U.S. patent application number 11/481190 was filed with the patent office on 2006-10-26 for 75k rna regulated transcription.
This patent application is currently assigned to Regents of the University of California. Invention is credited to Kunxin Luo, Zhiyuan Yang, Qiang Zheu, Qingwei Zhu.
Application Number | 20060239976 11/481190 |
Document ID | / |
Family ID | 31887453 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060239976 |
Kind Code |
A1 |
Zheu; Qiang ; et
al. |
October 26, 2006 |
75K RNA regulated transcription
Abstract
A method for altering transcription in a cell comprising an
amount of active CDK9/cyclin, comprises the steps of: (a)
introducing in the cell an agent which modulates the amount of
active CDK9/cyclin in the cell, and thereby alters transcription in
the cell, wherein the agent comprises an RNA selected from the
group consisting of an RNA aptamer that specifically binds
CDK9/cyclin, a CDK9/cyclin-binding domain of 7SK RNA, a 7SK
RNA-binding antisense 7SK RNA domain, and a 7SK RNA-specific RNAi,
and (b) detecting a resultant altering of transcription in the
cell. Methods for screening for an agent which modulates 7SK
RNA-CDK9/cyclin binding generally comprise the steps of (a)
incubating a mixture of 7SK RNA, CDK9/cyclin and a candidate agent
under conditions wherein but for the presence of the agent, the 7SK
RNA and CDK9/cyclin engage in a reference binding; and (b)
detecting an agent-biased binding of the 7SK RNA to the
CDK9/cyclin.
Inventors: |
Zheu; Qiang; (Berkeley,
CA) ; Yang; Zhiyuan; (Berkeley, CA) ; Zhu;
Qingwei; (Berkeley, CA) ; Luo; Kunxin;
(Berkeley, CA) |
Correspondence
Address: |
RICHARD ARON OSMAN;SCIENCE AND TECHNOLOGY LAW GROUP
242 AVE VISTA DEL OCEANO
SAN CLEMEMTE
CA
92672
US
|
Assignee: |
Regents of the University of
California
|
Family ID: |
31887453 |
Appl. No.: |
11/481190 |
Filed: |
July 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10227367 |
Aug 25, 2002 |
7087433 |
|
|
11481190 |
Jul 1, 2006 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
435/456; 514/44A |
Current CPC
Class: |
C12N 15/113
20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 435/456 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/867 20060101 C12N015/867 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. A141757 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1-20. (canceled)
21. A method for increasing a CDK9/cyclin-dependent transcription
in a cell, the method comprising the steps of: incubating a human
cell comprising human 7SK RNA (SEQ ID NO: 1) and comprising an
amount of active CDK9/cyclin, and in which a CDK9/cyclin-dependent
transcription is to be increased; introducing in the cell a human
7SK RNA-binding antisense 7SK RNA which increases the amount of
active CDK9/cyclin in the cell, and thereby increases said
CDK9/cyclin-dependent transcription in the cell, wherein the
antisense RNA consists of antisense of nucleotides 221-241 or
95-114 of human 7SK RNA (SEQ ID NO: 1); and detecting a resultant
increase in CDK9/cyclin-dependent transcription in the cell,
wherein the CDK9/cyclin-dependent transcription is selected from
the group consisting of transcription from a recombinant construct,
LTR promoter-controlled transcription and HIV transcription.
22. The method of claim 21, wherein the transcription is from a
recombinant construct.
23. The method of claim 21, wherein the transcription is an LTR
promoter-controlled transcription.
24. The method of claim 21, wherein the transcription is HIV
transcription.
25. The method of claim 21, wherein the antisense RNA consists of
antisense of nucleotides 221-241 or 95-114 of human 7SK RNA (SEQ ID
NO:1).
26. The method of claim 22, wherein the antisense RNA consists of
antisense of nucleotides 221-241 or 95-114 of human 7SK RNA (SEQ ID
NO:1).
27. The method of claim 23, wherein the antisense RNA consists of
antisense of nucleotides 221-241 or 95-114 of human 7SK RNA (SEQ ID
NO:1).
28. The method of claim 24, wherein the antisense RNA consists of
antisense of nucleotides 221-241 or 95-114 of human 7SK RNA (SEQ ID
NO:1).
29. The method of claim 21, wherein the antisense RNA consists of
antisense of nucleotides 95-114 of human 7SK RNA (SEQ ID NO:1).
30. The method of claim 22, wherein the antisense RNA consists of
antisense of nucleotides 95-114 of human 7SK RNA (SEQ ID NO:1).
31. The method of claim 23, wherein the antisense RNA consists of
antisense of nucleotides 95-114 of human 7SK RNA (SEQ ID NO:1).
32. The method of claim 24, wherein the antisense RNA consists of
antisense of nucleotides 95-114 of human 7SK RNA (SEQ ID NO:1).
Description
[0001] This application is a continuation of Ser. No. 10/227,367
filed Aug. 25, 2002, now U.S. Pat. No. 0,000,000.
INTRODUCTION
[0003] 1. Field of the Invention
[0004] The field of the invention is 7SK small nuclear RNA
regulated gene transcription.
[0005] 2. Background of the Invention
[0006] The human positive transcription elongation factor P-TEFb,
consisting of a CDK9/cyclin T1 heterodimer, functions as both a
general and an HIV-1 Tat-specific transcription factor (1,2).
P-TEFb activates transcription by phosphorylating RNA polymerase
(Pol) II, leading to the formation of processive elongation
complexes. As a Tat cofactor, P-TEFb stimulates HIV-1 transcription
by interacting with Tat and the transactivating responsive (TAR)
RNA structure located at the 5' end of the nascent viral transcript
(3). We identified 7SK (SEQ ID NO:1), an abundant and
evolutionarily conserved small nuclear RNA (snRNA) of previously
unknown function (4,5), as a specific P-TEFb-associated factor. 7SK
inhibits general and HIV-1 Tat-specific transcriptional activities
of P-TEFb in vivo and in vitro by inhibiting the kinase activity of
CDK9 and preventing recruitment of P-TEFb to the HIV-1 promoter
(Yang et al, Nature 414, 317-322, 2001; see also, Nguyen et al.,
Nature 414, 322-325, 2001; Blencowe, Current Biol 12, R147-9,
2002). 7SK is efficiently dissociated from P-TEFb by treatment of
cells with ultraviolet irradiation and actinomycin D. As these two
agents have been shown to significantly enhance HIV-1 transcription
and phosphorylation of Pol II (6,7,8), our data provide a
mechanistic explanation for their stimulatory effects. The
disclosed inventions exploit our finding that the 7SK/P-TEFb
interaction serves as a principal control point for the induction
of cellular and HIV-1 viral gene expression, particularly during
stress-related responses.
