A method for determining whether a compound up-regulates or
down-regulates expression of a CGI-69 gene in a cell, comprising:
(a) contacting the cell with said compound; and (b) detecting
expression of the gene.
26. The method of claim 25, wherein mRNA encoding CGI-69 is
detected.
27. The method of claim 25, wherin a CGI-69 polypeptide is
detected.
28. The method of claim 25, wherin said composition is a cell.
29. A transgenic non-human animal, having a disrupted CGI-69
gene.
30. The transgenic non-human animal of claim 29, wherein the
non-human animal is a mouse.
31. A transgenic non-human animal, comprising a transgene having at
least 80% sequence identity to the sequence of SEQ ID NO:1 or a
complement of said sequence.
32. A method of screening for a mutation in CGI-69 comprising
comparing a nucleic acid sequence to the sequence of SEQ ID Nos:1
or 2.
33. A method of measuring CGI-69 agonist or antagonist activity of
a compound comprising: (a) contacting a composition comprising
CGI-69 activity with the compound; and (b) determining a change in
the CGI-69 activity.
34. The method of claim 33, wherein the composition is a cell.
Description
RELATED APPLICATION
[0001] This application claims priority to U.S. provisional
application 60/213,307, filed Jun. 22, 2000, which is incorporated
herein by reference in its entirety.
BACKGROUND
[0002] Metabolic diseases represent a serious public health concern
worldwide.
[0003] Obese and overweight individuals account for up to half of
the population of the United States, and the incidence of obesity
has risen at an alarming rate over the last decade. Compared to
lean individuals, overweight persons are particularly susceptible
to an array of disorders, including heart disease, high blood
pressure, Type II diabetes/insulin resistance, stroke, and others
(Must et al., 1999). The etiology of excess weight gain is complex
and incompletely understood, but in general, approximately equal
contribution of genetics and environmental/social factors occurs
with respect to weight gain or loss (Bray, 1997; Hill and Peters,
1998).
[0004] In persons developing overweight or obese conditions, these
factors underlie an imbalance between appetite/caloric intake and
energy expenditure. Thus, novel strategies that improve energy
balance through modulation of appetite and/or metabolic rate are
useful to correct relevant diseases including obesity-related
disorders such as insulin resistance/diabetes, circulatory system
anomalies, etc. Strategies to thwart excessive adipose tissue
growth and accretion may also help in the treatment of obesity, or
in the treatment of other diseases associated with the presence of
inappropriately high amounts of this tissue globally or locally
(i.e., lipomas, phaeochromocytomas, hibemomas). Innovative clinical
treatments, including improving the circulating or tissue levels of
triglycerides, cholesterol, glucose, insulin, leptin or other
metabolically-relevant molecules, that help normalize one or more
of the altered factors concomitant with metabolic derangement would
also have tremendous value.
[0005] Increased risks of mortality and morbidity associated with
perturbations of metabolism are not confined to the obese,
overweight, or diabetic states, however. Cachexia, the loss of
appetite, leading to fewer calories taken in compared to caloric
requirements, is a feature of numerous disease states, including
certain cancers, some viral infections (i.e., acquired
immunodeficiency syndrome, AIDS), or bacterial infections (i.e.,
during some stages of sepsis). The clinical prognosis is poor for
patients who drift into negative energy balance (Tisdale, 1997),
and thus there is a need for new treatments that counteract
cachexia. Furthermore, conditions in which energy expenditure is
abnormally elevated can benefit from novel modalities that modulate
metabolism. In severe bums, for instance, the metabolic rate can
rise almost two-fold, making administration of appropriate
nutrition a tremendous challenge (Goldstein and Elwyn, 1989; Kinney
et al., 1970). Finally, poor outcomes from diseases characterized
by excessive adipose tissue loss (i.e., lipoatrophic disorders) are
common.
[0006] Treatment of metabolic disease may be in part effected
through the innovative use of certain molecules as drugs or as
targets of pharmaceutical intervention. However, there is also a
pressing need to discover molecules that may be used in creative
diagnostic and/or predictive strategies associated with metabolic
disease. For instance, alterations in the expression of certain
genes and proteins may underlie or mark the progression of
metabolic diseases such as obesity. Thus, analysis of the
expression of certain genes and proteins in afflicted patients
compared to a normal population will assist in unraveling the
etiology of their disease, thereby helping in the design of
effective therapeutic strategies. Normal patients may be screened
for expression in cases in which expression is altered prior to the
onset of disease, thus allowing for pre-emptive treatments that can
limit metabolic or other disease progression. Furthermore, changes
in the gene or protein sequences in certain populations may be
associated with disease, hence illustrating the need to discover
genes/proteins relevant to metabolic disorders and whose sequences
lead to biological changes that predispose to metabolic disease, or
are in fact predictive of the progression of disease. Finally,
knowledge of such unique genes/proteins will enable tracking of the
efficacy of therapeutic modalities designed to treat metabolic
diseases. The same case may be made for the use of expression and
sequence analysis to monitor or predict the outcome of other
diseases associated with excessive fat accretion, or those
associated with abnormal fat loss. Discovery of fat-specific genes
and proteins are especially attractive in this regard.
[0007] The bulk of animal tissue oxygen consumption is driven by a
finely-balanced system in which the rate of mitochondrial
catabolism of fuels is regulated largely by the flow of electrons
along the electron-transport chain. Concomitant pumping of protons
outward across the mitochondrial inner membrane establishes a
proton electrochemical gradient or protonmotive force (.DELTA.p),
which drives ATP synthesis via inward flow of protons through
F.sub.1F.sub.0 ATP synthase. Thus, fuel combustion, electron
transport, proton flux, and ATP turnover are intimately coupled.
However, a portion of the .DELTA.p is dissipated as protons flow
inward independent of ATP synthase, a phenomenon termed "proton
leak" or "uncoupling." Fuel combustion and electron
transport/outward proton pumping increase in response to
dissipation of .DELTA.p; thus, innate mitochondrial proton leak may
account for a significant amount of daily energy expenditure
(estimated at between 20-40% of tissue metabolic rate) (Brand, et
al., 1994; Rolfe, et al., 1999). Clarifying the molecular basis of
proton leak is an active area of research, and holds promise in
uncovering target pathways for pharmaceutical intervention to treat
obesity and other diseases arising from perturbations of energy
balance.
[0008] The first clue that specific proteins may underlie mammalian
mitochondrial proton leak emerged from studies of brown adipose
tissue (BAT), a specialized tissue in which a large proportion of
mitochondrial oxygen consumption is uncoupled from ATP synthesis
under conditions in which adaptational thermogenesis is triggered
(i.e. cold-exposure in rodents)(Nichols, et al., 1984).
[0009] Mitochondrial carrier proteins (MCPs) are proteins localized
to the mitochondrial membrane. MCPs facilitate the transport of
molecules across the membrane, such as citrate, protons, and other
ions. The level of activity of the MCPs can affect the level of
animal tissue oxygen consumption. For example, transportation of
protons inward across the membrane would decrease the proton
electrochemical gradient. Although dependent on additional factors,
this decrease would tend to have the effect of decreasing the net
efficiency of ATP synthesis (fuel consumed/ATP yield). An increase
in the activity of a MCP with this functionality would result in a
further decrease in the net efficiency of ATP synthesis. In a
similar fashion, a decrease in the activity of such a MCP would
tend to result in an increase in .DELTA.p, and a corresponding
increase in ATP synthesis. Additionally, an increase in the level
of such a MCP within the mitochondria, for example due to an
up-regulation of mRNA level, would also tend to decrease energetic
efficiency.
[0010] Brown adipose tissue (BAT) is a specialized tissue in which
a large proportion of mitochondrial oxygen consumption is uncoupled
from ATP synthesis under conditions in which adaptational
thermogenesis is triggered (i.e. cold-exposure in rodents)
(Nichols, et al., 1984). The heat-generating futile cycling of the
BAT mitochondrial proton circuit was found to be associated with a
specific protein termed uncoupling protein (UCP, subsequently named
UCP1) (Nichols, et al., 1984; Ricquier, et al., 1991). Uncoupling
proteins (UCPs) are one class of MCPs. Despite confinement of UCP1
to BAT under most conditions, significant proton leak occurs in all
tissues in which it has been measured (Rolfe, et al., 1994),
leading to the possibility that UCPs are present body-wide and
impact whole-animal metabolic rate. To date, four putative UCP
homologs have been identified, with homolog-specific tissue
expression patterns (see Adams, 2000).
SUMMARY
[0011] Using differential display mRNA expression analysis, a
previously-uncharacterized gene was found to be upregulated 2-fold
in brown adipose tissue (BAT) of mice exposed to cold (4.degree.
C.) for 48 hr. Contig and homology analysis revealed that the gene
represents the murine ortholog to a public database sequence
encoding a putative human protein (CGI-69). Isolation of CGI-69
cDNA from human liver revealed variants of CGI-69, including a
previously undescribed nucleic acid encoding a "long version" of
CGI-69 (CGI-69.sub.L). CGI-69 and CGI-69.sub.L are shown herein to
be localized in the mitochondrial membrane. Furthermore, a
carboxy-terminal tagged CGI-69 is shown herein to act as an
uncoupling protein (UCP).
[0012] In one embodiment, the invention provides an isolated
nucleic acid encoding human CGI-69, a CGI-69 variant, a CGI-69
fusion protein, or fragments thereof.
[0013] In one aspect, the invention provides isolated polypeptides
comprising CGI-69, a CGI-69 variant, a CGI-69 fusion protein, or
fragments thereof.
[0014] In another aspect of the invention, isolated CGI-69 nucleic
acids or polypeptides are used in methods to alter respiration in a
cell containing such isolated nucleic acid or polypeptide.
[0015] In yet another aspect of the invention, cells expressing
CGI-69 nucleic acids or polypeptides are used in methods of
screening compounds for the ability to alter CGI-69 acitvity. Such
compounds may be useful in altering respiration in a cell.
[0016] In another embodiment, CGI-69 nucleic acids or polypeptides
are used in methods of altering the metabolism of a patient. Such
methods may be useful for treating metabolic disorders.
[0017] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. In the case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
DESCRIPTION OF THE FIGURE
[0018] FIG. 1 depicts an alignment of human CGI-69 (SEQ ID NO:4),
CGI-69.sub.L (SEQ ID NO:3), and the mouse CGI-69 (SEQ ID NO:18).
Boxes correspond to regions of mitochondrial energy transfer
signature motifs found in UCP homologs that are reasonably
preserved in CGI-69. Mitochodrial carrier domains (Pfam) in human
CGI-69 correspond to residues 10-48, 97-144, 152-276, and 298-340.
Subsequent to the filing of the priority document, the sequence of
human CGI-69.sub.L has been assigned GenBank accession numbers
AF317711 (cDNA) and AF317711.sub.--b 1 (protein).
DETAILED DESCRIPTION
[0019] The present invention relates to a novel characterization of
the putative protein CGI-69 as a mitochondrial carrier protein, the
discovery of the novel splice variant, CGI-69.sub.L, and the
discovery of the alteration of the mitochondrial membrane potential
(.DELTA..psi..sub.m) through the overexpression of the
carboxy-FLAG-tagged CGI-69. Also discussed is the use of the
protein, CGI-69 and CGI-69-encoding nucleic acids in diagnosing or
treating metabolic diseases in humans. The term "CGI-69" includes
splice variants such as CGI-69.sub.L. Novel evidence for an
important function for CGI-69 in modifying the .DELTA..psi..sub.m
in BAT is presented. The white and brown adipose tissue of mammals
are known to play a central role in energy storage and metabolic
signaling, thus impacting appetite, metabolic rate, and energy
balance.
[0020] The mouse ortholog to CGI-69 is up-regulated in mouse BAT
upon exposure to cold ambient temperature (T.sub.a). Based on this
finding, the localization of CGI-69 to the mitochondrial membrane,
and the domains shared with other mitochondrial proteins, it has
now been discovered that CGI-69 is involved in regulating cellular
metabolism. While not being bound by any particular theory, CGI-69
may be involved in cellular thermogenic uncoupling and, therefore,
may be utilized to diagnose and treat specific perturbations in
metabolic pathways underlying obesity and other metabolic
disorders.
[0021] Altering the expression of CGI-69 in a mammal, such as a
human, through gene therapy provides a method of treating metabolic
diseases, such as obesity or cachexia, or of increasing or of
decreasing body weight. The term "metabolic disease" means a
condition in which a disruption of normal energy homeostasis is a
causative or exacerbating factor underlying disease. For example,
metabolic disease includes obesity, wasting disorders such cachexia
(associated with, for example, HIV infection, sepsis and trauma,
and cancer), and diabetes (including Type II diabetes and insulin
resistance).
[0022] Because of the metabolic role of CGI-69 in fat tissues and
metabolism, compounds that have the property of increasing or
decreasing CGI-69 activity are useful. This increase in activity
may be produced in a variety of ways, for example: (1) by
increasing or decreasing the copies of the gene in the cell
(amplifiers and deamplifiers); (2) by increasing or decreasing
transcription of the CGI-69 gene (transcription up-regulators and
down-regulators); (3) by increasing or decreasing the translation
of CGI-69 mRNA into protein (translation up-regulators and
down-regulators); or (4) by increasing or decreasing the activity
of CGI-69 itself (agonists and antagonists).
[0023] Compounds that are amplifiers and deamplifiers of CGI-69 can
be identified by contacting cells or organisms with the compound;
and, then measuring the amount of DNA present that encodes CGI-69
(Ausubel et al., 1987). Compounds that are transcription
up-regulators and down-regulators are identified by contacting
cells or organisms with the compound, and then measuring the amount
of mRNA produced that encodes CGI-69 (Ausubel et al., 1987).
Compounds that are translation up-regulators and down-regulators
are identified by contacting cells or organisms with the compound,
and then measuring the amount of CGI-69 polypeptide produced
(Ausubel et al., 1987).
[0024] Compounds that are amplifiers, transcription up-regulators,
translation up-regulators or agonists, are effective to combat
metabolic diseases that can be ameliorated by increasing CGI-69 or
CGI-69 activity, such as obesity or to decrease weight gain.
Conversely, compounds that are deamplifiers, transcription
down-regulators, translation down-regulators or antagonists, are
effective to combat metabolic diseases that can be ameliorated by
decreasing CGI-69 or CGI-69 activity, such as cachexia, or to
increase weight gain. Gene therapy is another way to up-regulate or
down-regulate transcription and/or translation.
[0025] Both CGI-69 polypeptides and the polynucleotides can be used
in clinical screens to test for metabolic disease etiology, or to
assess the level of risk for these disorders. Tissue samples of a
patient can be examined for the amount of CGI-69 polypeptide or
CGI-69 mRNA. When amounts significantly smaller or larger than
normal are found, they are indicative of metabolic disease or risk
of metabolic disease. A mutated CGI-69 can yield altered activity,
expression/protein levels, and a patient with such a mutation may
have a metabolic disease or be at risk for a metabolic disease.
Finally, determining the amount of expression of CGI-69 in a
mammal, in a tissue sample, or in a tissue culture, can be used to
discover inducers or repressors of the gene.
[0026] High expression of CGI-69 in mouse BAT indicates an
important role for this protein in regulating adipose tissue
physiological function and hence metabolic homeostasis. Modulation
of the protein's activity through agonists or antagonists can
therefore be useful in treating metabolic disorders and disorders
associated with changes in adipose tissue physiological function or
mass. A determination of the abundance of CGI-69 mRNA, CGI-69
protein or CGI-69 biological activity is useful to detect the
progression of diseases characterized by perturbations in cellular
metabolic function. The efficacy of drugs designed to modulate
metabolism, may also be monitored by assessing CGI-69
mRNA/protein/activity levels in clinical samples, which would be
expected to change relative to alterations in metabolism. Finally,
detection of polymorphisms or mutations in the gene or protein in a
patient can be used to predict: (1) the propensity of a person to
develop metabolic disease, (2) disease progression rate/prognosis
for said disorders, or (3) responses to therapeutic modalities
designed to combat metabolic disease.
[0027] Determination of CGI-69 mRNA, proteins or CGI-69 activity
levels in clinical samples has predictive value for tracking
progression of metabolic disorders or in cases in which therapeutic
modalities are applied to correct said disorders. Such
determinations of CGI-69 are useful in screening assays designed to
discover drugs or other therapeutic modalites that modulate
metabolic function.
[0028] Variation in the 3'UTR of gene transcripts can modify mRNA
stability and hence translation. It is possible that patients
suffering from metabolic disorders, or patients destined to have
such afflictions, may display disease-specific patterns of variant
expression. Thus, determination of the CGI-69 variant profile in
clinical samples is of value in diagnosis or prediction of disease
progression, or in assessment of the efficacy of therapeutic
modalites to treat said disorders.
[0029] These embodiments are accomplished by known methods
including those described below.
[0030] Definitions
[0031] Unless defined otherwise, all technical and scientific terms
have the same meaning as is commonly understood by one of skill in
the art to which this invention belongs. The definitions below are
presented for clarity.
[0032] The recommendations of (Demerec et al., 1966) where these
are relevant to genetics are adapted herein. To distinguish between
genes (and related nucleic acids) and the proteins that they
encode, the abbreviations for genes are indicated by italicized (or
underlined) text while abbreviations for the proteins are not
italicized. Thus, CGI-69 or CGI-69 refers to the nucleotide
sequence that encodes CGI-69.
[0033] "Isolated," when referred to a molecule, refers to a
molecule that has been identified and separated and/or recovered
from a component of its natural environment. Contaminant components
of its natural environment are materials that interfere with
diagnostic or therapeutic use.
[0034] 1. Nucleic acid-related definitions
[0035] (a) control sequences
[0036] Control sequences are DNA sequences that enable the
expression of an operably-linked coding sequence in a particular
host organism. Prokaryotic control sequences include promoters,
operator sequences, and ribosome binding sites. Eukaryotic cells
utilize promoters, polyadenylation signals, and enhancers.
[0037] (b) operably-linked
[0038] Nucleic acid is operably-linked when it is placed into a
functional relationship with another nucleic acid sequence. For
example, a promoter or enhancer is operably-linked to a coding
sequence if it affects the transcription of the sequence, or a
ribosome-binding site is operably-linked to a coding sequence if
positioned to facilitate translation. Generally, "operably-linked"
means that the DNA sequences being linked are contiguous, and, in
the case of a secretory leader, contiguous and in reading phase.
However, enhancers do not have to be contiguous. Linking can be
accomplished by conventional recombinant DNA methods.
[0039] (c) isolated nucleic acids
[0040] An isolated nucleic acid molecule is purified from the
setting in which it is found in nature and is separated from at
least one contaminant nucleic acid molecule. Isolated CGI-69
molecules are distinguished from the specific CGI-69 molecule, as
it exists in cells. However, an isolated CGI-69 molecule includes
CGI-69 molecules contained in cells that ordinarily express CGI-69
where, for example, the nucleic acid molecule is in a chromosomal
location different from that of natural cells.
[0041] CGI-69 nucleic acids include those provided in Tables 1
(CGI-69L) and 2 (CGI-69) or a fragment thereof. The start and stop
codons for the encoded protein is bolded and underlined.
1TABLE 1 CGI-69L nucleotide sequence (SEQ ID NO:1) CTGAAGCTTC
AAGATGGCTG ACCAGGACCC TGCGGGCATC AGCCCCCTCC AGCAAATGGT 60
GGCCTCAGGC ACCGGGGCTG TGGTTACCTC TCTCTTCATG ACACCCCTGG ACGTGGTGAA
120 GGTTCGCCTG CAGTCTCAGC GGCCCTCCAT GGCCAGCGAG CTGATGCCTT
CCTCCAGACT 180 GTGGAGCCTC TCCTATACCA AATTGCCCTC CTCTCTCCAA
TCCACAGGGA AGTGCCTCCT 240 GTATTGCAAT GGTGTCCTGG AGCCTCTGTA
CCTGTGCCCA AATGGTGCCC GCTGTGCCAC 300 CTGGTTTCAA GACCCTACCC
GCTTCACTGG CACCATGGAT GCCTTCGTGA AGATCGTGAG 360 GCACGAGGGC
ACCAGGACCC TCTGGAGCGG CCTCCCCGCC ACCCTGGTGA TGACTGTGCC 420
AGCTACCGCC ATCTACTTCA CTGCCTATGA CCAACTGAAG GCCTTCCTGT GTGGTCGAGC
480 CCTGACCTCT GACCTCTACG CACCCATGGT GGCTGGCGCG CTGGCCCGCC
TGGGCACCGT 540 GACTGTGATC AGCCCCCTGG AGCTTATGCG GACAAAGCTG
CAGGCTCAGC ATGTGTCGTA 600 CCGGGAGCTG GGTGCCTGTG TTCGAACTGC
AGTGGCTCAG GGTGGCTGGC GCTCACTGTG 660 GCTGGGCTGG GGCCCCACTG
CCCTTCGAGA TGTGCCCTTC TCAGCCCTGT ACTGGTTCAA 720 CTATGAGCTG
GTGAAGAGCT GGCTCAATGG GCTCAGGCCG AAGGACCAGA CTTCTGTGGG 780
CATGAGCTTT GTGGCTGGTG GCATCTCAGG GACGGTGGCT GCAGTGCTGA CTCTACCCTT
840 TGACGTGGTA AAGACCCAAC GCCAGGTCGC TCTGGGAGCG ATGGAGGCTG
TGAGAGTGAA 900 CCCCCTGCAT GTGGACTCCA CCTGGCTGCT GCTGCGGAGG
ATCCGGGCCG AGTCGGGCAC 960 CAAGGGACTC TTTGCAGGCT TCCTTCCTCG
GATCATCAAG GCTGCCCCCT CCTGTGCCAT 1020 CATGATCAGC ACCTATGAGT
TCGGCAAAAG CTTCTTCCAG AGGCTGAACC AGGACCGGCT 1080 TCTGGGCGGC
TGAAAGGGGC AAGGAGGCAA GGAC 1114
[0042]
2TABLE 2 CGI-69 nucleotide sequence (SEQ ID NO:2) GGCTAGGTGC
GCTGCGAGCG CGCGGAGCCA CGAGGGCGGA CGGACGTAAT GGGCCCGCCT 60
GGCCCTGGGC GCCGCGCCGC ACGAGCACCA GCCTAGAGCC AGGACTGAAG CTTCAAGATG
120 GCTGACCAGG ACCCTGCGGG CATCAGCCCC CTCCAGCAAA TGGTGGCCTC
AGGCACCGGG 180 GCTGTGGTTA CCTCTCTCTT CATGACACCC CTGGACGTGG
TGAAGGTTCG CCTGCAGTCT 240 CAGCGGCCCT CCATGGCCAG CGAGCTGATG
CCTTCCTCCA GACTGTGGAG CCTCTCCTAT 300 ACCAAATGGA AGTGCCTCCT
GTATTGCAAT GGTGTCCTGG AGCCTCTGTA CCTGTGCCCA 360 AATGGTGCCC
GCTGTGCCAC CTGGTTTCAA GACCCTACCC GCTTCACTGG CACCATGGAT 420
GCCTTCGTGA AGATCGTGAG GCACGAGGGC ACCAGGACCC TCTGGAGCGG CCTCCCCGCC
480 ACCCTGGTGA TGACTGTGCC AGCTACCGCC ATCTACTTCA CTGCCTATGA
CCAACTGAAG 540 GCCTTCCTGT GTGGTCGAGC CCTGACCTCT GACCTCTACG
CACCCATGGT GGCTGGCGCG 600 CTGGCCCGCC TGGGCACCGT GACTGTGATC
AGCCCCCTGG AGCTTATGCG GACAAAGCTG 660 CAGGCTCAGC ATGTGTCGTA
CCGGGAGCTG GGTGCCTGTG TTCGAACTGC AGTGGCTCAG 720 GGTGGCTGGC
GCTCACTGTG GCTGGGCTGG GGCCCCACTG CCCTTCGAGA TGTGCCCTTC 780
TCAGCCCTGT ACTGGTTCAA CTATGAGCTG GTGAAGAGCT GGCTCAATGG GTTCAGGCCG
840 AAGGACCAGA CTTCTGTGGG CATGAGCTTT GTGGCTGGTG GCATCTCAGG
GACGGTGGCT 900 GCAGTGCTGA CTCTACCCTT TGACGTGGTA AAGACCCAAC
GCCAGGTCGC TCTGGGAGCG 960 ATGGAGGCTG TGAGAGTGAA CCCCCTGCAT
GTGGACTCCA CCTGGCTGCT GCTGCGGAGG 1020 ATCCGGGCCG AGTCGGGCAC
CAAGGGACTC TTTGCAGGCT TCCTTCCTCG GATCATCAAG 1080 GCTGCCCCCT
CCTGTGCCAT CATGATCAGC ACCTATGAGT TCGGCAAAAG CTTCTTCCAG 1140
AGGCTGAACC AGGACCGGCT TCTGGGCGGC TGAAAGGGGC AAGGAGGCAA GGACCCCGTC
1200 TCTCCCACGG ATGGGGAGAG GGCAGGAGGA GACCCAGCCA AGTGCCTTTT
CCTCAGCACT 1260 GAGGGAGGGG GCTTGTTTCC CTTCCCTCCC GGCGACAAGC
TCCAGGGCAG GGCTGTCCCT 1320 CTGGGCGGCC CAGCACTTCC TCAGACACAA
CTTCTTCCTG CTGCTCCAGT CGTGGGGATC 1380 ATCACTTACC CACCCCCCAA
GTTCAAGACC AAATCTTCCA GCTGCCCCCT TCGTGTTTCC 1440 CTGTGTTTGC
TGTAGCTGGG CATGTCTCCA GGAACCAAGA AGCCCTCAGC CTGGTGTAGT 1500
CTCCCTGACC CTTGTTAATT CCTTAAGTCT AAAGATGATG AACTTC 1556
[0043] 2. Protein-related definitions
[0044] (a) purified polypeptide
[0045] When the molecule is a purified polypeptide, the polypeptide
will be purified (1) to obtain at least 15 residues of N-terminal
or internal amino acid sequence using a sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions
using Coomassie blue or silver stain. Isolated polypeptides include
those expressed heterologously in genetically-engineered cells or
expressed in vitro, since at least one component of the CGI-69
natural environment will not be present. Ordinarily, isolated
polypeptides are prepared by at least one purification step.
