U.S. patent application number 11/717572 was filed with the patent office on 2008-07-10 for assays for tert promoter modulatory agents.
Invention is credited to William H. Andrews, Laura A. Briggs, Lancer K. Brown, Christopher A. Foster, Mieczyslaw A. Piatyszek.
Application Number | 20080166711 11/717572 |
Document ID | / |
Family ID | 36653703 |
Filed Date | 2008-07-10 |
United States Patent
Application |
20080166711 |
Kind Code |
A1 |
Andrews; William H. ; et
al. |
July 10, 2008 |
Assays for TERT promoter modulatory agents
Abstract
Methods and compositions for assaying an agent for TERT promoter
modulatory activity are provided. In the subject methods, an agent
is contacted with a cell comprising a mutant telomerase structural
RNA component (TR) that results in a detectable phenotype in the
presence of telomerase reverse transcriptase (TERT). Also provided
are compositions, systems and kits thereof, as well as devices,
that find use in practicing the subject methods. The subject
invention finds use in assaying agents for TERT promoter modulatory
activity, such as in a high throughput format.
Inventors: |
Andrews; William H.; (Reno,
NV) ; Briggs; Laura A.; (Reno, NV) ; Foster;
Christopher A.; (Carmichael, CA) ; Piatyszek;
Mieczyslaw A.; (Morgan Hill, CA) ; Brown; Lancer
K.; (Sparks, NV) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE, SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
36653703 |
Appl. No.: |
11/717572 |
Filed: |
March 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11085872 |
Mar 21, 2005 |
7226744 |
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11717572 |
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60643649 |
Jan 12, 2005 |
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Current U.S.
Class: |
435/6.18 |
Current CPC
Class: |
C12Q 1/6897
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of determining whether an agent modulates transcription
control activity of a TERT promoter, said method comprising: (a)
contacting said agent with a cell comprising a mutant telomerase
structural RNA component (TR) that results in a detectable
phenotype in the presence of telomerase reverse transcriptase
(TERT); and (b) evaluating said cell for said detectable phenotype
to determine whether said agent modulates transcription control
activity of said TERT promoter nucleic acid.
2. The method according to claim 1, wherein said TERT promoter
nucleic acid is a human TERT promoter nucleic acid.
3. The method according to claim 1, wherein said cell comprises an
expression cassette that expresses said mutant telomerase
structural RNA component (TR).
4. The method according to claim 3, wherein said expression
cassette is episomally maintained in said cell.
5. The method according to claim 3, wherein said expression
cassette is chromosomally integrated in said cell.
6. The method according to claim 5, wherein said expression
cassette is not chromosomally integrated into a chromosome of said
cell that includes a TERT coding sequence.
7. The method according to claim 6, wherein said cell is a human
cell and said expression cassette is not integrated into chromosome
5.
8. The method according to claim 1, wherein said detectable
phenotype is cell death.
9. The method according to claim 1, wherein said cell is a mutant
cell that expresses telomerase and said method is a method for
determining whether said agent inhibits expression controlled by a
TERT promoter nucleic acid.
10. The method according to claim 1, wherein said cell is a normal
cell and said method is a method of determining whether said agent
enhances expression controlled by a TERT promoter nucleic acid.
11. The method according to claim 1, wherein said method comprises
determining the modulatory activity of at least two different
agents.
12. The method according to claim 11, wherein said method is a
high-throughput method.
13. The method according to claim 1, wherein said agent is a small
molecule.
14. A method of determining whether a small molecule agent can
derepress transcription repression activity of a TERT promoter,
said method comprising: (a) contacting said agent with a cell
comprising a mutant telomerase structural RNA component (TR) that
results in a detectable phenotype in the presence of telomerase
reverse transcriptase (TERT); and (b) evaluating said cell for said
detectable phenotype to determine whether said agent derepresses
transcription repression activity of said TERT promoter nucleic
acid.
15. The method according to claim 14, wherein said cell is a human
cell.
16. The method according to claim 15, wherein said TERT promoter
nucleic acid is a human TERT promoter nucleic acid.
17. The method according to claim 14, wherein said cell comprises
an expression cassette that expresses said mutant telomerase
structural RNA component (TR).
18. The method according to claim 17, wherein said expression
cassette is episomally maintained in said cell.
19. The method according to claim 17, wherein said expression
cassette is chromosomally integrated in said cell.
20. The method according to claim 19, wherein said expression
cassette is not integrated into chromosome 5.
21. The method according to claim 14, wherein said detectable
phenotype is cell death.
22. The method according to claim 14, wherein said method comprises
determining the activity of at least two different agents.
23. The method according to claim 22, wherein said method is a
high-throughput method.
24. The method according to claim 14, wherein said agent is a small
molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn. 119 (e), this application
claims priority to the filing date of U.S. Provisional Patent
Application Ser. No. 60/643,649 filed Jan. 12, 2005; the disclosure
of which application is herein incorporated by reference.
INTRODUCTION
Background of the Invention
[0002] Telomeres, which define the ends of chromosomes, consist of
short, tandemly repeated DNA sequences loosely conserved in
eukaryotes. For example, human telomeres consist of many kilobases
of (TTAGGG)n together with various associated proteins. Small
amounts of these terminal sequences or telomeric DNA are lost from
the tips of the chromosomes during S phase because of incomplete
DNA replication. Many human cells progressively lose terminal
sequence with cell division, a loss that correlates with the
apparent absence of telomerase in these cells. The resulting
telomeric shortening has' been demonstrated to limit cellular
lifespan.
[0003] Telomerase is a ribonucleoprotein that synthesizes telomeric
DNA. In general, telomerase is made up of two components: (1) an
essential structural RNA (TR or TER) (where the human component is
referred to in the art as hTR or hTER); and (2) a catalytic protein
(telomerase reverse transcriptase or TERT) (where the human
component is referred to in the art as hTERT). Telomerase works by
recognizing the 3' end of DNA, e.g., telomeres, and adding multiple
telomeric repeats to its 3' end with the catalytic protein
component, e.g., hTERT, which has polymerase activity, and hTR
which serves as the template for nucleotide incorporation. Both the
catalytic protein component and the RNA template component are
activity-limiting components.
[0004] Because of its role in cellular senescence and
immortalization, there is much interest in the development of
protocols and compositions for regulating telomerase activity. Of
particular interest is the development of assays that detect agents
that directly regulate the endogenous TERT, and specifically hTERT,
promoter, e.g., in a high-throughput format.
Literature of Interest
[0005] U.S. Pat. Nos. 5,972,605; 6,610,839 and 6,664,046 and
published United States Application 2004/0072787; as well as WO
02/070668; WO 03/016474; WO 03/000916; WO 02/101010; WO 02/090571;
WO 02/090570; WO 02/072787; WO 02/070668; WO 02/16658; WO 02/16657;
and the references cited therein. Also of interest is Li et al.,
Rapid Inhibition of Cancer Cell Growth Induced by Lentiviral
Delivery and Expression of Mutant-Template Telomerase RNA and
Anti-Telomerase Short-Interfering RNA," Cancer Res. (Jul. 15, 2004)
64:4833-4840.
SUMMARY OF THE INVENTION
[0006] Methods and compositions for assaying an agent for TERT
promoter modulatory activity are provided. In the subject methods,
an agent is contacted with a cell comprising a mutant telomerase
structural RNA component (TR) that results in a detectable
phenotype in the presence of telomerase reverse transcriptase
(TERT). Also provided are compositions, systems and kits thereof,
as well as devices, that find use in practicing the subject
methods. The subject invention finds use in assaying agents for
TERT promoter modulatory activity, such as in a high throughput
format.
[0007] Aspects of the invention include methods of determining
whether an agent modulates transcription control activity of a TERT
promoter, where the method includes:
[0008] (a) contacting the agent with a cell that includes a mutant
telomerase structural RNA component (TR) that results in a
detectable phenotype in the presence of telomerase reverse
transcriptase (TERT); and
[0009] (b) evaluating the cell for the detectable phenotype to
determine whether the agent modulates transcription control
activity of the TERT promoter nucleic acid. In certain embodiments,
TERT promoter nucleic acid is a human TERT promoter nucleic acid.
In certain embodiments, the cell comprises an expression cassette
that expresses the mutant telomerase structural RNA component (TR).
In certain embodiments, the expression cassette is episomally
maintained in the cell. In certain embodiments, the expression
cassette is chromosomally integrated in the cell. In certain
embodiments, the expression cassette is not chromosomally
integrated into a chromosome of the cell that includes a TERT
coding sequence. In certain embodiments, the cell is a human cell
and the expression cassette is not integrated into chromosome 5. In
certain embodiments, the detectable phenotype is cell death. In
certain embodiments, the cell is a mutant cell that expresses
telomerase and the method is a method for determining whether the
agent inhibits expression controlled by a TERT promoter nucleic
acid. In certain embodiments, the cell is a normal cell and the
method is a method of determining whether the agent enhances
expression controlled by a TERT promoter nucleic acid. In certain
embodiments, the method includes determining the modulatory
activity of at least two different agents. In certain embodiments,
the method is a high-throughput method. In certain embodiments, the
agent is a small molecule.
[0010] Additional aspects of the invention include methods of
determining whether a small molecule agent can de-repress
transcription repression activity of a TERT promoter. In these
embodiments, the method includes:
[0011] (a) contacting an agent with a cell that includes a mutant
telomerase structural RNA component (TR) that results in a
detectable phenotype in the presence of telomerase reverse
transcriptase (TERT); and
[0012] (b) evaluating the cell for said detectable phenotype to
determine whether said agent de-represses transcription repression
activity of said TERT promoter nucleic acid. Variations of this
embodiment include those summarized above.
[0013] Also provided are systems for determining whether an agent
modulates transcription control activity of a TERT promoter, where
the systems include:
[0014] (a) a cell comprising a mutant telomerase structural RNA
component (TR) that results in a detectable phenotype in the
presence of telomerase reverse transcriptase (TERT); and
[0015] (b) said agent.
[0016] Also provided are high throughput assay devices that include
a cell comprising a mutant telomerase structural RNA component (TR)
that results in a detectable phenotype in the presence of
telomerase reverse transcriptase (TERT).
DEFINITIONS
[0017] As used herein, the term "TERT promoter" includes any TERT
genomic sequences capable of driving transcription in a telomerase
activity positive cell. Thus, TERT promoters of the invention
include without limitation cis-acting transcriptional control
elements and regulatory sequences that are involved in regulating
or modulating the timing and/or rate of transcription of a TERT
gene. For example, the TERT promoter of the invention comprises
cis-acting transcriptional control elements, including enhancers,
promoters, transcription terminators, origins of replication,
chromosomal integration sequences, 5' and 3' untranslated regions,
exons and introns, which are involved in transcriptional
regulation. These cis-acting sequences typically interact with
proteins or other biomolecules to carry out (turn on/off, regulate,
modulate, etc.) transcription.
