U.S. patent application number 10/350798 was filed with the patent office on 2004-05-06 for methods and compositions for rna interference.
This patent application is currently assigned to Genetica, Inc.. Invention is credited to Beach, David H., Bernstein, Emily, Caudy, Amy, Conklin, Douglas, Hammond, Scott, Hannon, Gregory J., Paddison, Patrick J..
Application Number | 20040086884 10/350798 |
Document ID | / |
Family ID | 26885458 |
Filed Date | 2004-05-06 |
United States Patent
Application |
20040086884 |
Kind Code |
A1 |
Beach, David H. ; et
al. |
May 6, 2004 |
Methods and compositions for RNA interference
Abstract
The present invention provides methods for attenuating gene
expression in a cell using gene-targeted double stranded RNA
(dsRNA). The dsRNA contains a nucleotide sequence that hybridizes
under physiologic conditions of the cell to the nucleotide sequence
of at least a portion of the gene to be inhibited (the "target"
gene).
Inventors: |
Beach, David H.; (Boston,
MA) ; Bernstein, Emily; (Huntington, NY) ;
Caudy, Amy; (St. Louis, NY) ; Hammond, Scott;
(Pittsboro, NC) ; Hannon, Gregory J.; (Huntington,
NY) ; Paddison, Patrick J.; (Cold Spring Harbor,
NY) ; Conklin, Douglas; (Centerport, NY) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Genetica, Inc.
Cambridge
MA
Cold Spring Harbor Laboratory
Cold Spring Harbor
NY
|
Family ID: |
26885458 |
Appl. No.: |
10/350798 |
Filed: |
January 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10350798 |
Jan 24, 2003 |
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10055797 |
Jan 22, 2002 |
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10055797 |
Jan 22, 2002 |
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PCT/US01/08435 |
Mar 16, 2001 |
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10350798 |
Jan 24, 2003 |
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09866557 |
May 24, 2001 |
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10350798 |
Jan 24, 2003 |
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09858862 |
May 16, 2001 |
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60243097 |
Oct 24, 2000 |
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60189739 |
Mar 16, 2000 |
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Current U.S.
Class: |
435/6.14 ;
435/455 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 2320/12 20130101; C12N 15/102 20130101; C12N 2310/53 20130101;
C12N 15/1034 20130101; C12N 15/111 20130101; C12N 2310/14
20130101 |
Class at
Publication: |
435/006 ;
435/455 |
International
Class: |
C12Q 001/68; C12N
015/85 |
Goverment Interests
[0002] Work described herein was supported by National Institutes
of Health Grant R01-GM62534. The United States Government may have
certain rights in the invention.
Claims
We claim:
1. A method for attenuating expression of a target gene in host
cells, comprising introducing double stranded RNA (dsRNA) into the
host cells in an amount sufficient to attenuate expression of the
target gene, wherein the dsRNA comprises a nucleotide sequence that
hybridizes under stringent conditions to an untranslated sequence
of the target gene.
2. A method for attenuating expression of a target gene in host
cells, comprising introducing double stranded RNA (dsRNA) into the
host cells in an amount sufficient to attenuate expression of the
target gene, wherein the dsRNA comprises a nucleotide sequence that
hybridizes under stringent conditions to an intronic sequence of
the target gene.
3. A composition for attenuating expression of a target gene,
comprising a double stranded RNA (dsRNA) having a nucleotide
sequence that hybridizes under stringent conditions to an
untranslated sequence of the target gene.
4. A composition for attenuating expression of a target gene,
comprising an expression vector which, when expressed, produces a
double stranded RNA (dsRNA) having a nucleotide sequence that
hybridizes under stringent conditions to an untranslated sequence
of the target gene.
5. A composition for attenuating expression of a target gene,
comprising a double stranded RNA (dsRNA) having a nucleotide
sequence that hybridizes under stringent conditions to an intronic
sequence of the target gene.
6. A composition for attenuating expression of a target gene,
comprising an expression vector which, when expressed, produces a
double stranded RNA (dsRNA) having a nucleotide sequence that
hybridizes under stringent conditions to an intronic sequence of
the target gene.