[0046] (b) active polypeptide
[0047] An active CGI-69 CGI-69 fragment retains a biological
activity of native or naturally-occurring CGI-69. Biological
activity or refers to a function, either inhibitory or stimulatory,
caused by a native CGI-69. A biological activity of CGI-69
includes, for example, mitochondrial localization.
[0048] CGI-69 polypeptides include the amino acids whose sequences
are provided in Tables 3 (CGI-69L) and 4 (CGI-69) or a fragment
thereof.
[0049] A polypeptide encoded by SEQ ID NO: 1 is presented in Table
3.
3TABLE 3 CGI-69.sub.L polypeptide sequence (SEQ ID NO:3)
MADQDPAGISPLQQMVASGTGAVVTSLFMTPLDVVKVRLQSQR- PSMASELMPS
SRLWSLSYTKLPSSLQSTGKCLLYCNGVLEPLYLCPNGARCATWF- QDPTRFTG
TMDAFVKIVRHEGTRTLWSGLPATLVMTVPATAIYFTAYDQLKAFLC- GRALTS
DLYAPMVAGALARLGTVTVISPLELMRTKLQAQHVSYRELGACVRTAVA- QGGW
RSLWLGWGPTALRDVPFSALYWFNYELVKSWLNGLRPKDQTSVGMSFVAGG- IS
GTVAAVLTLPFDVVKTQRQVALGAMEAVRVNPLHVDSTWLLLRRIRAESGTKG
LFAGFLPRIIKAAPSCAIMISTYEFGKSFFQRLNQDRLLGG
[0050] A polypeptide encoded by SEQ ID NO:2 is presented in Table
4.
4TABLE 4 CGI-69 polypeptide sequence (SEQ ID NO:4)
MADQDPAGISPLQQMVASGTGAVVTSLFMTPLDVVKVRLQSQRPSMASE- LMPS
SRLWSLSYTKWKCLLYCNGVLEPLYLCPNGARCATWFQDPTRFTGTMDAFV- KI
VRHEGTRTLWSGLPATLVMTVPATAIYFTAYDQLKAFLCGRALTSDLYAPMVA
GALARLGTVTVISPLELMRTKLQAQHVSYRELGACVRTAVAQGGWRSLWLGWG
PTALRDVPFSALYWFNYELVKSWLNGFRPKDQTSVGMSFVAGGISGTVAAVLT
LPFDVVKTQRQVALGAMEAVRVNPLHVDSTWLLLRRIRAESGTKGLFAGFLPR
IIKAAPSCAIMISTYEFGKSFFQRLNQDRLLGG
[0051] 1. probes
[0052] Probes are nucleic acid sequences of variable length,
preferably between at least about 10 nucleotides (nt), 100 nt, or
many (e.g., 6,000 nt) depending on the specific use. Probes are
used to detect identical, similar, or complementary nucleic acid
sequences. Longer length probes can be obtained from a natural or
recombinant source, are highly specific, and much slower to
hybridize than shorter-length oligomer probes. Probes may be
single- or double-stranded and designed to have specificity in PCR,
membrane-based hybridization technologies, or ELISA-like
technologies. Probes are substantially purified oligonucleotides
that will hybridize under stringent conditions to at least
optimally 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400
consecutive sense strand nucleotide sequence of SEQ ID NOS:1 or 2;
or an anti-sense strand nucleotide sequence of SEQ ID NOS:1 or 2;
or of a naturally occurring mutant of SEQ ID NOS:1 or 2.
[0053] The full- or partial length native sequence CGI-69 may be
used to "pull out" similar (homologous) sequences (Ausubel et al.,
1987; Sambrook, 1989), such as: (1) full-length or fragments of
CGI-69 cDNA from a cDNA library from any species (e.g. human,
murine, feline, canine, bacterial, viral, retroviral, yeast), (2)
from cells or tissues, (3) variants within a species, and (4)
homologues and variants from other species. To find related
sequences that may encode related genes, the probe may be designed
to encode unique sequences or degenerate sequences. Sequences may
also be genomic sequences including promoters, enhancer elements
and introns of native sequence CGI-69.
[0054] For example, a CGI-69 coding region in another species may
be isolated using such probes. A probe of about 40 bases is
designed, based on CGI-69, and made. To detect hybridizations,
probes are labeled using, for example, radionucleotides such as
.sup.32P or .sup.35S, or enzymatic labels such as alkaline
phosphatase coupled to the probe via avidin-biotin systems. Labeled
probes are used to detect nucleic acids having a complementary
sequence to that of CGI-69 in libraries of cDNA, genomic DNA or
mRNA of a desired species.
[0055] Such probes can be used as a part of a diagnostic test kit
for identifying cells or tissues which mis-express a CGI-69, such
as by measuring a level of CGI-69 in a sample of cells from a
subject e.g., detecting CGI-69 mRNA levels or determining whether a
genomic CGI-69 has been mutated or deleted.
[0056] 2. isolated nucleic acid
[0057] An isolated nucleic acid molecule is separated from other
nucleic acid molecules that are present in the natural source of
the nucleic acid. Preferably, an isolated nucleic acid is free of
sequences that naturally flank the nucleic acid (i.e., sequences
located at the 5'- and 3'-termini of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, isolated CGI-69 molecules can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1
kb of nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell/tissue from which the nucleic
acid is derived (e.g., brain, heart, liver, spleen, etc.).
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 of
chemical precursors or other chemicals when chemically
synthesized.
[0058] A nucleic acid molecule of the CGI-69, e.g., a nucleic acid
molecule having the nucleotide sequence of SEQ ID NOS:1 or 2, or a
complement, can be isolated using standard molecular biology
techniques and the provided sequence information. Using all or a
portion of the nucleic acid sequence of SEQ ID NOS:1 or 2 as a
hybridization probe, CGI-69 molecules can be isolated using
standard hybridization and cloning techniques (Ausubel et al.,
1987; Sambrook, 1989).
[0059] PCR amplification techniques can be used to amplify CGI-69
using cDNA, mRNA or alternatively, genomic DNA, as a template and
appropriate oligonucleotide primers. Such nucleic acids can be
cloned into an appropriate vector and characterized by DNA sequence
analysis. Furthermore, oligonucleotides corresponding to CGI-69
sequences can be prepared by standard synthetic techniques, e.g.,
an automated DNA synthesizer.
[0060] 3. oligonucleotide
[0061] An oligonucleotide comprises a series of linked nucleotide
residues, which oligonucleotide has a sufficient number of
nucleotide bases to be used in a PCR reaction or other application.
A short oligonucleotide sequence may be based on, or designed from,
a genomic or cDNA sequence and is used to amplify, confirm, or
reveal the presence of an identical, similar or complementary DNA
or RNA in a particular cell or tissue. Oligonucleotides comprise
portions of a nucleic acid sequence having about 10 nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one
embodiment of the invention, an oligonucleotide comprising a
nucleic acid molecule less than 100 nt in length would further
comprise at least 6 contiguous nucleotides of SEQ ID NOS: 1 or 2,
or a complement thereof. Oligonucleotides may be chemically
synthesized and may also be used as probes.
[0062] 4. complementary nucleic acid sequences; binding
[0063] In another embodiment, an isolated nucleic acid molecule of
CGI-69 comprises a nucleic acid molecule that is a complement of
the nucleotide sequence shown in SEQ ID NOS:1 or 2, or a portion of
this nucleotide sequence (e.g., a fragment that can be used as a
probe or primer or a fragment encoding a biologically-active
portion of a CGI-69). A nucleic acid molecule that is complementary
to the nucleotide sequence shown in SEQ ID NOS:1 or 2, is one that
is sufficiently complementary to the nucleotide sequence shown in
SEQ ID NOS:1 or 2, that it can hydrogen bond with little or no
mismatches to the nucleotide sequence shown in SEQ ID NOS:1 or 2,
thereby forming a stable duplex.
[0064] "Complementary" refers to Watson-Crick or Hoogsteen base
pairing between nucleotides units of a nucleic acid molecule, and
the term "binding" means the physical or chemical interaction
between two polypeptides or compounds or associated polypeptides or
compounds or combinations thereof. Binding includes ionic,
non-ionic, van der Waals, hydrophobic interactions, and the like. A
physical interaction can be either direct or indirect. Indirect
interactions may be through or due to the effects of another
polypeptide or compound. Direct binding refers to interactions that
do not take place through, or due to, the effect of another
polypeptide or compound, but instead are without other substantial
chemical intermediates.
[0065] 5. Fragments
[0066] Nucleic acid fragments are at least 6 (contiguous) nucleic
acids or at least 4 (contiguous) amino acids, a length sufficient
to allow for specific hybridization in the case of nucleic acids or
for specific recognition of an epitope in the case of amino acids,
respectively, and are at most some portion less than a full-length
sequence. Of course, any length in between is contemplated.
Fragments may be derived from any contiguous portion of a nucleic
acid or amino acid sequence of choice.
[0067] 6. derivatives and analogs
[0068] Derivatives are nucleic acid sequences or amino acid
sequences formed from the native compounds either directly or by
modification or partial substitution. Analogs are nucleic acid
sequences or amino acid sequences that have a structure similar to,
but not identical to, the native compound but differ from it in
respect to certain components or side chains. Analogs may be
synthetic or from a different evolutionary origin and may have a
similar or opposite metabolic activity compared to wild type.
Homologs are nucleic acid sequences or amino acid sequences of a
particular gene that are derived from different species.
[0069] Derivatives and analogs may be full length or other than
full length, if the derivative or analog contains a modified
nucleic acid or amino acid. Derivatives or analogs of the nucleic
acids or proteins of CGI-69 include, but are not limited to,
molecules comprising regions that are substantially homologous to
the nucleic acids or proteins of CGI-69, in various embodiments, by
at least about 70%, 80%, or 95% identity (with a preferred identity
of 80-95%) over a nucleic acid or amino acid sequence of identical
size or when compared to an aligned sequence in which the alignment
is done by a computer homology program known in the art, or whose
encoding nucleic acid is capable of hybridizing to the complement
of a sequence encoding the aforementioned proteins under stringent,
moderately stringent, or low stringent conditions (Ausubel et al.,
1987).
[0070] 7. homology
[0071] A "homologous nucleic acid sequence" or "homologous amino
acid sequence," or variations thereof, refer to sequences
characterized by a homology at the nucleotide level or amino acid
level as discussed above. Homologous nucleotide sequences encode
those sequences coding for isoforms of CGI-69. Isoforms can be
expressed in different tissues of the same organism as a result of,
for example, alternative splicing of RNA. For example, CGI-69.sub.L
is an isoform of CGI-69. Alternatively, different genes can encode
isoforms. For CGI-69, homologous nucleotide sequences include
nucleotide sequences encoding for an CGI-69 of species other than
humans, including, but not limited to: vertebrates, e.g., frog,
mouse, (see FIG. 2), rat, rabbit, dog, cat, cow, horse, and other
organisms. Homologous nucleotide sequences also include, but are
not limited to, naturally occurring allelic variations and
mutations of the nucleotide sequences set forth herein. A
homologous nucleotide sequence does not, however, include the exact
nucleotide sequence encoding human CGI-69. Homologous nucleic acid
sequences include those nucleic acid sequences that encode
conservative amino acid substitutions (see below) in SEQ ID NOS:3
or 4, as well as a polypeptide possessing CGI-69 biological
activity. Various biological activities of the CGI-69 are described
below.
[0072] 8. open reading frames
[0073] The open reading frame (ORF) of a CGI-69 gene encodes CGI-69
or CGI-.sup.69.sub.L. An ORF is a nucleotide sequence that has a
start codon (ATG) and terminates with one of the three "stop"
codons (TAA, TAG, or TGA). In this invention, however, an ORF may
be any part of a coding sequence that may or may not comprise a
start codon and a stop codon. To achieve a unique sequence,
preferable CGI-69 ORFs encode at least 50 amino acids.
[0074] CGI-69 polypeptides
[0075] 1. mature
[0076] A CGI-69 can encode a mature CGI-69. A "mature" form of a
polypeptide or protein disclosed in the present invention is the
product of a naturally occurring polypeptide or precursor form or
proprotein. The naturally occurring polypeptide, precursor or
proprotein includes, by way of nonlimiting example, the full-length
gene product, encoded by the corresponding gene. Alternatively, it
may be defined as the polypeptide, precursor or proprotein encoded
by an open reading frame described herein. The product "mature"
form arises, again by way of nonlimiting example, as a result of
one or more naturally occurring processing steps as they may take
place within the cell, or host cell, in which the gene product
arises. Examples of such processing steps leading to a "mature"
form of a polypeptide or protein include the cleavage of the
N-terminal methionine residue encoded by the initiation codon of an
open reading frame, or the proteolytic cleavage of a signal peptide
or leader sequence. Thus a mature form arising from a precursor
polypeptide or protein that has residues 1 to N, where residue 1 is
the N-terminal methionine, would have residues 2 through N
remaining after removal of the N-terminal methionine.
Alternatively, a mature form arising from a precursor polypeptide
or protein having residues 1 to N, in which an N-terminal signal
sequence from residue 1 to residue M is cleaved, would have the
residues from residue M+1 to residue N remaining. Further as used
herein, a "mature" form of a polypeptide or protein may arise from
a step of post-translational modification other than a proteolytic
cleavage event. Such additional processes include, by way of
non-limiting example, glycosylation, myristoylation or
phosphorylation. In general, a mature polypeptide or protein may
result from the operation of only one of these processes, or a
combination of any of them.
[0077] 2. active
[0078] An active CGI-69 polypeptide or CGI-69 polypeptide fragment
retains a biological activity similar, but not necessarily
identical, to an activity of a naturally-occuring (wild-type)
CGI-69 polypeptide, including mature forms. A particular biological
assay, with or without dose dependency, can be used to determine
CGI-69 activity. A nucleic acid fragment encoding a
biologically-active portion of CGI-69 can be prepared by isolating
a portion of SEQ ID NOS:1 or 2 that encodes a polypeptide having an
CGI-69 biological activity (the biological activities of the CGI-69
are described below), expressing the encoded portion of CGI-69
(e.g., by recombinant expression in vitro) and assessing the
activity of the encoded portion of CGI-69. Biological activity
refers to a function, either inhibitory or stimulatory, caused by a
native CGI-69, for example, mitochondrial localization.
[0079] CGI-69 nucleic acid variants and hybridization
[0080] 1. variant polynucleotides, genes and recombinant genes
[0081] In addition to the CGI-69 sequences shown in SEQ ID NOS:1 or
2, DNA sequence polymorphisms that change the amino acid sequences
of the CGI-69 may exist within a population. For example, allelic
variation among individuals will exhibit genetic polymorphism in
CGI-69. The terms "gene" and "recombinant gene" refer to nucleic
acid molecules comprising an open reading frame (ORF) encoding
CGI-69, preferably a vertebrate CGI-69. Such natural allelic
variations can typically result in 1-5% variance in CGI-69. "CGI-69
variant polynucleotide" or "CGI-69 variant nucleic acid sequence"
means a nucleic acid molecule which encodes an active CGI-69 that
(1) has at least about 80% nucleic acid sequence identity with a
nucleotide acid sequence encoding a full-length native CGI-69, (2)
a full-length native CGI-69 lacking the signal peptide, or (3) any
other fragment of a full-length CGI-69. Ordinarily, an CGI-69
variant polynucleotide will have at least about 80% nucleic acid
sequence identity, more preferably at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% nucleic acid sequence identity and yet more preferably at
least about 99% nucleic acid sequence identity with the nucleic
acid sequence encoding a full-length native CGI-69. A CGI-69
variant polynucleotide may encode full-length native CGI-69 lacking
the signal peptide, or any other fragment of a full-length CGI-69.
Variants do not encompass the native nucleotide sequence.
[0082] Ordinarily, CGI-69 variant polynucleotides are at least
about 30 nucleotides in length, often at least about 60, 90, 120,
150, 180, 210, 240, 270, 300, 450, 600 nucleotides in length, more
often at least about 900 nucleotides in length, or more.
[0083] "Percent (%) nucleic acid sequence identity" with respect to
CGI-69-encoding nucleic acid sequences identified herein is defined
as the percentage of nucleotides in CGI-69 that are identical with
the nucleotides in a candidate sequence of interest, after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of
determining % nucleic acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared.
[0084] When nucleotide sequences are aligned, the % nucleic acid
sequence identity of a given nucleic acid sequence C to, with, or
against a given nucleic acid sequence D (which can alternatively be
phrased as a given nucleic acid sequence C that has or comprises a
certain % nucleic acid sequence identity to, with, or against a
given nucleic acid sequence D) can be calculated as follows:
% nucleic acid sequence identity=W/Z.multidot.100
[0085] where
[0086] W is the number of nucleotides scored as identical matches
by the sequence alignment program's or algorithm's alignment of C
and D
[0087] and
[0088] Z is the total number of nucleotides in D.
[0089] When the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence D, the % nucleic acid sequence
identity of C to D will not equal the % nucleic acid sequence
identity of D to C.
[0090] 2. Stringency
[0091] Homologs (i.e., nucleic acids encoding CGI-69 derived from
species other than human) or other related sequences (e.g.,
paralogs) can be obtained by low, moderate or high stringency
hybridization with all or a portion of the particular human
sequence as a probe using methods well known in the art for nucleic
acid hybridization and cloning.
[0092] The specificity of single stranded DNA to hybridize
complementary fragments is determined by the "stringency" of the
reaction conditions. Hybridization stringency increases as the
propensity to form DNA duplexes decreases. In nucleic acid
hybridization reactions, the stringency can be chosen to either
favor specific hybridizations (high stringency), which can be used
to identify, for example, full-length clones from a library.
Less-specific hybridizations (low stringency) can be used to
identify related, but not exact, DNA molecules (homologous, but not
identical) or segments.
[0093] DNA duplexes are stabilized by: (1) the number of
complementary base pairs, (2) the type of base pairs, (3) salt
concentration (ionic strength) of the reaction mixture, (4) the
temperature of the reaction, and (5) the presence of certain
organic solvents, such as formamide which decreases DNA duplex
stability. In general, the longer the probe, the higher the
temperature required for proper annealing. A common approach is to
vary the temperature: higher relative temperatures result in more
stringent reaction conditions. (Ausubel et al., 1987) provide an
excellent explanation of stringency of hybridization reactions.
[0094] To hybridize under "stringent conditions" describes
hybridization protocols in which nucleotide sequences at least 60%
homologous to each other remain hybridized. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength and pH. The Tm is the temperature (under defined
ionic strength, pH and nucleic acid concentration) at which 50% of
the probes complementary to the target sequence hybridize to the
target sequence at equilibrium. Since the target sequences are
generally present at excess, at Tm, 50% of the probes are occupied
at equilibrium.
[0095] (a) high stringency
[0096] "Stringent hybridization conditions" conditions enable a
probe, primer or oligonucleotide to hybridize only to its target
sequence. Stringent conditions are sequence-dependent and will
differ. Stringent conditions comprise: (1) low ionic strength and
high temperature washes (e.g. 15 mM sodium chloride, 1.5 mM sodium
citrate, 0.1% sodium dodecyl sulfate at 50.degree. C.); (2) a
denaturing agent during hybridization (e.g. 50% (v/v) formamide,
0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone,
50 mM sodium phosphate buffer (pH 6.5; 750 mM sodium chloride, 75
mM sodium citrate at 42.degree. C.); or (3) 50% formamide. Washes
typically also comprise 5.times.SSC (0.75 M NaCl, 75 mM sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C. Preferably, the conditions are
such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%,
98%, or 99% homologous to each other typically remain hybridized to
each other. These conditions are presented as examples and are not
meant to be limiting.
[0097] (b) moderate stringency
[0098] "Moderately stringent conditions" use washing solutions and
hybridization conditions that are less stringent (Sambrook, 1989),
such that a polynucleotide will hybridize to the entire, fragments,
derivatives or analogs of SEQ ID NOS:1 or 2. One example comprises
hybridization in 6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS
and 100 mg/ml denatured salmon sperm DNA at 55.degree. C., followed
by one or more washes in 1.times.SSC, 0.1% SDS at 37.degree. C. The
temperature, ionic strength, etc., can be adjusted to accommodate
experimental factors such as probe length. Other moderate
stringency conditions have been described (Ausubel et al., 1987;
Kriegler, 1990).
[0099] (c) low stringency
[0100] "Low stringent conditions" use washing solutions and
hybridization conditions that are less stringent than those for
moderate stringency (Sambrook, 1989), such that a polynucleotide
will hybridize to the entire, fragments, derivatives or analogs of
SEQ ID NOS:1 or 2,. A non-limiting example of low stringency
hybridization conditions are hybridization in 35% formamide,
5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40.degree. C., followed by one or more
washes in 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%
SDS at 50.degree. C. Other conditions of low stringency, such as
those for cross-species hybridizations are well-described (Ausubel
et al., 1987; Kriegler, 1990; Shilo and Weinberg, 1981).
[0101] 3. Conservative mutations
[0102] In addition to naturally-occurring allelic variants of
CGI-69, changes can be introduced by mutation into SEQ ID NOS:1 or
2 that incur alterations in the amino acid sequences of the encoded
CGI-69 that do not alter CGI-69 function. For example, nucleotide
substitutions leading to amino acid substitutions at
"non-essential" amino acid residues can be made in the sequence of
SEQ ID NOS:3 or 4. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequences of the
CGI-69 without altering biological activity, whereas an "essential"
amino acid residue is required for such biological activity. For
example, amino acid residues that are conserved among the CGI-69 of
the invention are predicted to be particularly non-amenable to
alteration. Amino acids for which conservative substitutions can be
made are well known in the art.
[0103] Useful conservative substitutions are shown in Table A,
"Preferred substitutions." Conservative substitutions whereby an
amino acid of one class is replaced with another amino acid of the
same type fall within the scope of the invention so long as the
substitution does not materially alter the biological activity of
the compound. If such substitutions result in a change in
biological activity, then more substantial changes, indicated in
Table B as exemplary, are introduced and the products screened for
CGI-69 polypeptide biological activity.
5TABLE A Preferred substitutions Original residue Exemplary
substitutions Preferred substitutions Ala (A) Val, Leu, Ile Val Arg
(R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu
Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro,
Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala,
Phe, Leu Norleucine Leu (L) Norleucine, Ile, Val, Met, Ile Ala, Phe
Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu,
Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser
Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V)
Ile, Leu, Met, Phe, Ala, Leu Norleucine
[0104] Non-conservative substitutions that effect (1) the structure
of the polypeptide backbone, such as a .beta.-sheet or
.alpha.-helical conformation, (2) the charge (3) hydrophobicity, or
(4) the bulk of the side chain of the target site can modify CGI-69
polypeptide function. Residues are divided into groups based on
common side-chain properties as denoted in Table B.
Non-conservative substitutions entail exchanging a member of one of
these classes for another class. Substitutions may be introduced
into conservative substitution sites or more preferably into
non-conserved sites.