[0018] As used herein, the terms "allele" or "allelic sequence"
refer to an alternative form of a nucleic acid sequence (i.e., a
nucleic acid corresponding to a TERT promoter, particularly, an
hTERT promoter). Alleles result from mutations (i.e., changes in
the nucleic acid sequence), and can produce differently regulated
mRNAs. Common mutational changes that give rise to alleles are
generally ascribed to natural deletions, additions, or
substitutions of nucleotides. Each of these types of changes may
occur alone, in combination with the others, or one or more times
within a given gene, chromosome or other cellular nucleic acid.
Thus, the term "TERT promoter" includes allelic forms of TERT
promoter sequences, i.e., TERT cis-acting transcriptional control
elements, including, e.g., the exemplary human and mouse sequences
described herein. In alternative embodiments, the TERT promoter
sequence comprises TERT sequences 5' (upstream) of the
translational start site (ATG). For example, in one embodiment, the
hTERT promoter comprises residues 44 to 13545 of SEQ ID NO:01.
Other embodiments include sequences starting within about one to 5
nucleotides of a translation start codon (for example in SEQ ID
NO:01) and ending at about 50, 100, 150, 200, 250, 500, 1000, 2500
or 13500 nucleotides upstream of the translation start codon. Such
embodiments can optionally include other regulatory sequences, such
as, exon and/or intron sequences hTERT promoters of the invention
also include sequences substantially identical (as defined herein)
to an exemplary hTERT promoter sequence of the invention, having
the sequence set forth by SEQ ID NO:01. Similarly, mTERT promoters
of the invention also include sequences substantially identical to
an exemplary mTERT promoter sequence of the invention, having the
sequence set forth by SEQ ID NO:02.
[0019] The term "heterologous" when used with reference to portions
of a nucleic acid, indicates that the nucleic acid comprises two or
more subsequences which are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged in a manner not found in nature; e.g., a promoter
sequence of the invention operably linked to a polypeptide coding
sequence that,. when operably linked, does not reform the naturally
occurring TERT gene. For example, the invention provides
recombinant constructs (expression cassettes, vectors, viruses, and
the like) comprising various combinations of promoters of the
invention, or subsequences thereof, and heterologous coding
sequences, many examples of which are described in detail
below.
[0020] As used herein, "isolated," when referring to a molecule or
composition, such as, e.g., an hTERT promoter sequence, means that
the molecule or composition is separated from at least one other
compound, such as a protein, DNA, RNA, or other contaminants with
which it is associated in vivo or in its naturally occurring state.
Thus, a nucleic acid sequence is considered isolated when it has
been isolated from any other component with which it is naturally
associated. An isolated composition can, however, also be
substantially pure. An isolated composition can be in a homogeneous
state. It can be in a dry or an aqueous solution. Purity and
homogeneity can be determined, e.g., using analytical chemistry
techniques such as, e.g., polyacrylamide gel electrophoresis
(PAGE), agarose gel electrophoresis or high pressure liquid
chromatography (HPLC).
[0021] As used herein, the terms "nucleic acid" and
"polynucleotide" are used interchangeably, and include
oligonucleotides (i.e., short polynucleotides). They also refer to
synthetic and/or non-naturally occurring nucleic acids (i.e.,
comprising nucleic acid analogues or modified backbone residues or
linkages). The terms also refer to deoxyribonucleotide or
ribonucleotide oligonucleotides in either single- or
double-stranded form. The terms encompass nucleic acids containing
known analogues of natural nucleotides. The term also encompasses
nucleic acid-like structures with synthetic backbones. DNA backbone
analogues provided by the invention include phosphodiester,
phosphorothioate, phosphorodithioate, methyl-phosphonate,
phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal,
methylene (methylimino), 3'-N-carbamate, morpholino carbamate, and
peptide nucleic acids (PNAs); see Oligonucleotides and Arialogues,
a Practical Approach, edited by F. Eckstein, IRL Press at Oxford
University Press (1991); Antisense Strategies, Annals of the New
York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt
(NTYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense
Research and Applications (1993, CRC Press). PNAs contain non-ionic
backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate
linkages are described in WO 97/03211; WO 96/39154; Mata (1997)
Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones
encompassed by the term include methyl-phosphonate linkages or
alternating methylphosphonate and phosphodiester linkages
(Strauss-Soukup (1997) Biochemistry 36:8692-8698), and
benzyl-phosphonate linkages (Samstag (1996) Antisense Nucleic Acid
Drug Dev 6:153-156).
[0022] As used herein, the term "operably linked" refers to a
functional relationship between two or more nucleic acid (e.g.,
DNA) segments. Typically, it refers to the functional relationship
of a transcriptional regulatory sequence to a transcribed sequence.
For example, a promoter sequence is operably linked to a coding
sequence if it stimulates or modulates the transcription of the
coding sequence in an appropriate host cell or other expression
system. Generally, promoter transcriptional regulatory sequences
that are operably linked to a transcribed sequence are physically
contiguous to the transcribed sequence, i.e., they are cis-acting.
However, some transcriptional regulatory sequences, such as
enhancers, need not be physically contiguous or located in close
proximity to the coding sequences whose transcription they
enhance.
[0023] As used herein, "recombinant" refers to a polynucleotide
synthesized or otherwise manipulated in vitro (e.g., "recombinant
polynucleotide"), to methods of using recombinant polynucleotides
to produce gene products in cells or other biological systems, or
to a polypeptide ("recombinant protein") encoded by a recombinant
polynucleotide. "Recombinant means" also encompass the ligation of
nucleic acids having coding or promoter sequences from different
sources into an expression cassette or vector for expression of,
e.g., a fusion protein; or, inducible, constitutive expression of a
protein (i.e., a TERT promoter of the invention operably linked to
a heterologous nucleotide, such as a polypeptide coding
sequence).
[0024] As used herein, the "sequence" of a gene (unless
specifically stated otherwise) or nucleic acid refers to the order
of nucleotides in the polynucleotide, including either or both
strands of a double-stranded DNA molecule, e.g., the sequence of
both the coding strand and its complement, or of a single-stranded
nucleic acid molecule. For example, in alternative embodiments, the
TERT promoter of the invention comprises untranscribed,
untranslated, and intronic TERT sequences, e.g., as set forth in
the exemplary SEQ ID NO:01 and SEQ ID NO:02.
[0025] As used herein, the term "transcribable sequence" refers to
any sequence which, when operably linked to a cis-acting
transcriptional control element, e.g., a promoter, and when placed
in the appropriate conditions, is capable of being transcribed to
generate RNA, e.g., messenger RNA (mRNA).
[0026] The term "assessing" includes any form of measurement, and
includes determining if an element is present or not. The terms
"determining", "measuring", "evaluating", "assessing" and
"assaying" are used interchangeably and include quantitative and
qualitative determinations. Assessing may be relative or absolute.
"Assessing the presence of" includes determining the amount of
something present, and/or determining whether it is present or
absent. As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations.
[0027] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of nucleotides (or amino acid residues) that
are the same, when compared and aligned for maximum correspondence
over a comparison window, as measured using one of the following
sequence comparison algorithms or by manual alignment and visual
inspection. This definition also refers to the complement of a
sequence. For example, in alternative embodiments, nucleic acids
within the scope of the invention include those with a nucleotide
sequence identity that is at least about 60%, at least about
75-80%, about 90%, and about 95% of the exemplary TERT promoter
sequence set forth in SEQ ID NO:01 (including residues 44 to 13544
of SEQ ID NO:01) or SEQ ID NO:02. Two sequences with these levels
of identity are "substantially identical." Thus, if a sequence has
the requisite sequence identity to a TERT promoter sequence or
subsequence of the invention, it also is a TERT promoter sequence
within the scope of the invention. Preferably, the percent identity
exists over a region of the sequence that is at least about 25
nucleotides in length, more preferably over a region that is at
least about 50-100 nucleotides in length.
[0028] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithms test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s) relative to the reference sequence, based on the
designated or default program parameters. A "comparison window", as
used herein, includes reference to a segment of any one of the
number of contiguous positions selected from the group consisting
of from 25 to 600, usually about 50 to about 200, more usually
about 100 to about 150 in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are well-known in the art. Optimal
alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl.
Math. 2:482 (1981), by the homology alignment algorithm of
Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson & Lipman, Proc. Natl. Acad.
Sci. USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., supra).
[0029] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendrogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by 5 designating
the program parameters. Using PILEUP, a reference sequence (e.g., a
TERT promoter sequence of the invention as set forth by. e.g., SEQ
ID NO:01 or SEQ ID NO:02) is compared to another sequence to
determine the percent sequence identity relationship (i.e., that
the second sequence is substantially identical and within the scope
of the invention) using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps. PILEUP can be obtained from the GCG sequence analysis
software package, e.g., version 7.0 (Devereaux (1984) Nuc. Acids
Res. 12:387-395).
[0030] Another example of algorithm that is suitable for
determining percent sequence identity (i.e., substantial similarity
or identity) is the BLAST algorithm, which is described in Altschul
(1990) J. Mol. Biol. 215:403-410. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul (1990) supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues, always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. In one embodiment, to determine if a
nucleic acid sequence is within the scope of the invention, the
BLASTN program (for nucleotide sequences) is used incorporating as
defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,
N=4, and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as default parameters a wordlength (W) of
3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see,
e.g., Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
[0031] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin (1993)
Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0032] Methods and compositions for assaying an agent for TERT
promoter modulatory activity are provided. In the subject methods,
an agent is contacted with a cell comprising a mutant telomerase
structural RNA component (TR) that results in a detectable
phenotype in the presence of telomerase reverse transcriptase
(TERT). Also provided are compositions, systems and kits thereof,
as well as devices, that find use in practicing the subject
methods. The subject invention finds use in assaying agents for
TERT promoter modulatory activity, such as in a high throughput
format.
[0033] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0034] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0035] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events.
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0037] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0038] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. It is
further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0039] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0040] In further describing the invention, the subject methods are
described first in greater detail, followed by a review of
representative applications in which the subject methods find use,
as well as a discussion of representative systems and kits that
find use in practicing the subject methods.
Methods
[0041] As summarized above, the subject invention provides methods
of determining whether an agent has TERT promoter modulatory
activity. An agent is considered to have TERT promoter modulatory
activity if its interaction with a TERT promoter causes a change in
transcription activity, e.g., level (for example, in terms of
transcribed copies of a coding sequence for a given period of time)
of a nucleic acid sequence (e.g., a transcribable sequence, such as
the coding sequence for TERT) operably linked to the promoter,
e.g., as compared to a control (e.g., the transcription activity of
an analogous TERT promoter/reporter nucleic acid construct not
contacted with the agent of interest). The change that is observed
may be an increase or decrease of transcription of the operably
linked nucleic acid, e.g., TERT coding sequence. In other words,
the agent may enhance or inhibit transcription of the nucleic acid
sequence operably linked to the TERT promoter. By enhance is meant
that the expression level of the operably linked reporter nucleic
acid sequence is increased by at least about 2 fold, usually by at
least about 5 fold and sometimes by at least 25, 50, 100 fold and
in particular about 300 fold or higher, as compared to a control,
i.e., expression from an analogous or identical expression system
that is not contacted with the agent in question. Alternatively, in
cases where expression of the operably linked nucleic acid is so
low that it is undetectable, expression of the operably linked
nucleic acid is considered to be enhanced if expression is
increased to a level that is easily detectable. By inhibit is meant
that the expression level of the operably linked nucleic acid
sequence is decreased by at least about 2 fold, usually by at least
about 5 fold and sometimes by at least 25, 50, 100 fold and in
particular about 300 fold or higher, as compared to a control,
i.e., expression from an analogous or identical expression system
that is not contacted with the agent in question. Alternatively, in
cases where expression of the operably linked nucleic acid is
detectable, expression of the operably linked nucleic acid is
considered to be inhibited if expression is decreased to a level
that is not detectable.