7. The method of claim 1 or the composition of claim 3 or 4,
wherein the untranslated sequence of the target gene to which the
dsRNA hybridizes is selected from the group consisting of a
promoter sequence and an enhancer sequence.
8. The method of any of claims 1 and 2, wherein the host cells are
suspended in culture.
9. The method of any of claims 1 and 2, wherein the host cells are
in a whole animal.
10. The method of any of claims 1 and 2, wherein the target gene is
a genomically integrated gene of the host cells.
11. The method of any of claims 1 and 2, wherein the target gene is
a heterologous gene
12. The method of claim 11, wherein the target gene is a viral
gene.
13. The method of any of claims 1 and 2, wherein the host cells is
a primate cells.
14. The method of claim 13, wherein the host cells are human
cells.
15. The method of any of claims 1 and 2, or the composition of
claim 3 or 5, wherein the dsRNA is short interfering double
stranded RNA which does not produce a significant PKR-dependent
response in the host cells at concentrations effective for
attenuating expression of the target gene.
16. The method or the composition of claim 15, wherein the dsRNA is
15-45 basepairs in length.
17. The method or the composition of claim 15, wherein the dsRNA is
19-30 basepairs in length.
18. The method of any of claims 1 and 2, wherein the dsRNA is
produced in the cell by an expression vector having a coding
sequence that is transcribed to produce one or more transcriptional
products that are processed to an siRNA species by said host
cells.
19. The method of any of claims 1 and 2, or the composition of
claim 3 or 5, wherein the dsRNA is a hairpin RNA that is processed
to an siRNA species by said host cells.
20. The method of any of claims 1 and 2, wherein expression of the
target gene is attenuated by at least 10 fold.
21. A pharmaceutical package comprising: a pharmaceutically
preparation of a double stranded RNA (DSRNA) that is, or gives rise
to, a short interfering double stranded RNA (siRNA) in a mammalian
cell and attenuates expression of a target gene, which dsRNA does
not produce a significant PKR-dependent response in the mammalian
cell at concentrations effective for attenuating expression of the
target gene; and label or instructions (written and/or pictorial)
for administering the preparation to a patient.
22. The pharmaceutical package of claim 21, wherein the siRNA
hybridizes to an untranscribed sequence or an non-coding sequence
of the target gene.
23. A method of attenuating expression of a target gene in the
cells of a mammal, comprising administering the mammal a
composition of any of claims 3-6.
24. The method of claim 23, wherein attenuating expression of the
target gene reduces unwanted growth or differentiation of the
cells.
25. A method for generating short interfering double-stranded RNA
(siRNA), comprising: (i) providing an in vitro transcription system
including: a double-stranded nucleic acid having complementary
sense and antisense target sequences, which target sequence is
flanked by promoters for an RNA polymerase, the RNA polymerase,
wherein the sense and antisense target sequences are transcribed
and can anneal to form an siRNA; and (ii) isolating said siRNA from
said in vitro transcription system.
26. The method of claim 25, wherein the RNA polymerase is a
bacteriophage RNA polymerase.
27. The method of claim 26, wherein the RNA polymerase is selected
from the group consisting of T3 polymerase, T7 polymerase and SP6
polymerase.
28. The method of claim 25, wherein the in vitro transcription
system includes a variegated library of target sequences to produce
a variegated library of siRNA species.
29. A method for performing a business of distributing short
interfering double-stranded RNA (siRNA), comprising: (i) accepting
orders for siRNA species having a sequence designated by user; (ii)
providing an in vitro transcription system including: a
double-stranded nucleic acid having a target sequence of
complementary sense and antisense sequences for said user
designated siRNA species, which target sequence is flanked by
promoters for an RNA polymerase, and the RNA polymerase, wherein
the sense and antisense target sequences are transcribed and can
anneal to form an siRNA; (iii) isolating said siRNA from said in
vitro transcription system; and (iv) packaging and shipping said
siRNA to said user.
30. A method for altering the MHC phenotype of a donor stem cell or
the progeny thereof, comprising introducing a double stranded RNA
(dsRNA) into a stem cell in an amount sufficient to attenuate
expression of an MHC gene otherwise expressed by said stem cell or
the progeny thereof.