SUMMARY OF THE INVENTION
[0007] The invention provides methods and compositions for
regulating transcription. The inventors have found that they can
modulate transcription by modulating sequestration of P-TEFb (a
CDK9/cyclin complex) by RNA. Hence, the invention provides methods
for altering transcription in a cell comprising an amount of active
CDK9/cyclin, comprising the steps of: (a) introducing in the cell
an agent which modulates the amount of active CDK9/cyclin in the
cell, and thereby alters transcription in the cell, wherein the
agent comprises an RNA selected from the group consisting of an RNA
aptamer that specifically binds CDK9/cyclin, a CDK9/cyclin-binding
domain of 7SK RNA, a 7SK RNA-binding antisense 7SK RNA domain, a
7SK RNA-specific ribozyme, and a 7SK RNA-specific RNAi, and (b)
detecting a resultant altering of transcription in the cell.
[0008] Depending on the selected agent, the method can decrease the
amount of active CDK9/cyclin in the cell and thereby reduce said
transcription, or increase the amount of active CDK9/cyclin in the
cell and thereby increase said transcription. For example, in
particular embodiments, (a) the RNA is an RNA aptamer that
specifically binds CDK9/cyclin, decreases the amount of active
CDK9/cyclin in the cell, and thereby reduces said transcription;
(b) the RNA comprises a CDK9/cyclin-binding domain of 7SK RNA,
decreases the amount of active CDK9/cyclin in the cell, and thereby
reduces said transcription; (c) the RNA comprises a 7SK RNA-binding
antisense 7SK RNA domain, increases the amount of active
CDK9/cyclin in the cell, and thereby increases said transcription;
(d) the RNA comprises a 7SK RNA-specific ribozyme, increases the
amount of active CDK9/cyclin in the cell, and thereby increases
said transcription; or (e) the RNA comprises a 7SK RNA-specific
RNAi, increases the amount of active CDK9/cyclin in the cell, and
thereby increases said transcription.
[0009] A wide variety of transcriptions may be targeted; in
particular embodiments, the transcription is (a) a recombinant
protein transcription; (b) an LTR promoter-controlled
transcription; or (c) HIV transcription.
[0010] The invention also provides methods for screening for an
agent which modulates 7SK RNA-CDK9/cyclin binding. In general,
these methods comprise the steps of (a) incubating a mixture of 7SK
RNA, CDK9/cyclin and a candidate agent under conditions wherein but
for the presence of the agent, the 7SK RNA and CDK9/cyclin engage
in a reference binding; and (b) detecting an agent-biased binding
of the 7SK RNA to the CDK9/cyclin, wherein a difference between the
reference binding and the agent-biased binding indicates that the
agent modulates 7SK RNA-CDK9/cyclin binding.
[0011] Essentially any candidate agent or library of agents may be
screened; in particular embodiments, the agent comprises (a) an RNA
aptamer that specifically binds CDK9/cyclin; (b) a
CDK9/cyclin-binding domain of 7SK RNA; (c) a 7SK RNA-binding
antisense 7SK RNA domain; (d) a 7SK RNA-specific ribozyme; or (e) a
7SK RNA-specific RNAi. In addition, a wide variety of formats may
be used; in particular embodiments, the binding is detected (a)
directly in a coprecipitation binding assay; (b) directly in a
solid-phase binding assay; (c) indirectly in a transcriptional
readout assay; or (d) indirectly in a viral replication assay.
Furthermore, a wide variety of mixtures may be used, depending on
the selected assay format; for example, the mixture may be a
cell-free lysate or solution, or a cell in vitro or in situ.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
[0012] Our general method for altering transcription in a cell
comprising an amount of transcriptionally active CDK9/cyclin,
comprises introducing in the cell an agent which modulates the
amount of active CDK9/cyclin in the cell by modulating the amount
of CDK9/cyclin bound and sequestered by 7SK RNA, and thereby alters
CDK9/cyclin-dependent transcription in the cell. Active CDK9/cyclin
is unsequestered by 7SK RNA and provides demonstrable kinase and
transcriptional activity, as shown below. Accordingly, subject
agents simulate, promote or inhibit 7SK RNA binding to CDK9/cyclin.
Agents include regulators of endogenous 7SK RNA expression,
exogenous 7SK RNA molecules, agonistic (e.g. simulatory) or
antagonistic (e.g. dominant negative) domains thereof, 7SK
RNA-specific ribozymes, RNAi's and antisense RNAs, and agents
identified or characterized in the subject 7SK RNA-CDK9/cyclin
binding assays. In particular embodiments, the agent comprises, and
preferably consists or consists essentially of, an RNA selected
from the group consisting of an RNA aptamer that specifically binds
CDK9/cyclin, a CDK9/cyclin-binding domain of 7SK RNA, a 7SK
RNA-binding antisense 7SK RNA domain, a 7SK RNA-specific ribozyme,
and a 7SK RNA-specific RNAi.
[0013] Depending on the selected agent, the method can decrease the
amount of active CDK9/cyclin in the cell and thereby reduce said
transcription, or increase the amount of active CDK9/cyclin in the
cell and thereby increase said transcription. For example, domains
and derivatives of 7SK RNA, including SELEX-derived aptamers, can
bind CDK9/cyclin as an agonist and decrease the amount of active
CDK9/cyclin in the cell, or as an antagonist, interfering with
endogenous 7SK RNA binding while not interfering with
CDK9/cyclin-dependent transcription.