[0042] In practicing the subject methods, an agent to be tested or
assayed for TERT promoter modulatory activity (sometimes referred
to herein as a candidate agent) is contacted with a cell that
includes a mutant (i.e., modified) telomerase structural RNA
component (TR) that results in a detectable phenotype in the
presence of telomerase reverse transcriptase (TERT).
[0043] Depending on the particular assay, the cell (also referred
to herein as the target cell or test cell) with which the agent is
contacted during practice of the subject methods may be a normal
cell that provides wild type conditions, e.g., a cell that normally
lacks telomerase activity, e.g., an MRC5 cell, etc.; or the cell
may be mutant cell in which telomerase activity is present, e.g., a
cancerous cell. In representative embodiments, the cell is a
mammalian cell, where mammalian cells, of interest include, but are
not limited to: murine, porcine, ovine, equine, rat, ungulates,
dog, cat, monkey, and human cells, and the like. In many
embodiments, the cell will be a human cell.
[0044] A feature of the cells employed in the subject methods is
that they include a modified telomerase RNA (TR) component, i.e., a
modified TR component. By "modified" is meant that the RNA
component differs by at least one base as compared to the
corresponding wild-type RNA component present in the cells of the
organism from which the test cell was originally obtained. As such,
the modified TR component is also properly referred to as a mutant
TR component. For example, where the test cell is a mouse cell, the
cell includes a modified TR component as compared to the wild-type
mouse TR (i.e., mTR) component. Likewise, where the test cell is a
human cell, the cell includes a modified TR component as compared
to the wild type human TR (i.e., hTR) component. Wild type
sequences of TR components as well as the sequences of DNAs
encoding the same are known for multiple species, including but not
limited to: human, mouse, rat, hamster, cow, etc. See e.g., U.S.
Pat. Nos. 6,013,468 and 5,876,979, the disclosures of which are
herein incorporated by reference. As indicated above, the sequence
of the modified TR component present in the test cells employed in
the subject invention differs from the corresponding wild type TR
component of the test cell by at least one base, where the number
of bases that differ may vary. As such, sequence identity between
the modified and corresponding TR component will be less than 100%
(as determined using an alignment program, such as an alignment
program specified above), where the sequence identity may be less
than about 98%, less than about 95%, less than about 90%, less than
about 85% etc in certain embodiments.
[0045] In certain representative embodiments, the modified TR
component is a modified hTR component. In certain of these
representative embodiments, the modified hTR component differs from
the sequence of the wild type hTR component, as described above.
The sequence of the wild-type hTR component for this embodiment is
deposited with Genbank and has been assigned an accession no. of
NR.sub.--001566.
[0046] A feature of the modified TR component present in the test
cells employed in the subject methods is that the modified TR
component imparts a detectable phenotype to the cell in the
presence of telomerase reverse transcriptase (TERT). In other
words, when TERT is present in the cell along with the modified TR
component, the cell has a detectable phenotype that is attributable
to the presence of these two components. The detectable phenotype
may vary, where representative phenotypes include, but are not
limited to: cell death; cell growth/proliferation; cell morphology;
the production of new telomere sequences; changes in levels of
telomere binding proteins; the presence or absence of binding
proteins, e.g., transcriptions factors, that recognize new telomere
sequences; changes in gene expression factors (e.g., resulting from
presence of new telomere sequences and use of transcription factors
as a result thereof, which modulates expression of other gene
products); presence of restriction sites in new telomere sequences;
changes in fluorescence polarization of the TR component (e.g.,
when bound to TERT); presence of a reporter gene; presence of a
reporter sequence (e.g., directly or indirectly detectable) in a
newly synthesized telomere sequence; detection of a TR/TERT
complex, either directly or indirectly; and the like.
[0047] In representative embodiments, the modified TR component is
one that causes cell death (e.g., via apoptosis) in the presence of
telomerase. A number of different such modified TR components have
been reported in the literature, including, but not limited to,
those reported in: Feng et al, Science (1995) 269(5228):1236-41,
Kim et al., Proc. Nat'l Acad. Sci. USA (2001) 98: 7982-7; Marusic
et al., Mol. Cell Biol. (1997) 17:6394-401; and Li et al., Cancer
Res. (2004) 64: 4833-4840.
[0048] As the test cell employed in the subject assays comprises a
modified TR component. In certain embodiments, an expression
cassette that includes a coding sequence for the modified TR
component operably linked to suitable promoter is present in the
cell.
[0049] In certain embodiments, the expression cassette is present
on a vector that is episomally (i.e., extrachomosomally) maintained
in the host cell. Expression vectors of interest generally contain
a promoter that is recognized by the host organism and is operably
linked to the coding sequence for the modified TR component.
Promoters are untranslated sequences located upstream (5') to the
start codon of a structural gene (generally within about 100 to
1000 bp) that control the transcription and translation of
particular nucleic acid sequence to which they are operably linked.
Such promoters typically fall into two classes, inducible and
constitutive. Inducible promoters are promoters that initiate
increased levels of transcription from DNA under their control in
response to some change in culture conditions, e.g., the presence
or absence of a nutrient or a change in temperature. A large number
of promoters recognized by a variety of potential host cells are
well known. Both a native promoter sequence, e.g., the promoter
sequence operably linked to the wild type TR coding sequence, and
many heterologous promoters may be used to direct expression of the
coding sequence for the modified TR component.
[0050] Transcription from vectors in mammalian cells may be
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase),
or an immunoglobulin promoter, from heat-shock promoters, provided
such promoters are compatible with the host cell systems. The early
and late promoters of the SV40 virus are conveniently obtained as
an SV40 restriction fragment that also contains the SV40 viral
origin of replication. The immediate early promoter of the human
cytomegalovirus is conveniently obtained as a HindIII E restriction
fragment. Also of interest are promoters for snRNAs, e.g. U1 and
U6.
[0051] Transcription by higher eukaryotes is often increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp, which
act on a promoter to increase its transcription. Enhancers are
relatively orientation and position independent, having been found
5' and 3' to the transcription unit, within an intron, as well as
within the coding sequence itself. Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin,
a-fetoprotein, and insulin). Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin, the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
The enhancer may be spliced into the expression vector at a
position 5' or 3' to the coding sequence, but is preferably located
at a site 5' from the promoter.
[0052] Expression vectors used in eukaryotic host cells may also
contain sequences necessary for the termination of transcription
and for stabilizing the mRNA. Such sequences are commonly available
from the 5' and, occasionally 3', untranslated regions of
eukaryotic or viral DNAs or cDNAs.
[0053] In certain embodiments, the expression cassette may be
genomically integrated in the target cell, i.e., integrated onto a
chromosome of the target cell. A variety of integrating vectors and
methodologies for using the same are known in the art. For such
embodiments, the expression cassette maybe placed into a vector
that is suitable for use in integrating the expression cassette
into the target cell genome, where representative vectors include,
but are not limited to: plasmid DNA vectors, retroviral vectors;
adeno-associated vectors, adenoviral vectors, double stranded DNA
vectors, etc. For example, viral vector delivery vehicles may be
employed to integrate an expression cassette into a target cell
genome. A variety of viral vector delivery vehicles are known to
those of skill in the art and include, but are not limited to:
adenovirus, herpesvirus, lentivirus, vaccinia virus and
adeno-associated virus (AAV).
[0054] In representative embodiments where the expression cassette
encoding the modified TR component is chromosomally integrated and
a stable clone of a cell containing this integration is isolated,
clones containing integrations into the same chromosome that
includes the TERT coding sequence are excluded. For example, where
the target cell is a human cell, since the TERT coding sequence is
present on Chromosome 5, the modified TR expression cassette is
integrated into a chromosome other than Chromosome 5. In cases
where the modified TR is chromosomally integrated and used as a
pool (i.e., no clonal isolation), then it is not possible to
exclude integrations into chromosome 5.
[0055] For vector construction, any convenient method may be
employed. Construction of suitable vectors containing one or more
of the above-listed components may employ standard ligation
techniques. Isolated plasmids or DNA fragments are cleaved,
tailored, and re-ligated in the form desired to generate the
plasmids required. For analysis to confirm correct sequences in
plasmids constructed, the ligation mixtures are used to transform
host cells, and successful transformants selected by ampicillin or
tetracycline resistance where appropriate. Plasmids from the
transformants are prepared, analyzed by restriction endonuclease
digestion, and/or sequenced.
[0056] Yet another feature of certain embodiments of the subject
invention is that the target cell is one in which the native TERT
gene is present, where by TERT gene is meant the TERT coding
sequences as well as transcription control sequences, e.g.,
promoters, etc. In certain embodiments where the cell is a human
cell, the cell includes the native hTERT gene. Yet another feature
of certain embodiments of the invention is that the test cell lacks
a functional gene for wild type TR, such that wild-type TR is not
present in the test cell. In certain embodiments, wild-type TR is
present.
[0057] In practicing the subject methods, the target cell, as
described above, is contacted with the candidate agent whose
activity is to be tested. Contact of the candidate agent is
achieved using any convenient protocol, such as introducing the
agent into cell culture medium in which the target cell is present,
etc.
[0058] The conditions under which the cell and the candidate agent
are contacted may vary depending on the nature of the assay and the
nature of the candidate agent modulatory activity to be determined.
For example, where the assay is employed to screen candidate agents
for TERT promoter de-repression activity, i.e., activation
activity, the conditions under which the expression system and the
agent are contacted are generally wild type conditions, where the
conditions may be described as an environment in which, in the
absence of the candidate agent, TERT expression is repressed.
[0059] Alternatively, where the assay is employed to screen
candidate agents for TERT promoter repression activity, i.e.,
inhibition activity, the conditions under which the cell and the
agent are contacted are generally mutant conditions, where the
conditions may be described as an environment in which, in the
absence of the candidate agent, TERT expression occurs. Such
conditions may be found in target cells that constitutively express
TERT, e.g., cancerous cells.
[0060] Following contact of the candidate agent and the target
cell, the phenotype of cell is evaluated or assessed to determine
the promoter modulatory activity of the candidate agent. This step
of assessing or evaluating the phenotype of the cell will
necessarily vary depending on the nature of the phenotype that is
induced by the presence of both TERT and the modified TR component
in the cell. This step of the subject methods may include either a
qualitative or quantitative evaluation of the phenotype, and may or
may not include use of one or more reference or controls, as may be
desired.