31. The method of claim 30, wherein the dsRNA reduces expression of
one or more human leukocyte antigens (HLA) otherwise expressed by
said stem cell or the progeny thereof.
32. A culture of donor stem cells or the progeny thereof, having an
altered MHC phenotype resulting from stable attenuation of
expression of one or more MHC genes by introduction of double
stranded RNA (dsRNA) into the donor stem cells.
33. A method of transplanting a patient, comprising: (i) generating
an ex vivo cell or tissue culture of donor stem cells, or the
progeny thereof, having an altered MHC phenotype resulting from
stable attenuation of expression of one or more MHC genes by
introduction of double stranded RNA (dsRNA) into the donor stem
cells; and (ii) transplanting said patient with said cell or tissue
culture.
34. Use of a stem cell in the manufacture of a cellular medicament
for transplantation to a patient, which cellular medicament
comprises stem cells or the progeny thereof which have an altered
MHC phenotype resulting from stable attenuation of expression of
one or more MHC genes by introduction of double stranded RNA
(dsRNA) into the stem cells.
35. Use of the compositions of any of claims 3-6 in the manufacture
of a medicament for attenuating expression of one or more genes in
vivo.
36. A method for reducing the susceptibility of host cells to
infection by pathogen, comprising introducing a double stranded RNA
(dsRNA) into said host cells in an amount sufficient to attenuate
expression of one or more genes necessary for expression by said
pathogen.
37. The method of claim 36, wherein the pathogen is a virus, and
the dsRNA attenuates expression of a cellular surface protein
necessary for infection of the host cell by the virus.
38. A non-human transgenic mammal having germline and/or somatic
cells comprising a transgene encoding a double stranded RNA
transcriptional produce that is processed to an short interfering
double-stranded RNA (siRNA) species, wherein transcription of said
transgene attenuates expression of an endogenous target gene in at
least one cell-type of the animal.
39. The transgenic animal of claim 38, which is chimeric for said
transgene.
40. The transgenic animal of claim 38, wherein said transgene is
chromosomally incorporated.
41. The transgenic animal of claim 38, wherein the transgene
transcribes separate complementary transcripts that anneal to form
said siRNA, which siRNA does not produce a significant
PKR-dependent response at concentrations effective for attenuating
expression of the target gene.
42. The transgenic animal of claim 38, wherein the transgene
transcribes a hairpin RNA that is processed to said siRNA.
43. A hairpin RNA comprising a first nucleotide sequence that
hybridizes under stringent conditions to a nucleotide sequence of a
target gene, and a second nucleotide sequence which is a
complementary inverted repeat of said first nucleotide sequence and
hybridizes to said first nucleotide sequence to form a hairpin
structure, which hairpin RNA attenuates expression of said target
gene and does not produce a significant PKR-dependent response at
concentrations effective for attenuating expression of the target
gene.
44. The hairpin RNA of claim 43, wherein the hairpin RNA is
chemically synthesized.
45. The hairpin RNA of claim 43, wherein the hairpin RNA is
enzymatically synthesized in vitro.
46. The hairpin RNA of claim 45, wherein the hairpin RNA is
synthesized by T7 RNA polymerase in vitro.
47. The hairpin RNA of claim 43, wherein the hairpin RNA is
enzymatically synthesized in vivo.
48. The hairpin RNA of claim 47, wherein the hairpin RNA is
synthesized by RNA polymerase III in vivo.
49. The hairpin RNA of claim 43, wherein the hairpin RNA is
produced by a vector.
50. The hairpin RNA of claim 43, wherein the hairpin RNA has a size
of about 20-50 nucleotides.
51. The hairpin RNA of claim 43, wherein the hairpin RNA has a size
of about 50-100 nucleotides.
52. The hairpin RNA of claim 43, wherein the hairpin RNA has a size
of about 100-500 nucleotides.
53. The hairpin RNA of claim 43, which includes a restriction
enzyme site in the loop of the hairpin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/055,797, filed Jan. 22, 2002, which is a
continuation-in-part of International Application PCT/US01/08435,
filed Mar. 16, 2001, which claims the benefit of priority from U.S.