[0014] Hence, in particular embodiments, the selected agent
promotes CDK9/cyclin sequestration by promoting or stabilizing
binding to endogenous 7SK RNA, or providing additional 7SK RNA or
domains or derivatives thereof which bind CDK9/cyclin and thereby
inhibit CDK9/cyclin-dependent transcription. For example, we
identified a variety of distinct binding promoters and stabilizers
in our 7SK RNA-CDK9/cyclin binding assays (below). Similarly, by
deletion and mutation analysis, we identified and characterized a
number of CDK9/cyclin binding domains of RNAs, including domain
mutants, including recombined 7SK RNA domains, sufficient to bind
and thereby inhibit CDK9/cyclin-dependent transcription. Table 1
provides a number of exemplary 7SK RNA domains sufficient to bind
CDK9/cyclin and thereby inhibit CDK9/cyclin-dependent
transcription. 7SKD1 for example, is an internal deletion, wherein
nucleotides 161-300 of native 7SK RNA (SEQ ID NO:1) are deleted. By
convention, RNA sequences are presented herein by their
corresponding DNA sequences. TABLE-US-00001 TABLE 1 Exemplary 7SK
RNA domains sufficient to bind CDK9/cyclin and thereby inhibit
CDK9/cyclin-dependent transcription. CDK9/cyclin Transcription RNA
Structure binding Inhibition 7SKD1 SEQ ID NO: 2 +++ +++ 7SKD2 SEQ
ID NO: 3 +++ +++ 7SKD3 SEQ ID NO: 4 +++ +++ 7SKD4 SEQ ID NO: 5 +++
+++ 7SKM2 SEQ ID NO: 6 +++ +++ 7SKM3 SEQ ID NO: 7 +++ +++ 7SKM2 SEQ
ID NO: 8 +++ +++ 7SKM3 SEQ ID NO: 9 +++ +++
[0015] We selected several CDK9/cyclin-binding domains of 7SK RNA
for SELEX (systematic evolution of ligands by exponential
enrichment; Turek et al. Science 249, 505-10, 1990; Martell et al.,
Mol Ther. July 2002; 6(1):30-4) to generate a panel of
CDK9/cyclin-specific RNA aptamers. Our exemplary protocol for
preparing SELEX-derived RNA aptamers specific for 7SK RNA is
similar to that described in Joshi et al. J Virol July 2002;
76(13):6545-57. Briefly, to assay HIV-1 transcriptional inhibition,
aptamers are expressed with flanking, self-cleaving ribozymes to
generate aptamer RNA transcripts with minimal flanking sequences.
From these we select aptamers based on binding constants (K(d)) and
the degree of inhibition of HIV transcription in vitro (50%
inhibitory concentration [IC(50)]). These aptamers are each stably
expressed in 293T cells followed by transfection of a molecular
clone of HIV. Selected aptamers demonstrate consistently reduced
viral transcription and particle production.
[0016] Subsequently, we designed luciferase transcriptional
reporter assays to identify a panel of transcriptional regulators
of 7SK RNA expression. In our assay, 100 ng 7SK RNA promoter (e.g.
U.S. Pat. No. 5,624,803; Boyd et al., Mol Biol. Nov. 10,
1995;253(5):677-90; Boyd et al., Gene Apr. 18,
2000;247(1-2):33-44)-luciferase reporter constructs are transfected
into HeLa cells pre-seeded at 2.times.105 cells per well in a
6-well dish. After 44 hr incubation, candidate transcriptional
regulators are provided to discrete cultures in logarithmic dosages
at hourly time points. After 48 hr total incubation, cell lysates
are analysed for luciferase activity. Trans-acting candidates are
subject to CDK9/cyclin binding and HIV-1 transcription assays
(supra). Table 2 shows the effect of exemplary transcriptional
regulators on 7SK RNA gene expression, CDK9/cyclin-specific binding
and specific inhibition of CDK9/cyclin-dependent transcription.
[0017] Table 2. The effect of exemplary transcriptional regulators
on 7SK RNA gene expression, CDK9/cyclin-specific binding and
specific inhibition of CDK9/cyclin-dependent transcription ---, and
+++ indicate significant decreases and increases, respectively over
three experiments. TABLE-US-00002 Transcriptional Luciferase
CDK9/cyclin Transcription Regulator Expression binding Inhibition
ADH368 -45% --- --- ADH042 -83% --- --- ADH947 -27% --- --- ADH506
+59% +++ +++ ADH487 +662% +++ +++ ADH122 +134% +++ +++
[0018] We also demonstrated our method with 7SK RNA-binding
antisense 7SK RNA domains, with and without supplemental RNase H
cleavage. In our initial experiments, antisense and scrambled
oligonucleotides specifically targeting various 7SK RNA regions
were transfected into HeLa cells together with an HIV-1 LTR
luciferase construct. We found that antisense 7SK RNA domains could
significantly increase transcription, and that these increases
correlated with their ability to inhibit 7SK-CDK9/cyclin
binding.
[0019] Our 7SK RNA-specific RNAi and 7SK RNA-specific ribozyme
assays are constructed similarly. 7SK RNA-specific RNAi (Elbashir
et al., Methods February 2002; 26(2): 199-213; Hannon, 2002, Nature
418, 244-5 1), and trans-cleaving, 7SK RNA-specific ribozymes
(Lyngstadaas, 2001, Crit Rev Oral Biol Med 12(6):469-78; Doudna et
al., 2002 Nature 418, 222-228) specifically targeting various 7SK
RNA regions are transfected into HeLa cells together with an HIV-1
LTR luciferase construct. Results indicate that our 7SK
RNA-specific RNAi and 7SK RNA-specific ribozymes can significantly
increase transcription, and that these increases correlate with
their ability to inhibit 7SK-CDK9/cyclin binding. Similar results
are obtained using retroviral vectors to introduce expression
cassettes for 7SK RNA-specific ribozymes into CD4+ lymphocytes or
CD34+ haematopoeietic precursors ex vivo, isolated from infected
patients. To enhance therapeutic persistence, nuclease resistant
synthetic ribozymes may be used (e.g. Usman et al., J Clin Invest
106, 1197-1202, 2000).
[0020] A wide variety of transcriptions may be targeted by our
methods. For example, the method is particularly suited to
increasing target viral or recombinant protein transcription. While
lentivirus LTR-promoted transcription is particularly sensitive,
CDK9/cyclin is often transcriptionally limiting in mammalian cells.
Hence, the method may be used to increase the yield of proteins
recombinantly expressed in mammalian cells, particularly under
lentivirus LTR promoters. In a particular application, the methods
are used to therapeutically increase HIV transcription,
accelerating viremia and host immune responsiveness.