[0061] In certain embodiments, the cell is assayed for cell-death
or apoptosis. Any convenient apoptosis assay may be employed,
including but not limited to, those described in: (Note: Feng at
all describes altered telomeres but no apoptosis assay) Kim et al.,
Proc. Nat'l Acad. Sci. USA (2001) 98: 7982-7; Marusic et al., Mol.
Cell Biol. (1997) 17:6394-401; and Li et al., Cancer Res. (2004)
64: 4833-4840.
[0062] Where the phenotype to be evaluated is cell death, any
convenient assay for such a phenotype may be employed, where a
number of different such assays are known to those of skill in the
art. Specific representative assays of interest are reviewed in
greater detail below.
[0063] In certain embodiments, the assay can be one the employs a
mixed population of test and non-test cells, where the assay looks
at changes in the proportion of each type of cell in the population
as an indication of the presence of cell death in the test cell.
For example, a first population of test cells may be produced by
transfecting the mutant TR expression construct into normal cells
along with reporter construct, e.g., a gene expressing the Green
Fluorescent Protein, where the reporter construct may be on the
same vector as, or a different vector from, the mutant hTR
expression construct, e.g., on the plasmid or on a separate
plasmid. A second population of control cells may be transfected
with a second reporter construct distinct from the first, e.g., a
reporter construct that expresses a non-Green Fluorescent Protein,
such as Red, Yellow, Blue, etc. In certain embodiments, the second
reporter construct may be on the same vector as a wild type hTR
expression construct, e.g., on the plasmid. As a result, two
different populations of cells, e.g., cell lines, are produced,
where the cell lines are identical except that the first test cell
line expresses mutant hTR and a first reporter molecule, e.g., GFP,
while the second control cell line expresses wild type hTR and a
second reporter molecule, e.g., RFP. Following production of the
test and control cell populations, a mixed population is produced
in which the test and control cells are mixed together in known
amounts, e.g., in equal amounts, such that a mixture of cells is
produced in which the proportion of test and control cells is
known. Application of a test compound to such a mixed population
provides for a ready determination of whether the compound has a
modulatory effect on TERT promoter activity. For example, if a
compound has no effect on the cells (e.g., it does not activate
telomerase expression) then both test and control cells should grow
normally and the ratio of signal from the first and second reporter
construct, e.g., ratio of GFP to RFP should remain constant, e.g. 1
where the population includes equal amounts of cells.
Alternatively, if a compound activates hTERT expression and
incorporation of the mutant hTR negatively effects the health of
the cells, then the ratio of GFP to RFP should decrease relative to
the starting ratio, e.g., to less than 1, such as to less then 0.1.
In these representative embodiments, if a compound is toxic to all
cells, then the ratio of GFP to RFP should remain 1, but the total
fluorescent signal should be significantly less since neither cell
grew.
[0064] Other assays for dead or live cells that can be included in
at least some of the assays described above include: Caspase assays
for the presence of apoptosis (such as Caspase 3 and 7 activity
measurement: Caspase-Glo 3/7 Assay, cat. # G8092 (Promega); Caspase
8 activity measurement: Caspase-Glo 8 Assay, cat. # G8202
(Promega); Caspase 9 activity measurement: Caspase-Glo 9 Assay,
cat. # G8212 (Promega); etc.); viability and proliferation assays,
such as ATPlite, cell viability homogenous assay cat. # 6016947
(PerkinElmer), etc.; cell death detection assays, such as Cell
Death Detection ELISAPLUS cat. # 1 920 685 (Roche Applied Science);
Propidium Iodide assay (MTG, Inc. Product number M0795); etc.
[0065] In yet other embodiments, the assay employed may include
molecular probing for new telomere sequences. Such embodiments
include those situations where the presence of new telomere
sequences is used as the indication of TERT promoter activity. The
presence of new telomere sequences may be detected using any of a
number of different protocols, e.g., by hybridization, PCR, FRET,
or antibody, etc. In yet other embodiments, the evaluation step may
include probing for lack of telomere binding proteins. In yet other
embodiments, the evaluation step may include probing for binding
proteins (e.g., transcription factors) that recognize new telomere
sequences. In yet other embodiments, the evaluation step may
include assaying for altered regulation of genes whose
transcription factors are recruited by the new telomere sequences,
if present, where such recruitments results in a modulation of the
expression pattern of one or more additional genes. In certain
embodiments, the evaluation step includes a step of probing for
reporter sequences that are present in a newly synthesized telomere
sequence, where representative reporter sequences of interest
include restriction sites. In certain embodiments, cells are
evaluated for an alteration of fluorescence polarization of the TR
component, e.g., mutant hTR (or hTER), when bound to telomerase,
e.g., (hTERT). In certain embodiments, cells are assayed for the
presence of a reporter gene (e.g., where the template region of hTR
is altered to express a reporter gene, such as luciferase (or
adenovirus VA RNA sequence), where the new telomere sequence would
contain repeats of the luciferase gene and synthesis of new
telomeres would be detected by luciferase activity. In certain
embodiments, evaluation includes a FISH assay. In certain
embodiments, evaluating includes assaying for virus integration
into an integration site within a new telomere sequence. In certain
embodiments, evaluating includes assaying for site-specific
recombination of a reporter gene (e.g., Luciferase) into a
recombination site within a new telomere sequence. In certain
embodiments, evaluating includes detection of a reporter sequence
in a new telomere sequence, e.g., via an engineered Zinc Finger
Protein. In certain embodiments, evaluating includes assaying
directly for complex formation of the TR and TERT components, e.g.,
via FRET using antibody to hTERT and oligo to mutant template of
hTR. The above assays are merely representative.
[0066] In certain embodiments, the subject methods are performed in
a high throughput (HT) format. In the subject HT embodiments of the
subject invention, a plurality of different compounds are
simultaneously tested. By simultaneously tested is meant that each
of the compounds in the plurality are tested at substantially the
same time. Thus, at least some, if not all, of the compounds in the
plurality are assayed for their effects in parallel. The number of
compounds in the plurality that are simultaneously tested is
typically at least about 10, where in certain embodiments the
number may be at least about 100 or at least about 1000, where the
number of compounds tested may be higher. In general, the number of
compounds that are tested simultaneously in the subject HT methods
ranges from about 10 to 10,000, usually from about 100 to 10,000
and in certain embodiments from about 1000 to 5000. A variety of
high throughput screening assays for determining the activity of
candidate agent are known in the art and are readily adapted to the
present invention, including those described in e.g., Schultz
(1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) Mol Divers.
3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603;
Sittampalam (1997) Curr Opin Chem Biol 1:384-91; as well as those
described in published United States application 20040072787 and
issued U.S. Pat. No. 6,127,133; the disclosures of which are herein
incorporated by reference.
[0067] Testing of a candidate agent according to the invention as
described above readily determines whether or not an agent has TERT
promoter modulatory activity. As mentioned above, an agent is
considered to have TERT promoter modulatory activity if its
interaction with TERT promoter causes a change in transcription
activity, e.g., level (for example, in terms of transcribed copies
for a given period of time), of a nucleic acid sequence (i.e.,
transcribable sequence) operably linked to the promoter, e.g., as
compared to a control (e.g., the transcription activity of an
analogous TERT promoter/reporter nucleic acid construct not
contacted with the agent of interest). The change that is observed
may be an increase or decrease of TERT transcription. In other
words, the agent may enhance or inhibit transcription of TERT. By
enhance is meant that the expression level of TERT is increased by
at least about 2 fold, usually by at least about 5 fold and
sometimes by at least 25, 50, 100 fold and in particular about 300
fold or higher, as compared to a control, i.e., expression from an
analogous or identical expression system that is not contacted with
the agent in question. Alternatively, in cases where expression of
TERT is so low that it is undetectable, expression of TERT is
considered to be enhanced if expression is increased to a level
that is easily detectable. By inhibit is meant that the expression
level of the TERT is decreased by at least about 2 fold, usually by
at least about 5 fold and sometimes by at least 25, 50, 100 fold
and in particular about 300 fold or higher, as compared to a
control, i.e., expression from an analogous or identical expression
system that is not contacted with the agent in question.
Utility
[0068] A variety of different candidate agents may be screened by
the above methods. Candidate agents encompass numerous chemical
classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 50
and less than about 2,500 daltons. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0069] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0070] The subject assays find use in any application where it is
desired to determine whether a candidate agent has TERT promoter
modulatory activity. Specifically, the subject assays find use in
applications where one wishes to determine whether an agent has
TERT promoter repressor activity and in applications where one
wishes to determine whether an agent has TERT promoter activator or
enhancer activity. In representative embodiments, the methods
provide for identification of agents which have human TERT promoter
modulatory activity.
[0071] Agents identified in the above screening assays that inhibit
repression of TERT transcription find use in the methods of
enhancement of TERT expression, e.g., in the treatment of disease
conditions, in research applications, etc., where representative
specific applications include those described in United States
Published Applications: 20030211965; 20030171326; 20030104420;
20030050264; and 20020193289; the disclosures of which are herein
incorporated by reference. Alternatively, agents identified in the
above screening assays that enhance repression find use in
applications where inhibition of TERT expression is desired, e.g.,
in the treatment of disease conditions characterized by the
presence of unwanted TERT expression, such as cancer and other
diseases characterized by the presence of unwanted cellular
proliferation, where such methods are described in, for example,
U.S. Pat. Nos. 5,645,986; 5,656,638; 5,703,116; 5,760,062;
5,767,278; 5,770,613; and 5,863,936; the disclosures of which are
herein incorporated by reference.
Kits
[0072] Also provided are kits that find use in practicing the
subject methods, as described above. For example, in some
embodiments, kits for practicing the subject methods include at
least a test cell as described above, or elements for constructing
the same, e.g., expression vectors, etc. Furthermore, additional
reagents that are required or desired in the protocol to be
practiced with the kit components may be present, which additional
reagents include, but are not limited to: aqueous mediums, culture
mediums, and the like. The kits may also include reference or
control elements, e.g., that provide calibration signals or values
for use in assessing the observed signal generated by an assay
performed with the kit components. The kit components may be
present in separate containers, or one or more of the components
may be present in the same container, where the containers may be
storage containers and/or containers that are employed during the
assay for which the kit is designed.
[0073] In addition to the above components, the subject kits may
further include instructions for practicing the subject methods.
These instructions may be present in the subject kits in a variety
of forms, one or more of which may be present in the kit. One form
in which these instructions may be present is as printed
information on a suitable medium or substrate, e.g., a piece or
pieces of paper on which the information is printed, in the
packaging of the kit, in a package insert, etc. Yet another means
would be a computer readable medium, e.g., diskette, CD, etc., on
which the information has been recorded. Yet another means that may
be present is a website address which may be used via the internet
to access the information at a removed site. Any convenient means
may be present in the kits.