Provisional Application Nos. 60/189,739, filed Mar. 16, 2000, and
60/243,097, filed Oct. 24, 2000. This application is also a
continuation-in-part of U.S. application Ser. No. 09/866,557 filed
May 24, 2001 and a continuation-in-part of U.S. application Ser.
No. 09/858,862 filed May 16, 2001. The specifications of such
applications are incorporated by reference herein. International
Application PCT/US01/08435 was published under PCT Article 21(2) in
English.
BACKGROUND OF THE INVENTION
[0003] "RNA interference", "post-transcriptional gene silencing",
"quelling"--these different names describe similar effects that
result from the overexpression or misexpression of transgenes, or
from the deliberate introduction of double-stranded RNA into cells
(reviewed in Fire A (1999) Trends Genet 15: 358-363; Sharp P A
(1999) Genes Dev 13: 139-141; Hunter C (1999) Curr Biol 9:
R440-R442; Baulcombe D C (1999) Curr Biol 9: R599-R601; Vaucheret
et al. (1998) Plant J 16: 651-659). The injection of
double-stranded RNA into the nematode Caenorhabditis elegans, for
example, acts systemically to cause the post-transcriptional
depletion of the homologous endogenous RNA (Fire et al. (1998)
Nature 391: 806-811; and Montgomery et al. (1998) PNAS 95:
15502-15507). RNA interference, commonly referred to as RNAi,
offers a way of specifically and potently inactivating a cloned
gene, and is proving a powerful tool for investigating gene
function. Although the phenomenon is interesting in its own right;
the mechanism has been rather mysterious, but recent research--for
example that recently reported by Smardon et al. (2000) Curr Biol
10: 169-178--is beginning to shed light on the nature and evolution
of the biological processes that underlie RNAi.
[0004] RNAi was discovered when researchers attempting to use the
antisense RNA approach to inactivate a C. elegans gene found that
injection of sense-strand RNA was actually as effective as the
antisense RNA at inhibiting gene function (Guo et al. (1995) Cell
81: 611-620). Further investigation revealed that the active agent
was modest amounts of double-stranded RNA that contaminate in vitro
RNA preparations. Researchers quickly determined the `rules` and
effects of RNAi which have become the paradigm for thinking about
the mechanism which mediates this affect. Exon sequences are
required, whereas introns and promoter sequences, while
ineffective, do not appear to compromise RNAi (though there may be
gene-specific exceptions to this rule). RNAi acts
systemically--injection into one tissue inhibits gene function in
cells throughout the animal. The results of a variety of
experiments, in C. elegans and other organisms, indicate that RNAi
acts to destabilize cellular RNA after RNA processing.
[0005] The potency of RNAi inspired Timmons and Fire (1998 Nature
395: 854) to do a simple experiment that produced an astonishing
result. They fed to nematodes bacteria that had been engineered to
express double-stranded RNA corresponding to the C. elegans unc-22
gene. Amazingly, these nematodes developed a phenotype similar to
that of unc-22 mutants that was dependent on their food source. The
ability to conditionally expose large numbers of nematodes to
gene-specific double-stranded RNA formed the basis for a very
powerful screen to select for RNAi-defective C. elegans mutants and
then to identify the corresponding genes.
[0006] Double-stranded RNAs (dsRNAs) can provoke gene silencing in
numerous in vivo contexts including Drosophila, Caenorhabditis
elegans, planaria, hydra, trypanosomes, fungi and plants. However,
the ability to recapitulate this phenomenon in higher eukaryotes,
particularly mammalian cells, has not been accomplished in the art.