6TABLE B Amino acid classes Class Amino acids hydrophobic
Norleucine, Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser,
Thr acidic Asp, Glu basic Asn, Gln, His, Lys, Arg disrupt chain
Gly, Pro conformation aromatic Trp, Tyr, Phe
[0105] The variant polypeptides can be made using methods known in
the art such as oligonucleotide-mediated (site-directed)
mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette
mutagenesis, restriction selection mutagenesis (Wells et al., 1985)
or other known techniques can be performed on the cloned DNA to
produce the CGI-69 variant DNA (Ausubel et al., 1987; Sambrook,
1989).
[0106] In one embodiment, the isolated nucleic acid molecule
comprises a nucleotide sequence encoding a protein, wherein the
protein comprises an amino acid sequence at least about 45%,
preferably 60%, more preferably 70%, 80%, 90%, and most preferably
about 95% homologous to SEQ ID NOS:3 or 4.
[0107] 4. Anti-sense nucleic acids
[0108] Using antisense and sense CGI-69 oligonucleotides can
prevent CGI-69 polypeptide expression. These oligonucleotides bind
to target nucleic acid sequences, forming duplexes that block
transcription or translation of the target sequence by enhancing
degradation of the duplexes, terminating prematurely transcription
or translation, or by other means.
[0109] Antisense or sense oligonucleotides are singe-stranded
nucleic acids, either RNA or DNA, which can bind target CGI-69 mRNA
(sense) or CGI-69 DNA (antisense) sequences. Anti-sense nucleic
acids can be designed according to Watson and Crick or Hoogsteen
base pairing rules. The anti-sense nucleic acid molecule can be
complementary to the entire coding region of CGI-69 mRNA, but more
preferably, to only a portion of the coding or noncoding region of
CGI-69 mRNA. For example, the anti-sense oligonucleotide can be
complementary to the region surrounding the translation start site
of CGI-69 mRNA. Antisense or sense oligonucleotides may comprise a
fragment of the CGI-69 DNA coding region of at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. In
general, antisense RNA or DNA molecules can comprise at least 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100 bases in length or more. Among others, (Stein and Cohen,
1988; van der Krol et al., 1988b) describe methods to derive
antisense or a sense oligonucleotides from a given cDNA
sequence.
[0110] Examples of modified nucleotides that can be used to
generate the anti-sense nucleic acid include: 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the anti-sense nucleic acid can
be produced biologically using an expression vector into which a
nucleic acid has been subcloned in an anti-sense orientation such
that the transcribed RNA will be complementary to a target nucleic
acid of interest.
[0111] To introduce antisense or sense oligonucleotides into target
cells (cells containing the target nucleic acid sequence), any gene
transfer method may be used. Examples of gene transfer methods
include (1) biological, such as gene transfer vectors like
Epstein-Barr virus or conjugating the exogenous DNA to a
ligand-binding molecule, (2) physical, such as electroporation and
injection, and (3) chemical, such as CaPO.sub.4 precipitation and
oligonucleotide-lipid complexes.
[0112] An antisense or sense oligonucleotide is inserted into a
suitable gene transfer retroviral vector. A cell containing the
target nucleic acid sequence is contacted with the recombinant
retroviral vector, either in vivo or ex vivo. Examples of suitable
retroviral vectors include those derived from the murine retrovirus
M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy
vectors designated DCT5A, DCT5B and DCT5C (WO 90/13641, 1990). To
achieve sufficient nucleic acid molecule transcription, vector
constructs in which the transcription of the anti-sense nucleic
acid molecule is controlled by a strong pol II or pol III promoter
are preferred. Alternatively, inducible promoters may be preferred
when it is desired to control the expression of the construct.
[0113] To specify target cells in a mixed population of cells, cell
surface receptors that are specific to the target cells can be
exploited. Antisense and sense oligonucleotides are conjugated to a
ligand-binding molecule, as described in (WO 91/04753, 1991).
Ligands are chosen for receptors that are specific to the target
cells. Examples of suitable ligand-binding molecules include cell
surface receptors, growth factors, cytokines, or other ligands that
bind to cell surface receptors or molecules. Preferably,
conjugation of the ligand-binding molecule does not substantially
interfere with the ability of the receptors or molecule to bind the
ligand-binding molecule conjugate, or block entry of the sense or
antisense oligonucleotide or its conjugated version into the
cell.
[0114] Liposomes efficiently transfer sense or an antisense
oligonucleotide to cells (WO 90/10448, 1990). The sense or
antisense oligonucleotide-lipid complex is preferably dissociated
within the cell by an endogenous lipase.
[0115] The anti-sense nucleic acid molecule of CGI-69 may be an
.alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .alpha.-units,
the strands run parallel to each other (Gautier et al., 1987). The
anti-sense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al., 1987a) or a chimeric
RNA-DNA analogue (Inoue et al., 1987b).
[0116] In one embodiment, an anti-sense nucleic acid of CGI-69 is a
ribozyme.
[0117] Ribozymes are catalytic RNA molecules with ribonuclease
activity that are capable of cleaving a single-stranded nucleic
acid, such as an mRNA, to which they have a complementary region.
Thus, ribozymes, such as hammerhead ribozymes (Haseloff and
Gerlach, 1988) can be used to catalytically cleave CGI-69 mRNA
transcripts and thus inhibit translation. A ribozyme specific for
an CGI-69-encoding nucleic acid can be designed based on the
nucleotide sequence of an CGI-69 cDNA (i.e., SEQ ID NOS:lor 2). For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in an
CGI-69-encoding mRNA (Cech et al., U.S. Pat. No. 5,116,742, 1992;
Cech et al., U.S. Pat. No. 4,987,071, 1991). CGI-69 mRNA can also
be used to select a catalytic RNA having a specific ribonuclease
activity from a pool of RNA molecules (Bartel and Szostak,
1993).
[0118] Alternatively, CGI-69 expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the CGI-69 (e.g., the CGI-69 promoter and/or enhancers)
to form triple helical structures that prevent transcription of the
CGI-69 in target cells (Helene, 1991; Helene et al., 1992; Maher,
1992).
[0119] Modifications of antisense and sense oligonucleotides can
augment their effectiveness. Modified sugar-phosphodiester bonds or
other sugar linkages (WO 91/06629, 1991), increase in vivo
stability by conferring resistance to endogenous nucleases without
disrupting binding specificity to target sequences. Other
modifications can increase the affinities of the oligonucleotides
for their targets, such as covalently linked organic moieties (WO
90/10448, 1990) or poly-(L)-lysine. Other attachments modify
binding specificities of the oligonucleotides for their targets,
including metal complexes or intercalating (e.g. ellipticine) and
alkylating agents.
[0120] For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids
(Hyrup and Nielsen, 1996). "Peptide nucleic acids" or "PNAs" refer
to nucleic acid mimics (e.g., DNA mimics) in that the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs allows for specific hybridization to DNA and RNA under
conditions of low ionic strength. The synthesis of PNA oligomers
can be performed using standard solid phase peptide synthesis
protocols (Hyrup and Nielsen, 1996; Perry-O'Keefe et al.,
1996).
[0121] PNAs of CGI-69 can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as anti-sense or
antigene agents for sequence-specific modulation of gene expression
by inducing transcription or translation arrest or inhibiting
replication. CGI-69 PNAs may also be used in the analysis of single
base pair mutations (e.g., PNA directed PCR clamping; as artificial
restriction enzymes when used in combination with other enzymes,
e.g., S.sub.1 nucleases (Hyrup and Nielsen, 1996); or as probes or
primers for DNA sequence and hybridization (Hyrup and Nielsen,
1996; Perry-O'Keefe et al., 1996).
[0122] PNAs of CGI-69 can be modified to enhance their stability or
cellular uptake. Lipophilic or other helper groups may be attached
to PNAs, PNA-DNA dimmers formed, or the use of liposomes or other
drug delivery techniques. For example, PNA-DNA chimeras can be
generated that may combine the advantageous properties of PNA and
DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and
DNA polymerases) to interact with the DNA portion while the PNA
portion provides high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup and Nielsen, 1996). The
synthesis of PNA-DNA chimeras can be performed (Finn et al., 1996;
Hyrup and Nielsen, 1996). For example, a DNA chain can be
synthesized on a solid support using standard phosphoramidite
coupling chemistry, and modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thy- midine phosphoramidite, can
be used between the PNA and the 5' end of DNA (Finn et al., 1996;
Hyrup and Nielsen, 1996). PNA monomers are then coupled in a
stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al., 1996). Alternatively,
chimeric molecules can be synthesized with a 5' DNA segment and a
3' PNA segment (Petersen et al., 1976).
[0123] The oligonucleotide may include other appended groups such
as peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (Lemaitre et
al., 1987; Letsinger et al., 1989; Tullis, U.S. Pat. No. 4,904,582,
1988) or the blood-brain barrier (e.g., (Pardridge and Schimmel,
WO89/10134,1989)). In addition, oligonucleotides can be modified
with hybridization-triggered cleavage agents (van der Krol et al.,
1988a) or intercalating agents (Zon, 1988). The oligonucleotide may
be conjugated to another molecule, e.g., a peptide, a hybridization
triggered cross-linking agent, a transport agent, a
hybridization-triggered cleavage agent, and the like.
[0124] CGI-69 polypeptides
[0125] One aspect of the invention pertains to isolated CGI-69, and
biologically-active portions derivatives, fragments, analogs or
homologs thereof. In one embodiment, native CGI-69 can be isolated
from cells or tissue sources by an appropriate purification scheme
using standard protein purification techniques. In another
embodiment, CGI-69 molecules are produced by recombinant DNA
techniques. Alternative to recombinant expression, a CGI-69 or
polypeptide can be synthesized chemically using standard peptide
synthesis techniques.
[0126] 1. Polypeptides
[0127] A CGI-69 polypeptide includes the amino acid sequence of
CGI-69 which sequences are provided in SEQ ID NOS:3 or 4. The
invention also includes a mutant or variant protein any of which
residues may be changed from the corresponding residues shown in
SEQ ID NOS:3 or 4, while still encoding a protein that maintains
CGI-69 activities and physiological functions, or a functional
fragment thereof.
[0128] 2. Variant CGI-69polypeptides
[0129] In general, an CGI-69 variant preserves CGI-69 -like
function and includes any variant in which residues at a particular
position in the sequence have been substituted by other amino
acids, and further includes the possibility of inserting an
additional residue or residues between two residues of the parent
protein as well as the possibility of deleting one or more residues
from the parent sequence. Any amino acid substitution, insertion,
or deletion is encompassed by the invention. In favorable
circumstances, the substitution is a conservative substitution as
defined above.
[0130] "CGI-69 polypeptide variant" means an active CGI-69
polypeptide having at least: (1) about 80% amino acid sequence
identity with a full-length native sequence CGI-69 polypeptide
sequence or (2) any fragment of a full-length CGI-69 polypeptide
sequence. For example, CGI-69 polypeptide variants include CGI-69
polypeptides wherein one or more amino acid residues are added or
deleted at the N- or C-terminus of the full-length native amino
acid sequence. An CGI-69 polypeptide variant will have at least
about 80% amino acid sequence identity, preferably at least about
81% amino acid sequence identity, more preferably at least about
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% amino acid sequence identity and most preferably
at least about 99% amino acid sequence identity with a full-length
native sequence CGI-69 polypeptide sequence. Ordinarily, CGI-69
variant polypeptides are at least about 10 amino acids in length,
often at least about 20 amino acids in length, more often at least
about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids
in length, or more.
[0131] "Percent (%) amino acid sequence identity" is defined as the
percentage of amino acid residues in CGI-69 that are identical with
amino acid residues in a candidate sequence when the two sequences
are aligned. To determine % amino acid identity, sequences are
aligned and if necessary, gaps are introduced to achieve the
maximum % sequence identity; conservative substitutions are not
considered as part of the sequence identity. Amino acid sequence
alignment procedures to determine percent identity are well known
to those of skill in the art.
[0132] Often publicly available computer software such as BLAST,
BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align
peptide sequences. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared.
[0133] When amino acid sequences are aligned, the % amino acid
sequence identity of a given amino acid sequence A to, with, or
against a given amino acid sequence B (which can alternatively be
phrased as a given amino acid sequence A that has or comprises a
certain % amino acid sequence identity to, with, or against a given
amino acid sequence B) can be calculated as:
% amino acid sequence identity=X/Y.multidot.100
[0134] where
[0135] X is the number of amino acid residues scored as identical
matches by the sequence alignment program's or algorithm's
alignment of A and B
[0136] and
[0137] Y is the total number of amino acid residues in B.
[0138] If the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the % amino acid sequence identity
of A to B will not equal the % amino acid sequence identity of B to
A.
[0139] 3. Isolated/purified polypeptides
[0140] An "isolated" or "purified" polypeptide, protein or
biologically active fragment is separated and/or recovered from a
component of its natural environment. Contaminant components
include materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous materials.
Preferably, the polypeptide is purified to a sufficient degree to
obtain at least 15 residues of N-terminal or internal amino acid
sequence. To be substantially isolated, preparations having less
than 30% by dry weight of non-CGI-69 contaminating material
(contaminants), more preferably less than 20%, 10% and most
preferably less than 5% contaminants. An isolated,
recombinantly-produced CGI-69 or biologically active portion is
preferably substantially free of culture medium, i.e., culture
medium represents less than 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
CGI-69 preparation. Examples of contaminants include cell debris,
culture media, and substances used and produced during in vitro
synthesis of CGI-69.
[0141] 4. Biologically active
[0142] Biologically active portions of CGI-69 include peptides
comprising amino acid sequences sufficiently homologous to or
derived from the amino acid sequences of CGI-69 (SEQ ID NOS:3 or 4)
that include fewer amino acids than the full-length CGI-69, and
exhibit at least one activity of an CGI-69. Biologically active
portions comprise a domain or motif with at least one activity of
native CGI-69. A biologically active portion of a CGI-69 can be a
polypeptide that is, for example, 10, 25, 50, 100 or more amino
acid residues in length. Other biologically active portions, in
which other regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of a native CGI-69.
[0143] Biologically active portions of CGI-69 may have an amino
acid sequence shown in SEQ ID NOS:3 or 4, or substantially
homologous to SEQ ID NOS:3 or 4, and retains the functional
activity of the protein of SEQ ID NOS:3 or 4, yet differs in amino
acid sequence due to natural allelic variation or mutagenesis.
Other biologically active CGI-69 may comprise an amino acid
sequence at least 45% homologous to the amino acid sequence of SEQ
ID NOS:3 or 4, and retains the functional activity of native CGI-69
.
[0144] 5. Chimeric and fusion proteins
[0145] Fusion polypeptides are useful in expression studies,
cell-localization, bioassays, and CGI-69 purification. A CGI-69
"chimeric protein" or "fusion protein" comprises CGI-69 fused to a
non-CGI-69 polypeptide. A non-CGI-69 polypeptide is not
substantially homologous to CGI-69 (SEQ ID NOS:3 or 4). A CGI-69
fusion protein may include any portion to the entire CGI-69,
including any number of the biologically active portions. CGI-69
may be fused to the C-terminus of the GST (glutathione
S-transferase) sequences. Such fusion proteins facilitate the
purification of recombinant CGI-69. In certain host cells, (e.g.
mammalian), heterologous signal sequences fusions may ameliorate
CGI-69 expression and/or secretion. Additional exemplary fusions
are presented in Table C.
[0146] Fusion proteins can be easily created using recombinant
methods. A nucleic acid encoding CGI-69 can be fused in-frame with
a non-CGI-69 encoding nucleic acid, to the CGI-69 NH.sub.2-- or
COO-- -terminus, or internally. Fusion genes may also be
synthesized by conventional techniques, including automated DNA
synthesizers. PCR amplification using anchor primers that give rise
to complementary overhangs between two consecutive gene fragments
that can subsequently be annealed and reamplified to generate a
chimeric gene sequence (Ausubel et al., 1987) is also useful. Many
vectors are commercially available that facilitate sub-cloning
CGI-69 in-frame to a fusion moiety.
[0147] Fusion proteins can be used to give CGI-69 compositions a
novel characteristic not present in native CGI-69. For example,
amino-FLAG-tagged CGI-69 and native CGI-69 have not been shown to
exhibit uncoupling activity. In contrast, overexpression of
carboxy-FLAG-tagged CGI-69 in 293 cells diminished the
mitochondrial membrane potential to a similar magnitude as an
uncoupling protein (UCP3). Other negatively charged sequences may
have a similar biological function.
7TABLE C Useful non-CGI-69 fusion polypeptides Reporter in vitro in
vivo Notes Reference Human Radio- none Expensive, (Selden growth
immuno- insensitive, et al., hormone assay narrow linear 1986)
(hGH) range. .beta.-glucu- Colorimetric, colorimetric sensitive,
(Gallagher, ronidase fluorescent, (histo- broad linear 1992) (GUS)
or chemi- chemical range, non- luminescent staining with iostopic.
X-gluc) Green Fluorescent fluorescent can be used in (Chalfie et
fluorescent live cells; al., 1994) protein resists photo- (GFP) and
bleaching related molecules (RFP, BFP, CGI-69, etc.) Luciferase
bioluminse- Bio- protein is (de Wet et (firefly) cent luminescent
unstable, al., 1987) difficult to reproduce, signal is brief
Chloram- Chromato- none Expensive (Gorman et phenic oal graphy,
radioactive al., 1982) acetyltrans- differential substrates, ferase
(CAT) extraction, time- fluorescent, consuming, or insensitive,
immuno- narrow linear assay range .beta.-galacto- colorimetric,
colorimetric sensitive, (Alam and sidase fluorescence, (histo-
broad linear Cook, 1990) chemi- chemical range; some luminscence
staining with cells have X-gal), bio- high luminescent endogenous
in live cells activity Secrete colorimetric, none Chem- (Berger et
alkaline biolumines- iluminscence al., 1988) phosphatase cent,
chemi- assay is (SEAP) luminescent sensitive and broad linear
range; some cells have endogenouse alkaline phosphatase activity
Tat from Mediates Mediates Exploits (Frankel HIV delivery into
delivery into amino acid et al., cytoplasm cytoplasm residues of
U.S. Pat. and nuclei and nuclei HIV tat No. protein. 5,804,604,
1998)
[0148] Therapeutic applications of CGI-69
[0149] 1. Agonists and antagonists
[0150] "Antagonist" includes any molecule that partially or fully
blocks, inhibits, or neutralizes a biological activity of
endogenous CGI-69. Similarly, "agonist" includes any molecule that
mimics a biological activity of endogenous CGI-69. Biologic
activity biologic and/or immunologic activities of native or
naturally-occurring human OGC. A preferred activity is
mitochondrial localization. Molecules that can act as agonists or
antagonists include fragments or variants of endogenous CGI-69,
peptides, antisense oligonucleotides, small organic molecules,
etc.
[0151] 2. Identifying antagonists and agonists
[0152] To assay for antagonists, CGI-69 is added to, or expressed
in, a cell along with the compound to be screened for a particular
activity. If the compound inhibits the activity of interest in the
presence of the CGI-69, that compound is an antagonist to the
CGI-69; if CGI-69 activity is enhanced, the compound is an
agonist.
[0153] (a) Specific examples ofpotential antagonists and
agonist
[0154] Any molecule that alters CGI-69 cellular effects is a
candidate antagonist or agonist. Screening techniques well known to
those skilled in the art can identify these molecules. Examples of
antagonists and agonists include: (1) small organic and inorganic
compounds, (2) small peptides, (3) Abs and derivatives, (4)
polypeptides closely related to CGI-69, (5) antisense DNA and RNA,
(6) ribozymes, (7) triple DNA helices and (8) nucleic acid
aptamers.
[0155] Small molecules that bind to the CGI-69 active site or other
relevant part of the polypeptide and inhibit the biological
activity of the CGI-69 are antagonists. Examples of small molecule
antagonists include small peptides, peptide-like molecules,
preferably soluble, and synthetic non-peptidyl organic or inorganic
compounds. These same molecules, if they enhance CGI-69 activity,
are examples of agonists.
[0156] Alternatively, a potential antagonist or agonist may be a
closely related protein, for example, a mutated form of the CGI-69
that recognizes an CGI-69-interacting protein but imparts no
effect, thereby competitively inhibiting CGI-69 action.
Alternatively, a mutated CGI-69 may be constitutively activated and
may act as an agonist.
[0157] Antisense RNA or DNA constructs can be effective
antagonists. Antisense RNA or DNA molecules block function by
inhibiting translation by hybridizing to targeted mRNA. Antisense
technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
depend on polynucleotide binding to DNA or RNA. For example, the 5'
coding portion of the CGI-69 sequence is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription (triple helix) (Beal
and Dervan, 1991; Cooney et al., 1988; Lee et al., 1979), thereby
preventing transcription and the production of the CGI-69. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the CGI-69 (antisense)
(Cohen, 1989; Okano et al., 1991). These oligonucleotides can also
be delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of the CGI-69. When
antisense DNA is used, oligodeoxyribonucleotides derived from the
translation-initiation site, e.g., between about -10 and +10
positions of the target gene nucleotide sequence, are
preferred.
[0158] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques (WO
97/33551, 1997; Rossi, 1994).
[0159] To inhibit transcription, triple-helix nucleic acids that
are single-stranded and comprise deoxynucleotides are useful
antagonists. These oligonucleotides are designed such that
triple-helix formation via Hoogsteen base-pairing rules is
promoted, generally requiring stretches of purines or pyrimidines
(WO 97/33551, 1997).
[0160] Aptamers are short oligonucleotide sequences that can be
used to recognize and specifically bind almost any molecule. The
systematic evolution of ligands by exponential enrichment (SELEX)
process (Ausubel et al., 1987; Ellington and Szostak, 1990; Tuerk
and Gold, 1990) is powerful and can be used to find such aptamers.
Aptamers have many diagnostic and clinical uses; almost any use in
which an antibody has been used clinically or diagnostically,
aptamers too may be used. In addition, aptamers are cheaper to make
once they have been identified, and can be easily applied in a
variety of formats, including administration in pharmaceutical
compositions, bioassays and diagnostic tests (Jayasena, 1999).
[0161] CGI-69 recombinant expression vectors and host cells
[0162] Vectors are tools used to shuttle nucleic acids between host
cells or as a means to express a nucleotide sequence. Some vectors
function only in prokaryotes, while others function in both
prokaryotes and eukaryotes, enabling large-scale DNA preparation
from prokaryotes for expression in eukaryotes. Inserting a DNA of
interest, such as CGI-69 nucleotide sequence or a fragment, is
accomplished by ligation techniques and/or mating protocols well
known to the skilled artisan. Such DNA is inserted such that its
integration does not disrupt any necessary components of the
vector. In the case of vectors that are used to express the
inserted DNA protein, the introduced DNA is operably-linked to the
vector elements that govern its transcription and translation.
[0163] Vectors can be divided into two general classes: Cloning
vectors are replicating plasmid or phage with regions that are
non-essential for propagation in an appropriate host cell, and into
which foreign DNA can be inserted; the foreign DNA is replicated
and propagated as if it were a component of the vector. An
expression vector (such as a plasmid, yeast, or animal virus
genome) is used to introduce foreign genetic material into a host
cell or tissue in order to transcribe and translate the foreign
DNA. In expression vectors, the introduced DNA is operably-linked
to elements, such as promoters, that signal to the host cell to
transcribe the inserted DNA. Some promoters are exceptionally
useful, such as inducible promoters that control gene transcription
in response to specific factors. Operably-linking CGI-69 or
anti-sense construct to an inducible promoter can control the
expression of CGI-69 or fragments, or anti-sense constructs.
Examples of classic inducible promoters include those that are
responsive to .alpha.-interferon, heat-shock, heavy metal ions, and
steroids such as glucocorticoids (Kaufman, 1990) and tetracycline.
Other desirable inducible promoters include those that are not
endogenous to the cells in which the construct is being introduced,
but, however, is responsive in those cells when the induction agent
is exogenously supplied.
[0164] Vectors have many difference manifestations. A "plasmid" is
a circular double stranded DNA molecule into which additional DNA
segments can be introduced. Viral vectors can accept additional DNA
segments into the viral genome. Certain vectors are capable of
autonomous replication in a host cell (e.g., episomal mammalian
vectors or bacterial vectors having a bacterial origin of
replication). Other vectors (e.g., non-episomal mammalian vectors)
are integrated into the genome of a host cell upon introduction
into the host cell, and thereby are replicated along with the host
genome. In general, useful expression vectors are often plasmids.