Systems
[0074] Also provided are systems that find use in practicing the
subject methods, as described above. For example, in some
embodiments, systems for practicing the subject methods include at
least a test cell as described above. Furthermore, additional
reagents that are required or desired in the protocol to be
practiced with the system components may be present, which
additional reagents include, but are not limited to: aqueous
mediums, culture mediums, and the like. The systems may also
include reference or control elements, e.g., that provide
calibration signals or values for use in assessing the observed
signal generated by an assay performed with the system components.
The systems generally also include one or more candidate
agents.
Devices
[0075] Also provided are high throughput (HT) devices that find use
in practicing the subject methods, particularly HT embodiments
thereof. The high throughput devices may have any convenient
configuration, and generally include a plurality of two or more
fluid containment elements in which assays can take place, agent
administration elements and signal detection elements. For example,
representative HT devices of the subject invention include a plate
or substrate having a plurality of fluid-containing wells,
reagent-adding equipment responsive to a computer for adding
reagent, e.g., candidate agent, to the wells, measurement equipment
for measuring at least one attribute of the sample or cells
contained by the wells (e.g., for phenotype evaluation) and moving
equipment which is responsive to the computer for aligning one of
the wells first with the reagent-adding component, then with the
measurement device, as further described in U.S. Pat. No.
6,127,133, the disclosure of which is herein incorporated by
reference. Also of interest are the devices described in U.S. Pat.
Nos. 6,468,736 and 5,989,835; as well as U.S. Provisional
Application Ser. No. 60/618,484; the disclosures of which are
herein incorporated by reference. A feature of the HT devices of
the present invention is that they include in at least one fluid
containment element containing a target cell as described
above.
[0076] It is evident from the above results and discussion that the
subject invention provides for greatly improved assays for
determining the TERT promoter modulatory activity of a candidate
agent. A particularly important advantage provided by certain
embodiments of the subject invention is that the modulatory
activity of the candidate agent is assessed on a native TERT gene
present in a cell. Accordingly, the subject invention represents a
significant contribution to the art.
[0077] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
Sequence CWU 1
1
2115418DNAhuman 1gcggccgcga gctctaatac gactcactat agggcgtcga
ctcgatcaat ggaagatgag 60gcattgccga agaaaagatt aatggatttg aacacacagc
aacagaaact acatgaagtg 120aaacacagga aaaaaaagat aaagaaacga
aaagaaaagg gcatcagtga gcttcagcag 180aagttccatc ggccttacat
atgtgtaagc agaggccctg taggagcaga ggcaggggga 240aaatacttta
agaaataatg tctaaaagtt tttcaaatat gaggaaaaac ataaaaccac
300agatccaaga agctcaacaa aacaaagcac aagaaacagg aagaaattaa
aagttatatc 360acagtcaaat tgctgaaaac cagcaacaaa gagaatatct
taagagtatc agaggaaaag 420agattaatga caggccaaga aacaatgaaa
acaatacaga tttcttgtag gaaacacaag 480acaaaagaca ttttttaaaa
ccaaaaggaa aaaaaatgct acattaaaat gttttttacc 540cactgaaagt
atatttcaaa acatatttta ggccaggctt ggtggctcac acctgtaatc
600ccagcacttt gggaggccaa ggtgggtgga tcgcttaagg tcaggagttc
gagaccagcc 660tggccaatat agcgaaaccc catctgtact aaaaacacaa
aaattagctg ggtgtggtga 720cacatgcctg taatcccagg tactcaggag
gctaaggcag gagaattgct tgaactggga 780ggcagaggtg gtgagccaag
attgcaccag tgcactccag ccttggtgac agagtgaaac 840tccatctcaa
aaacaaacaa acaaaataca tatacataaa tatatatgca catatatata
900catatataaa tatatataca catatataaa tctatataca tatatacata
tatacacata 960tataaatcta tatacatata tatacatata taatatattt
acatatataa atatatacat 1020atataaatat acatatataa atacatatat
aaatatacat atataaatat acatatataa 1080atatacatat ataaatatat
acatatataa atatacatat ataaatatat atacatatat 1140aaatatataa
atatacaagt atatacaaat atatacatat ataaatgtat atacgtatat
1200acatatatat ataaatatat aaaaaaactt ttggctgggc acctttccaa
atctcatggc 1260acatataagt ctcatggtaa cctcaaataa aaaaacatat
aacagataca ccaaaaataa 1320aaaccaataa attaaatcat gccaccagaa
gaaattacct tcactaaaag gaacacagga 1380aggaaagaaa gaaggaagag
aagaccatga aacaaccaga aaacaaacaa caaaacagca 1440ggagtaattc
ctgacttatc aataataatg ctgggtgtaa atggactaaa ctctccaatc
1500aaaagacata gagtggctga atggacgaaa aaaacaagac tcaataatct
gttgcctaca 1560agaatatact tcacctataa agggacacat agactgaaaa
taaaaggaag gaaaaatatt 1620ctatgcaaat ggaaaccaaa aaaagaacag
aactagctac acttatatca gacaaaatag 1680atttcaagac aaaaagtaca
aaaagagaca aagtaattat ataataataa agcaaaaaga 1740tataacaatt
gtgaatttat atgcgcccaa cactgggaca cccagatata tacagcaaat
1800attattagaa ctaaggagag agagagatcc ccatacaata atagctggag
acttcacccc 1860gcttttagca ttggacagat catccagaca gaaaatcaac
caaaaaattg gacttaatct 1920ataatataga acaaatgtac ctaattgatg
tttacaagac atttcatcca gtagttgcag 1980aatatgcatt ttttcctcag
catatggatc attctcaagg atagaccata tattaggcca 2040cagaacaagc
cattaaaaat tcaaaaaaat tgagccaggc atgatggctt atgcttgtaa
2100ttacagcact ttggggaggg tgaggtggga ggatgtcttg agtacaggag
tttgagacca 2160gcctgggcaa aatagtgaga ccctgtctct acaaactttt
ttttttaatt agccaggcat 2220agtggtgtgt gcctgtagtc ccagctactt
aggaggctga agtgggagga tcacttgagc 2280ccaagagttc aaggctacgg
tgagccatga ttgcaacacc acacaccagc cttggtgaca 2340gaatgagacc
ctgtctcaaa aaaaaaaaaa aaaattgaaa taatataaag catcttctct
2400ggccacagtg gaacaaaacc agaaatcaac aacaagagga attttgaaaa
ctatacaaac 2460acatgaaaat taaacaatat acttctgaat aaccagtgag
tcaatgaaga aattaaaaag 2520gaaattgaaa aatttattta agcaaatgat
aacggaaaca taacctctca aaacccacgg 2580tatacagcaa aagcagtgct
aagaaggaag tttatagcta taagcagcta catcaaaaaa 2640gtagaaaagc
caggcgcagt ggctcatgcc tgtaatccca gcactttggg aggccaaggc
2700gggcagatcg cctgaggtca ggagttcgag accagcctga ccaacacaga
gaaaccttgt 2760cgctactaaa aatacaaaat tagctgggca tggtggcaca
tgcctgtaat cccagctact 2820cgggaggctg aggcaggata accgcttgaa
cccaggaggt ggaggttgcg gtgagccggg 2880attgcgccat tggactccag
cctgggtaac aagagtgaaa ccctgtctca agaaaaaaaa 2940aaaagtagaa
aaacttaaaa atacaaccta atgatgcacc ttaaagaact agaaaagcaa
3000gagcaaacta aacctaaaat tggtaaaaga aaagaaataa taaagatcag
agcagaaata 3060aatgaaactg aaagataaca atacaaaaga tcaacaaaat
taaaagttgg ttttttgaaa 3120agataaacaa aattgacaaa cctttgccca
gactaagaaa aaaggaaaga agacctaaat 3180aaataaagtc agagatgaaa
aaagagacat tacaactgat accacagaaa ttcaaaggat 3240cactagaggc
tactatgagc aactgtacac taataaattg aaaaacctag aaaaaataga
3300taaattccta gatgcataca acctaccaag attgaaccat gaagaaatcc
aaagcccaaa 3360cagaccaata acaataatgg gattaaagcc ataataaaaa
gtctcctagc aaagagaagc 3420ccaggaccca atggcttccc tgctggattt
taccaatcat ttaaagaaga atgaattcca 3480atcctactca aactattctg
aaaaatagag gaaagaatac ttccaaactc attctacatg 3540gccagtatta
ccctgattcc aaaaccagac aaaaacacat caaaaacaaa caaacaaaaa
3600aacagaaaga aagaaaacta caggccaata tccctgatga atactgatac
aaaaatcctc 3660aacaaaacac tagcaaacca aattaaacaa caccttcgaa
agatcattca ttgtgatcaa 3720gtgggattta ttccagggat ggaaggatgg
ttcaacatat gcaaatcaat caatgtgata 3780catcatccca acaaaatgaa
gtacaaaaac tatatgatta tttcacttta tgcagaaaaa 3840gcatttgata
aaattctgca cccttcatga taaaaaccct caaaaaacca ggtatacaag
3900aaacatacag gccaggcaca gtggctcaca cctgcgatcc cagcactctg
ggaggccaag 3960gtgggatgat tgcttgggcc caggagtttg agactagcct