Nor has the prior art demonstrated that this phenomena can be
observed in cultured eukaryotic cells. Additionally, the `rules`
established by the prior art have taught that RNAi requires exon
sequences, and thus constructs consisting of intronic or promoter
sequences were not believed to be effective reagents in mediating
RNAi. The present invention aims to address each of these
deficiencies in the prior art and provides evidence both that RNAi
can be observed in cultured eukaryotic cells and that RNAi
constructs consisting of non-exon sequences can effectively repress
gene expression.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention provides a method for
attenuating expression of a target gene in cultured cells,
comprising introducing double stranded RNA (dsRNA) into the cells
in an amount sufficient to attenuate expression of the target gene,
wherein the dsRNA comprises a nucleotide sequence that hybridizes
under stringent conditions to a nucleotide sequence of the target
gene.
[0008] Another aspect of the present invention provides a method
for attenuating expression of a target gene in a mammalian cell,
comprising
[0009] (i) activating one or both of a Dicer activity or an
Argonaut activity in the cell, and
[0010] (ii) introducing into the cell a double stranded RNA (dsRNA)
in an amount sufficient to attenuate expression of the target gene,
wherein the dsRNA comprises a nucleotide sequence that hybridizes
under stringent conditions to a nucleotide sequence of the target
gene.
[0011] In certain embodiments, the cell is suspended in culture;
while in other embodiments the cell is in a whole animal, such as a
non-human mammal.
[0012] In certain preferred embodiments, the cell is engineered
with (i) a recombinant gene encoding a Dicer activity, (ii) a
recombinant gene encoding an Argonaut activity, or (iii) both. For
instance, the recombinant gene may encode, for a example, a protein
which includes an amino acid sequence at least 50 percent identical
to SEQ ID No. 2 or 4; or be defined by a coding sequence which
hybridizes under wash conditions of 2.times.SSC at 22.degree. C. to
SEQ ID No. 1 or 3. In certain embodiments, the recombinant gene may
encode, for a example, a protein which includes an amino acid
sequence at least 50 percent identical to the Argonaut sequence
shown in FIG. 24. In certain embodiments, the recombinant gene may
encode a protein which includes an amino acid sequence at least 60
percent, 70 percent, 80 percent, 85 percent, 90 percent, or 95
percent identical to SEQ ID No. 2 or 4. In certain embodiments, the
recombinant gene may be defined by a coding sequence which
hybridizes under stringent conditions, including a wash step
selected from 0.2.times.-2.0.times.SSC at from 50.degree.
C.-65.degree. C., to SEQ ID No. 1 or 3.
[0013] In certain embodiments, rather than use a heterologous
expression construct(s), an endogenous Dicer gene or Argonaut gene
can be activated, e.g., by gene activation technology, expression
of activated transcription factors or other signal transduction
protein(s), which induces expression of the gene, or by treatment
with an endogenous factor which upregulates the level of expression
of the protein or inhibits the degradation of the protein.
[0014] In certain preferred embodiments, the target gene is an
endogenous gene of the cell. In other embodiments, the target gene
is a heterologous gene relative to the genome of the cell, such as
a pathogen gene, e.g., a viral gene.
[0015] In certain embodiments, the cell is treated with an agent
that inhibits protein kinase RNA-activated (PKR) apoptosis, such as
by treatment with agents which inhibit expression of PKR, cause its
destruction, and/or inhibit the kinase activity of PKR.
[0016] In certain preferred embodiments, the cell is a primate
cell, such as a human cell.
[0017] In certain preferred embodiments, the length of the dsRNA is
at least 20, 21 or 22 nucleotides in length, e.g., corresponding in
size to RNA products produced by Dicer-dependent cleavage. In
certain embodiments, the dsRNA construct is at least 25, 50, 100,
200, 300 or 400 bases. In certain embodiments, the dsRNA construct
is 400-800 bases in length.
[0018] In certain preferred embodiments, expression of the target
gene is attenuated by at least 5 fold, and more preferably at least
10, 20 or even 50 fold, e.g., relative to the untreated cell or a
cell treated with a dsRNA construct which does not correspond to
the target gene.