However, other forms of expression vectors, such as viral vectors
(e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses) are contemplated.
[0165] Recombinant expression vectors that comprise CGI-69 (or
fragments) regulate CGI-69 transcription by exploiting one or more
host cell-responsive (or that can be manipulated in vitro)
regulatory sequences that is operably-linked to CGI-69.
"Operably-linked" indicates that a nucleotide sequence of interest
is linked to regulatory sequences such that expression of the
nucleotide sequence is achieved.
[0166] Vectors can be introduced in a variety of organisms and/or
cells (Table D). Alternatively, the vectors can be transcribed and
translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase.
8TABLE D Examples of hosts for cloning or expression Organisms
Examples Sources and References* Prokaryotes Enterobacteriaceae E.
coli K 12 strain MM294 ATCC 31,446 X1776 ATCC 31,537 W3110 ATCC
27,325 K5 772 ATCC 53,635 Enterobacter Erwinia Klebsiella Proteus
Salmonella (S. tyhpimurium) Serratia (S. marcescans) Shigella
Bacilli (B. subtilis and B. licheniformis) Pseudomonas (P.
aeruginosa) Streptomyces Eukaryotes Yeasts Saccharomyces cerevisiae
Schizosaccharomyces pombe Kluyveromyces (Fleer et al., 1991) K.
lactis MW98-8C, (de Louvencourt et al., CBS683, CBS4574 1983) K.
fragilis ATCC 12,424 K. bulgaricus ATCC 16,045 K. wickeramii ATCC
24,178 K. waltii ATCC 56,500 K. drosophilarum ATCC 36,906 K.
thermotolerans K. marxianus; yarrowia (EPO 402226, 1990) Pichia
pastoris (Sreekrishna et al., 1988) Candida Trichoderma reesia
Neurospora crassa (Case et al., 1979) Torulopsis Rhodotorula
Schwanniomyces (S. occidentalis) Filamentous Neurospora Fungi
Penicillium Tolypocladium (WO 91/00357, 1991) Aspergillus (A.
nidulans (Kelly and Hynes, 1985; and A. niger) Tilbum et al., 1983;
Yelton et al., 1984) Invertebrate Drosophila S2 cells Spodoptera
Sf9 Vertebrate Chinese Hamster Ovary cells (CHO) simian COS ATCC
CRL 1651 COS-7 HEK 293 *Unreferenced cells are generally available
from American Type Culture Collection (Manassas, VA).
[0167] Vector choice is dictated by the organism or cells being
used and the desired fate of the vector. Vectors may replicate once
in the target cells, or may be "suicide" vectors. In general,
vectors comprise signal sequences, origins of replication, marker
genes, enhancer elements, promoters, and transcription termination
sequences. The choice of these elements depends on the organisms in
which the vector will be used and are easily determined. Some of
these elements may be conditional, such as an inducible or
conditional promoter that is turned "on" when conditions are
appropriate. Examples of inducible promoters include those that are
tissue-specific, which relegate expression to certain cell types,
steroid-responsive, or heat-shock reactive. Some bacterial
repression systems, such as the lac operon, have been exploited in
mammalian cells and transgenic animals (Fieck et al., 1992;
Wyborski et al., 1996; Wyborski and Short, 1991). Vectors often use
a selectable marker to facilitate identifying those cells that have
incorporated the vector. Many selectable markers are well known in
the art for the use with prokaryotes, usually antibiotic-resistance
genes or the use of autotrophy and auxotrophy mutants.
[0168] Using antisense and sense CGI-69 oligonucleotides can
prevent CGI-69 polypeptide expression. These oligonucleotides bind
to target nucleic acid sequences, forming duplexes that block
transcription or translation of the target sequence by enhancing
degradation of the duplexes, terminating prematurely transcription
or translation, or by other means.
[0169] Antisense or sense oligonucleotides are single-stranded
nucleic acids, either RNA or DNA, which can bind target CGI-69 mRNA
(sense) or CGI-69 DNA (antisense) sequences. According to the
present invention, antisense or sense oligonucleotides comprise a
fragment of the CGI-69 DNA coding region of at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. In
general, antisense RNA or DNA molecules can comprise at least 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100 bases in length or more. Among others, (Stein and Cohen,
1988; van der Krol et al., 1988b) describe methods to derive
antisense or a sense oligonucleotides from a given cDNA
sequence.
[0170] Modifications of antisense and sense oligonucleotides can
augment their effectiveness. Modified sugar-phosphodiester bonds or
other sugar linkages (WO 91/06629, 1991), increase in vivo
stability by conferring resistance to endogenous nucleases without
disrupting binding specificity to target sequences. Other
modifications can increase the affinities of the oligonucleotides
for their targets, such as covalently linked organic moieties (WO
90/10448, 1990) or poly-(L)-lysine. Other attachments modify
binding specificities of the oligonucleotides for their targets,
including metal complexes or intercalating (e.g. ellipticine) and
alkylating agents.
[0171] To introduce antisense or sense oligonucleotides into target
cells (cells containing the target nucleic acid sequence), any gene
transfer method may be used and are well known to those of skill in
the art. Examples of gene transfer methods include 1) biological,
such as gene transfer vectors like Epstein-Barr virus or
conjugating the exogenous DNA to a ligand-binding molecule (WO
91/04753, 1991), 2) physical, such as electroporation, and 3)
chemical, such as CaPO.sub.4 precipitation and
oligonucleotide-lipid complexes (WO 90/10448, 1990).
[0172] The terms "host cell" and "recombinant host cell" are used
interchangeably. Such terms refer not only to a particular subject
cell but also to the progeny or potential progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term.
[0173] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are well known in the art. The choice of host cell
will dictate the preferred technique for introducing the nucleic
acid of interest. Table E, which is not meant to be limiting,
summarizes many of the known techniques in the art. Introduction of
nucleic acids into an organism may also be done with ex vivo
techniques that use an in vitro method of transfection, as well as
established genetic techniques, if any, for that particular
organism.
9TABLE E Methods to introduce nucleic acid into cells Cells Methods
References Notes Prokaryotes Calcium (Cohen et al., 1972;
(bacteria) chloride Hanahan, 1983; Mandel and Higa, 1970)
Electropor- (Shigekawa and Dower, ation 1988) Eukaryotes Calcium
N-(2- Cells may be Mammalian phosphate Hydroxyethyl)piperazine
"shocked" with cells transfection N'-(2-ethanesulfonic acid
glycerol or (HEPES) buffered saline dimethylsulfox- solution (Chen
and ide (DMSO) to Okayama, 1988; Graham increase and van der Eb,
1973; transfection Wigler et al., 1978) efficiency BBS (N,N-bis(2-
(Ausubel et al., hydroxyethyl)-2- 1987). aminoethanesulfonic acid)
buffered solution (Ishiura et al., 1982) Diethyl- (Fujita et al.,
1986; Lopata Most useful for aminoethyl et al., 1984; Selden et
al., transient, but (DEAE)- 1986) not stable, Dextran
transfections. transfection Chloroquine can be used to increase
efficiency. Electropor- (Neumann et al., 1982; Especially ation
Potter, 1988; Potter et al., useful for hard- 1984; Wong and
to-transfect Neumann, 1982) lymphocytes. Cationic (Elroy-Stein
Applicable to lipid reagent and Moss, both in vivo transfection
1990; Felgner et al., 1987; and in vitro Rose et al., 1991; Whitt
et transfection. al., 1990) Retroviral Production exemplified by
Lengthy pro- (Cepko et al., 1984; Miller cess, many and Buttimore,
1986; Pear packaging lines et al., 1993) available at Infection in
vitro and in ATCC. vivo: (Austin and Cepko, Applicable to 1990;
Bodine et al., 1991; both in vivo Fekete and Cepko, 1993; and in
vitro Lemischka et al., 1986; transfection. Turner et al., 1990;
Williams et al., 1984) Polybrene (Chaney et al., 1986; Kawai and
Nishizawa, 1984) Micro- (Capecchi, 1980) Can be used to injection
establish cell lines carrying integrated copies of CGI-69 DNA
sequences. Protoplast (Rassoulzadegan et al., fusion 1982;
Sandri-Goldin et al., 1981; Schaffner, 1980) Insect cells
Baculovirus (Luckow, 1991; Miller, Useful for in (in vitro) systems
1988; O'Reilly et al., vitro produc- 1992) tion of proteins with
eukaryotic modifications. Yeast Electropor- (Becker and Guarente,
ation 1991) Lithium (Gietz et al., 1998; Ito et acetate al., 1983)
Spheroplast (Beggs, 1978; Hinnen et Laborious, can fusion al.,
1978) produce aneuploids. Plant cells Agro- (Bechtold and
Pelletier, [general bacterium 1998; Escudero and Hohn, reference:
trans- 1997; Hansen and Chilton, (Hansen formation 1999; Touraev
and al., and Wright, 1997) 1999)] Biolistics (Finer et al., 1999;
Hansen (micro- and Chilton, 1999; projectiles) Shillito, 1999)
Electropor- (Fromm et al., 1985; Ou- ation Lee et al., 1986; Rhodes
et (protoplasts) al., 1988; Saunders et al., 1989) May be combined
with liposomes (Trick and al., 1997) Polyethylene (Shillito, 1999)
glycol (PEG) treatment Liposomes May be combined with
electroporation (Trick and al., 1997) in planta (Leduc and al.,
1996; micro- Zhou and al., 1983) injection Seed (Trick and al.,
1997) imbibition Laser beam (Hoffman, 1996) Silicon (Thompson and
al., 1995) carbide whiskers
[0174] Vectors often use a selectable marker to facilitate
identifying those cells that have incorporated the vector. Many
selectable markers are well known in the art for the use with
prokaryotes, usually antibiotic-resistance genes or the use of
autotrophy and auxotrophy mutants. Table F lists often-used
selectable markers for mammalian cell transfection.
10TABLE F Useful selectable markers for eukaryote cell transfection
Selectable Marker Selection Action Reference Adenosine Media
includes 9- Conversion of Xyl-A (Kaufman deaminase
.beta.-D-xylofuran- to Xyl-ATP, which et al., 1986) (ADA) osyl
adenine incorporates into (Xyl-A) nucleic acids, killing cells. ADA
detoxifies Dihydrofolate Methotrexate MTX competitive (Simonsen
reductase (MTX) and inhibitor of DHFR. and (DHFR) dialyzed serum In
absence of Levinson, (purine-free exogenous purines, 1983) media)
cells require DHFR, a necessary enzyme in purine biosynthesis.
Aminoglyco- G418 G418, an (Southern side phospho- aminoglycoside
and Berg, transferase detoxified by APH, 1982) ("APH", "neo",
interferes with "G418") ribosomal function and consequently,
translation. Hygromycin-B- hygromycin-B Hygromycin-B, an (Palmer et
phospho- aminocyclitol al., 1987) transferase detoxified by HPH,
(HPH) disrupts protein translocation and promotes mistranslation.
Thymidine Forward selection Forward: (Littlefield, kinase (TK+):
Media Aminopterin forces 1964) (TK) (HAT) incorpor- cells to
synthesze ates aminopterin. dTTP from Reverse selection thymidine,
a pathway (TK-): Media requiring TK. incorporates 5- Reverse: TK
bromodeoxyur- phosphorylates idine (BrdU). BrdU, which incorporates
into nucleic acids, killing cells.
[0175] A prokaryotic or eukaryotic host cell in culture can be used
to produce CGI-69. Accordingly, CGI-69 provides methods for
producing CGI-69 using the host cells. In one embodiment, the
method comprises culturing the host cell (into which a recombinant
expression vector encoding CGI-69 has been introduced) in a
suitable medium, such that CGI-69 is produced. In another
embodiment, the method further comprises isolating CGI-69 from the
medium or the host cell.
[0176] Transgenic CGI-69 animals
[0177] Transgenic animals are useful for studying the function
and/or activity of CGI-69 and for identifying and/or evaluating
modulators of CGI-69 activity. "Transgenic animals" are non-human
animals, preferably mammals, more preferably rodents such as rats
or mice, in which one or more of the cells include a transgene.
Other transgenic animals include primates, sheep, dogs, cows,
goats, chickens, amphibians, etc. A "transgene" is exogenous DNA
that is integrated into the genome of a cell from which a
transgenic animal develops and remains in the genome of the mature
animal. Transgenes preferably direct the expression of an encoded
gene product in one or more cell types or tissues of the transgenic
animal with the purpose of preventing expression of a naturally
encoded gene product in one or more cell types or tissues (a
"knockout" transgenic animal), or serving as a marker or indicator
of an integration, chromosomal location, or region of recombination
(e.g. cre/loxP mice). A "homologous recombinant animal" is a
non-human animal, such as a rodent, in which endogenous CGI-69 has
been altered by an exogenous DNA molecule that recombines
homologously with endogenous CGI-69 in a (e.g. embryonic) cell
prior to development the animal. Host cells with exogenous CGI-69
can be used to produce non-human transgenic animals, such as
fertilized oocytes or embryonic stem cells into which CGI-69-coding
sequences have been introduced. Such host cells can then be used to
create non-human transgenic animals or homologous recombinant
animals.
[0178] 1. Approaches to transgenic animal production
[0179] A transgenic animal can be created by introducing CGI-69
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 (pffa). The CGI-69
cDNA sequences (SEQ ID NO: 1 or 2) can be introduced as a transgene
into the genome of a non-human animal. Alternatively, a homologue
of CGI-69, such as the naturally-occuring variant of CGI-69, can be
used as a transgene. Intronic sequences and polyadenylation signals
can also be included in the transgene to increase transgene
expression. Tissue-specific regulatory sequences can be
operably-linked to the CGI-69 transgene to direct expression of
CGI-69 to particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection, particularly
animals such as mice, have become conventional in the art, e.g.
(Evans et al., U.S. Pat. No. 4,870,009, 1989; Hogan, 0879693843,
1994; Leder and Stewart, U.S. Pat. No. 4,736,866, 1988; Wagner and
Hoppe, U.S. Pat. No. 4,873,191, 1989). Other non-mice transgenic
animals may be made by similar methods. A transgenic founder
animal, which can be used to breed additional transgenic animals,
can be identified based upon the presence of the transgene in its
genome and/or expression of the transgene mRNA in tissues or cells
of the animals. Transgenic (e.g. CGI-69) animals can be bred to
other transgenic animals carrying other transgenes.
[0180] A CGI-69 transgenic animal that is heterozyhous for the
transgene may be bred with another heterozyhous CGI-69 transgenic
animal to produce animals that are homozygous for the transgene. In
certain instances, such homozygous transgenic animals may display
different characteristics than the heterozygous parents. Thus, in
situations where the heterozygous transgenic animal lacks a
particular phenotype, such animals may still have substantial
utility as sources of the homozyhous transgenic animals displaying
a particular characteristic.
[0181] 2. Vectors for transgenic animal production
[0182] To create a homologous recombinant animal, a vector
containing at least a portion of CGI-69 into which a deletion,
addition or substitution has been introduced to thereby alter,
e.g., functionally disrupt, CGI-69. CGI-69 can be a murine gene, or
other CGI-69 homologue, such as the human homolog (SEQ ID NOS:1 or
2). In one approach, a knockout vector functionally disrupts the
endogenous CGI-69 gene upon homologous recombination, and thus a
non-functional CGI-69 protein, if any, is expressed.
[0183] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous CGI-69 is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of endogenous CGI-69). In this type of homologous
recombination vector, the altered portion of the CGI-69 is flanked
at its 5'- and 3'-termini by additional nucleic acid of the CGI-69
to allow for homologous recombination to occur between the
exogenous CGI-69 carried by the vector and an endogenous CGI-69 in
an embryonic stem cell. The additional flanking CGI-69 nucleic acid
is sufficient to engender homologous recombination with endogenous
CGI-69. Typically, several kilobases of flanking DNA (both at the
5'- and 3'-termini) are included in the vector (Thomas and
Capecchi, 1987). The vector is then introduced into an embryonic
stem cell line (e.g., by electroporation), and cells in which the
introduced CGI-69 has homologously-recombined with the endogenous
CGI-69 are selected (Li et al., 1992).
[0184] 3. Introduction of CGI-69 transgene cells during
development
[0185] Selected cells are then injected into a blastocyst of an
animal (e.g., a mouse) to form aggregation chimeras (Bradley,
1987). A chimeric embryo can then be implanted into a suitable pffa
and the embryo brought to term. Progeny harboring the
homologously-recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the
homologously-recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described (Berns et
al., WO 93/04169, 1993; Bradley, 1991; Kucherlapati et al., WO
91/01140, 1991; Le Mouellic and Brullet, WO 90/11354, 1990).
[0186] Alternatively, transgenic animals that contain selected
systems that allow for regulated expression of the transgene can be
produced. An example of such a system is the cre/loxP recombinase
system of bacteriophage P1 (Lakso et al., 1992). Another
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al., 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 are required. Such animals can be produced as
"double" transgenic animals, by mating an animal containing a
transgene encoding a selected protein to another containing a
transgene encoding a recombinase.
[0187] Clones of transgenic animals can also be produced (Wilmut et
al., 1997). In brief, a cell from a transgenic animal can be
isolated and induced to exit the growth cycle and enter G.sub.0
phase. The quiescent cell can then be fused to an enucleated oocyte
from an animal of the same species from which the quiescent cell is
isolated. The reconstructed oocyte is then cultured to develop to a
morula or blastocyte and then transferred to a pffa. The offspring
borne of this female foster animal will be a clone of the "parent"
transgenic animal.
[0188] Pharmaceutical compositions
[0189] Agonists or antagonists of CGI-69 can be incorporated into
pharmaceutical compositions. Such compositions typically comprise
the agonists or antagonists and a pharmaceutically acceptable
carrier. A "pharmaceutically acceptable carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration (Gennaro,
2000). Preferred examples of such carriers or diluents include, but
are not limited to, water, saline, Finger's solutions, dextrose
solution, and 5% human serum albumin. Liposomes and non-aqueous
vehicles such as fixed oils may also be used. Except when a
conventional media or agent is incompatible with an active
compound, use of these compositions is contemplated. Supplementary
active compounds can also be incorporated into the
compositions.
[0190] 1. General considerations
[0191] A pharmaceutical composition of the agonist or antagonist is
formulated to be compatible with its intended route of
administration, including intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (i.e., topical), transmucosal,
and rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA); buffers such as acetates, citrates or phosphates, and
agents for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. The parenteral preparation
can be enclosed in ampules, disposable syringes or multiple dose
vials made of glass or plastic.
[0192] 2. Injectable formulations
[0193] Pharmaceutical compositions suitable for injection include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. For intravenous administration,
suitable carriers include physiological saline, bacteriostatic
water, CREMOPHOR EL.TM. (BASF, Parsippany, N.J.) or phosphate
buffered saline (PBS). In all cases, the composition must be
sterile and should be fluid so as to be administered using a
syringe. Such compositions should be stable during manufacture and
storage and must be preserved against contamination from
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (such as glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures. Proper fluidity can be
maintained, for example, by using a coating such as lecithin, by
maintaining the required particle size in the case of dispersion
and by using surfactants. Various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, ascorbic
acid, and thimerosal, can contain microorganism contamination.
Isotonic agents, for example, sugars, polyalcohols such as manitol,
sorbitol, and sodium chloride can be included in the composition.
Compositions that can delay absorption include agents such as
aluminum monostearate and gelatin.
[0194] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients as
required, followed by sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium, and the other
required ingredients. Sterile powders for the preparation of
sterile injectable solutions, methods of preparation include vacuum
drying and freeze-drying that yield a powder containing the active
ingredient and any desired ingredient from a sterile solutions.
[0195] 3. Oral compositions
[0196] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally. Pharmaceutically compatible binding agents, and/or
adjuvant materials can be included. Tablets, pills, capsules,
troches and the like can contain any of the following ingredients,
or compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, PRIMOGEL,
or corn starch; a lubricant such as magnesium stearate or STEROTES;
a glidant such as colloidal silicon dioxide; a sweetening agent
such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl salicylate, or orange flavoring.
[0197] 4. Compositions for inhalation
[0198] For administration by inhalation, the compounds are
delivered as an aerosol spray from a nebulizer or a pressurized
container that contains a suitable propellant, e.g., a gas such as
carbon dioxide.
[0199] 5. Systemic administration
[0200] Systemic administration can also be transmucosal or
transdermal. For transmucosal or transdermal administration,
penetrants that can permeate the target barrier(s) are selected.
Transmucosal penetrants include, detergents, bile salts, and
fusidic acid derivatives. Nasal sprays or suppositories can be used
for transmucosal administration. For transdermal administration,
the active compounds are formulated into ointments, salves, gels,
or creams.
[0201] The compounds can also be prepared in the form of
suppositories (e.g., with bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery.
[0202] 6. Carriers
[0203] In one embodiment, the active compounds are prepared with
carriers that protect the compound against rapid elimination from
the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable or
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Such materials can be obtained commercially from
ALZA Corporation (Mountain View, Calif.) and NOVA Pharmaceuticals,
Inc. (Lake Elsinore, Calif.), or prepared by one of skill in the
art. Liposomal suspensions can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, such as in (Eppstein et al.,
U.S. Pat. No. 4,522,811, 1985).
[0204] 7. Unit dosage
[0205] Oral formulations or parenteral compositions in unit dosage
form can be created to facilitate administration and dosage
uniformity. Unit dosage form refers to physically discrete units
suited as single dosages for the subject to be treated, containing
a therapeutically effective quantity of active compound in
association with the required pharmaceutical carrier. The
specification for the unit dosage forms of the invention are
dictated by, and directly dependent on, the unique characteristics
of the active compound and the particular desired therapeutic
effect, and the inherent limitations of compounding the active
compound.
[0206] 8. Gene therapy compositions
[0207] The nucleic acid molecules of CGI-69 can be inserted into
vectors and used as gene therapy vectors. Gene therapy vectors can
be delivered to a subject by, for example, intravenous injection,
local administration (Nabel and Nabel, U.S. Pat. No. 5,328,470,
1994), or by stereotactic injection (Chen et al., 1994). The
pharmaceutical preparation of a gene therapy vector can include an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells that produce the gene
delivery system.
[0208] 9. Dosage
[0209] The pharmaceutical composition may further comprise other
therapeutically active compounds as noted herein which are usually
applied in the treatment of CGI-69 -related conditions.
[0210] In the treatment or prevention of conditions which require
CGI-69 modulation an appropriate dosage level of an agonist or
antagonist will generally be about 0.01 to 500 mg per kg patient
body weight per day which can be administered in single or multiple
doses. Preferably, the dosage level will be about 0.1 to about 250
mg/kg per day; more preferably about 0.5 to about 100 mg/kg per
day. A suitable dosage level may be about 0.01 to 250 mg/kg per
day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per
day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5
to 50 mg/kg per day. For oral administration, the compositions are
preferably provided in the form of tablets containing 1.0 to 1000
milligrams of the active ingredient, particularly 1.0, 5.0, 10.0,
15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0,
400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of
the active ingredient for the symptomatic adjustment of the dosage
to the patient to be treated. The compounds may be administered on
a regimen of 1 to 4 times per day, preferably once or twice per
day.
[0211] However, the specific dose level and frequency of dosage for
any particular patient may be varied and will depend upon a variety
of factors including the activity of the specific compound
employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and
time of administration, rate of excretion, drug combination, the
severity of the particular condition, and the host undergoing
therapy.
[0212] 10. Kits for pharmaceutical compositions
[0213] The pharmaceutical compositions can be included in a kit,
container, pack, or dispenser together with instructions for
administration to treat a metabolic disorder or disease. When the
invention is supplied as a kit, the different components of the
composition may be packaged in separate containers and admixed
immediately before use. Such packaging of the components separately
may permit long-term storage without losing the active components'
functions.
[0214] Kits may also include reagents in separate containers that
facilitate the execution of a specific test, such as diagnostic
tests or tissue typing. For example, CGI-69 DNA templates and
suitable primers may be supplied for internal controls.
[0215] (a) Containers or vessels
[0216] The reagents included in kits can be supplied in containers
of any sort such that the life of the different components are
preserved, and are not adsorbed or altered by the materials of the
container. For example, sealed glass ampules may contain
lyophilized CGI-69 or buffer that have been packaged under a
neutral, non-reacting gas, such as nitrogen. Ampules may consist of
any suitable material, such as glass, organic polymers, such as
polycarbonate, polystyrene, etc., ceramic, metal or any other
material typically employed to hold reagents. Other examples of
suitable containers include simple bottles that may be fabricated
from similar substances as ampules, and envelopes, that may consist
of foil-lined interiors, such as aluminum or an alloy. Other
containers include test tubes, vials, flasks, bottles, syringes, or
the like. Containers may have a sterile access port, such as a
bottle having a stopper that can be pierced by a hypodermic
injection needle. Other containers may have two compartments that
are separated by a readily removable membrane that upon removal
permits the components to mix. Removable membranes may be glass,
plastic, rubber, etc.