gggcaacaaa atgagacctg 4020gtctacaaaa aactttttta aaaaattagc
caggcatgat ggcatatgcc tgtagtccca 4080gctagtctgg aggctgaggt
gggagaatca cttaagccta ggaggtcgag gctgcagtga 4140gccatgaaca
tgtcactgta ctccagccta gacaacagaa caagacccca ctgaataaga
4200agaaggagaa ggagaaggga gaaaggaggg agaagggagg aggaggagaa
ggaggaggtg 4260gaggagaagt ggaaggggaa ggggaaggga aagaggaaga
agaagaaaca tatttcaaca 4320taataaaagc cctatatgac agaccgaggt
agtattatga ggaaaaactg aaagcctttc 4380ctctaagatc tggaaaatga
caagggccca ctttcaccac tgtgattcaa catagtacta 4440gaagtcctag
ctagagcaat cagataagag aaagaaataa aaggcatcca aactggaaag
4500gaagaagtca aattatcctg tttgcagatg atatgatctt atatctggaa
aagacttaag 4560acaccactaa aaaactatta gagctgaaat ttggtacagc
aggatacaaa atcaatgtac 4620aaaaatcagt agtatttcta tattccaaca
gcaaacaatc tgaaaaagaa accaaaaaag 4680cagctacaaa taaaattaaa
cagctaggaa ttaaccaaag aagtgaaaga tctctacaat 4740gaaaactata
aaatattgat aaaagaaatt gaagagggca caaaaaaaga aaagatattc
4800catgttcata gattggaaga ataaatactg ttaaaatgtc catactaccc
aaagcaattt 4860acaaattcaa tgcaatccct attaaaatac taatgacgtt
cttcacagaa atagaagaaa 4920caattctaag atttgtacag aaccacaaaa
gacccagaat agccaaagct atcctgacca 4980aaaagaacaa aactggaagc
atcacattac ctgacttcaa attatactac aaagctatag 5040taacccaaac
tacatggtac tggcataaaa acagatgaga catggaccag aggaacagaa
5100tagagaatcc agaaacaaat ccatgcatct acagtgaact catttttgac
aaaggtgcca 5160agaacatact ttggggaaaa gataatctct tcaataaatg
gtgctggagg aactggatat 5220ccatatgcaa aataacaata ctagaactct
gtctctcacc atatacaaaa gcaaatcaaa 5280atggatgaaa ggcttaaatc
taaaacctca aactttgcaa ctactaaaag aaaacaccgg 5340agaaactctc
caggacattg gagtgggcaa agacttcttg agtaattccc tgcaggcaca
5400ggcaaccaaa gcaaaaacag acaaatggga tcatatcaag ttaaaaagct
tctgcccagc 5460aaaggaaaca atcaacaaag agaagagaca acccacagaa
tgggagaata tatttgcaaa 5520ctattcatct aacaaggaat taataaccag
tatatataag gagctcaaac tactctataa 5580gaaaaacacc taataagctg
attttcaaaa ataagcaaaa gatctgggta gacatttctc 5640aaaataagtc
atacaaatgg caaacaggca tctgaaaatg tgctcaacac cactgatcat
5700cagagaaatg caaatcaaaa ctactatgag agatcatctc accccagtta
aaatggcttt 5760tattcaaaag acaggcaata acaaatgcca gtgaggatgt
ggataaaagg aaacccttgg 5820acactgttgg tgggaatgga aattgctacc
actatggaga acagtttgaa agttcctcaa 5880aaaactaaaa ataaagctac
catacagcaa tcccattgct aggtatatac tccaaaaaag 5940ggaatcagtg
tatcaacaag ctatctccac tcccacattt actgcagcac tgttcatagc
6000agccaaggtt tggaagcaac ctcagtgtcc atcaacagac gaatggaaaa
agaaaatgtg 6060gtgcacatac acaatggagt actacgcagc cataaaaaag
aatgagatcc tgtcagttgc 6120aacagcatgg ggggcactgg tcagtatgtt
aagtgaaata agccaggcac agaaagacaa 6180acttttcatg ttctccctta
cttgtgggag caaaaattaa aacaattgac atagaaatag 6240aggagaatgg
tggttctaga ggggtggggg acagggtgac tagagtcaac aataatttat
6300tgtatgtttt aaaataacta aaagagtata attgggttgt ttgtaacaca
aagaaaggat 6360aaatgcttga aggtgacaga taccccattt accctgatgt
gattattaca cattgtatgc 6420ctgtatcaaa atatctcatg tatgctatag
atataaaccc tactatatta aaaattaaaa 6480ttttaatggc caggcacggt
ggctcatgtc cataatccca gcactttggg aggccgaggc 6540ggtggatcac
ctgaggtcag gagtttgaaa ccagtctggc caccatgatg aaaccctgtc
6600tctactaaag atacaaaaat tagccaggcg tggtggcaca tacctgtagt
cccaactact 6660caggaggctg agacaggaga attgcttgaa cctgggaggc
ggaggttgca gtgagccgag 6720atcatgccac tgcactgcag cctgggtgac
agagcaagac tccatctcaa aacaaaaaca 6780aaaaaaagaa gattaaaatt
gtaattttta tgtaccgtat aaatatatac tctactatat 6840tagaagttaa
aaattaaaac aattataaaa ggtaattaac cacttaatct aaaataagaa
6900caatgtatgt ggggtttcta gcttctgaag aagtaaaagt tatggccacg
atggcagaaa 6960tgtgaggagg gaacagtgga agttactgtt gttagacgct
catactctct gtaagtgact 7020taattttaac caaagacagg ctgggagaag
ttaaagaggc attctataag ccctaaaaca 7080actgctaata atggtgaaag
gtaatctcta ttaattacca ataattacag atatctctaa 7140aatcgagctg
cagaattggc acgtctgatc acaccgtcct ctcattcacg gtgctttttt
7200tcttgtgtgc ttggagattt tcgattgtgt gttcgtgttt ggttaaactt
aatctgtatg 7260aatcctgaaa cgaaaaatgg tggtgatttc ctccagaaga
attagagtac ctggcaggaa 7320gcaggtggct ctgtggacct gagccacttc
aatcttcaag ggtctctggc caagacccag 7380gtgcaaggca gaggcctgat
gacccgagga caggaaagct cggatgggaa ggggcgatga 7440gaagcctgcc
tcgttggtga gcagcgcatg aagtgccctt atttacgctt tgcaaagatt
7500gctctggata ccatctggaa aaggcggcca gcgggaatgc aaggagtcag
aagcctcctg 7560ctcaaaccca ggccagcagc tatggcgccc acccgggcgt
gtgccagagg gagaggagtc 7620aaggcacctc gaagtatggc ttaaatcttt
ttttcacctg aagcagtgac caaggtgtat 7680tctgagggaa gcttgagtta
ggtgccttct ttaaaacaga aagtcatgga agcacccttc 7740tcaagggaaa
accagacgcc cgctctgcgg tcatttacct ctttcctctc tccctctctt
7800gccctcgcgg tttctgatcg ggacagagtg acccccgtgg agcttctccg
agcccgtgct 7860gaggaccctc ttgcaaaggg ctccacagac ccccgccctg
gagagaggag tctgagcctg 7920gcttaataac aaactgggat gtggctgggg
gcggacagcg acggcgggat tcaaagactt 7980aattccatga gtaaattcaa
cctttccaca tccgaatgga tttggatttt atcttaatat 8040tttcttaaat
ttcatcaaat aacattcagg agtgcagaaa tccaaaggcg taaaacagga
8100actgagctat gtttgccaag gtccaaggac ttaataacca tgttcagagg
gatttttcgc 8160cctaagtact ttttattggt tttcataagg tggcttaggg
tgcaagggaa agtacacgag 8220gagaggactg ggcggcaggg ctatgagcac
ggcaaggcca ccggggagag agtccccggc 8280ctgggaggct gacagcagga
ccactgaccg tcctccctgg gagctgccac attgggcaac 8340gcgaaggcgg
ccacgctgcg tgtgactcag gaccccatac cggcttcctg ggcccaccca
8400cactaaccca ggaagtcacg gagctctgaa cccgtggaaa cgaacatgac
ccttgcctgc 8460ctgcttccct gggtgggtca agggtaatga agtggtgtgc
aggaaatggc catgtaaatt 8520acacgactct gctgatgggg accgttcctt
ccatcattat tcatcttcac ccccaaggac 8580tgaatgattc cagcaacttc
ttcgggtgtg acaagccatg acaacactca gtacaaacac 8640cactctttta
ctaggcccac agagcacggc ccacacccct gatatattaa gagtccagga
8700gagatgaggc tgctttcagc caccaggctg gggtgacaac agcggctgaa
cagtctgttc 8760ctctagacta gtagaccctg gcaggcactc ccccagattc
tagggcctgg ttgctgcttc 8820ccgagggcgc catctgccct ggagactcag
cctggggtgc cacactgagg ccagccctgt 8880ctccacaccc tccgcctcca
ggcctcagct tctccagcag cttcctaaac cctgggtggg 8940ccgtgttcca
gcgctactgt ctcacctgtc ccactgtgtc ttgtctcagc gacgtagctc
9000gcacggttcc tcctcacatg gggtgtctgt ctccttcccc aacactcaca
tgcgttgaag 9060ggaggagatt ctgcgcctcc cagactggct cctctgagcc
tgaacctggc tcgtggcccc 9120cgatgcaggt tcctggcgtc cggctgcacg
ctgacctcca tttccaggcg ctccccgtct 9180cctgtcatct gccggggcct
gccggtgtgt tcttctgttt ctgtgctcct ttccacgtcc 9240agctgcgtgt
gtctctgtcc gctagggtct cggggttttt ataggcatag gacgggggcg
9300tggtgggcca gggcgctctt gggaaatgca acatttgggt gtgaaagtag
gagtgcctgt 9360cctcacctag gtccacgggc acaggcctgg ggatggagcc
cccgccaggg acccgccctt 9420ctctgcccag cacttttctg cccccctccc
tctggaacac agagtggcag tttccacaag 9480cactaagcat cctcttccca
aaagacccag cattggcacc cctggacatt tgccccacag 9540ccctgggaat
tcacgtgact acgcacatca tgtacacact cccgtccacg accgaccccc
9600gctgttttat tttaatagct acaaagcagg gaaatccctg ctaaaatgtc
ctttaacaaa 9660ctggttaaac aaacgggtcc atccgcacgg tggacagttc
ctcacagtga agaggaacat 9720gccgtttata aagcctgcag gcatctcaag
ggaattacgc tgagtcaaaa ctgccacctc 9780catgggatac gtacgcaaca
tgctcaaaaa gaaagaattt caccccatgg caggggagtg 9840gttggggggt
taaggacggt gggggcagca gctgggggct actgcacgca ccttttacta
9900aagccagttt cctggttctg atggtattgg ctcagttatg ggagactaac
cataggggag 9960tggggatggg ggaacccgga ggctgtgcca tctttgccat
gcccgagtgt cctgggcagg 10020ataatgctct agagatgccc acgtcctgat
tcccccaaac ctgtggacag aacccgcccg 10080gccccagggc ctttgcaggt
gtgatctccg tgaggaccct gaggtctggg atccttcggg 10140actacctgca
ggcccgaaaa gtaatccagg ggttctggga agaggcgggc aggagggtca
10200gaggggggca gcctcaggac gatggaggca gtcagtctga ggctgaaaag
ggagggaggg 10260cctcgagccc aggcctgcaa gcgcctccag aagctggaaa
aagcggggaa gggaccctcc 10320acggagcctg cagcaggaag gcacggctgg