[0021] Alternatively, by using agents which promote CDK9/cyclin
sequestration by promoting or stabilizing binding to endogenous 7SK
RNA, or providing additional 7SK RNA or domains or derivatives
thereof which bind CDK9/cyclin and thereby inhibit
CDK9/cyclin-dependent transcription, the methods may be used to
decrease target viral or recombinant protein transcription. Targets
of transcriptional inhibition are generally pathogenic
transcriptions, including expression of pathogenic viral and host
genes.
[0022] In many applications, particularly for increasing expression
of recombinant proteins, the target cells are in vitro. This aspect
of the invention is useful for providing enhance expression of
virtually any recombinant protein, particularly commercial and
therapeutic proteins. For example, we modified a commercial
protocol (Cosgrove et al., Protein Expr Purif December 1995;
6(6):789-98) for large-scale production of insulin receptor by
incubating with a high-affinity CDK9/cyclin binding, SELEX-derived
7SK RNA aptamer. Our aptamer is further protected from RNase
degradation by condensation with the polycationic peptide
protamine, which also promotes intracellular delivery. Briefly,
ectodomain of the exon 11 + form of the human insulin receptor
(hIR) is expressed in the mammalian cell secretion vector
pEE6.HCMV-GS, containing the glutamine synthetase gene. Following
transfection of the hIR ectodomain gene into Chinese hamster ovary
(CHO-K1) cells, clones are isolated by selecting for glutamine
synthetase expression with methionine sulphoximine. The expression
levels of ectodomain are subsequently increased by gene
amplification. Production is scaled up using a 40-liter airlift
fermenter in which the transfected CHO-K1 cells were cultured on
microcarrier beads in the presence of our 7SK RNA aptamer,
initially in medium containing 10% fetal calf serum (FCS). By
continuous perfusion of serum-free medium supplemented with aptamer
into the bioreactor, cell viability is maintained during reduction
of FCS, which enabled soluble hIR ectodomain to be harvested for at
least 22 days. Our results demonstrate the successful production
and purification of hIR ectodomain by processes amenable to large
scale-up.
[0023] In other embodiments, the target cell is in situ,
particularly a cell of a patient determined to be in need of
specific transcriptional modulation, particularly one determined to
be subject to pathogenic transcription, particularly of protein
pathogenic or expressed at a pathogenic level in the host. The
patient is typically a human patient, but also includes animal,
particularly mammalian patients such as dogs and cats encountered
in veterinary applications, and rats and mice encountered in
biomedical research applications. Hence, the need for
transcriptional modulation will generally originate with the
patient, but may also be that imposed by the biomedical researcher.
In a particular embodiment, the patient is a human predetermined to
be in medical need of transcriptional modulation, more
particularly, a patient suffering from a lentivirus, particularly
an HIV infection.
[0024] Protocols for delivering the agent and effective dosages are
known in the art and/or readily determined empirically by those
skilled in the art guided by the selected agent and the present
disclosure. As noted and exemplified herein, a wide variety of
alternative agents may be employed, guided by efficacy,
physiological compatibility and convenience. For example, a variety
of applicable delivery protocols have been shown to effectively
deliver therapeutic RNA agents to mammalian cells and animals, see
Sullenger et al., 2002, Nature 418, 252-58, and references therein.
For example, expression cassette SELEX greatly facilitates the use
of aptamers for a variety of gene therapy applications. Martell et
al., Mol Ther. July 2002; 6(1):30-4; and for various protocols for
therapeutic aptamer delivery, see, White et al., 2000, J Clin
Invest 106, 929-34; Hicke et al., 2000, J Clin Invest 106,
923-28.
[0025] In a particular embodiment, retroviral vectors are used to
mediate transfer of 7SK RNA-specific ribozyme and a control
neo.sup.R gene into CD4.sup.+ T-cells selected from apheresis
samples of HIV-infected patients. In patients analyzed to date,
transduced cells of each population (ribozyme and control) were
found up to 10 months post-infusion. A related study using an
HIV-specific ribozyme in CD34+ cells found multilineage gene
presence after at 3 months (Amado et al., 6.sup.th Conf
Retroviruses Opportunistic Infections, Chicago, Ill., Abstr #17,
1999). Another study utilized an anti-HIV-1 tat and rev double
hammerhead in a similar CD34.sup.+ cell study. Long-term bone
marrow cultures of ribozyme transduced cells showed protection from
HIV challenge compared to control-transduced cells. In half of
patients observed for at least 12 months, vector sequences were
detectable by DNA PCR in PBMC and/or marrow at 6 months.
[0026] The agents are typically administered in the form of a
pharmaceutical composition comprising at least one recited agent
and a carrier, vehicle or excipient suitable for use in
pharmaceutical compositions. Without being limited thereto, such
materials include diluents, binders and adhesives, lubricants,
plasticizers, disintegrants, colorants, bulking substances,
flavorings, sweeteners and miscellaneous materials such as buffers
and adsorbents in order to prepare a particular medicated
composition. Such carriers are well known in the pharmaceutical art
as are procedures for preparing pharmaceutical compositions.
Depending on the intended route of delivery, the compositions may
be administered in one or more dosage form(s) including, without
limitation, liquid, solution, suspension, emulsion, tablet,
multi-layer tablet, bi-layer tablet, capsule, gelatin capsule,
caplet, lozenge, chewable lozenge, bead, powder, granules,
dispersible granules, cachets, douche, suppository, cream, topical,
inhalant, aerosol inhalant, patch, particle inhalant, implant,
depot implant, ingestible, injectable, or infusion. The dosage
forms may include a variety of other ingredients, including
binders, solvents, bulking agents, plasticizers etc. Preferred
agents are orally administrable to human patients, meaning they are
both safe and effective when orally administered. The above
described components for orally administrable or injectable
compositions are merely representative. Other materials as well as
processing techniques and the like are set forth in Part 8 of
Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack
Publishing Company, Easton, Pa., which is incorporated herein by
reference.
[0027] The dosage forms of the present invention involve the
administration of an active therapeutic substance or multiple
active therapeutic substances in a single dose during a 24 hour
period of time or multiple doses during a 24 hour period of time.
The doses may be uneven in that each dose is different from at
least one other dose. The subject compositions may be administered
to effect various forms of release, which include, without
limitation, immediate release, extended release, controlled
release, timed release, sustained release, delayed release, long
acting, pulsatile delivery, etc., using well known procedures and
techniques available to the ordinary skilled artisan. A description
of representative sustained release materials can be found in the
incorporated materials in Remington's Pharmaceutical Sciences.