[0019] Yet another aspect of the present invention provides a
method for attenuating expression of a target gene in cultured
cells, comprising introducing an expression vector having a "coding
sequence" which, when transcribed, produces double stranded RNA
(dsRNA) in the cell in an amount sufficient to attenuate expression
of the target gene, wherein the dsRNA comprises a nucleotide
sequence that hybridizes under stringent conditions to a nucleotide
sequence of the target gene. In certain embodiments, the vector
includes a single coding sequence for the dsRNA which is operably
linked to (two) transcriptional regulatory sequences which cause
transcription in both directions to form complementary transcripts
of the coding sequence. In other embodiments, the vector includes
two coding sequences which, respectively, give rise to the two
complementary sequences which form the dsRNA when annealed. In
still other embodiments, the vector includes a coding sequence
which forms a hairpin. In certain embodiments, the vectors are
episomal, e.g., and transfection is transient. In other
embodiments, the vectors are chromosomally integrated, e.g., to
produce a stably transfected cell line. Preferred vectors for
forming such stable cell lines are described in U.S. Pat. No.
6,025,192 and PCT publication WO/9812339, which are incorporated by
reference herein.
[0020] Another aspect of the present invention provides a method
for attenuating expression of a target gene in cultured cells,
comprising introducing an expression vector having a "non-coding
sequence" which, when transcribed, produces double stranded RNA
(dsRNA) in the cell in an amount sufficient to attenuate expression
of the target gene. The non-coding sequence may include intronic or
promoter sequence of the target gene of interest, and the dsRNA
comprises a nucleotide sequence that hybridizes under stringent
conditions to a nucleotide sequence of the promoter or intron of
the target gene. In certain embodiments, the vector includes a
single sequence for the dsRNA which is operably linked to (two)
transcriptional regulatory sequences which cause transcription in
both directions to form complementary transcripts of the sequence.
In other embodiments, the vector includes two sequences which,
respectively, give rise to the two complementary sequences which
form the dsRNA when annealed. In still other embodiments, the
vector includes a coding sequence which forms a hairpin. In certain
embodiments, the vectors are episomal, e.g., and transfection is
transient. In other embodiments, the vectors are chromosomally
integrated, e.g., to produce a stably transfected cell line.
Preferred vectors for forming such stable cell lines are described
in U.S. Pat. No. 6,025,192 and PCT publication WO/9812339, which
are incorporated by reference herein.
[0021] Another aspect the present invention provides a double
stranded (ds) RNA for inhibiting expression of a mammalian gene.
The dsRNA comprises a first nucleotide sequence that hybridizes
under stringent conditions, including a wash step of 0.2.times.SSC
at 65.degree. C., to a nucleotide sequence of at least one
mammalian gene and a second nucleotide sequence which is
complementary to the first nucleotide sequence.
[0022] In one embodiment, the first nucleotide sequence of said
double-stranded RNA is at least 20, 21, 22, 25, 50, 100, 200, 300,
400, 500, 800 nucleotides in length.
[0023] In another embodiment, the first nucleotide sequence of said
double-stranded RNA is identical to at least one mammalian gene. In
another embodiment, the first nucleotide sequence of said
double-stranded RNA is identical to one mammalian gene. In yet
another embodiment, the first nucleotide sequence of said
double-stranded RNA hybridizes under stringent conditions to at
least one human gene. In still another embodiment, the first
nucleotide sequence of said double-stranded RNA is identical to at
least one human gene. In still another embodiment, the first
nucleotide sequence of said double-stranded RNA is identical to one
human gene.
[0024] The double-stranded RNA may be an siRNA or a hairpin, and
may be expressed transiently or stably. In one embodiment, the
double-stranded RNA is a hairpin comprising a first nucleotide
sequence that hybridizes under stringent conditions to a nucleotide
sequence of at least one mammalian gene, and a second nucleotide
sequence which is a complementary inverted repeat of said first
nucleotide sequence and hybridizes to said first nucleotide
sequence to form a hairpin structure.
[0025] The first nucleotide sequence of said double-stranded RNA
can hybridize to either coding or non-coding sequence of at least
one mammalian gene. In one embodiment, the first nucleotide
sequence of said double-stranded RNA hybridizes to a coding
sequence of at least one mammalian gene. In another embodiment, the
first nucleotide sequence of said double-stranded RNA hybridizes to
a coding sequence of at least one human gene. In another
embodiment, the first nucleotide sequence of said double-stranded
RNA is identical to a coding sequence of at least one mammalian
gene. In still another embodiment, the first nucleotide sequence of
said double-stranded RNA is identical to a coding sequence of at
least one human gene.