cccttagccc accagggccc atcgtggacc 10380tccggcctcc gtgccatagg
agggcactcg cgctgccctt ctagcatgaa gtgtgtgggg 10440atttgcagaa
gcaacaggaa acccatgcac tgtgaatcta ggattatttc aaaacaaagg
10500tttacagaaa catccaagga cagggctgaa gtgcctccgg gcaagggcag
ggcaggcacg 10560agtgatttta tttagctatt ttattttatt tacttacttt
ctgagacaga gttatgctct 10620tgttgcccag gctggagtgc agcggcatga
tcttggctca ctgcaacctc cgtctcctgg 10680gttcaagcaa ttctcgtgcc
tcagcctccc aagtagctgg gatttcaggc gtgcaccacc 10740acacccggct
aattttgtat ttttagtaga gatgggcttt caccatgttg gtcaggctga
10800tctcaaaatc ctgacctcag gtgatccgcc cacctcagcc tcccaaagtg
ctgggattac 10860aggcatgagc cactgcacct ggcctattta accattttaa
aacttccctg ggctcaagtc 10920acacccactg gtaaggagtt catggagttc
aatttcccct ttactcagga gttaccctcc 10980tttgatattt tctgtaattc
ttcgtagact ggggatacac cgtctcttga catattcaca 11040gtttctgtga
ccacctgtta tcccatggga cccactgcag gggcagctgg gaggctgcag
11100gcttcaggtc ccagtggggt tgccatctgc cagtagaaac ctgatgtaga
atcagggcgc 11160gagtgtggac actgtcctga atctcaatgt ctcagtgtgt
gctgaaacat gtagaaatta 11220aagtccatcc ctcctactct actgggattg
agccccttcc ctatcccccc ccaggggcag 11280aggagttcct ctcactcctg
tggaggaagg aatgatactt tgttattttt cactgctggt 11340actgaatcca
ctgtttcatt tgttggtttg tttgttttgt tttgagaggc ggtttcactc
11400ttgttgctca ggctggaggg agtgcaatgg cgcgatcttg gcttactgca
gcctctgcct 11460cccaggttca agtgattctc ctgcttccgc ctcccatttg
gctgggatta caggcacccg 11520ccaccatgcc cagctaattt tttgtatttt
tagtagagac gggggtgggg gtggggttca 11580ccatgttggc caggctggtc
tcgaacttct gacctcagat gatccacctg cctctgcctc 11640ctaaagtgct
gggattacag gtgtgagcca ccatgcccag ctcagaattt actctgttta
11700gaaacatctg ggtctgaggt aggaagctca ccccactcaa gtgttgtggt
gttttaagcc 11760aatgatagaa tttttttatt gttgttagaa cactcttgat
gttttacact gtgatgacta 11820agacatcatc agcttttcaa agacacacta
actgcaccca taatactggg gtgtcttctg 11880ggtatcagcg atcttcattg
aatgccggga ggcgtttcct cgccatgcac atggtgttaa 11940ttactccagc
ataatcttct gcttccattt cttctcttcc ctcttttaaa attgtgtttt
12000ctatgttggc ttctctgcag agaaccagtg taagctacaa cttaactttt
gttggaacaa 12060attttccaaa ccgccccttt gccctagtgg cagagacaat
tcacaaacac agccctttaa 12120aaaggcttag ggatcactaa ggggatttct
agaagagcga cccgtaatcc taagtattta 12180caagacgagg ctaacctcca
gcgagcgtga cagcccaggg agggtgcgag gcctgttcaa 12240atgctagctc
cataaataaa gcaatttcct ccggcagttt ctgaaagtag gaaaggttac
12300atttaaggtt gcgtttgtta gcatttcagt gtttgccgac ctcagctaca
gcatccctgc 12360aaggcctcgg gagacccaga agtttctcgc cccttagatc
caaacttgag caacccggag 12420tctggattcc tgggaagtcc tcagctgtcc
tgcggttgtg ccggggcccc aggtctggag 12480gggaccagtg gccgtgtggc
ttctactgct gggctggaag tcgggcctcc tagctctgca 12540gtccgaggct
tggagccagg tgcctggacc ccgaggctgc cctccaccct gtgcgggcgg
12600gatgtgacca gatgttggcc tcatctgcca gacagagtgc cggggcccag
ggtcaaggcc 12660gttgtggctg gtgtgaggcg cccggtgcgc ggccagcagg
agcgcctggc tccatttccc 12720accctttctc gacgggaccg ccccggtggg
tgattaacag atttggggtg gtttgctcat 12780ggtggggacc cctcgccgcc
tgagaacctg caaagagaaa tgacgggcct gtgtcaagga 12840gcccaagtcg
cggggaagtg ttgcagggag gcactccggg aggtcccgcg tgcccgtcca
12900gggagcaatg cgtcctcggg ttcgtcccca gccgcgtcta cgcgcctccg
tcctcccctt 12960cacgtccggc attcgtggtg cccggagccc gacgccccgc
gtccggacct ggaggcagcc 13020ctgggtctcc ggatcaggcc agcggccaaa
gggtcgccgc acgcacctgt tcccagggcc 13080tccacatcat ggcccctccc
tcgggttacc ccacagccta ggccgattcg acctctctcc 13140gctggggccc
tcgctggcgt ccctgcaccc tgggagcgcg agcggcgcgc gggcggggaa
13200gcgcggccca gacccccggg tccgcccgga gcagctgcgc tgtcggggcc
aggccgggct 13260cccagtggat tcgcgggcac agacgcccag gaccgcgctt
cccacgtggc ggagggactg 13320gggacccggg cacccgtcct gccccttcac
cttccagctc cgcctcctcc gcgcggaccc 13380cgccccgtcc cgacccctcc
cgggtccccg gcccagcccc ctccgggccc tcccagcccc 13440tccccttcct
ttccgcggcc ccgccctctc ctcgcggcgc gagtttcagg cagcgctgcg
13500tcctgctgcg cacgtgggaa gccctggccc cggccacccc cgcgatgccg
cgcgctcccc 13560gctgccgagc cgtgcgctcc ctgctgcgca gccactaccg
cgaggtgctg ccgctggcca 13620cgttcgtgcg gcgcctgggg ccccagggct
ggcggctggt gcagcgcggg gacccggcgg 13680ctttccgcgc gctggtggcc
cagtgcctgg tgtgcgtgcc ctgggacgca cggccgcccc 13740ccgccgcccc
ctccttccgc caggtgggcc tccccggggt cggcgtccgg ctggggttga
13800gggcggccgg ggggaaccag cgacatgcgg agagcagcgc aggcgactca
gggcgcttcc 13860cccgcaggtg tcctgcctga aggagctggt ggcccgagtg
ctgcagaggc tgtgcgagcg 13920cggcgcgaag aacgtgctgg ccttcggctt
cgcgctgctg gacggggccc gcgggggccc 13980ccccgaggcc ttcaccacca
gcgtgcgcag ctacctgccc aacacggtga ccgacgcact 14040gcgggggagc
ggggcgtggg ggctgctgct gcgccgcgtg ggcgacgacg tgctggttca
14100cctgctggca cgctgcgcgc tctttgtgct ggtggctccc agctgcgcct
accaggtgtg 14160cgggccgccg ctgtaccagc tcggcgctgc cactcaggcc
cggcccccgc cacacgctag 14220tggaccccga aggcgtctgg gatgcgaacg
ggcctggaac catagcgtca gggaggccgg 14280ggtccccctg ggcctgccag
ccccgggtgc gaggaggcgc gggggcagtg ccagccgaag 14340tctgccgttg
cccaagaggc ccaggcgtgg cgctgcccct gagccggagc ggacgcccgt
14400tgggcagggg tcctgggccc acccgggcag gacgcgtgga ccgagtgacc
gtggtttctg 14460tgtggtgtca cctgccagac ccgccgaaga agccacctct
ttggagggtg cgctctctgg 14520cacgcgccac tcccacccat ccgtgggccg
ccagcaccac gcgggccccc catccacatc 14580gcggccacca cgtccctggg
acacgccttg tcccccggtg tacgccgaga ccaagcactt 14640cctctactcc
tcaggcgaca aggagcagct gcggccctcc ttcctactca gctctctgag
14700gcccagcctg actggcgctc ggaggctcgt ggagaccatc tttctgggtt
ccaggccctg 14760gatgccaggg actccccgca ggttgccccg cctgccccag
cgctactggc aaatgcggcc 14820cctgtttctg gagctgcttg ggaaccacgc
gcagtgcccc tacggggtgc tcctcaagac 14880gcactgcccg ctgcgagctg
cggtcacccc agcagccggt gtctgtgccc gggagaagcc 14940ccagggctct
gtggcggccc ccgaggagga ggacacagac ccccgtcgcc tggtgcagct
15000gctccgccag cacagcagcc cctggcaggt gtacggcttc gtgcgggcct
gcctgcgccg 15060gctggtgccc ccaggcctct ggggctccag gcacaacgaa
cgccgcttcc tcaggaacac 15120caagaagttc atctccctgg ggaagcatgc
caagctctcg ctgcaggagc tgacgtggaa 15180gatgagcgtg cgggactgcg
cttggctgcg caggagccca ggtgaggagg tggtggccgt 15240cgagggccca
ggccccagag ctgaatgcag taggggctca gaaaaggggg caggcagagc
15300cctggtcctc ctgtctccat cgtcacgtgg gcacacgtgg cttttcgctc
aggacgtcga 15360gtggacacgg tgatcgagtc gactcccttt agtgagggtt
aattgagctc gcggccgc 1541827498DNAmouse 2aagcttccag caaaccagtt
agagctgagt tgatgctctg aagaagagaa aatgtagaga 60cggtactgaa caaataatgt
ctgggcaaac ctcagacatg aaaatggaag acgtggaaat 120ccagagaact
ctgagggaaa ataaaacaca actccaggtc atcacgggac tcatcaaact
180gctgaggtgc agccacagag aaaaatctta aaatagccta gaacgatgca
tgacacataa 240agcacagaga agacgaagct gagtctgtct tgtaggaaca
acttgagaag acctaaacca 300ctgcaatgag tgcattctgc taacttagaa
tttgctaccc agttcagatc caaaaagggt 360ttcacaaagt tcaacacaaa
acagtagcag gagtggctaa gggggacaca ctgataggaa 420ttcagagaag
tagggaatgc tcatatgggg acattacaaa atgtactttc atgttgctta
480aatcatttta attgtcaacc acatcaagct aaataatgct ttgaggttca
taacatttgg 540agattatgtc tacactagca gagaaggcac caataacatc
ccaattgcta gattctcata 600gaatcatgag tcacaatggc agagacaggt
tctgagagtg tgtccttgtt gtaaacagta 660tgctctacaa actaagttgg
ctgcaatatc actaggcagt gttgtcccat aagacaacta 720tcacatatgt
ggtccagtga tgaccaaagc atcttttagc attttgcaaa tgaagctcaa
780atcgaatatg actaagctca tgcagtacaa atcaaaggta cactgggata
gtttaaaaga 840tacatacttg tactggttag ttttgtgtca gcttgacaca
gctggagtta tcacagagaa 900aagagcttca gttgaggaaa ttcctccatg
agatccagct atagggcatt ttctcaatta 960gtgatcaagg ggggaaggcc
ccttgtgggt gggaccatct ctgggctggt agtcttggtt 1020ctataagaga
gcaggctgag caagccagga gaagcaagcc agtaaagaac atccctccat
1080ggcttctgca tcagctcctg ctccctgacc tgcttgagtt ccagttctaa
cttctttcag 1140tgatgaacag caatgtggaa atgaaagctg aataaaccct