[0028] Our methods generally also comprise the step of detecting or
confirming a resultant altering of transcription in the target
cell. Transcriptional modulation may be detected in any convenient
manner, including directly, such as by reporter expression, or
indirectly, such as by a target transcription dependent change in
host cell or animal physiology (e.g. viremia, pathology, or other
ultimate indication of transcriptional change).
[0029] The invention also provides methods for screening for an
agent which modulates 7SK RNA-CDK9/cyclin binding. In general,
these methods comprise the steps of (a) incubating a mixture of 7SK
RNA, CDK9/cyclin and a candidate agent under conditions wherein but
for the presence of the agent, the 7SK RNA and CDK9/cyclin engage
in a reference binding; and (b) detecting an agent-biased binding
of the 7SK RNA to the CDK9/cyclin, wherein a difference between the
reference binding and the agent-biased binding indicates that the
agent modulates 7SK RNA-CDK9/cyclin binding. Essentially any
candidate agent or library of agents may be screened in any of the
cell or animal systems described or cited herein, that provide a
measure of 7SK RNA-CDK9/cyclin binding, CDK9/cyclin kinase
activity, CDK9/cyclin-dependent transcription, etc. In a particular
embodiment, we used an in vitro, cell-based transcriptional
reporter assay to identify a variety of distinct 7SK
RNA-CDK9/cyclin binding promoters and stabilizers (Table 3).
TABLE-US-00003 TABLE 3 Exemplary 7SK RNA - CDK9/cyclin binding
promoters and stabilizers. Binding CDK9/cyclin Luciferase
Transcription promoter binding Expression Inhibition ALM726 +++
+107% +++ ALM488 +++ +372% +++ ALM045 +++ +94% +++ ALM362 +++ +405%
+++ ALM157 +++ +223% +++ ALM283 +++ 3.5 +++
EXEMPLARY EXPERIMENTAL PROTOCOLS
[0030] To identify nuclear factors that can interact with and
regulate the activity of P-TEFb, we affinity purified Flag-tagged
CDK9 and its associated factors from the nuclear extract of an
engineered HeLa cell line (F1C2 cells) that stably expressed
CDK9-Flag (9). Analysis of the purified material by silver staining
detected cyclin T1 and a novel band with a relative molecular mass
of 110,000 (Mr 110K) derived from F1C2 but not the parental HeLa
cells. Coomassie blue could not stain the 110K band, and the
yellowish, silver-stained color was different from that of the
brown color typical of proteins, indicating that the band may not
be a protein. Indeed, treatment of the affinity-purified CDK9-Flag
preparation with RNase A, but not DNase I, eliminated the 110K
band, indicating that it may contain a CDK9-associated RNA
molecule.
[0031] RNA was extracted from the CDK9-Flag preparation and
analysed on a denaturing gel with small RNAs recovered from HeLa
nuclear extract as markers. The identities of some of these HeLa
RNAs were pre-determined by oligonucleotide-directed RNase H
digestion (10). The CDK9-Flag preparation contained a single RNA
species that co-migrated with the 7SK RNA, comprising 330
nucleotides, in HeLa extract. On transcription by RNA Pol III, the
mammalian 7SK RNA is an abundant (approximately 2.times.105 per
cell) and evolutionarily conserved snRNA of unknown function
(4,11). Using full-length 7SK antisense RNA as a probe, northern
hybridization was performed to confirm that the CDK9-associated
110K RNA was 7SK. Sequencing of the complementary DNA copy of this
RNA revealed a complete match with the published 7SK sequences
(12).
[0032] To investigate the role of 7SK in transcription, it was
quantitatively removed from HeLa nuclear extract using an
immobilized 2'-O-methyl (2'-OMe) RNA oligonucleotide complementary
to an exposed region in 7SK (residues 221-241) (4). Notably, the
7SK small nuclear ribonucloprotein particle (snRNP) associated with
the 2'-OMe RNA beads contained both cyclin T1 and CDK9, indicating
an association of 7SK with the CDK9/cyclin T1 heterodimer in HeLa
nuclear extract. Approximately twice the amount of cyclin T1 and
CDK9 was detected in the mock-depleted extract than in the extract
that was depleted of 7SK, indicating that about 50% of P-TEFb may
be stably associated with 7SK.
[0033] We next compared the abilities of the mock- and 2'-OMe
RNA-depleted HeLa nuclear extracts to transcribe templates
pSV40EP-G400 and pHIVTAR-G100 (13) in the same reaction. Removal of
7SK and its associated P-TEFb had no effect on transcription
proximal to the promoter of the 400-nucleotide G-less cassette
(G400), which was driven by the SV40 early promoter, or on
transcription distal to the promoter of a G100 cassette, driven by
the HIV-1 promoter; furthermore, there was no effect on Tat
activation of HIV-1 transcription. Thus, the P-TEFb bound to 7SK
did not contribute to the transcriptional activity of HeLa nuclear
extract.
[0034] If the P-TEFb bound to 7SK is inactive in transcription, we
asked whether this might be due to 7SK having an inhibitory effect
on the function of P-TEFb. To disrupt 7SK, oligonucleotide-directed
RNase H digestion (10) of 7SK was performed in the nuclear extract
of F1C2 cells. Treatment with 221-241A-an antisense
deoxyoligonucleotide targeted against 7SK-but not a control
oligonucleotide, caused cleavage of full-length 7SK (330
nucleotides) into two fragments of approximately 220 and 90
nucleotides, respectively. The integrity of the targeted region
(nucleotides 221-241) appeared to be critical for the binding of
7SK to P-TEFb, as very little of the cleaved 7SK fragments were
associated with the affinity-purified CDK9-Flag/cyclin T1. Thus,
treatment with 221-241A effectively created more P-TEFb -that was
not bound to 7SK (free P-TEFb), in the extract. Compared with both
untreated and control oligonucleotide-treated extracts, the extract
treated with 221-241A consistently yielded 2-3-fold more basal and
Tat-activated HIV-1 transcription from templates pHIV+TAR-G400
(which contained the wild-type TAR element) and pHIVTAR-G100 (with
a mutant TAR) (13). Given that only about 50% of P-TEFb associated
with 7SK, the 2-3-fold increase in transcription was significant,
and it indicated that 7SK was suppressing the transcriptional
activity of P-TEFb in vitro.