[0026] In another embodiment, the first nucleotide sequence of said
double-stranded RNA hybridizes to a non-coding sequence of at least
one mammalian gene. In another enbodiment, the first nucleotide
sequence of said double-stranded RNA hybridizes to a non-coding
sequence of at least one human gene. In another embodiment, the
first nucleotide sequence of said double-stranded RNA is identical
to a non-coding sequence of at least one mammalian gene. In still
another embodiment, the first nucleotide sequence of said
double-stranded RNA is identical to a non-coding sequence of at
least one human gene. In any of the foregoing embodiments, the
non-coding sequence may be a non-transcribed sequence.
[0027] Still another aspect of the present invention provides an
assay for identifying nucleic acid sequences, either coding or
non-coding sequences, responsible for conferring a particular
phenotype in a cell, comprising
[0028] (i) constructing a variegated library of nucleic acid
sequences from a cell in an orientation relative to a promoter to
produce double stranded DNA;
[0029] (ii) introducing the variegated dsRNA library into a culture
of target cells;
[0030] (iii) identifying members of the library which confer a
particular phenotype on the cell, and identifying the sequence from
a cell which correspond, such as being identical or homologous, to
the library member.
[0031] Yet another aspect of the present invention provides a
method of conducting a drug discovery business comprising:
[0032] (i) identifying, by the subject assay, a target gene which
provides a phenotypically desirable response when inhibited by
RNAi;
[0033] (ii) identifying agents by their ability to inhibit
expression of the target gene or the activity of an expression
product of the target gene;
[0034] (iii) conducting therapeutic profiling of agents identified
in step (b), or further analogs thereof, for efficacy and toxicity
in animals; and
[0035] (iv) formulating a pharmaceutical preparation including one
or more agents identified in step (iii) as having an acceptable
therapeutic profile.
[0036] The method may include an additional step of establishing a
distribution system for distributing the pharmaceutical preparation
for sale, and may optionally include establishing a sales group for
marketing the pharmaceutical preparation.
[0037] Another aspect of the present invention provides a method of
conducting a target discovery business comprising:
[0038] (i) identifying, by the subject assay, a target gene which
provides a phenotypically desirable response when inhibited by
RNAi;
[0039] (ii) (optionally) conducting therapeutic profiling of the
target gene for efficacy and toxicity in animals; and
[0040] (iii) licensing, to a third party, the rights for further
drug development of inhibitors of the target gene.
[0041] Another aspect of the invention provides a method for
inhibiting RNAi by inhibiting the expression or activity of an RNAi
enzyme. Thus, the subject method may include inhibiting the
activity of Dicer and/or the 22-mer RNA.
[0042] Still another aspect relates to a method for altering the
specificity of an RNAi by modifying the sequence of the RNA
component of the RNAi enzyme.
[0043] In another aspect, gene expression in an undifferentiated
stem cell, or the differentiated progeny thereof, is altered by
introducing dsRNA of the present invention. In one embodiment, the
stem cells are embryonic stem cells. Preferably, the embryonic stem
cells are derived from mammals, more preferably from non-human
primates, and most preferably from humans.
[0044] The embryonic stem cells may be isolated by methods known to
one of skill in the art from the inner cell mass (ICM) of
blastocyst stage embryos. In one embodiment the embryonic stem
cells are obtained from previously established cell lines. In a
second embodiment, the embryonic stem cells are derived de novo by
standard methods.