ttcctcccca ttttgcttct 1200tggtcatgat gtttgtgcag gaatagaaac
cctgactaag acaatactat aaaccctaaa 1260agttgtaaac caaacacatg
tgtttccatt aagccatcgt agaacaataa gtactcaacc 1320ccaagtcaca
taactataat cccagccttt gaaaaccggg atcaggaatt caaggctagc
1380ctcatctata tgtaagatta aagcctgttt gggctgcatg agactttgtt
tcaaaaaaaa 1440aaaaaaaaaa gcaaacaggc aaaaacaaac acaagacaag
acagatgtaa aatgaaggag 1500gggtagatgg gtcaagtaga aaatagcata
ggaaacgagt caagtataga agaggtggta 1560gtaaccagat catgcagaag
gactcaaggc catctcctca cagtggctta ggtaggcctt 1620cctctgctct
tgagcagggg cagagttgcc gctttaagga ggggatcagt cacctttaag
1680aactgaaaag ctgaacagtc ttctcaagtc agaagccagt ggcttcatct
tacacctctc 1740ttccttccct tgctactcat attggatctg atgatttgcc
caacttggaa gaaacatctc 1800ttctgaaggg tttcacagac accccatctt
tccgagaaag gaccgcatag gctggccatc 1860cctgtgctta caaaaggaat
aattaagaaa cttaattcca taagcaaata caacctttcc 1920aagccccaag
tggatgattt tatcttactg tttttttata tctcatcaaa taacttccaa
1980gggctcaaaa atccaaagat gtaaaaaagg aactgagctc tgtttgccaa
gccatgagga 2040ttaaataatg acattcaaag agatttttgt gccctaagta
ctttttattg gttttcatag 2100atggtttaat gtgcaagatg aagcaaacag
agatgggagt ggtatcagca tggattaagg 2160tggcagttgt gagggagggg
tactgagaga acaggacaag gtaacctatc taaggagagg 2220ccaagttggc
aagtgccagg gacttctaag cccagaacta gtacacattc cttaggtgct
2280gtttgggaag tcagggagtc accagccttg ggatctataa aagtgcatgg
tggcattcac 2340tcacatactt cctgagctgt tcgatgttga tgaagtcgtg
ggtatgagac tgttgtgtca 2400gtgacaaact atgtaaatga gaatgattgt
ttccatcttg accactaaga cgtaaaccgg 2460ttccagtgat ctccaaacat
ggcaagctac agcagagcag cagccccatc cagagccttg 2520ccctggttct
gaatggggga gaatccagtg ggagtcggtt gctgccagca tgttggggta
2580gaaggctgga gcatgacagg tccccgagga tttcctgctt cctatatggg
tagggatact 2640tgaggtcctc tcttctacct ccttccctgc agggtttata
acctctacca ctgtctgtct 2700ctgggatagc tcctagggtg cagcccctcc
ccaaaaaggc ctctccctgg cctcatgtct 2760ctaagaacag ctttctaaag
caggcctgtt acacaaaggc tcccttttcc tggcttcatc 2820gttgctggta
gacaacttcc actcgttttc cacttcagtt tcttctactc tgttgttatt
2880tgattctgat gcttgaaccc agggttgtgt agtcagcaag tgctaccccc
tccctcctct 2940tctttgtttt tttgaggcag ggtctcattt tgcccaagtg
gacctaaatt tcagcatgta 3000gctggcctgg ttttgaatgc cttctcatcc
tgcctctact tcccaagagt agcttacaag 3060tgtgcaccac catgccccgc
gatattctta tttttgagac tgttttctat gctggtttct 3120ttggggaact
acactaaggt agcttacaag tgtgcaccac catgccccgc gatattctta
3180tttttgagac tgttttctat gctggtttct ttggggaact acactaaggt
agcttcattg 3240ttggcataaa tttctcagtt caggcccata tctcctaagt
agcagaacta agcaaatctc 3300aaacaaaccc ctcaaaaaga ctgatgtcca
ctaaacggac ttctaaaata gctcctgtaa 3360tcctgagcat ttacaaggcg
gcagacctcc tataagggag taaatatgaa aacgcgcctg 3420ttcaaatgct
aggtcggtgg atagaagcaa tttcctcaga aagctgaagg caccaaaggt
3480tatatttgtt agcatttcag tgtttgccaa actcagctac agtagagatc
acagattccc 3540tatttcccag agattcaaaa ttcagcagcc cctctctaac
tatggctcag agtcgtgtca 3600ttacatatgc cccaacaaca acccccaccc
ctatcctacc cccgcctcac acgtgcaagt 3660actatcacag ttgccaacct
agcagagctg ccatcctaag gtcgaggtcg ccgctttggc 3720tgtgtgcaca
ggcaagcgcc ctcacccaat ggccctggcc ttgctatggg tgcgtgagtt
3780gagatgatgc tctggactct gaggtgaagg ccactggaac agtgaaaaaa
gctaacgcag 3840ggcttttacc tagtcccctt cctttggtgg tgggtgttta
cggaacatat ttgggatctg 3900agtgtatggt cgcaccacaa taaagcctta
acctatatag tagaatttca gctgtaatca 3960ttaagaactg agattgccac
cacccacctc actgtctgtg tcaaccacag caggctggag 4020cagtcagctc
aggaacaggc aaaaccttag gtccctccgc ctacctaacc ttcaatacat
4080caaggatagg cttctttgct tgcccaaacc tcgccccagt ctagaccacc
tggggattcc 4140cagctcaggg cgaaaaggaa gcccgagaag cattctgtag
agggaaatcc tgcatgagtg 4200cgcccccttt cgttactcca acacatccag
caaccactga acttggccgg ggaacacacc 4260tggtcctcat gcaccagcat
tgtgaccatc aacggaaaag tactattgct gcgaccccgc 4320cccttccgct
acaacgcttg gtccgcctga atcccgcccc ttcctccgtt cccagcctca
4380tctttttcgt cgtggactct cagtggcctg ggtcctggct gttttctaag
cacacccttg 4440catcttggtt cccgcacgtg ggaggcccat cccggccttg
agcacaatga cccgcgctcc 4500tcgttgcccc gcggtgcgct ctctgctgcg
cagccgatac cgggaggtgt ggccgctggc 4560aacctttgtg cggcgcctgg
ggcccgaggg caggcggctt gtgcaacccg gggacccgaa 4620gatctaccgc
actttggttg cccaatgcct agtgtgcatg cactggggct cacagcctcc
4680acctgccgac ctttccttcc accaggtggg cctccaggcg ggatccccat
gggtcagggg 4740cggaaagccg ggaggacgtg ggatagtgcg tctagctcat
gtgtcaagac cctcttctcc 4800ttaccaggtg tcatccctga aagagctggt
ggccagggtt gtgcagagac tctgcgagcg 4860caacgagaga aacgtgctgg
cttttggctt tgagctgctt aacgaggcca gaggcgggcc 4920tcccatggcc
ttcactagta gcgtgcgtag ctacttgccc aacactgtta ttgagaccct
4980gcgtgtcagt ggtgcatgga tgctactgtt gagccgagtg ggcgacgacc
tgctggtcta 5040cctgctggca cactgtgctc tttatcttct ggtgcccccc
agctgtgcct accaggtgtg 5100tgggtctccc ctgtaccaaa tttgtgccac
cacggatatc tggccctctg tgtccgctag 5160ttacaggccc acccgacccg
tgggcaggaa tttcactaac cttaggttct tacaacagat 5220caagagcagt
agtcgccagg aagcaccgaa acccctggcc ttgccatctc gaggtacaaa
5280gaggcatctg agtctcacca gtacaagtgt gccttcagct aagaaggcca
gatgctatcc 5340tgtcccgaga gtggaggagg gaccccacag gcaggtgcta
ccaaccccat caggcaaatc 5400atgggtgcca agtcctgctc ggtcccccga
ggtgcctact gcagagaaag atttgtcttc 5460taaaggaaag gtgtctgacc
tgagtctctc tgggtcggtg tgctgtaaac acaagcccag 5520ctccacatct
ctgctgtcac caccccgcca aaatgccttt cagctcaggc catttattga
5580gaccagacat ttcctttact ccaggggaga tggccaagag cgtctaaacc
cctcattcct 5640actcagcaac ctccagccta acttgactgg ggccaggaga
ctggtggaga tcatctttct 5700gggctcaagg cctaggacat caggaccact
ctgcaggaca caccgtctat cgcgtcgata 5760ctggcagatg cggcccctgt
tccaacagct gctggtgaac catgcagagt gccaatatgt 5820cagactcctc
aggtcacatt gcaggtttcg aacagcaaac caacaggtga cagatgcctt
5880gaacaccagc ccaccgcacc tcatggattt gctccgcctg cacagcagtc
cctggcaggt 5940atatggtttt cttcgggcct gtctctgcaa ggtggtgtct
gctagtctct ggggtaccag 6000gcacaatgag cgccgcttct ttaagaactt
aaagaagttc atctcgttgg ggaaatacgg 6060caagctatca ctgcaggaac
tgatgtggaa gatgaaagta gaggattgcc actggctccg 6120cagcagcccg
ggtgagcatg gctggtctcc agctgaatgc attaggggcc cagaaaaggg
6180agacaatggg tggcagtaac ccaggtcccc agtggtgtgg tggctttatg
cagtccgtgg 6240ttggatgagt tccatcttat ggtctctgac tccaagctcc
ctccagctcg ccttgcacaa 6300actaagattc ttgtccaagc cctgggcagg
ttctcagggc tggggacatt gtggtgaaca 6360gataagcaga cggggagcat
ggtggatagg agttctggca cagtgcacca gagagagtct 6420ggaagcgcta
gtgagagcta atgtaagggc ccgtggttcg ccaaagaatg ataaccccgg
6480actcaaatag tatgccaaag caaggagcat ttcattctgc agaaatcaag
catgcaggtg 6540gggggggggg gttgctctca ttccaagatg gagagacaac
caagtataga ttttaagggg 6600atcgggggcc tttatcttac tccatctcta
ggggcattcc attactgggg catggggttg 6660gaggttggaa actgttaatg
gggaggtctg gaaacttgct gccccattgt ccttgcttca 6720ggctaggtag
ctgagtagct tctaatggca ggatagtttc tgactagctg tctaaagtct
6780ggggtgtttg tttttttgtt ttttctagta acttacttgc ctgaacttgc
tcagttttta 6840ggcctggtct cctggactgc caatttgaag cctattaagg
agtcagcctg tctcactact 6900ccaggttatc tataatcccc ctgtagaacg
gtacctcact gataacaatg acagaccaac 6960ataggaaccc actatccttg
tggtgcatga gtttcaaagg ttcttctggt cctcccagtg 7020tgcagatcca
tgcttaagct atggtcctcc cagtgtgcag atccgtgctt aagctatggt
7080cttgcagctg ctcgatctac aaagggtagg gtgaacgaag gaaagataaa
tgaaaaaaaa 7140aaaactgttt cctacagtga agatcgctgc cccatcttag
ctatgagaag ggactgggga 7200gtggagcctg gtgcataaaa gaggattgtg
ttacttggaa ggctgcagag cctggactcc 7260tgtgccctcc ttgcctggtt
ttctgggttt aatgttgagg ttggccctct gtagtcacta 7320cctgacccct
tccctttcag ccaaccctcc ggttacaccc tgtgcatgta tggaaggggc
7380caaacgccct atcctgctct cccttcccca aaattcttag gatattaaca
acttatgggg 7440aaaagatggt agagctatgt ttacccacca tgtacttggg
aagctccgaa gtaagctt 7498
* * * * *
References