[0035] When 221-241A or a scrambled oligonucleotide, 221-241S, was
cotransfected with CDK9-Flag into HeLa cells, only 221-241A
significantly reduced the binding of 7SK to CDK9-Flag, effectively
creating more free P-TEFb in the cell. To determine whether 7SK
also suppresses P-TEFb activity in vivo, the effect of 221-241A on
the abilities of various promoters to transcribe a luciferase
reporter gene was examined. Transfection of 221-241A, but not
221-241 S, into HeLa cells increased transcription from all
promoters tested, with the largest increase (roughly 9.5-fold)
displayed by the HIV-1 long terminal repeat (LTR). Smaller
increases were displayed by the SV40 early promoter, and the
(Gal4)5-thymidine kinase promoter, the transforming growth factor
responsive promoter p3TP. Similar results were also obtained in
several other cell lines of diverse origins. Finally, transfection
of 221-241A into HeLa cells increased both basal and Tat-activated
HIV-1 transcription. These experiments and the above in vitro
transcriptional analyses reveal a general inhibitory effect of 7SK
on P-TEFb transcriptional activity. Notably, HIV-1 LTR seems to be
most sensitive to this inhibition, which is reasonable because it
is regulated mainly at the stage of elongation and requires P-TEFb
for both basal and Tat-activated transcription (1,2).
[0036] In addition to the region targeted by 221-241A, two other
regions of 7SK (residues 11-31 and 95-114) are also potentially
accessible to oligonucleotide-directed RNase H cleavage (4,14).
Antisense and scrambled deoxyoligonucleotides specifically
targeting these two regions were therefore transfected into HeLa
cells together with an HIV-1 LTR luciferase construct. Compared
with 221-241A, which increased significantly HIV transcription, a
smaller increase was observed with the antisense oligonucleotide
95-114A, and no increase with oligonucleotide 11-31A was observed.
The abilities of the three antisense oligonucleotides to increase
transcription correlated exactly with their abilities to induce 7SK
cleavage and to disrupt the interaction between 7SK and P-TEFb in
the nuclear extract, further documenting the inhibitory effect of
7SK on the transcriptional activity of P-TEFb.
[0037] 7SK could inhibit the activity of P-TEFb by suppressing the
kinase activity of CDK9/cyclin T1. Affinity-purified CDK9-Flag and
its associated factors were divided into two halves, incubated
respectively with RNase A and DNase I, and tested in kinase
reactions containing purified RNA Pol II as a substrate (9). RNase
A degraded the CDK9-Flag-associated 7SK, and increased the kinase
activity of CDK9-Flag by 3-4-fold, as seen by its increased
autophosphorylation and phosphorylation of Pol II. This increase
was significant given that only about 50% of the purified
CDK9-Flag/cyclin Ti was associated with 7SK. In addition to RNase
A, RNase H cleavage of 7SK directed by 221-241A also increased the
kinase activity of CDK9. To analyse more specifically the activity
of P-TEFb bound to 7SK, this complex was affinity purified from
HeLa nuclear extract using the 7SK antisense 2'-OMe RNA beads.
Eluted with a displacement deoxyoligonucleotide (15), the P-TEFb
bound to 7SK was divided into three equal portions, incubated
respectively with RNase A, DNase I or buffer alone, and analysed in
kinase reactions. Once again, degradation of 7SK by RNase A
significantly increased the kinase activity of CDK9, revealing the
inhibitory action of 7SK on CDK9/cyclin T1 kinase.
[0038] P-TEFb can be recruited to the pre-initiation complex (PIC)
at the HIV-1 promoter and then travel with the elongating Pol II
(16,17). The mechanism of recruitment is unclear although the
interaction of cyclin T 1 with the hypophosphorylated Pol II (18)
could be responsible. To study the effect of 7SK on the association
of P-TEFb with PIC, an immobilized HIV-1 promoter was incubated
with F1C2 nuclear extract to isolate the promoter-bound P-TEFb
(16,17). Northern blotting was performed to compare the level of
7SK associated with the promoter-bound P-TEFb with that in the
total P-TEFb affinity purified from the nuclear extract. When
normalized by their cyclin T1 and CDK9 levels, the promoter-bound
P-TEFb showed no 7SK, whereas abundant 7SK existed in the total
P-TEFb preparation, indicating that 7SK prevented the binding of
P-TEFb to the HIV-1 promoter in vitro. To verify this in vivo, a
chromatin immunoprecipitation (CHIP) (19) assay was performed to
examine the interaction of P-TEFb with an integrated HIV-1 promoter
in HeLa cells. Cells were cotransfected with CDK9-Flag and either
221-241A or 221-241S. As shown above, transfected 221-241A
disrupted the 7SK/P-TEFb interaction and increased HIV-1
transcription. Notably, it also increased the association of
CDK9-Flag with the HIV-1 promoter in these cells. Thus, the
7SK-P/TEFb interaction not only inhibited the kinase activity of
P-TEFb, but also blocked the recruitment of P-TEFb to the HIV-1
promoter.
[0039] Certain agents that elicit SOS-like stress responses in
mammalian cells can markedly enhance HIV-1 transcription in a
manner analogous to prophage induction in Escherichia coli
(6-8,20,21). For instance, treatment of HeLa cells with ultraviolet
irradiation or low levels of the global transcription inhibitor
actinomycin D enhances HIV-1 transcription to levels similar to
those obtained by Tat (6,8). Notably, these stimulatory effects
seemed to be caused by an enhanced phosphorylation of Pol II,
probably by the CDK9/cyclin T1 kinase (6). In light of these
observations, we investigated the effect of ultraviolet irradiation
and actinomycin D on the 7SK/P-TEFb interaction in nuclear extracts
of the treated F1 C2 cells. Consistent with their enhancement of
HIV-1 transcription and Pol II phosphorylation, both agents
significantly reduced the amount of 7SK associated with the
affinity-purified CDK9-Flag. Neither the level of total 7SK in the
nuclear extract nor the CDK9/cyclin Ti interaction was affected.