[0217] (b) Instructional materials
[0218] Kits may also be supplied with instructional materials.
Instructions may be printed on paper or other substrate, and/or may
be supplied as an electronic-readable medium, such as a floppy
disc, CD-ROM, DVD-ROM, Zip disc, videotape, laserdisc, audio tape,
etc. Detailed instructions may not be physically associated with
the kit; instead, a user may be directed to an internet web site
specified by the manufacturer or distributor of the kit, or
supplied as electronic mail.
[0219] Screening and detection methods
[0220] The isolated nucleic acid molecules of CGI-69 can be used to
express CGI-69 (e.g., via a recombinant expression vector in a host
cell in gene therapy applications), to detect CGI-69 mRNA (e.g., in
a biological sample) or a genetic lesion in a CGI-69, and to
modulate CGI-69 activity, as described below. In addition, CGI-69
polypeptides can be used to screen drugs or compounds that modulate
the CGI-69 activity or expression as well as to treat disorders
characterized by insufficient or excessive production of CGI-69 or
production of CGI-69 forms that have decreased or aberrant activity
compared to CGI-69 wild-type protein, or modulate biological
function that involve CGI-69. These reagents may be provided in the
form of a kit as described above for pharmaceutical administrations
optionally including instructions for assaying or screening CGI-69
involvment in a metabolic disease or disorder.
[0221] 1. Screening assays
[0222] The invention provides a method (screening assay) for
identifying modalities, i.e., candidate or test compounds or agents
(e.g., peptides, peptidomimetics, small molecules or other drugs),
foods, combinations thereof, etc., that effect CGI-69, a
stimulatory or inhibitory effect, including translation,
transcription, activity or copies of the gene in cells. The
invention also includes compounds identified in screening
assays.
[0223] Testing for compounds that increase or decrease CGI-69
activity are desirable. A compound may modulate CGI-69 activity by
affecting: (1) the number of copies of the gene in the cell
(amplifiers and deamplifiers); (2) increasing or decreasing
transcription of the CGI-69 (transcription up-regulators and
down-regulators); (3) by increasing or decreasing the translation
of CGI-69 mRNA into protein (translation up-regulators and
down-regulators); or (4) by increasing or decreasing the activity
of CGI-69 itself (agonists and antagonists).
[0224] (a) effects of compounds
[0225] To identify compounds that affect CGI-69 at the DNA, RNA and
protein levels, cells or organisms are contacted with a candidate
compound and the corresponding change in CGI-69 DNA, RNA or protein
is assessed (Ausubel et al., 1987). For DNA amplifiers and
deamplifiers, the amount of CGI-69 DNA is measured, for those
compounds that are transcription up-regulators and down-regulators,
the amount of CGI-69 mRNA is determined; for translational up- and
down-regulators, the amount of CGI-69 polypeptides is measured.
Compounds that are agonists or antagonists may be identified by
contacting cells or organisms with the compound.
[0226] Many assays for screening candidate or test compounds that
bind to or modulate the activity of CGI-69 or polypeptide or
biologically active portion are available. Test compounds can be
obtained using any of the numerous approaches in combinatorial
library methods, including: biological libraries; spatially
addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; the "one-bead
one-compound" library method; and synthetic library methods using
affinity chromatography selection. The biological library approach
is limited to peptides, while the other four approaches encompass
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, 1997).
[0227] (b) small molecules
[0228] A "small molecule" refers to a composition that has a
molecular weight of less than about 5 kD and more preferably less
than about 4 kD, and most preferably less than 0.6 kD. Small
molecules can be, nucleic acids, peptides, polypeptides,
peptidomimetics, carbohydrates, lipids or other organic or
inorganic molecules. Libraries of chemical and/or biological
mixtures, such as fungal, bacterial, or algal extracts, are known
in the art and can be screened with any of the assays of the
invention. Examples of methods for the synthesis of molecular
libraries have been described (Carell et al., 1994a; Carell et al.,
1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994;
Zuckermann et al., 1994).
[0229] Libraries of compounds may be presented in solution
(Houghten et al., 1992) or on beads (Lam et al., 1991), on chips
(Fodor et al., 1993), bacteria, spores (Ladner et al., U.S. Pat.
No. 5,223,409, 1993), plasmids (Cull et al., 1992) or on phage
(Cwirla et al., 1990; Devlin et al., 1990; Felici et al., 1991;
Ladner et al., U.S. Pat. No. 5,223,409, 1993; Scott and Smith,
1990). A cell-free assay comprises contacting CGI-69 or
biologically-active fragment with a known compound that binds
CGI-69 to form an assay mixture, contacting the assay mixture with
a test compound, and determining the ability of the test compound
to interact with CGI-69, where determining the ability of the test
compound to interact with CGI-69 comprises determining the ability
of the CGI-69 to preferentially bind to or modulate the activity of
an CGI-69 target molecule.
[0230] (c) cell-free assays
[0231] The cell-free assays of the invention may be used with the
membrane-bound forms of CGI-69. Cell-free assays comprising the
membrane-bound form, a solubilizing agent may be used to maintain
CGI-69 in solution. Examples of such solubilizing agents include
non-ionic detergents such as n-octylglucoside, n-dodecylglucoside,
n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamid- e, polyoxyethylene ethers (e.g.,
t-Octylphenoxypolyethoxyethanol (TRITON.RTM. X-100) and others from
the TRITON.RTM. series), polyoxyethylene 9 lauryl ether
(THESIT.RTM.), Isotridecypoly(ethylene glycol ether).sub.n,
N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,
3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane
sulfonate (CHAPSO).
[0232] (d) immobilization of target molecules to facilitate
screening
[0233] In more than one embodiment of the assay methods,
immobilizing either CGI-69 or its partner molecules can facilitate
separation of complexed from uncomplexed forms of one or both of
the proteins, as well as to accommodate high throughput assays.
Binding of a test compound to CGI-69, or interaction of CGI-69 with
a target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants, such as microtiter plates, test tubes, and
micro-centrifuge tubes. A fusion protein can be provided that adds
a domain that allows one or both of the proteins to be bound to a
matrix. For example, GST (glutathione S-transferase)-CGI-69 fusion
proteins or GST-target fusion proteins can be adsorbed onto
glutathione sepharose beads (SIGMA Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates that are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or CGI-69, and the mixture is incubated
under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly. Alternatively,
the complexes can be dissociated from the matrix, and the level of
CGI-69 binding or activity determined using standard
techniques.
[0234] Other techniques for immobilizing proteins on matrices can
also be used in screening assays. Either CGI-69 or its target
molecule can be immobilized using biotin-avidin or
biotin-streptavidin systems. Biotinylation can be accomplished
using many reagents, such as biotin-NHS (N-hydroxy-succinimide;
PIERCE Chemicals, Rockford, Ill.), and immobilized in wells of
streptavidin-coated 96 well plates (PIERCE Chemical).
Alternatively, Abs reactive with CGI-69 or target molecules but do
not interfere with binding of the CGI-69 to its target molecule can
be derivatized to the wells of the plate, and unbound target or
CGI-69 trapped in the wells by antibody conjugation. Methods for
detecting such complexes, in addition to those described for the
GST-immobilized complexes, include immunodetection of complexes
using Abs reactive with CGI-69 or its target, as well as
enzyme-linked assays that rely on detecting an enzymatic activity
associated with the CGI-69 or target molecule.
[0235] (e) screens to identify modulators
[0236] Modulators of CGI-69 expression can be identified in a
method where a cell is contacted with a candidate compound and the
expression of CGI-69 mRNA or protein in the cell is determined. The
expression level of CGI-69 mRNA or protein in the presence of the
candidate compound is compared to CGI-69 mRNA or protein levels in
the absence of the candidate compound. The candidate compound can
then be identified as a modulator of CGI-69 mRNA or protein
expression based upon this comparison. For example, when expression
of CGI-69 mRNA or protein is greater (i.e., statistically
significant) in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
CGI-69 mRNA or protein expression. Alternatively, when expression
of CGI-69 mRNA or protein is less (statistically significant) in
the presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of CGI-69 mRNA or
protein expression. The level of CGI-69 mRNA or protein expression
in the cells can be determined by methods described for detecting
CGI-69 mRNA or protein.
[0237] (f) hybrid assays
[0238] In yet another aspect of the invention, CGI-69 can be used
as "bait" in two-hybrid or three hybrid assays (Bartel et al.,
1993; Brent et al., WO94/10300, 1994; Iwabuchi et al., 1993; Madura
et al., 1993; Saifer et al., U.S. Pat. No. 5,283,317, 1994; Zervos
et al., 1993) to identify other proteins that bind or interact with
CGI-69 and modulate CGI-69 activity. Such CGI-69-interacting
partner proteins are also likely to be involved in the propagation
of signals by the CGI-69 as, for example, upstream or downstream
elements of a CGI-69 pathway.
[0239] The two-hybrid system is based on the modular nature of most
transcription factors, which consists of separable DNA-binding and
activation domains. The assay utilizes two different DNA
constructs. In one construct, the gene that codes for CGI-69 is
fused to a gene encoding the DNA binding domain of a known
transcription factor (e.g., GAL4). 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 CGI-69 -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) that 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 that encodes the CGI-69 -interacting
protein. The invention further pertains to novel agents identified
by the aforementioned screening assays and uses thereof for
treatments as described herein.
[0240] Predictive medicine
[0241] The invention also pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trials are used for
prognostic (predictive) purposes to treat an individual
prophylactically. Accordingly, one aspect of the invention relates
to diagnostic assays for determining CGI-69 and/or nucleic acid
expression as well as CGI-69 activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue) to determine
whether an individual is afflicted with a disease or disorder, or
is at risk of developing a disorder, associated with aberrant
CGI-69 expression or activity, including cancer. The invention also
provides for prognostic (or predictive) assays for determining
whether an individual is at risk of developing a disorder
associated with CGI-69, nucleic acid expression or activity. For
example, mutations in CGI-69 can be assayed in a biological sample.
Such assays can be used for prognostic or predictive purpose to
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with CGI-69 nucleic acid
expression, or biological activity.
[0242] Another aspect of the invention provides methods for
determining CGI-69 activity, or nucleic acid expression, in an
individual to select appropriate therapeutic or prophylactic agents
for that individual (referred to herein as "pharmacogenomics").
Pharmacogenomics allows for the selection of modalities (e.g.,
drugs, foods) for therapeutic or prophylactic treatment of an
individual based on the individual's genotype (e.g., the
individual's genotype to determine the individual's ability to
respond to a particular agent). Another aspect of the invention
pertains to monitoring the influence of modalities (e.g., drugs,
foods) on the expression or activity of CGI-69 in clinical
trials.
[0243] 1. Diagnostic assays
[0244] An exemplary method for detecting the presence or absence of
CGI-69 in a biological sample involves obtaining a biological
sample from a subject and contacting the biological sample with a
compound or an agent capable of detecting CGI-69 or CGI-69 (e.g.,
mRNA, genomic DNA) such that the presence of CGI-69 is confirmed in
the sample. An agent for detecting CGI-69 mRNA or genomic DNA is a
labeled nucleic acid probe that can hybridize to CGI-69 mRNA or
genomic DNA. The nucleic acid probe can be, for example, a
full-length CGI-69 nucleic acid, such as the nucleic acid of SEQ ID
NOS:1 or 2, or a portion thereof, such as an oligonucleotide of at
least 15, 30, 50, 100, 250 or 500 nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
CGI-69 mRNA or genomic DNA.
[0245] The term "biological sample" includes tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. The detection method of
the invention can be used to detect CGI-69 mRNA, protein, or
genomic DNA in a biological sample in vitro as well as in vivo. For
example, in vitro techniques for detection of CGI-69 mRNA include
Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of CGI-69 polypeptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations, and immunofluorescence. In vitro techniques
for detection of CGI-69 genomic DNA include Southern hybridizations
and fluorescent in situ hybridization (FISH).
[0246] In one embodiment, the biological sample from the subject
contains protein molecules, and/or mRNA molecules, and/or genomic
DNA molecules. Preferred biological samples are blood and adipose
tissue.
[0247] In another embodiment, the methods further involve obtaining
a biological sample from a subject to provide a control, contacting
the sample with a compound or agent to detect CGI-69, CGI-69 mRNA,
or genomic DNA, and comparing the presence of CGI-69, CGI-69 mRNA
or genomic DNA in the control sample with the presence of CGI-69,
CGI-69 mRNA or genomic DNA in the test sample.
[0248] The invention also encompasses kits for detecting CGI-69 in
a biological sample. For example, the kit can comprise: a labeled
compound or agent capable of detecting CGI-69 or CGI-69 mRNA in a
sample; reagent and/or equipment for determining the amount of
CGI-69 in the sample; and reagent and/or equipment for comparing
the amount of CGI-69 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 CGI-69 or nucleic
acid.
[0249] 2. Prognostic assays
[0250] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant CGI-69 expression or
activity. For example, the described assays can be used to identify
a subject having or at risk of developing a disorder associated
with CGI-69, nucleic acid expression or activity. Alternatively,
the prognostic assays can be used to identify a subject having or
at risk for developing a disease or disorder. The invention
provides a method for identifying a disease or disorder associated
with aberrant CGI-69 expression or activity in which a test sample
is obtained from a subject and CGI-69 or nucleic acid (e.g., mRNA,
genomic DNA) is detected. A test sample is a biological sample
obtained from a subject. For example, a test sample can be a
biological fluid (e.g., serum), cell sample, or tissue.
[0251] Prognostic assays can be used to determine whether a subject
can be administered a modality (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule,
food, etc.) to treat a disease or disorder associated with aberrant
CGI-69 expression or activity. Such methods can be used to
determine whether a subject can be effectively treated with an
agent for a disorder. The invention provides methods for
determining whether a subject can be effectively treated with an
agent for a disorder associated with aberrant CGI-69 expression or
activity in which a test sample is obtained and CGI-69 or nucleic
acid is detected (e.g., where the presence of CGI-69 or nucleic
acid is diagnostic for a subject that can be administered the agent
to treat a disorder associated with aberrant CGI-69 expression or
activity).
[0252] The methods of the invention can also be used to detect
genetic lesions in a CGI-69 to determine if a subject with the
genetic lesion is at risk for a disorder. Methods include
detecting, in a sample from the subject, the presence or absence of
a genetic lesion characterized by at an alteration affecting the
integrity of a gene encoding a CGI-69 polypeptide, or the
mis-expression of CGI-69. Such genetic lesions can be detected by
ascertaining: (1) a deletion of one or more nucleotides from
CGI-69; (2) an addition of one or more nucleotides to CGI-69; (3) a
substitution of one or more nucleotides in CGI-69, (4) a
chromosomal rearrangement of an CGI-69 gene; (5) an alteration in
the level of an CGI-69 mRNA transcripts, (6) aberrant modification
of an CGI-69, such as a change genomic DNA methylation, (7) the
presence of a non-wild-type splicing pattern of an CGI-69 mRNA
transcript, (8) a non-wild-type level of CGI-69, (9) allelic loss
of CGI-69, and/or (10) inappropriate post-translational
modification of CGI-69 polypeptide. There are a large number of
known assay techniques that can be used to detect lesions in
CGI-69. Any biological sample containing nucleated cells may be
used.
[0253] In certain embodiments, lesion detection may use a
probe/primer in a polymerase chain reaction (PCR) (e.g., (Mullis,
U.S. Pat. No. 4,683,202, 1987; Mullis et al., U.S. Pat. No.
4,683,195, 1987), such as anchor PCR or rapid amplification of cDNA
ends (RACE) PCR, or, alternatively, in a ligation chain reaction
(LCR) (e.g., (Landegren et al., 1988; Nakazawa et al., 1994), the
latter is particularly useful for detecting point mutations in
CGI-69-genes (Abravaya et al., 1995). This method may include
collecting a sample from a patient, isolating nucleic acids from
the sample, contacting the nucleic acids with one or more primers
that specifically hybridize to CGI-69 under conditions such that
hybridization and amplification of the CGI-69 (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. It is anticipated that PCR and/or
LCR may be desirable to use as a preliminary amplification step in
conjunction with any of the techniques used for detecting mutations
described herein.
[0254] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al., 1990), transcriptional
amplification system (Kwoh et al., 1989); Q.beta. Replicase
(Lizardi et al., 1988), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules present in low abundance.
[0255] Mutations in CGI-69 from a sample can be identified by
alterations in restriction enzyme cleavage patterns. For example,
sample and control DNA is isolated, amplified (optionally),
digested with one or more restriction endonucleases, and fragment
length sizes are determined by gel electrophoresis and compared.
Differences in fragment length sizes between sample and control DNA
indicates mutations in the sample DNA. Moreover, the use of
sequence specific ribozymes can be used to score for the presence
of specific mutations by development or loss of a ribozyme cleavage
site.
[0256] Hybridizing a sample and control nucleic acids, e.g., DNA or
RNA, to high-density arrays containing hundreds or thousands of
oligonucleotides probes, can identify genetic mutations in CGI-69
(Cronin et al., 1996; Kozal et al., 1996). For example, genetic
mutations in CGI-69 can be identified in two-dimensional arrays
containing light-generated DNA probes as described (Cronin et al.,
1996). Briefly, a first hybridization array of probes can be used
to scan through long stretches of DNA in a sample and control to
identify base changes between the sequences by making linear arrays
of sequential overlapping probes. This step allows the
identification of point mutations. This is followed by a second
hybridization array that allows the characterization of specific
mutations by using smaller, specialized probe arrays complementary
to all variants or mutations detected. Each mutation array is
composed of parallel probe sets, one complementary to the wild-type
gene and the other complementary to the mutant gene.
[0257] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
CGI-69 and detect mutations by comparing the sequence of the sample
CGI-69 with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on classic
techniques (Maxam and Gilbert, 1977; Sanger et al., 1977). Any of a
variety of automated sequencing procedures can be used when
performing diagnostic assays (Naeve et al., 1995) including
sequencing by mass spectrometry (Cohen et al., 1996; Griffin and
Griffin, 1993; Koster, WO94/16101, 1994).
[0258] Other methods for detecting mutations in the CGI-69 include
those in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et
al., 1985). In general, the technique of "mismatch cleavage" starts
by providing heteroduplexes formed by hybridizing (labeled) RNA or
DNA containing the wild-type CGI-69 sequence with potentially
mutant RNA or DNA obtained from a sample. The double-stranded
duplexes are treated with an agent that cleaves single-stranded
regions of the duplex such as those that arise from base pair
mismatches between the control and sample strands. For instance,
RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with S.sub.1 nuclease to enzymatically digest the
mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA
duplexes can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions. The digested
material is then separated by size on denaturing polyacrylamide
gels to determine the mutation site (Grompe et al., 1989; Saleeba
and Cotton, 1993). The control DNA or RNA can be labeled for
detection.
[0259] Mismatch cleavage reactions may employ one or more proteins
that recognize mismatched base pairs in double-stranded DNA (DNA
mismatch repair) in defined systems for detecting and mapping point
mutations in CGI-69 cDNAs obtained from samples of cells. For
example, the mutY enzyme of E. coli cleaves A at G/A mismatches and
the thymidine DNA glycosylase from HeLa cells cleaves T at G/T
mismatches (Hsu et al., 1994). According to an exemplary
embodiment, a probe based on a wild-type CGI-69 sequence is
hybridized to a cDNA or other DNA product from a test cell(s). The
duplex is treated with a DNA mismatch repair enzyme, and the
cleavage products, if any, can be detected from electrophoresis
protocols or the like (Modrich et al., U.S. Pat. No. 5,459,039,
1995).
[0260] Electrophoretic mobility alterations can be used to identify
mutations in CGI-69. For example, single strand conformation
polymorphism (SSCP) may be used to detect differences in
electrophoretic mobility between mutant and wild type nucleic acids
(Cotton, 1993; Hayashi, 1992; Orita et al., 1989). Single-stranded
DNA fragments of sample and control CGI-69 nucleic acids are
denatured and then renatured. The secondary structure of
single-stranded nucleic acids varies according to sequence; the
resulting alteration in electrophoretic mobility allows detection
of even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be
enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a sequence changes. The method may
use heteroduplex analysis to separate double stranded heteroduplex
molecules on the basis of changes in electrophoretic mobility (Keen
et al., 1991).
[0261] The migration of mutant or wild-type fragments can be
assayed using denaturing gradient gel electrophoresis (DGGE; (Myers
et al., 1985). In DGGE, DNA is modified to prevent complete
denaturation, for example by adding a GC clamp of approximately 40
bp of high-melting GC-rich DNA by PCR. A temperature gradient may
also be used in place of a denaturing gradient to identify
differences in the mobility of control and sample DNA (Rossiter and
Caskey, 1990).
[0262] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions that permit hybridization only if a
perfect match is found (Saiki et al., 1986; Saiki et al., 1989).
Such allele-specific oligonucleotides are hybridized to
PCR-amplified target DNA or a number of different mutations when
the oligonucleotides are attached to the hybridizing membrane and
hybridized with labeled target DNA.
[0263] Alternatively, allele specific amplification technology that
depends on selective PCR amplification may be used. Oligonucleotide
primers for specific amplifications may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization (Gibbs et al., 1989)) or at
the extreme 3'-terminus of one primer where, under appropriate
conditions, mismatch can prevent, or reduce polymerase extension
(Prosser, 1993). Novel restriction site in the region of the
mutation may be introduced to create cleavage-based detection
(Gasparini et al., 1992). Certain amplification may also be
performed using Taq ligase for amplification (Barany, 1991). In
such cases, ligation occurs only if there is a perfect match at the
3'-terminus of the 5' sequence, allowing detection of a known
mutation by scoring for amplification.
[0264] The described methods may be performed, for example, by
using pre-packaged kits comprising at least one probe (nucleic acid
or antibody) that may be conveniently used, for example, in
clinical settings to diagnose patients exhibiting symptoms or
family history of a disease or illness involving CGI-69.
[0265] Furthermore, any cell type or tissue in which CGI-69 is
expressed may be utilized in the prognostic assays described
herein.
[0266] 3. Pharmacogenomics
[0267] Agents, or modulators that have a stimulatory or inhibitory
effect on CGI-69 activity or expression, as identified by a
screening assay, can be administered to individuals to treat
prophylactically or therapeutically disorders. In conjunction with
such treatment, the pharmacogenomics (i.e., the study of the
relationship between a subject's genotype and the subject's
response to a foreign modality, such as a food, compound or drug)
may be considered. Metabolic differences of therapeutics can lead
to severe toxicity or therapeutic failure by altering the relation
between dose and blood concentration of the pharmacologically
active drug. Thus, the pharmacogenomics of the individual permits
the selection of effective agents (e.g., drugs) for prophylactic or
therapeutic treatments based on a consideration of the individual's
genotype. Pharmacogenomics can further be used to determine
appropriate dosages and therapeutic regimens. Accordingly, the
activity of CGI-69, expression of CGI-69, or CGI-69 mutation(s) in
an individual can be determined to guide the selection of
appropriate agent(s) for therapeutic or prophylactic treatment.
[0268] Pharmacogenomics deals with clinically significant
hereditary variations in the response to modalities due to altered
modality disposition and abnormal action in affected persons
(Eichelbaum and Evert, 1996; Linder et al., 1997). In general, two
pharmacogenetic conditions can be differentiated: (1) genetic
conditions transmitted as a single factor altering the interaction
of a modality with the body (altered drug action) or (2) genetic
conditions transmitted as single factors altering the way the body
acts on a modality (altered drug metabolism). These pharmacogenetic
conditions can occur either as rare defects or as nucleic acid
polymorphisms. For example, glucose-6-phosphate dehydrogenase
(G6PD) deficiency is a common inherited enzymopathy in which the
main clinical complication is hemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0269] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) explains the
phenomena of some patients who show exaggerated drug response
and/or serious toxicity after taking the standard and safe dose of
a drug. These polymorphisms are expressed in two phenotypes in the
population, the extensive metabolizer (EM) and poor metabolizer
(PM). The prevalence of PM is different among different
populations. For example, the CYP2D6 gene is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers due to mutant
CYP2D6 and CYP2CJ9 frequently experience exaggerated drug responses
and side effects when they receive standard doses. If a metabolite
is the active therapeutic moiety, PM shows no therapeutic response,
as demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. At the other extreme are the
so-called ultra-rapid metabolizers who are unresponsive to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0270] The activity of CGI-69, expression of CGI-69 nucleic acid,
or mutation content of CGI-69 in an individual can be determined to
select appropriate agent(s) for therapeutic or prophylactic
treatment of the individual. In addition, pharmacogenetic studies
can be used to apply genotyping of polymorphic alleles encoding
drug-metabolizing enzymes to the identification of an individual's
drug responsiveness phenotype. This knowledge, when applied to
dosing or drug selection, can avoid adverse reactions or
therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when treating a subject with an CGI-69 modulator, such
as a modulator identified by one of the described exemplary
screening assays.