Actinomycin D is known to intercalate into duplex DNA and perhaps
also RNA; however, direct incubation of this drug with nuclear
extract did not disrupt the 7SK/P-TEFb interaction, ruling out a
direct effect. As for ultraviolet irradiation, 7SK was dissociated
from P-TEFb as early as 15-30 min after treatment, long before any
signs of apoptosis appeared. Thus, stress signals such as
ultraviolet irradiation and actinomycin D can cause fast and
efficient 7SK dissociation from P-TEFb, which explains their
positive effects on HIV-1 transcription and Pol II
phosphorylation.
[0040] Constructs and stable CDK9-Flag-expressing cell line. In
vitro transcription template pSV40EP-G400 was generated by cloning
a 400-nucleotide G-less cassette (G400)(13) into the HindIII and
EcoRV sites downstream of the SV40 early promoter in pGL-2
(Promega). We performed in in vitro transcription assay as
described (13). To generate the F1C2 cell line expressing
CDK9-Flag, HeLa cells were stably transfected with
pBabe-puro-CDK9-Flag, which expresses Flag-tagged CDK9 and confers
puromycin resistance. We selected clone F1C2 because CDK9-Flag is
expressed at a similar level as the endogenous CDK9. CDK9-Flag and
its associated factors were affinity purified from F1C2 nuclear
extract using anti-Flag agarose beads (Sigma). After extensive
washes with buffer D (20 mM HEPES, pH 7.9, 15% glycerol, 0.2 mM
EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethyl sulphonyl fluoride)
containing 0.4 M KCl and 0.2% NP40, the materials were eluted by
Flag peptide as described (9).
[0041] Transfection of cells with deoxyoligonucleotides. For
luciferase assays, HeLa cells were seeded at 2.times.105 cells per
well in a 6-well dish one day before transfection. Using the
Lipofectamine-Plus Kit (Invitrogen), cells were cotransfected with
100 ng of the indicated luciferase reporter constructs, 2 ug
high-performance liquid chromatography-pure deoxyoligonucleotides,
and 20 ng Tat-expressing construct when indicated. Cell lysates
were analysed for luciferase activity at 48 h after transfection.
For chromatin immunoprecipitation assays (CHIP), a total of
6.times.106 HeLa cells containing an integrated HIV-l LTR was
seeded one day before transfection into five 10-cm dishes. Cells
were cotransfected with 12 ug per dish of the indicated
oligonucleotides and 4 ug per dish of a construct expressing
CDK9-Flag. At 36 h after transfection, cells were processed for
crosslinking and CHIP with anti-Flag agarose beads as described
(19). The HIV-1 promoter region between -168 and +82 was amplified
by polymerase chain reaction from the precipitated chromatin.
[0042] Depletion, purification and cleavage of 7SK. Depletion of
the 7SK RNP from HeLa nuclear extract was performed as described
(4) with some modifications. Briefly, 300 ul HeLa extract in buffer
D plus 0.1 M KCl, 0.05% NP40 and 0.2 U ul-l RNasin was incubated at
30.degree. C. for 30 min with 1.8 uM of the biotinylated antisense
2'-O-methyl RNA oligonucleotide that is complementary to a region
in 7SK from nucleotide 221 to 241. The reaction mixture was then
incubated for 1 h at 4.degree. C. with streptavidin agarose beads
(Sigma). After repeating the procedure twice, the beads were washed
with buffer D containing 0.4 M KCl and 0.2% NP40, and the
associated 7SK RNP was analysed. To elute 7SK RNP from the beads,
we used a 2.5-fold excess displacement deoxyoligonucleotide in
buffer D (0.1 M KCl). We performed oligonucleotide-directed RNase H
cleavage of 7SK as described (10).
REFERENCES
[0043] 1. Jones, K. A. Taking a new TAK on Tat transactivation.
Genes Dev. 11, 2593-2599 (1997). [0044] 2. Price, D. H. P-TEFb, a
cyclin-dependent kinase controlling elongation by RNA polymerase
II. Mol. Cell. Biol. 20, 2629-2634 (2000). [0045] 3. Wei, P.,
Garber, M. E., Fang, S. M., Fischer, W. H. & Jones, K. A. A
novel CDK9-associated C-type cyclin interacts directly with HIV-1
Tat and mediates its high-affinity, loop-specific binding to TAR
RNA. Cell 92, 451-462 (1998). [0046] 4. Wassarman, D. A. &
Steitz, J. A. Structural analyses of the 7SK ribonucleoprotein
(RNP), the most abundant human small RNP of unknown function. Mol.
Cell. Biol. 11, 3432-3445 (1991). [0047] 5. Zieve, G. & Penman,
S. Small RNA species of the HeLa cell: metabolism and subcellular
localization. Cell 8, 19-31 (1976). [0048] 6. Cass.THETA., C.,
Giannoni, F., Nguyen, V. T., Dubois, M. F. & Bensaude, 0. The
transcriptional inhibitors, actinomycin D and -amanitin, activate
the HIV-1 promoter and favor phosphorylation of the RNA polymerase
II C-terminal domain. J. Biol. Chem. 274, 16097-16106 (1999).
[0049] 7. Kumar, S. et al. Activation of the HIV-1 long terminal
repeat by cytokines and environmental stress requires an active
CSBP/p38 MAP kinase. J. Biol. Chem. 271, 30864-30869 (1996). [0050]
8. Valerie, K. et al. Activation of human immunodeficiency virus
type 1 by DNA damage in human cells. Nature 333, 78-81 (1988).
[0051] 9. Zhou, Q., Chen, D., Pierstorff, E. & Luo, K.
Transcription elongation factor P-TEFb mediates Tat activation of
HIV-1 transcription at multiple stages. EMBO J. 17, 3681-3691
(1998). [0052] 10. Black, D. L., Chabot, B. & Steitz, J. A. U2
as well as U1 small nuclear ribonucleoproteins are involved in
premessenger RNA splicing. Cell 42, 737-750 (1985). [0053] 11.
Zieve, G., Benecke, B. J. & Penman, S. Synthesis of two classes
of small RNA species in vivo and in vitro. Biochemistry 16,
4520-4525 (1977). [0054] 12. Murphy, S. et al. DNA sequences
complementary to human 7 SK RNA show structural similarities to the
short mobile elements of the mammalian genome. J. Mol. Biol. 177,
575-590 (1984). [0055] 13. Zhou, Q. & Sharp, P. A. Novel
mechanism and factor for regulation by HIV-1 Tat. EMBO J. 14,
321-328 (1995). [0056] 14. Luo, Y., Kurz, J., MacAfee, N. &
Krause, M. 0. C-myc deregulation during transformation induction:
involvement of 7SK RNA. J. Cell. Biochem. 64, 313-327 (1997).
[0057] 15. Schnapp, G., Rodi, H. P., Rettig, W. J., Schnapp, A.
& Damm, K. One-step affinity purification protocol for human
telomerase. Nucleic Acids Res. 26, 3311-3313 (1998). [0058] 16.
Ping, Y. H. & Rana, T. M. Tat-associated kinase (P-TEFb): a
component of transcription preinitiation and elongation complexes.
J. Biol. Chem. 274, 7399-7404 (1999). [0059] 17. Zhou, M. et al.
Tat modifies the activity of CDK9 to phosphorylate serine 5 of the
RNA polymerase II carboxyl-terminal domain during human
immunodeficiency virus type 1 transcription. Mol. Cell. Biol. 20,
5077-5086 (2000). [0060] 18. Fong, Y. W. & Zhou, Q. Relief of
two built-in autoinhibitory mechanisms in P-TEFb is required for
assembly of a multicomponent transcription elongation complex at
the human immunodeficiency virus type 1 promoter. Mol. Cell. Biol.
20, 5897-5907 (2000). [0061] 19. Boyd, K. E., Wells, J., Gutman,
J., Bartley, S. M. & Farnham, P. J. c-Myc target gene
specificity is determined by a post-DNA binding mechanism. Proc.
Natl Acad. Sci. USA 95, 13887-13892 (1998). [0062] 20. Kretz-Remy,
C. & Arrigo, A. P. The kinetics of HIV-1 long terminal repeat
transcriptional activation resemble those of hsp70 promoter in
heat-shock treated HeLa cells. FEBS Lett. 353, 339-344 (1994).
[0063] 21. Vlach, J. et al. Induction of Sp 1 phosphorylation and
NF-B-independent HIV promoter domain activity in T lymphocytes
stimulated by okadaic acid. Virology 208, 753-761 (1995).
[0064] The foregoing descriptions of particular embodiments and
examples are offered by way of illustration and not by way of
limitation. Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be readily apparent to those of
ordinary skill in the art in light of the teachings of this
invention that certain changes and modifications may be made
thereto without departing from the spirit or scope of the appended
claims.
[0065] All publications and patent applications cited in this
specification and all references cited therein are herein
incorporated by reference as if each individual publication or
patent application or reference were specifically and individually
indicated to be incorporated by reference. Any material
accompanying this application on compact disc or other recorded
medium is incorporated by reference.
Sequence CWU 1
1
9 1 330 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Sequence 1 ggatgtgagg gcgatctggc tgcgacatct gtcaccccat
tgatcgccag ggttgattcg 60 gctgatctgg ctggctaggc gggtgtcccc
ttcctccctc accgctccat gtgcgtccct 120 cccgaagctg cgcgctcggt
cgaagaggac gaccatcccc gatagaggag gaccggtctt 180 cggtcaaggg
tatacgagta gctgcgctcc cctgctagaa cctccaaaca agctctcaag 240
gtccatttgt aggagaacgt agggtagtca agcttccaag actccagaca catccaaatg
300 aggcgctgca tgtggcagtc tgcctttctt 330 2 191 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Sequence 2
ggatgtgagg gcgatctggc tgcgacatct gtcaccccat tgatcgccag ggttgattcg
60 gctgatctgg ctggctaggc gggtgtcccc ttcctccctc accgctccat
gtgcgtccct 120 cccgaagctg cgcgctcggt cgaagaggac gaccatcccc
gaggcgctgc atgtggcagt 180 ctgcctttct t 191 3 191 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Sequence 3
ggaagtgagg gcgatctggc tgcgacatct gtcaccccat tgatcgccag ggttgattcg
60 gctgatctgg ctggctaggc gggtgtcccc ttcctccctc accgctccat
gtgcgtccct 120 cccgaagctg cgcgctcggt cgaagaggac gaccatcccc
gaggcgttgc atgtggcagt 180 cttccttttt t 191 4 171 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Sequence 4
ggatgtgagg gcgatctggc tgcgacatct gtcaccccat tgatcgccag ggttgattcg
60 gctgatctgg ctggctaggc gggtgtcccc ttcctccctc accgctccat
gtgcgtccct 120 cccgaagctg cgcgctcggt cgaagaggac gaccatcccc
gaggcgttct t 171 5 161 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Sequence 5 gcgatctggc tgcgacatct
gtcaccccat tgatcgccag ggttgattcg gctgatctgg 60 ctggctaggc
gggtgtcccc ttcctccctc accgctccat gtgcgtccct cccgaagctg 120
cgcgctcggt cgaagaggac gaccatcccc gaggcgttct t 161 6 181 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Sequence 6 ggatgtgagg gcgatctggc tgcgacatct gtcaccccat tgatcgccag
ggttgattcg 60 gctgatctgg ctggctaggc gggtgtcccc ttcctccctc
accgctccat gtgcgtccct 120 cccgaagctg cgcgctcggt cgaagaggac
gaccatcccc gaggcgctgc atgcctttct 180 t 181 7 179 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Sequence 7
gagggcgatc tggctgcgac atctgtcacc ccattgatcg ccagggttga ttcggctgat
60 ctggctggct aggcgggtgt ccccttcctc cctcaccgct ccatgtgcgt
ccctcccgaa 120 gctgcgcgct cggtcgaaga ggacgaccat ccccttgcat
gtggcagtct ttttttttt 179 8 167 DNA Artificial Sequence Description
of Artificial Sequence Synthetic Sequence 8 gagggcgatc tggctgcgac
atctgtcacc ccattgatcg ccagggttga ttcggctgat 60 ctggctggct
aggcgggtgt ccccttcctc cctcaccgct ccatgtgcgt ccctcccgaa 120
gctgcgcgct cggtcgaaga ggacgaccat ccccgttttt ttttttt 167 9 148 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Sequence 9 gatctggctg cgacatctgt caccccattg atcgccaggg ttgattcggc
tgatctggct 60 ggctaggcgg gtgtcccctt cctccctcac cgctccatgt
gcgtgaagct gcgcgctcgg 120 tcgaagagga cgaccatccc cgtttttt 148
* * * * *