[0271] 4. Monitoring effects during clinical trials
[0272] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of CGI-69 can be applied not only in
basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay to
increase CGI-69 expression, protein levels, or up-regulate CGI-69
activity can be monitored in clinical trails of subjects exhibiting
decreased CGI-69 expression, protein levels, or down-regulated
CGI-69 activity. Alternatively, the effectiveness of an agent
determined to decrease CGI-69 expression, protein levels, or
down-regulate CGI-69 activity, can be monitored in clinical trails
of subjects exhibiting increased CGI-69 expression, protein levels,
or up-regulated CGI-69 activity. In such clinical trials, the
expression or activity of CGI-69 and, preferably, other genes that
have been implicated in, for example, cancer can be used as a "read
out" or markers for a particular cell's responsiveness.
[0273] For example, genes, including CGI-69, that are modulated in
cells by treatment with a modality (e.g., food, compound, drug or
small molecule) can be identified. To study the effect of agents on
metabolic disorders or disorders associated with changes in adipose
tissue physiological function or mass, for example, in a clinical
trial, cells can be isolated and RNA prepared and analyzed for the
levels of expression of CGI-69 and other genes implicated in the
disorder. The gene expression pattern can be quantified by Northern
blot analysis, nuclear run-on or RT-PCR experiments, or by
measuring the amount of protein, or by measuring the activity level
of CGI-69 or other gene products. In this manner, the gene
expression pattern itself can serve as a marker, indicative of the
cellular physiological response to the agent. Accordingly, this
response state may be determined before, and at various points
during, treatment of the individual with the agent.
[0274] The invention provides a method for monitoring the
effectiveness of treatment of a subject with an agent (e.g., an
agonist, antagonist, protein, peptide, peptidomimetic, nucleic
acid, small molecule, food or other drug candidate identified by
the screening assays described herein) comprising the steps of (1)
obtaining a pre-administration sample from a subject; (2) detecting
the level of expression of an CGI-69, CGI-69 mRNA, or genomic DNA
in the preadministration sample; (3) obtaining one or more
post-administration samples from the subject; (4) detecting the
level of expression or activity of the CGI-69, CGI-69 mRNA, or
genomic DNA in the post-administration samples; (5) comparing the
level of expression or activity of the CGI-69, CGI-69 mRNA, or
genomic DNA in the pre-administration sample with the CGI-69,
CGI-69 mRNA, or genomic DNA in the post administration sample or
samples; and (6) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
CGI-69 to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
CGI-69 to lower levels than detected, i.e., to decrease the
effectiveness of the agent.
[0275] 5. Methods of treatment
[0276] The invention provides for both prophylactic and therapeutic
methods of treating a subject at risk of (or susceptible to) a
disorder or having a disorder associated with aberrant CGI-69
expression or activity. Examples include disorders in which cell
metabolic demands (and consequently, demands on mitochondria and
endoplasmic reticulum) are high, such as during rapid cell growth.
Examples of such disorders and diseases include metabolic disorders
or disorders associated with changes in adipose tissue
physiological function or mass.
[0277] 6. Disease and disorders
[0278] A number of diseases and disorders can be treated or
diagnosed using the CGI-69 compositions of the present invention.
Such diseases and disorders include obesity, cachexia, tumors,
cancers, and fever associated with viral infections and bacterial
infections.
[0279] Diseases and disorders that are characterized by increased
CGI-69 levels or biological activity may be treated with
therapeutics that antagonize (i.e., reduce or inhibit) activity.
Antagonists may be administered in a therapeutic or prophylactic
manner. Therapeutics that may be used include: (1) CGI-69 peptides,
or analogs, derivatives, fragments or homologs thereof; (2) CGI-69;
(3) administration of antisense nucleic acid and nucleic acids that
are "dysfunctional" (i.e., due to a heterologous insertion within
the coding sequences) that are used to eliminate endogenous
function of by homologous recombination (Capecchi, 1989); or (4)
modulators (i.e., inhibitors, agonists and antagonists, including
additional peptide mimetic of the invention ) that alter the
interaction between CGI-69 and its binding partner.
[0280] Diseases and disorders that are characterized by decreased
CGI-69 levels or biological activity, such as obesity, may be
treated with therapeutics that increase (i.e., are agonists to)
activity. Therapeutics that upregulate activity may be administered
therapeutically or prophylactically. Therapeutics that may be used
include peptides, or analogs, derivatives, fragments or homologs
thereof; or an agonist that increases bioavailability.
[0281] Increased or decreased levels can be readily detected by
quantifying peptide and/or RNA, by obtaining a patient tissue
sample (e.g., from biopsy tissue) and assaying in vitro for RNA or
peptide levels, structure and/or activity of the expressed peptides
(or CGI-69 mRNAs). Methods include, but are not limited to,
immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis, immunocytochemistry, etc.) and/or hybridization
assays to detect expression of mRNAs (e.g., Northern assays, dot
blots, in situ hybridization, and the like).
[0282] 7. Prophylactic methods
[0283] The invention provides a method for preventing, in a
subject, a disease or condition associated with an aberrant CGI-69
expression or activity, by administering an agent that modulates
CGI-69 expression or at least one CGI-69 activity. Subjects at risk
for a disease that is caused or contributed to by aberrant CGI-69
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays. Administration of a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of the CGI-69 aberrancy, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of CGI-69 aberrancy, for
example, a CGI-69 agonist or CGI-69 antagonist can be used to treat
the subject. The appropriate agent can be determined based on
screening assays.
[0284] 8. Therapeutic methods
[0285] Another aspect of the invention pertains to methods of
modulating CGI-69 expression or activity for therapeutic purposes.
The modulatory method of the invention involves contacting a cell
with an agent that modulates one or more of the activities of
CGI-69 activity associated with the cell. An agent that modulates
CGI-69 activity can be a nucleic acid or a protein, a naturally
occurring cognate ligand of CGI-69, a peptide, a CGI-69
peptidomimetic, or other small molecule. The agent may stimulate
CGI-69 activity. Examples of such stimulatory agents include active
CGI-69 and a CGI-69 that has been introduced into the cell. In
another embodiment, the agent inhibits CGI-69 activity. An example
of an inhibitory agent includes antisense CGI-69 nucleic acids.
Modulatory methods can be performed in vitro (e.g., by culturing
the cell with the agent) or, alternatively, in vivo (e.g., by
administering the agent to a subject). As such, the invention
provides methods of treating an individual afflicted with a disease
or disorder characterized by aberrant expression or activity of a
CGI-69 or nucleic acid molecule. In one embodiment, the method
involves administering an agent (e.g., an agent identified by a
screening assay), or combination of agents that modulates (e.g.,
up-regulates or down-regulates) CGI-69 expression or activity. In
another embodiment, the method involves administering a CGI-69 or
nucleic acid molecule as therapy to compensate for reduced or
aberrant CGI-69 expression or activity.
[0286] Stimulation of CGI-69 activity is desirable in situations in
which CGI-69 is abnormally down-regulated and/or in which increased
CGI-69 activity is likely to have a beneficial effect; for example,
in treating obesity. Conversely, diminished CGI-69 activity is
desired in conditions in which CGI-69 activity is abnormally
up-regulated and/or in which decreased CGI-69 activity is likely to
to have a beneficial effect; for example, in treating cachexia.
[0287] 9. Determination of the biological effect of the
therapeutic
[0288] Suitable in vitro or in vivo assays can be performed to
determine the effect of a specific therapeutic and whether its
administration is indicated for treatment of the affected
tissue.
[0289] In various specific embodiments, in vitro assays may be
performed with representative cells of the type(s) involved in the
patient's disorder, to determine if a given therapeutic exerts the
desired effect upon the cell type(s). Modalities for use in therapy
may be tested in suitable animal model systems including, but not
limited to rats, mice, chicken, cows, monkeys, rabbits, dogs and
the like, prior to testing in human subjects. Similarly, for in
vivo testing, any of the animal model system known in the art may
be used prior to administration to human subjects.
[0290] 10. Prophylactic and therapeutic uses of the compositions of
the invention
[0291] CGI-69 nucleic acids and proteins are useful in potential
prophylactic and therapeutic applications implicated in a variety
of disorders including, but not limited to metabolic disorders or
disorders associated with changes in adipose tissue physiological
function or mass.
[0292] As an example, a cDNA encoding CGI-69 may be useful in gene
therapy, and the protein may be useful when administered to a
subject in need thereof. By way of non-limiting example, the
compositions of the invention will have efficacy for treatment of
patients suffering from metabolic disorders or disorders associated
with changes in adipose tissue physiological function or mass.
[0293] CGI-69 nucleic acids, or fragments thereof, may also be
useful in diagnostic applications, wherein the presence or amount
of the nucleic acid or the protein is to be assessed. A further use
could be as an anti-bacterial molecule (i.e., some peptides have
been found to possess anti-bacterial properties).
EXAMPLES
[0294] The following examples are included to demonstrate preferred
embodiments of the present invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples that follow represent techniques discovered by the
inventors to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments that are disclosed and still obtain a like
or similar result without departing form the spirit and scope of
the invention.
Example 1
Characterization of CGI-69
[0295] In this example, CGI-69 is characterized as a gene induced
by cold in brown adipose tissue (BAT) of mice by using differential
mRNA analysis. Through this result, along with the analysis of the
human CGI-69 protein, it was determined that CGI-69 is a
mitochondrial carrier protein (MCP).
[0296] All studies were done in accordance with guidelines set
forth by the Institutional Animal Care and Use Committee at
Genentech. Male FVB-N/J mice (Jackson Labs, Bar Harbor, Me., USA)
were received at 3 wk of age and housed at 2 mice/cage until tissue
harvest at 6 wk of age. All mice were fed rodent chow ad libitum
(Chow 5010, Ralston Purina, St. Louis, Mo., USA) and housed on a
12:12 light/dark cycle (lights on 06:00). Control and
Cold-Challenged mice were housed at 22.degree. C. during this 3 wk
period, whereas Warm-Acclimated mice were housed at 33.degree. C.,
within their thermoneutral zone (TNZ). For Cold-Challenged mice,
cages were transferred to a 4.degree. C. room for 48 hr prior to
tissue harvest. Following CO.sub.2-induced euthanasia in the
afternoon, interscapular BAT was excised, carefully cleared of
visible white adipose tissue (WAT), connective tissue, and blood
vessels, and snap-frozen in liquid nitrogen for subsequent RNA
preparation. For all treatment groups (Control, Cold-Challenged,
and Warm-Acclimated), 3 independent BAT samples were generated for
analysis; each sample was composed of BAT pooled from 10 mice.
[0297] Samples from each treatment group were transferred to
CuraGen Corp. (New Haven, Conn., USA), RNA prepared and
reverse-transcribed, and subjected to Quantitative Expression
Analysis (QEA), the details of which are presented elsewhere
(Shimkets, et al., 1999). Analyses focused on identification of
genes regulated at least 2-fold by changes in T.sub.a.
[0298] Full-length cDNAs of human CGI-69 were generated by PCR,
using primers [forward 5'CTGAAGCTTCAAGATGGCTGACCAG3' (SEQ ID NO:5)
and reverse 5'GTCCTTGCCTCCTTGCCCCTTTCAG3' (SEQ ID NO:6)] based on
the sequence deposited in the public database (GenBank accession
AF151827) and using human liver cDNA as a template (Clontech, Palo
Alto, Calif., USA). For subcellular localization studies, a
carboxy-terminus FLAG-tagged version (FLAG-huCGI-69) was generated
by PCR using the forward primer and a FLAG-reverse primer
[5'CTTGTCATCGTCGTCCTTGTAGTCGCCGCCCAGAAGCCGGTC 3' (SEQ ID NO:7)].
CGI-69 PCR products were subcloned from pCR2.1 (Invitrogen,
Carlsbad, Calif., USA) into pRK7 (Genentech, Inc.) for expression
analyses. Also for subcellular localization, MCF7 cells were
transfected with pcDNA3-Flag-UCP3 or pRK7-FLAG-huCGI-69, and fixed
in 3% formaldehyde as previously-described (Yu, et al., in press).
Incubations with anti-FLAG and anti-cytochrome c oxidase
antibodies, Cy3 and FITC-conjugated secondary antibodies, and
visualization via confocal microscopy were performed as detailed
elsewhere (Yu, et al., in press).
[0299] Analysis of BAT genes upregulated by cold identified a 348
bp gene fragment whose QEA profile indicated significant induction
in Cold-Challenged mice. Initial identification of this DNA as
corresponding to murine EST AA985996 was confirmed by sequencing.
Real-time RT-PCR using primers/probes specific to this sequence
validated the marked 2-fold cold-induction of the gene in the BAT
of Cold-Challenged mice. Using murine EST AA985996 as the template
for a contig analysis, and using mouse ESTs from the public
database (SeqExtend Program, Genentech, Inc.), a putative murine
full-length gene encoding a protein with high homology to the
putative human protein CGI-69 (86% identical/98% similar) was
discovered, thus confirming its identity as the mouse ortholog.
Analysis of the CGI-69 protein structure indicated the presence of
4 mitochondrial carrier domains, 6 potential transmembrane spanning
regions, a likely mitochondrial localization (NNPSL algorithm, The
Sanger Centre, Hinxton, UK), and 3 regions with reasonable
homologies to putative mitochondrial energy transfer signature
motifs present in known UCP homologs. The mitochondrial
localization of carboxy- and amino-FLAG-tagged CGI-69 indicates
that native CGI-69 is targeted to this organelle.
Example 2
Human CGI-69 Variants
[0300] In this example, a novel splice variant of human CGI-69 was
discovered (SEQ ID NO:3).
[0301] mRNA abundance was analyzed in total RNA samples treated
with DNAse per manufacturer's instructions (GIBCO BRL, Grand
Island, N.Y., USA). Real-time quantitative RT-PCR was employed as
described previously (Yu, et al., 2000a; Yu, et al., 2000b), using
species- and isoform-specific primers and probes recognizing
CGI-69. The isoform specificity of the human primer/probe sets were
tested against authentic plasmids containing said isoforms. The
sequences of primers and probes (5'43') are as follows:
11 Human CGI-69 (all isoforms): fwdCCACCTGGTTTCAAGACCCTAC;
probeCGCTTCACTGGCACCATGGATGC (SEQ ID NO:9); revTGCCTCACGATCTTCACGAA
(SEQ ID NO:10) Human CGI-69.sub.L: fwdAGCGAGCTGATGCCTTCCT (SEQ ID
NO:11); probeCAGACTGTGGAGCTTCTCCTATACCAAATTGCC (SEQ ID NO:12);
revCCCTGTGGATTGGAGAGAGG (SEQ ID NO:13) Mouse CGI-69:
fwdCTGGCTCCTGCTTCGCA (SEQ ID NO:14); probeTCCGGGCTGAATCTGGCACCA
(SEQ ID NO:15); revGGAAGCCTGCAAAGAGTCCC (SEQ ID NO:16)
[0302] All data were normalized using 18S mRNA abundance to account
for loading differences, using commercially-available 18S
primer/probe sets (PE Applied Biosystems, Foster City, Calif.,
USA).
[0303] A variety of CGI-69 clones were isolated from human liver
upon PCR amplification and cloning, one of which corresponded to
the original AF151827 sequence in GenBank ("CGI-69"). Numerous
clones derived from separate, independent PCR cloning efforts
diverged from the GenBank sequence in that they encoded an 8 amino
acid insert preceded by a W64L change: this "long version" isoform
was termed "CGI-69.sub.L." In addition, various additional clones
encoded proteins with an additional change (F239L in CGI-69; F247L
in CGI-69.sub.L). CGI-69 transcript was detected in numerous
tissues, with particularly strong abundance in testis and BAT of
mice, and testis and kidney of humans. In humans, both the short
form(s) and long form(s) of the gene were expressed at various
ratios. Transcripts for CGI-69 were widely-detected in human
tissues, with particularly high expression in testis and kidney.
All values are expressed relative to liver CGI-69 mRNA abundance,
and represent abundance of total CGI-69. The relative contribution
of CGI-69.sub.L (% of total CGI-69 transcript) in humans was: 23%
(skeletal muscle, SKM), .about.40-45% (heart, stomach, lung,
uterus), 59% (brain), .about.72% (liver, spleen), 80% (kidney), and
91% (testis).
Example 3
Alteration of .DELTA..psi..sub.m
[0304] This example demonstrates that overexpression of a CGI-69
fusion protein having a carboxy FLAG-tagged CGI-69 diminished
.DELTA..psi..sub.m. The FLAG tag contains the negatively charged
amino acid sequence DYKDDDDK (SEQ ID NO:17).
[0305] Transfections and measurements of .DELTA..psi..sub.m were
carried out using protocols described previously (Yu, et al.,
2000b). 293 cells were co-transfected with pGreen Lantern-1 (green
fluorescent protein, GFP; GIBCO BRL) along with pRK7 vector alone
(control) or expression vectors containing human CGI-69, OGC, or
UCP3. Approximately 24 hr later, treatment-related differences in
.DELTA..psi..sub.m were determined in green-fluorescent protein
(GFP) positive cells by monitoring changes in the fluorescence
intensity of the .DELTA..psi..sub.m-sensitive dye TMRE
(tetramethylrhodamine ethyl ester; Molecular Probes, Eugene, Oreg.,
USA). The degree of diminution of the .DELTA..psi..sub.m was
assessed by the shift in the relative number of cells displaying
lowered .DELTA..psi..sub.m. The transfection protocols employed
herein resulted in at least a 30-fold overexpression of each gene
as judged by real-time RT-PCR analysis of mRNA abundance.
[0306] Incubations with anti-FLAG and anti-cytochrome c oxidase
antibodies, Cy3 and FITC-conjugated secondary antibodies, and
visualization via confocal microscopy were performed as detailed
elsewhere (Mao, et al., 1999).
[0307] Overexpression of carboxy-FLAG-tagged CGI-69 in 293 cells
diminished .DELTA..psi..sub.m to a similar magnitude as did human
UCP3. Similar to untagged CGI-69, amino-FLAG-tagged CGI-69 had no
effect on .DELTA..psi..sub.m, despite mitochondrial localization in
MCF7 cells. Overexpression of human CGI-69 in 293 cells also did
not influence .DELTA..psi..sub.m.
EQUIVALENTS
[0308] Although particular embodiments have been disclosed herein
in detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims that follow. In particular, it
is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims. The choice of nucleic acid starting material, clone of
interest, or library type is believed to be a matter of routine for
a person of ordinary skill in the art with knowledge of the
embodiments described herein. Other aspects, advantages, and
modifications considered to be within the scope of the following
claims.
REFERENCES
[0309] U.S. Pat. No. 4,166,452. Apparatus for testing human
responses to stimuli. 1979.
[0310] U.S. Pat. No. 4,485,045. Synthetic phosphatidyl cholines
useful in forming liposomes. 1984.
[0311] U.S. Pat. No. 4,544,545. Liposomes containing modified
cholesterol for organ targeting. 1985.
[0312] U.S. Pat. No. 4,676,980. Target specific cross-linked
heteroantibodies. 1987.
[0313] U.S. Pat. No. 4,816,567. Recombinant immunoglobin
preparations. 1989.
[0314] U.S. Pat. No. 5,013,556. Liposomes with enhanced circulation
time. 1991.
[0315] U.S. Pat. No. 5,545,807. Production of antibodies from
transgenic animals. 1996.
[0316] U.S. Pat. No. 5,545,806. Ransgenic <sic> non-human
animals for producing heterologous antibodies. 1996.
[0317] U.S. Pat. No. 5,569,825. Transgenic non-human animals
capable of producing heterologous antibodies of various isotypes.
1996.
[0318] U.S. Pat. No. 5,633,425. Transgenic non-human animals
capable of producing heterologous antibodies. 1997.
[0319] U.S. Pat. No. 5,661,016. Transgenic non-human animals
capable of producing heterologous antibodies of various isotypes.
1997.
[0320] U.S. Pat. No. 5,625,126. Transgenic non-human animals for
producing heterologous antibodies. 1997.
[0321] U.S. Pat. No. 3,773,919. Polylactide-drug mixtures.
1973.
[0322] U.S. Pat. No. 5,116,742. RNA ribozyme restriction
endoribonucleases and methods. 1992.
[0323] U.S. Pat. No. 4,987,071. RNA ribozyme polymerases,
dephosphorylases, restriction endoribonucleases and methods.
1991.
[0324] U.S. Pat. No. 4,522,811. Serial injection of
muramyldipeptides and liposomes enhances the anti-infective
activity of muramyldipeptides Serial injection of muramyldipeptides
and liposomes enhances the anti-infective activity of
muramyldipeptides. 1985.
[0325] U.S. Pat. No. 4,870,009. Method of obtaining gene product
through the generation of transgenic animals. 1989.
[0326] U.S. Pat. No. 5,804,604. Tat-derived transport polypeptides
and fusion proteins. 1998.
[0327] U.S. Pat. No. 5,223,409. Directed evolution of novel binding
proteins. 1993.
[0328] U.S. Pat. No. 5,459,039. Methods for mapping genetic
mutations. 1995.
[0329] U.S. Pat. No. 4,683,202. Process for amplifying nucleic acid
sequences. 1987.
[0330] U.S. Pat. No. 4,683,195. Process for amplifying, detecting,
and/or cloning nucleic acid sequences. 1987.
[0331] U.S. Pat. No. 5,328,470. Treatment of diseases by
site-specific instillation of cells or site-specific transformation
of cells and kits therefor. 1994.
[0332] U.S. Pat. No. 5,283,317. Intermediates for conjugation of
polypeptides with high molecular weight polyalkylene glycols.
1994.
[0333] U.S. Pat. No. 5,272,057. Method of detecting a
predisposition to cancer by the use of restriction fragment length
polymorphism of the gene for human poly (ADP-ribose) polymerase.
1993.
[0334] U.S. Pat. No. 4,904,582. Novel amphiphilic nucleic acid
conjugates. 1988.
[0335] U.S. Pat. No. 4,873,191. Genetic transformation of zygotes.
1989.
[0336] U.S. Pat. No. 4,736,866. Transgenic non-human animals.
1988.
[0337] WO 90/10448. Covalent conjugates of lipid and
oligonucleotide. 1990.
[0338] WO 90/13641. Stably transformed eucaryotic cells comprisng a
foreign transcribable DNA under the control of a pol III promoter.
1990.
[0339] WO 91/00360. Bispecific reagents for AIDS therapy. 1991.
[0340] WO 91/04753. Conjugates of antisense oligonucleotides and
therapeutic uses thereof. 1991.
[0341] WO 91/00357. New strain with filamentous fungi mutants,
process for the production of recombinant proteins using said
strain, and strains and proteins. 1991.
[0342] WO 91/06629. Oligonucleotide analogs with novel linkages.
1991.
[0343] WO 92/20373. Heteroconjugate antibodies for treatment of HIV
infection. 1992.
[0344] WO 93/08829. Compositions that mediate killing of
HIV-infected cells. 1993.
[0345] WO 94/11026. Therapeutic application of chimeric and
radiolabeled antibodies to human B lymphocyte restricted
differentiation antigen for treatment of B cells. 1994.
[0346] WO 96/27011. A method for making heteromultimeric
polypeptides. 1996.
[0347] WO 97/33551. Compositions and methods for the diagnosis,
prevention, and treatment of neoplastic cell growth and
proliferation. 1997.
[0348] WO 089/10134. Chimeric peptides for neuropeptide delivery
through the blood-brain barrier. 1989.
[0349] WO 91/01140. HOMOLOGOUS RECOMBINATION FOR UNIVERSAL DONOR
CELLS AND CHIMERIC MAMMALIAN HOSTS. 1991.
[0350] WO 90/11354. Process for the specific replacement of a copy
of a gene present in the receiver genome via the integration of a
gene. 1990.
[0351] WO94/16101. DNA SEQUENCING BY MASS SPECTROMETRY. 1994.
[0352] WO94/10300. INTERACTION TRAP SYSTEM FOR ISOLATING NOVEL
PROTEINS. 1994.
[0353] WO 93/04169. GENE TARGETING IN ANIMAL CELLS USING ISOGENIC
DNA CONSTRUCTS. 1993.
[0354] EPO 402226. Transformation vectors for yeast Yarrowia.
1990.
[0355] Abravaya, K., J. J. Carrino, S. Muldoon, and H. H. Lee.
1995. Detection of point mutations with a modified ligase chain
reaction (Gap-LCR). Nucleic Acids Res. 23:675-82.
[0356] Adams, S. H. 2000. J. Nutr. 130: 711-714.
[0357] Alam, J., and J. L. Cook. 1990. Reporter genes: Application
to the study of mammalian gene transcription. Anal. Biochem.
188:245-254.
[0358] Austin, C. P., and C. L. Cepko. 1990. Cellular migration
patterns in the developing mouse cerebral cortex. Development.
110:713-732.
[0359] Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, et
al. 1987. Current protocols in molecular biology. John Wiley &
Sons, New York.
[0360] Barany, F. 1991. Genetic disease detection and DNA
amplification using cloned thermostable ligase. Proc Natl Acad Sci
USA. 88:189-93.
[0361] Bartel, D. P., and J. W. Szostak. 1993. Isolation of new
ribozymes from a large pool of random sequences [see comment].
Science. 261:1411-8.
[0362] Bartel, P., C. T. Chien, R. Stemglanz, and S. Fields. 1993.
Elimination of false positives that arise in using the two-hybrid
system. Biotechniques. 14:920-4.
[0363] Beal, P. A., and P. B. Dervan. 1991. Second structural motif
for recognition of DNA by oligonucleotide-directed triple-helix
formation. Science. 251:1360-3.
[0364] Bechtold, N., and G. Pelletier. 1998. In planta
Agrobacterium-mediated transformation of adult Arabidopsis thaliana
plants by vacuum infiltration. Methods Mol Biol. 82:259-66.
[0365] Becker, D. M., and L. Guarente. 1991. High-efficiency
transformation of yeast by electroporation. Methods Enzymol.
194:182-187.
[0366] Beggs, J. D. 1978. Transformation of yeast by a replicating
hybrid plasmid. Nature. 275:104-109.
[0367] Bently, D. R., and I. Dunham. 1995. Mapping human
chromosomes. Curr Opin Genet Dev. 5:328-34.
[0368] Berger, J., J. Hauber, R. Hauber, R. Geiger, et al. 1988.
Secreted placental alkaline phosphatase: A powerful new
qunatitative indicator of gene expression in eukaryotic cells.
Gene. 66:1-10.
[0369] Bodine, D. M., K. T. McDonagh, N. E. Seidel, and A. W.
Nienhuis. 1991. Survival and retrovirus infection of murine
hematopoietic stem cells in vitro: effects of 5-FU and method of
infection. Exp. Hematol. 19:206-212.
[0370] Boemer, P., R. Lafond, W. Z. Lu, P. Brams, et al. 1991.
Production of antigen-specific human monoclonal antibodies from in
vitro-primed human splenocytes. J Immunol. 147:86-95.
[0371] Bradley. 1987. Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach. Oxford University Press, Inc., Oxford. 268
pp.
[0372] Bradley, A. 1991. Modifying the mammalian genome by gene
targeting. Curr Opin Biotechnol. 2:823-9.
[0373] Brand, M. D., L. F. Chien, E. K. Ainscow, D. F. S. Rolfe,
and R. K. Porter. 1994. The causes and functions of mitochondrial
proton leak. Biochim. Biophys. Acta 1187: 132-139.
[0374] Bray, G. A. 1997. Progress in understanding the genetics of
obesity. J Nutr. 127:940S-942S.
[0375] Brennan, M., P. F. Davison, and H. Paulus. 1985. Preparation
of bispecific antibodies by chemical recombination of monoclonal
immunoglobulin G1 fragments. Science. 229:81-3.
[0376] Capecchi, M. R. 1980. High efficiency transformation by
direct microinjection of DNA into cultured mammalian cells. Cell.
22:479.
[0377] Capecchi, M. R. 1989. Altering the genome by homologous
recombination. Science. 244:1288-92.
[0378] Carell, T., E. A. Wintner, and J. Rebek Jr. 1994a. A novel
procedure for the synthesis of libraries containing small organic
molecules. Angewandte Chemie International Edition.
33:2059-2061.
[0379] Carell, T., E. A. Wintner, and J. Rebek Jr. 1994b. A
solution phase screening procedure for the isolation of active
compounds from a molecular library. Angewandte Chemie International
Edition. 33:2061-2064.
[0380] Caron, P. C., W. Laird, M. S. Co, N. M. Avdalovic, et al.
1992. Engineered humanized dimeric forms of IgG are more effective
antibodies. J Exp Med. 176:1191-5.
[0381] Carter, P. 1986. Site-directed mutagenesis. Biochem J.
237:1-7.
[0382] Case, M. E., M. Schweizer, S. R. Kushner, and N. H. Giles.
1979. Efficient transformation of Neurospora crassa by utilizing
hybrid plasmid DNA. Proc Natl Acad Sci USA. 76:5259-63.
[0383] Cepko, C. L., B. E. Roberts, and R. E. Mulligan. 1984.
Construction and applications of a highly transmissible murine
retrovirus shuttle vector. Cell. 37:1053-1062.
[0384] Chalfie, M., Y. tu, G. Euskirchen, W. W. Ward, et al. 1994.
Green fluorescent protein as a marker for gene expression. Science.
263:802-805.
[0385] Chaney, W. G., D. R. Howard, J. W. Pollard, S. Sallustio, et
al. 1986. High-frequency transfection of CHO cells using Polybrene.
Somatic Cell Mol. Genet. 12:237.
[0386] Chen, C., and H. Okayama. 1988. Calcium phosphate-mediated
gene transfer: A highly efficient system for stably transforming
cells with plasmid DNA. BioTechniques. 6:632-638.
[0387] Chen, S. H., H. D. Shine, J. C. Goodman, R. G. Grossman, et
al. 1994. Gene therapy for brain tumors: regression of experimental
gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl
Acad Sci USA. 91:3054-7.
[0388] Cho, C. Y., E. J. Moran, S. R. Cherry, J. C. Stephans, et
al. 1993. An unnatural biopolymer. Science. 261:1303-5.
[0389] Cohen, A. S., D. L. Smisek, and B. H. Wang. 1996. Emerging
technologies for sequencing antisense oligonucleotides: capillary
electrophoresis and mass spectrometry. Adv Chromatogr.
36:127-62.
[0390] Cohen, J. S. 1989. Oligodeoxynucleotides: Antisense
inhibitors of gene expression. CRC Press, Boca Raton, Fla. 255
pp.
[0391] Cohen, S. M. N., A. C. Y. Chang, and L. Hsu. 1972.
Nonchromosomal antibiotic resistance in bacteria: Genetic
transformation of Escherichia coli by R-factor DNA. Proc. Natl.
Acad. Sci. USA. 69:2110.
[0392] Cooney, M., G. Czemuszewicz, E. H. Postel, S. J. Flint, et
al. 1988. Site-specific oligonucleotide binding represses
transcription of the human c-myc gene in vitro. Science.
241:456-9.
[0393] Cotton, R. G. 1993. Current methods of mutation detection.
Mutat Res. 285:125-44.
[0394] Cronin, M. T., R. V. Fucini, S. M. Kim, R. S. Masino, et al.
1996. Cystic fibrosis mutation detection by hybridization to
light-generated DNA probe arrays. Hum Mutat. 7:244-55.
[0395] Cull, M. G., J. F. Miller, and P. J. Schatz. 1992. Screening
for receptor ligands using large libraries of peptides linked to
the C terminus of the lac repressor. Proc Natl Acad Sci USA.
89:1865-9.
[0396] Cwirla, S. E., E. A. Peters, R. W. Barrett, and W. J. Dower.
1990. Peptides on phage: a vast library of peptides for identifying
ligands. Proc Natl Acad Sci USA. 87:6378-82.
[0397] de Boer, A. G. 1994. Drug absorption enhancement: Concepts,
possibilities, limitations and trends. Harwood Academic Publishers,
Langhorne, Pa.
[0398] de Louvencourt, L., H. Fukuhara, H. Heslot, and M.
Wesolowski. 1983. Transformation of Kluyveromyces lactis by killer
plasmid DNA. J Bacteriol. 154:737-42.
[0399] de Wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, et al.
1987. Sturcture and expression in mammalian cells. Mol. Cell Biol.
7:725-737.
[0400] Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman.
1966. A proposal for a uniform nomenclature in bacterial genetics.
Genetics. 54:61-76.
[0401] Devlin, J. J., L. C. Panganiban, and P. E. Devlin. 1990.
Random peptide libraries: a source of specific protein binding
molecules. Science. 249:404-6.
[0402] DeWitt, S. H., J. S. Kiely, C. J. Stankovic, M. C.
Schroeder, et al. 1993. "Diversomers": an approach to nonpeptide,
nonoligomeric chemical diversity. Proc Natl Acad Sci USA.
90:6909-13.
[0403] Eichelbaum, M., and B. Evert. 1996. Influence of
pharmacogenetics on drug disposition and response. Clin Exp
Pharmacol Physiol. 23:983-5.
[0404] Ellington, A. D., and J. W. Szostak. 1990. In vitro
selection of RNA molecules that bind specific ligands. Nature.
346:818-22.
[0405] Elroy-Stein, O., and B. Moss. 1990. Cytoplasmic expression
system based on constitutive synthesis of bacteriophage T7 RNA
polymerase in mammalian cells. Proc. Natl. Acad. Sci. USA.
87:6743-6747.
[0406] Eppstein, D. A., Y. V. Marsh, M. van der Pas, P. L. Felgner,
et al. 1985. Biological activity of liposome-encapsulated murine
interferon gamma is mediated by a cell membrane receptor. Proc Natl
Acad Sci USA. 82:3688-92.
[0407] Escudero, J., and B. Hohn. 1997. Transfer and integration of
T-DNA without cell injury in the host plant. Plant Cell.
9:2135-2142.
[0408] Fekete, D. M., and C. L. Cepko. 1993. Retroviral infection
coupled with tissue transplantation limits gene transfer in the
chick embryo. Proc. Natl. Acad. Sci. USA. 90:2350-2354.
[0409] Feigner, P. L., T. R. Gadek, M. Holm, R. Roman, et al. 1987.
Lipofectin: A highly efficient, lipid-mediated DNA/transfection
procedure. Proc. Natl. Acad. Sci. USA. 84:7413-7417.
[0410] Felici, F., L. Castagnoli, A. Musacchio, R. Jappelli, et al.
1991. Selection of antibody ligands from a large library of
oligopeptides expressed on a multivalent exposition vector. J Mol
Biol. 222:301-10.
[0411] Fieck, A., D. L. Wyborski, and J. M. Short. 1992.
Modifications of the E. coli Lac repressor for expression in
eukaryotic cells: effects of nuclear signal sequences on protein
activity and nuclear accumulation. Nucleic Acids Res.
20:1785-91.
[0412] Finer, J. J., K. R. Finer, and T. Ponappa. 1999. Particle
bombardment-mediated transformation. Current Topics in microbiology
and immunology. 240:59-80.
[0413] Finn, P. J., N. J. Gibson, R. Fallon, A. Hamilton, et al.
1996. Synthesis and properties of DNA-PNA chimeric oligomers.
Nucleic Acids Res. 24:3357-63.
[0414] Fishwild, D. M., S. L. O'Donnell, T. Bengoechea, D. V.
Hudson, et al. 1996. High-avidity human IgG kappa monoclonal
antibodies from a novel strain of minilocus transgenic mice [see
comments]. Nat Biotechnol. 14:845-51.
[0415] Fleer, R., P. Yeh, N. Amellal, I. Maury, et al. 1991. Stable
multicopy vectors for high-level secretion of recombinant human
serum albumin by Kluyveromyces yeasts. Biotechnology (NY).
9:968-75.
[0416] Fodor, S. P., R. P. Rava, X. C. Huang, A. C. Pease, et al.
1993. Multiplexed biochemical assays with biological chips. Nature.
364:555-6.
[0417] Fromm, M., L. P. Taylor, and V. Walbot. 1985. Expression of
genes transferred into monocot and dicot plant cells by
electroporation. Proc. Natl. Acad. Sci. USA. 82:5824-5828.
[0418] Fujita, T., H. Shubiya, T. Ohashi, K. Yamanishi, et al.
1986. Regulation of human interleukin-2 gene: Functional DNA
sequences in the 5' flanking region for the gene expression in
activated T lymphocytes. Cell. 46:401-407.
[0419] Gabizon, A., R. Shiota, and D. Papahadjopoulos. 1989.
Pharmacokinetics and tissue distribution of doxorubicin
encapsulated in stable liposomes with long circulation times. J
Natl Cancer Inst. 81:1484-8.
[0420] Gallagher, S. R. 1992. GUS protocols: Using the GUS gene as
a reporter of gene expression. Academic Press, San Diego,
Calif.
[0421] Gallop, M. A., R. W. Barrett, W. J. Dower, S. P. Fodor, et
al. 1994. Applications of combinatorial technologies to drug
discovery. 1. Background and peptide combinatorial libraries. J Med
Chem. 37:1233-51.
[0422] Gasparini, P., A. Bonizzato, M. Dognini, and P. F. Pignatti.
1992. Restriction site generating-polymerase chain reaction
(RG-PCR) for the probeless detection of hidden genetic variation:
application to the study of some common cystic fibrosis mutations.
Mol Cell Probes. 6:1-7.
[0423] Gautier, C., F. Morvan, B. Rayner, T. Huynh-Dinh, et al.
1987. Alpha-DNA. IV: Alpha-anomeric and beta-anomeric
tetrathymidylates covalently linked to intercalating
oxazolopyridocarbazole. Synthesis, physicochemical properties and
poly (rA) binding. Nucleic Acids Res. 15:6625-41.
[0424] Gennaro, A. R. 2000. Remington: The science and practice of
pharmacy. Lippincott, Williams & Wilkins, Philadelphia, Pa.
[0425] Gibbs, R. A., P. N. Nguyen, and C. T. Caskey. 1989.
Detection of single DNA base differences by competitive
oligonucleotide priming. Nucleic Acids Res. 17:2437-48.
[0426] Gietz, R. D., R. A. Woods, P. Manivasakam, and R. H.
Schiestl. 1998. Growth and transformation of Saccharomyces
cerevisiae. In Cells: A laboratory manual. Vol. I. D. Spector, R.
Goldman, and L. Leinwand, editors. Cold Spring Harbor Press, Cold
Spring Harbor, N.Y.
[0427] Goding, J. W. 1996. Monoclonal antibodies: Principles and
Practice. Academic Press, San Diego. 492 pp.
[0428] Goldstein, S. A., and D. H. Elwyn. 1989. The effects of
injury and sepsis on fuel utilization. Annu Rev Nutr. 9:445-73.
[0429] Gong, D. W., S. Monemdjou, O. Gavrilova, L. R. Leon, B.
Marcus-Samuels, C. J. Chou, C. Everett, L. P. Kozak, C. Li, C.
Deng, M. E. Harper, and M. L. Reitman. 2000. Lack of obesity and
normal response to fasting and thyroid hormone in mice lacking
uncoupling protein-3. J. Biol. Chem. 275(21)16251-7.
[0430] Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982.
Recombinant genomes which express chloramphenicol acetyltransferase
in manunalian cells. Mol. Cell. Biol. 2:1044-1051.
[0431] Graham, F. L., and A. J. van der Eb. 1973. A new technique
for the assay of infectivity of human adenovirus 5 DNA. Virology.
52:456-.
[0432] Green, H., and M. Meuth. 1974. An established pre-adipose
cell line and its differentiation in culture. Cell. 3:127-33.
[0433] Griffin, H. G., and A. M. Griffin. 1993. DNA sequencing.
Recent innovations and future trends. Appl Biochem Biotechnol.
38:147-59.
[0434] Grompe, M., D. M. Muzny, and C. T. Caskey. 1989. Scanning
detection of mutations in human omithine transcarbamoylase by
chemical mismatch cleavage. Proc Natl Acad Sci USA. 86:5888-92.
[0435] Gruber, M., B. A. Schodin, E. R. Wilson, and D. M. Kranz.
1994. Efficient tumor cell lysis mediated by a bispecific single
chain antibody expressed in Escherichia coli. J Immunol.
152:5368-74.
[0436] Guatelli, J. C., K. M. Whitfield, D. Y. Kwoh, K. J.
Barringer, et al. 1990. Isothermal, in vitro amplification of
nucleic acids by a multienzyme reaction modeled after retroviral
replication. Proc Natl Acad Sci US A. 87:1874-8.
[0437] Hanahan, D. 1983. Studies on transformation of Escherichia
coli with plasmids. J. Mol. Biol. 166:557-580.
[0438] Hansen, G., and M.-D. Chilton. 1999. Lessons in gene
transfer to plants by a gifted microbe. Curr. Top. Microbiol.
Immunol. 240:21-57.
[0439] Hansen, G., and M. S. Wright. 1999. Recent advances in the
transformation of plants. Trends Plant Sci. 4:226-231.
[0440] Harlow, E., and D. Lane. 1988. Antibodies: A laboratory
manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
726 pp.
[0441] Harlow, E., and D. Lane. 1999. Using antibodies: A
laboratory manual. Cold Spring Harbor Laboratory PRess, Cold Spring
Harbor, N.Y.
[0442] Haseloff, J., and W. L. Gerlach. 1988. Simple RNA enzymes
with new and highly specific endoribonuclease activities. Nature.
334:585-91.
[0443] Hayashi, K. 1992. PCR-SSCP: A method for detection of
mutations. Genetic and Analytical Techniques Applications.
9:73-79.
[0444] Helene, C. 1991. The anti-gene strategy: control of gene
expression by triplex-forming-oligonucleotides. Anticancer Drug
Des. 6:569-84.
[0445] Helene, C., N. T. Thuong, and A. Harel-Bellan. 1992. Control
of gene expression by triple helix-forming oligonucleotides. The
antigene strategy. Ann NY Acad Sci. 660:27-36.
[0446] Hill, J. O., and J. C. Peters. 1998. Environmental
contributions to the obesity epidemic. Science. 280:1371-4.
[0447] Hinnen, A., J. B. Hicks, and G. R. Fink. 1978.
Transformation of yeast. Proc. Natl. Acad. Sci. USA.
75:1929-1933.
[0448] Hoffman, F. 1996. Laser microbeams for the manipulation of
plant cells and subcellular structures. Plant Sci. 113:1-11.
[0449] Hogan, B., Beddington, R., Costantini, F., Lacy, E. 1994.
Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring
Harbor Laboratory Press. 500 pp.
[0450] Holliger, P., T. Prospero, and G. Winter. 1993. "Diabodies":
small bivalent and bispecific antibody fragments. Proc Natl Acad
Sci USA. 90:6444-8.
[0451] Hoogenboom, H. R., A. D. Griffiths, K. S. Johnson, D. J.
Chiswell, et al. 1991. Multi-subunit proteins on the surface of
filamentous phage: methodologies for displaying antibody (Fab)
heavy and light chains. Nucleic Acids Res. 19:4133-7.
[0452] Houghten, R. A., J. R. Appel, S. E. Blondelle, J. H. Cuervo,
et al. 1992. The use of synthetic peptide combinatorial libraries
for the identification of bioactive peptides. Biotechniques.
13:412-21.
[0453] Hsu, I. C., Q. Yang, M. W. Kahng, and J. F. Xu. 1994.
Detection of DNA point mutations with DNA mismatch repair enzymes.
Carcinogenesis. 15:1657-62.
[0454] Hudson, T. J., L. D. Stein, S. S. Gerety, J. Ma, et al.
1995. An STS-based map of the human genome. Science.
270:1945-54.
[0455] Hwang, K. J., K. F. Luk, and P. L. Beaumier. 1980. Hepatic
uptake and degradation of unilamellar sphingomyelin/cholesterol
liposomes: a kinetic study. Proc Natl Acad Sci USA. 77:4030-4.
[0456] Hyrup, B., and P. E. Nielsen. 1996. Peptide nucleic acids
(PNA): synthesis, properties and potential applications. Bioorg Med
Chem. 4:5-23.
[0457] Inoue, H., Y. Hayase, A. Imura, S. Iwai, et al. 1987a.
Synthesis and hybridization studies on two complementary
nona(2'-O-methyl)ribonucle- otides. Nucleic Acids Res.
15:6131-48.
[0458] Inoue, H., Y. Hayase, S. Iwai, and E. Ohtsuka. 1987b.
Sequence-dependent hydrolysis of RNA using modified oligonucleotide
splints and RNase H. FEBS Lett. 215:327-30.
[0459] Ishiura, M., S. Hirose, T. Uchida, Y. Hamada, et al. 1982.
Phage particle-mediated gene transfer to cultured mammalian cells.
Molecular and Cellular Biology. 2:607-616.
[0460] Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983.
Transformation of intact yeast cells treated with alkali cations.
J. Bacteriol. 153:163-168.
[0461] Iwabuchi, K., B. Li, P. Bartel, and S. Fields. 1993. Use of
the two-hybrid system to identify the domain of p53 involved in
oligomerization. Oncogene. 8:1693-6.
[0462] Jayasena, S. D. 1999. Aptamers: an emerging class of
molecules that rival antibodies in diagnostics. Clin Chem.
45:1628-50.
[0463] Jones, P. T., P. H. Dear, J. Foote, M. S. Neuberger, et al.
1986. Replacing the complementarity-determining regions in a human
antibody with those from a mouse. Nature. 321:522-5.
[0464] Kaufmnan, R. J. 1990. Vectors used for expression in
mammalian cells. Methods Enzymol. 185:487-511.
[0465] Kaufman, R. J., P. Murtha, D. E. Ingolia, C.-Y. Yeung, et
al. 1986. Selection and amplification of heterologous genes
encoding adenosine deaminase in mammalian cells. Proc. Natl. Acad.
Sci. USA. 83:3136-3140.
[0466] Kawai, S., and M. Nishizawa. 1984. New procedure for DNA
transfection with polycation and dimethyl sulfoxide. Mol. Cell.
Biol. 4:1172.
[0467] Keen, J., D. Lester, C. Inglehearn, A. Curtis, et al. 1991.
Rapid detection of single base mismatches as heteroduplexes on
Hydrolink gels. Trends Genet. 7:5.
[0468] Kelly, J. M., and M. J. Hynes. 1985. Transformation of
Aspergillus niger by the amdS gene of Aspergillus nidulans. Embo J.
4:475-9.
[0469] Kinney, J., J. Duke, Jr., C. Long, and R. Gump. 1970. Tissue
fuel and weight loss after injury. J. Clin. Path. 23 (suppl.
4):65-72.
[0470] Kissebah, A. H., G. E. Sonnenberg, J. Myklebust, M.
Goldstein, et al. 2000. Quantitative trait loci on chromosomes 3
and 17 influence phenotypes of the metabolic syndrome. Proc Natl
Acad Sci USA. 97:14478-83.
[0471] Kostelny, S. A., M. S. Cole, and J. Y. Tso. 1992. Formation
of a bispecific antibody by the use of leucine zippers. J Immunol.
148:1547-53.
[0472] Kozal, M. J., N. Shah, N. Shen, R. Yang, et al. 1996.
Extensive polymorphisms observed in HIV-1 clade B protease gene
using high-density oligonucleotide arrays. Nat Med. 2:753-9.
[0473] Kozbor, D., P. Tripputi, J. C. Roder, and C. M. Croce. 1984.
A human hybrid myeloma for production of human monoclonal
antibodies. J Immunol. 133:3001-5.
[0474] Kriegler, M. 1990. Gene transfer and expression: A
laboratory manual. Stockton Press, New York. 242 pp.
[0475] Kwoh, D. Y., G. R. Davis, K. M. Whitfield, H. L. Chappelle,
et al. 1989. Transcription-based amplification system and detection
of amplified human immunodeficiency virus type 1 with a bead-based
sandwich hybridization format. Proc Natl Acad Sci USA.
86:1173-7.
[0476] Lakso, M., B. Sauer, B. Mosinger, E. J. Lee, et al. 1992.
Targeted oncogene activation by site-specific recombination in
transgenic mice. Proc Natl Acad Sci USA. 89:6232-6.
[0477] Lam, K. S. 1997. Application of combinatorial library
methods in cancer research and drug discovery. Anticancer Drug
Design. 12:145-167.
[0478] Lam, K. S., S. E. Salmon, E. M. Hersh, V. J. Hruby, et al.
1991. General method for rapid synthesis of multicomponent peptide
mixtures. Nature. 354:82-84.
[0479] Landegren, U., R. Kaiser, J. Sanders, and L. Hood. 1988. A
ligase-mediated gene detection technique. Science. 241:1077-80.
[0480] Leduc, N., and e. al. 1996. Isolated maize zygotes mimic in
vivo embryogenic development and express microinjected genes when
cultured in vitro. Dev. Biol. 10: 190-203.
[0481] Lee, J. S., D. A. Johnson, and A. R. Morgan. 1979. Complexes
formed by (pyrimidine)n.(purine)n DNAs on lowering the pH are
three-stranded. Nucleic Acids Res. 6:3073-91.
[0482] Lee, V. H. L. 1990. Peptide and protein drug delivery.
Marcel Dekker, New York, N.Y.
[0483] Lemaitre, M., B. Bayard, and B. Lebleu. 1987. Specific
antiviral activity of a poly(L-lysine)-conjugated
oligodeoxyribonucleotide sequence complementary to vesicular
stomatitis virus N protein mRNA initiation site. Proc Natl Acad Sci
USA. 84:648-52.
[0484] Lemischka, I. R., D. H. Raulet, and R. C. Mulligan. 1986.
Developmental potential and dynamic behavior of hematopoietic stem
cells. Cell. 45:917-927.
[0485] Letsinger, R. L., G. R. Zhang, D. K. Sun, T. Ikeuchi, et al.
1989. Cholesteryl-conjugated oligonucleotides: synthesis,
properties, and activity as inhibitors of replication of human
immunodeficiency virus in cell culture. Proc Natl Acad Sci USA.
86:6553-6.
[0486] Li, E., T. H. Bestor, and R. Jaenisch. 1992. Targeted
mutation of the DNA methyltransferase gene results in embryonic
lethality. Cell. 69:915-26.
[0487] Linder, M. W., R. A. Prough, and R. Valdes. 1997.
Pharmacogenetics: a laboratory tool for optimizing therapeutic
efficiency. Clin Chem. 43:254-66.
[0488] Littlefield, J. W. 1964. Selection of hybrids from matings
of fibroblasts in vitro and their presumed recombinants. Science.
145:709-710.
[0489] Lizardi, P. M., C. E. Guerra, H. Lomeli, I. Tussie-Luna, et
al. 1988. Exponential amplification of recombinant-RNA
hybridization probes. Biotechnology. 6:1197-1202.
[0490] Lonberg, N., and D. Huszar. 1995. Human antibodies from
transgenic mice. Int Rev Immunol. 13:65-93.
[0491] Lonberg, N., L. D. Taylor, F. A. Harding, M. Trounstine, et
al. 1994. Antigen-specific human antibodies from mice comprising
four distinct genetic modifications [see comments]. Nature.
368:856-9.
[0492] Lopata, M. A., D. W. Cleveland, and B. Sollner-Webb. 1984.
High-level expression of a chloramphenicol acetyltransferase gene
by DEAEdextran-mediated DNA traansfection couled with a
dimethylsulfoxide or glycerol shock treatment. Nucleic Acids
Research. 12:5707.
[0493] Luckow, V. A. 1991. Cloning and expression of heterologous
genes in insect cells with baculovirus vectors. In Recombinant DNA
technology and applications. A. Prokop, R. K. Bajpai, and C. Ho,
editors. McGraw-Hill, New York. 97-152.
[0494] Madura, K., R. J. Dohmen, and A. Varshavsky. 1993.
N-recognin/ubc2 interactions in the N-end rule pathway. J Biol
Chem. 268:12046-54.
[0495] Maher, L. J. 1992. DNA triple-helix formation: an approach
to artificial gene repressors? Bioessays. 14:807-15.
[0496] Mandel, M., and A. Higa. 1970. Calcium-dependent
bacteriophage DNA infection. J Mol biol. 53:159-162.
[0497] Mao, W., X. X. Yu, A. Zhong, W. Li, J. Brush, S. W.
Sherwood, S. H. Adams, and G. Pan. 1999. UCP4, a novel
brain-specific mitochondrial protein that reduces membrane
potential in mammalian cells. FEBS Letters. 443: 326-330.
[0498] Marasco, W. A., W. A. Haseltine, and S. Y. Chen. 1993.
Design, intracellular expression, and activity of a human
anti-human immunodeficiency virus type 1 gp120 single-chain
antibody. Proc Natl Acad Sci USA. 90:7889-93.
[0499] Marks, J. D., A. D. Griffiths, M. Malmqvist, T. P. Clackson,
et al. 1992. By-passing immunization: building high affinity human
antibodies by chain shuffling. Biotechnology (NY). 10:779-83.
[0500] Marks, J. D., H. R. Hoogenboom, T. P. Bonnert, J.
McCafferty, et al. 1991. By-passing immunization. Human antibodies
from V-gene libraries displayed on phage. J Mol Biol.
222:581-97.
[0501] Martin, F. J., and D. Papahadjopoulos. 1982. Irreversible
coupling of immunoglobulin fragments to preformed vesicles. An
improved method for liposome targeting. J Biol Chem. 257:286-8.
[0502] Maxam, A. M., and W. Gilbert. 1977. A new method for
sequencing DNA. Proc Natl Acad Sci USA. 74:560-4.
[0503] Miller, A. D., and C. Buttimore. 1986. Redesign of
retrovirus packaging cell lines to avoid recombination leading to
helper virus production. Mol. Cell biol. 6:2895-2902.
[0504] Miller, L. K. 1988. Baculoviruses as gene expression
vectors. Annu. Rev. Microbiol. 42:177-199.
[0505] Milstein, C., and A. C. Cuello. 1983. Hybrid hybridomas and
their use in immunohistochemistry. Nature. 305:537-40.
[0506] Mitchell, B. D., S. A. Cole, A. G. Comuzzie, L. Almasy, et
al. 1999. A quantitative trait locus influencing BMI maps to the
region of the beta-3 adrenergic receptor. Diabetes. 48:1863-7.
[0507] Morrison, S. L., L. Wims, S. Wallick, L. Tan, et al. 1987.
Genetically engineered antibody molecules and their application.
Ann NY Acad Sci. 507:187-98.
[0508] Munson, P. J., and D. Rodbard. 1980. Ligand: a versatile
computerized approach for characterization of ligand-binding
systems. Anal Biochem. 107:220-39.
[0509] Must, A., J. Spadano, E. H. Coakley, A. E. Field, et al.
1999. The disease burden associated with overweight and obesity.
Jama. 282:1523-9.
[0510] Myers, R. M., Z. Larin, and T. Maniatis. 1985. Detection of
single base substitutions by ribonuclease cleavage at mismatches in
RNA:DNA duplexes. Science. 230:1242-6.
[0511] Naeve, C. W., G. A. Buck, R. L. Niece, R. T. Pon, et al.
1995. Accuracy of automated DNA sequencing: a multi-laboratory
comparison of sequencing results. Biotechniques. 19:448-53.
[0512] Nakazawa, H., D. English, P. L. Randell, K. Nakazawa, et al.
1994. UV and skin cancer: specific p53 gene mutation in normal skin
as a biologically relevant exposure measurement. Proc Natl Acad Sci
USA. 91:360-4.
[0513] Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H.
Hofschneider. 1982. Gene transfer into mouse lyoma cells by
electroporation in high electric fields. EMBO J. 1:841-845.
[0514] Nicholls, D. G. and R. M. Locke. 1984. Thermogenic
mechanisms in brown fat. Physiol. Rev. 64: 1-64.
[0515] O'Gorman, S., D. T. Fox, and G. M. Wahl. 1991.
Recombinase-mediated gene activation and site-specific integration
in mammalian cells. Science. 251:1351-5.
[0516] Okano, H., J. Aruga, T. Nakagawa, C. Shiota, et al. 1991.
Myelin basic protein gene and the function of antisense RNA in its
repression in myelin-deficient mutant mouse. J Neurochem.
56:560-7.
[0517] O'Reilly, D. R., L. K. Miller, and V. A. Luckow. 1992.
Baculovirus expression vectors. W. H. Freeman and Company, New
York.
[0518] Orita, M., H. Iwahana, H. Kanazawa, K. Hayashi, et al. 1989.
Detection of polymorphisms of human DNA by gel electrophoresis as
single-strand conformation polymorphisms. Proc Natl Acad Sci USA.
86:2766-70.
[0519] Ou-Lee, T. M., R. Turgeon, and R. Wu. 1986. Uptake and
expression of a foreign gene linked to either a plant virus or
Drosophila promoter in protoplasts of rice, wheat and sorghum.
Proc. Natl. Acad. Sci. USA. 83:6815-6819.
[0520] Palmer, T. D., R. A. Hock, W. R. A. osbome, and A. D.
Miller. 1987. Efficient retrovirus-mediated transfer and expression
of a human adenosine deaminase gene in diploid skin fibroblasts
from an adenosie-deficient human. Proc. Natl. Acad. Sci. USA.
84:1055-1059.
[0521] Pear, W., G. Nolan, M. Scott, and D. Baltimore. 1993.
Production of high-titer helper-free retroviruses by transient
transfection. Proc. Natl. Acad. Sci. USA. 90:8392-8396.
[0522] Perry-O'Keefe, H., X. W. Yao, J. M. Coull, M. Fuchs, et al.
1996. Peptide nucleic acid pre-gel hybridization: an alternative to
southern hybridization. Proc Natl Acad Sci USA. 93:14670-5.
[0523] Petersen, K. H., D. K. Jensen, M. Egholm, 0. Buchardt, et
al. 1976. A PNA-DNA linker synthesis of
N-((4,4'-dimethoxytrityloxy)ehtyl)-N-(thymi- n-1-ylacetyl)glycine.
Biorganic and Medicianl Chemistry Letters. 5:1119-1124.
[0524] Potter, H. 1988. Electroporation in biology: Methods,
applications,, and instrumentation. Analytical Biochemistry.
174:361-373.
[0525] Potter, H., L. Weir, and P. Leder. 1984. Enhancer-dependent
expression of human kappa immunoglobulin genes introduced into
mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci.
USA. 81:7161-7165.
[0526] Presta, L. G. 1992. Antibody engineering. Curr Opin
Biotechnol. 3:394-8.
[0527] Prosser, J. 1993. Detecting single-base mutations. Trends
Biotechnol. 11:238-46.
[0528] Rassoulzadegan, M., B. Binetruy, and F. Cuzin. 1982. High
frequency of gene transfer after fusion between bacteria and
eukaryotic cells. Nature. 295:257.
[0529] Reisfeld, R. A., and S. Sell. 1985. Monoclonal antibodies
and cancer therapy: Proceedings of the Roche-UCLA symposium held in
Park City, Utah, Jan. 26-Feb. 2, 1985. Alan R. Liss, New York. 609
pp.
[0530] Rhodes, C. A., D. A. Pierce, I. J. Mettler, D. Mascarenhas,
et al. 1988. Genetically transformed maize plants from protoplasts.
Science. 240:204-207.
[0531] Ricquier, D., L. Casteilla, and F. Bouillaud. 1991.
Molecular studies of the uncoupling protein. FASEB J. 5:
2237-2242.
[0532] Riechmann, L., M. Clark, H. Waldmann, and G. Winter. 1988.
Reshaping human antibodies for therapy. Nature. 332:323-7.
[0533] Rolfe, D. F. S., A. J. Hulbert, and M. D. Brand. 1994.
Characteristics of mitochondrial proton leak and control of
oxidative phosphorylation in the major oxygen-consuming tissues of
the rat. Biochim. Biophys. Acta 1118: 405-416.
[0534] Rolfe, D. F. S., J. M. B. Newman, J. A. Buckingham, M. G.
Clark, and M. D. Brand. 1999. Contribution of mitochondrial proton
leak to respiration rate in working skeletal muscle and liver and
to SMR. Am. J. Physiol. 276: C692-C699.
[0535] Rose, J. K., L. Buonocore, and M. Whitt. 1991. A new
cationic liposome reagent mediating nearly quantitative
transfection of animal cells. BioTechniques. 10:520-525.
[0536] Rossi, J. J. 1994. Practical ribozymes. Making ribozymes
work in cells. Curr Biol. 4:469-71.
[0537] Rossiter, B. J., and C. T. Caskey. 1990. Molecular scanning
methods of mutation detection. J Biol Chem. 265:12753-6.
[0538] Saiki, R. K., T. L. Bugawan, G. T. Horn, K. B. Mullis, et
al. 1986. Analysis of enzymatically amplified beta-globin and
HLA-DQ alpha DNA with allele-specific oligonucleotide probes.
Nature. 324:163-6.
[0539] Saiki, R. K., P. S. Walsh, C. H. Levenson, and H. A. Erlich.
1989. Genetic analysis of amplified DNA with immobilized
sequence-specific oligonucleotide probes. Proc Natl Acad Sci USA.
86:6230-4.
[0540] Saleeba, J. A., and R. G. Cotton. 1993. Chemical cleavage of
mismatch to detect mutations. Methods Enzymol. 217:286-95.
[0541] Sambrook, J. 1989. Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor.
[0542] Sandri-Goldin, R. M., A. L. Goldin, J. C. Glorioso, and M.
Levine. 1981. High-frequency transfer of cloned herpes simjplex
virus type I sequences to mammalian cells by protoplast fusion.
Mol. Cell. Biol. 1:7453-752.
[0543] Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA
sequencing with chain-terminating inhibitors. Proc Natl Acad Sci
USA. 74:5463-7.
[0544] Saunders, J. A., B. F. Matthews, and P. D. Miller. 1989.
Plant gene transfer using electrofusion and electroporation. In
Electroporation and electrofusion in cell biology. E. Neumann, A.
E. Sowers, and C. A. Jordan, editors. Plenum Press, New York.
343-354.
[0545] Schade, R., C. Staak, C. Hendriksen, M. Erhard, et al. 1996.
The production of avian (egg yold) antibodies: IgY. The report and
recommendations of ECVAM workshop. Alternatives to Laboratory
Animals (ATLA). 24:925-934.
[0546] Schaffner, W. 1980. Direct transfer of cloned genes from
bacteria to mammalian cells. Proc. Natl. Acad. Sci. USA.
77:2163.
[0547] Schook, L. B. 1987. Monoclonal antibody production
techniques and applications. Marcel Dekker, Inc., New York. 336
pp.
[0548] Schwartz, D. C., and A. Samad. 1997. Optical mapping
approaches to molecular genomics. Curr Opin Biotechnol. 8:70-4.
[0549] Scott, J. K., and G. P. Smith. 1990. Searching for peptide
ligands with an epitope library. Science. 249:386-90.
[0550] Selden, R. F., K. Burke-Howie, M. E. Rowe, H. M. Goodman, et
al. 1986. Human growth hormone as a reporter gene in regulation
studies employing transient gene expression. Molecular and Cellular
Biololgy. 6:3173-3179.
[0551] Shalaby, M. R., H. M. Shepard, L. Presta, M. L. Rodrigues,
et al. 1992. Development of humanized bispecific antibodies
reactive with cytotoxic lymphocytes and tumor cells overexpressing
the HER2 protooncogene. J. Exp Med. 175:217-25.
[0552] Shigekawa, K., and W. J. Dower. 1988. Electroporation of
eukaryotes and prokaryotes: A general approach to the introduction
of macomolecules into cells. BioTechniques. 6:742-751.
[0553] Shillito, R. 1999. Methods of genetic transformations:
Electroporation and polyethylene glycol treatment. In Molecular
improvement of cereal crop. I. Vasil, editor. Kluwer, Dordrecht,
The Netherlands. 9-20.
[0554] Shilo, B. Z., and R. A. Weinberg. 1981. DNA sequences
homologous to vertebrate oncogenes are conserved in Drosophila
melanogaster. Proc Natl Acad Sci USA. 78:6789-92.
[0555] Shimkets, R. A., D. G. Lowe, J. T. Tai, P. Sehl, H. Jin, R.
Yang, P. F. Predki, B. E. Rothberg, M. T. Murtha, M. E. Roth, S. G.
Shenoy, A. Windemuth, J. W. Simpson, J. F. Simons, M. P. Daley, S.
A. Gold, M. P. McKenna, K. Hillan, G. T. Went, and J. M. Rothberg.
1999. Gene expression analysis by transcript profiling coupled to a
gene database query. Nature Biotech. 17(8): 798-803.
[0556] Shopes, B. 1992. A genetically engineered human IgG mutant
with enhanced cytolytic activity. J Immunol. 148:2918-22.
[0557] Simonsen, C. C., and A. D. Levinson. 1983. Isolation and
expression of an altered mouse dihydrofolate reductase cDNA. Proc.
Natl. Acad. Sci. USA. 80:2495-2499.
[0558] Skulachev, V. P. 1996. Role of uncoupled and non-coupled
oxidations in maintenance of safely low levels of oxygen and its
one-electron reductants. Quarterly Rev. Biophys. 29(2):
169-202.
[0559] Southern, P. J., and P. Berg. 1982. Transformation of
mammalian cells to antibiotic resistanced with a bacterial gene
under control of the SV40 early region promoter. J. Mol. Appl. Gen.
1:327-341.
[0560] Sreekrishna, K., R. H. Potenz, J. A. Cruze, W. R. McCombie,
et al. 1988. High level expression of heterologous proteins in
methylotrophic yeast Pichia pastoris. J Basic Microbiol.
28:265-78.
[0561] Stein, C. A., and J. S. Cohen. 1988. Oligodeoxynucleotides
as inhibitors of gene expression: a review. Cancer Res.
48:2659-68.
[0562] Stevenson, G. T., A. Pindar, and C. J. Slade. 1989. A
chimeric antibody with dual Fc regions (bisFabFc) prepared by
manipulations at the IgG hinge. Anticancer Drug Des. 3:219-30.
[0563] Stewart, E. A., K. B. McKusick, A. Aggarwal, E. Bajorek, et
al. 1997. An STS-based radiation hybrid map of the human genome.
Genome Res. 7:422-33.
[0564] Suresh, M. R., A. C. Cuello, and C. Milstein. 1986.
Bispecific monoclonal antibodies from hybrid hybridomas. Methods
Enzymol. 121:210-28.
[0565] Thomas, K. R., and M. R. Capecchi. 1987. Site-directed
mutagenesis by gene targeting in mouse embryo-derived stem cells.
Cell. 51:503-12.
[0566] Thompson, J. A., and e. al. 1995. Maize transformation
utilizing silicon carbide whiskers: A review. Euphytica.
85:75-80.
[0567] Tilburn, J., C. Scazzocchio, G. G. Taylor, J. H.
Zabicky-Zissman, et al. 1983. Transformation by integration in
Aspergillus nidulans. Gene. 26:205-21.
[0568] Tisdale, M. J. 1997. Cancer cachexia: metabolic alterations
and clinical manifestations. Nutrition. 13:1-7.
[0569] Touraev, A., and e. al. 1997. Plant male germ line
transformation. Plant J 12:949-956.
[0570] Traunecker, A., F. Oliveri, and K. Karjalainen. 1991.
Myeloma based expression system for production of large mammalian
proteins. Trends Biotechnol. 9:109-13.
[0571] Trick, H. N., and e. al. 1997. Recent advances in soybean
transformation. Plant Tissue Cult. Biotechnol. 3:9-26.
[0572] Tuerk, C., and L. Gold. 1990. Systematic evolution of
ligands by exponential enrichment: RNA ligands to bacteriophage T4
DNA polymerase. Science. 249:505-10.
[0573] Turner, D. L., E. Y. Snyder, and C. L. Cepko. 1990.
Lineage-independent determinationh of cell type in the embryonic
mouse retina. Neuron. 4:833-845.
[0574] Tutt, A., G. T. Stevenson, and M. J. Glennie. 1991.
Trispecific F(ab')3 derivatives that use cooperative signaling via
the TCR/CD3 complex and CD2 to activate and redirect resting
cytotoxic T cells. J Immunol. 147:60-9.
[0575] van der Krol, A. R., J. N. Mol, and A. R. Stuitje. 1988a.
Modulation of eukaryotic gene expression by complementary RNA or
DNA sequences. Biotechniques. 6:958-76.
[0576] van der Krol, A. R., J. N. Mol, and A. R. Stuitje. 1988b.
Modulation of eukaryotic gene expression by complementary RNA or
DNA sequences. Biotechniques. 6:958-76.
[0577] Verhoeyen, M., C. Milstein, and G. Winter. 1988. Reshaping
human antibodies: grafting an antilysozyme activity. Science.
239:1534-6.
[0578] Vidal-Puig, A. J., D. Grujic, C. Y. Zhang, T. Hagen, O.
Boss, Y. Ido, A. Szczepanik, J. Wade, V. Mootha, R. Cortright, D.
M. Muoio, and B. B. Lowell. 2000. Energy metabolism uncoupling
protein 3 gene knockout mice. J. Biol. Chem. 275(21)16258-66.
[0579] Vitetta, E. S., R. J. Fulton, R. D. May, M. Till, et al.
1987. Redesigning nature's poisons to create anti-tumor reagents.
Science. 238:1098-104.
[0580] Wells, J. A., M. Vasser, and D. B. Powers. 1985. Cassette
mutagenesis: an efficient method for generation of multiple
mutations at defined sites. Gene. 34:315-23.
[0581] Whitt, M. A., L. Buonocore, J. K. Rose, V. Ciccarone, et al.
1990. TransfectACE reagent promotes transient transfection
frequencies greater than 90%. Focus. 13:8-12.
[0582] Wigler, M., A. Pellicer, S. Silversttein, and R. Axel. 1978.
Biochemical transfer of single-copy eucaryotic genes using total
cellular DNA as donor. Cell. 14:725.
[0583] Williams, D. A., I. R. Lemiscbka, D. G. Nathan, and R. C.
Mulligan. 1984. Introduction of a new genetic material into
pluripotent haematopoietic stem cells of the mouse. Nature.
310:476-480.
[0584] Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind, et al.
1997. Viable offspring derived from fetal and adult mammalian
cells. Nature. 385:810-3.
[0585] Wolff, E. A., G. J. Schreiber, W. L. Cosand, and H. V. Raff.
1993. Monoclonal antibody homodimers: enhanced antitumor activity
in nude mice. Cancer Res. 53:2560-5.
[0586] Wong, T. K., and E. Neumann. 1982. Electric field mediated
gene transfer. Biochemical and Biophysical Research Communications.
107:584-587.
[0587] Wyborski, D. L., L. C. DuCoeur, and J. M. Short. 1996.
Parameters affecting the use of the lac repressor system in
eukaryotic cells and transgenic animals. Environ Mol Mutagen.
28:447-58.
[0588] Wyborski, D. L., and J. M. Short. 1991. Analysis of inducers
of the E.coli lac repressor system in mammalian cells and whole
animals. Nucleic Acids Res. 19:4647-53.
[0589] Yelton, M. M., J. E. Hamer, and W. E. Timberlake. 1984.
Transformation of Aspergillus nidulans by using a trpC plasmid.
Proc Natl Acad Sci USA. 81:1470-4.
[0590] Yu, X. X., J. L. Barger, B. B. Boyer, M. D. Brand, G. Pan,
and S. H. Adams. 2000a. Impact of endotoxin on UCP homolog mRNA
abundance, thermoregulation, and mitochondrial proton leak
kinetics. Am. J Physiol., 279(2):E433-46.
[0591] Yu, X. X., W. Mao, A. Zhong, P. Schow, J. Brush, S. W.
Sherwood, S. H. Adams, and G. Pan. 2000b. Characterization of novel
UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5
expression through dietary or temperature manipulation. FASEB J
14(11):1611-8.
[0592] Zervos, A. S., J. Gyuris, and R. Brent. 1993. Mxi1, a
protein that specifically interacts with Max to bind Myc-Max
recognition sites. Cell. 72:223-32.
[0593] Zhou, G., and e. al. 1983. Introduction of exogenous DNA
into cotton embryos. Methods Enzymol. 101:433-481.
[0594] Zoller, M. J., and M. Smith. 1987. Oligonucleotide-directed
mutagenesis: a simple method using two oligonucleotide primers and
a single-stranded DNA template. Methods Enzymol. 154:329-50.
[0595] Zon, G. 1988. Oligonucleotide analogues as potential
chemotherapeutic agents. Pharm Res. 5:539-49.
[0596] Zuckermann, R. N., E. J. Martin, D. C. Spellmeyer, G. B.
Stauber, et al 1994. Discovery of nanomolar ligands for
7-transmembrane G-protein-coupled receptors from a diverse
N-(substituted)glycine peptoid library. J Med Chem. 37:2678-85.
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