[0045] In another aspect, the embryonic stem cells are the result
of nuclear transfer. The donor nuclei are obtained from any adult,
fetal, or embryonic tissue by methods well known in the art. In one
embodiment, the donor nuclei is transferred to a recipient oocyte
which had previously been modified. In one embodiment, the oocyte
is modified using one or more dsRNAs. Exemplary modifications of
the recipient oocyte include any changes in gene or protein
expression that prevent an embryo derived from said modified oocyte
from successfully implanting in the uterine wall. Since
implantation in the uterine wall is essential for fertilized
mammalian embryos to progress from beyond the blastocyst stage,
embryos made from such modified oocytes could not give rise to
viable organisms. Non-limiting examples of such modifications
include those that decrease or eliminate expression of cell surface
receptors (i.e., integrins) required for the recognition between
the blastocyst and the uterine wall, modifications that decrease or
eliminate expression of proteases (i.e., collagenase, stromelysin,
and plasminogen activator) required to digest matrix in the uterine
lining and thus allow proper implantation, and modifications that
decrease or eliminate expression of proteases (i.e., strypsin)
necessary for the blastocyst to hatch from the zona pellucida. Such
hatching is required for implantation.
[0046] In another embodiment, embryonic stem cells, embryonic stem
cells obtained from fertilization of modified oocytes, or the
differentiated progeny thereof, can be modified or further modified
with one or more dsRNAs. In a preferred embodiment, the
modification decreases or eliminates MHC expression. Cells modified
in this way will be tolerated by the recipient, thus avoiding
complications arising from graft rejection. Such modified cells are
suitable for transplantation into a related or unrelated patient to
treat a condition characterized by cell damage or cell loss.
[0047] In another aspect of the invention, the undifferentiated
stem cell is an adult stem cell. Exemplary adult stem cells
include, but are not limited to, hematopoietic stem cells,
mesenchymal stem cells, cardiac stem cells, pancreatic stem cells,
and neural stem cells. Exemplary adult stem cells include any stem
cell capable of forming differentiated ectodermal, mesodermal, or
endodermal derivatives. Non-limiting examples of differentiated
cell types which arise from adult stem cells include: blood,
skeletal muscle, myocardium, endocardium, pericardium, bone,
cartilage, tendon, ligament, connective tissue, adipose tissue,
liver, pancreas, skin, neural tissue, lung, small intestine, large
intestine, gall bladder, rectum, anus, bladder, female or male
reproductive tract, genitals, and the linings of the body
cavity.
[0048] In one embodiment, an undifferentiated adult stem cell, or
the differentiated progeny thereof, is altered with one or more
dsRNAs to decrease or eliminate MHC expression. Cells modified in
this way will be tolerated by the recipient, thus avoiding
complications arising from graft rejection. Such modified cells are
suitable for transplantation into a related or unrelated patient to
treat a condition characterized by cell damage or cell loss.
[0049] In another aspect of the invention, an embryonic stem cell,
an undifferentiated adult stem cell, or the differentiated progeny
of either an embryonic or adult stem cell is altered with one or
more dsRNA to decrease or eliminate expression of genes required
for HIV infection. In a preferred embodiment, the stem cell is one
capable of giving rise to hematopoietic cells. Modified cells with
hematopoietic potential can be transplanted into a patient as a
preventative therapy or treatment for HIV or AIDS.
[0050] Another aspect of the invention relates to purified or
semi-purified preparations of the RNAi enzyme or components
thereof. In certain embodiments, the preparations are used for
identifying compounds, especially small organic molecules, which
inhibit or potentiate the RNAi activity. Small molecule inhibitors,
for example, can be used to inhibit dsRNA responses in cells which
are purposefully being transfected with a virus which produces
double stranded RNA.
[0051] The dsRNA construct may comprise one or more strands of
polymerized ribonucleotide. It may include modifications to either
the phosphate-sugar backbone or the nucleoside. The double-stranded
structure may be formed by a single self-complementary RNA strand
or two complementary RNA strands. RNA duplex formation may be
initiated either inside or outside the cell. The dsRNA construct
may be introduced in an amount which allows delivery of at least
one copy per cell. Higher doses of double-stranded material may
yield more effective inhibition. Inhibition is sequence-specific in
that nucleotide sequences corresponding to the duplex region of the
RNA are targeted for genetic inhibition. In certain embodiments,
dsRNA constructs containing a nucleotide sequences identical to a
portion of the target gene are preferred for inhibition. RNA
sequences with insertions, deletions, and single point mutations
relative to the target sequence (i.e., RNA sequences similar to the
target sequence) have also been found to be effective for
inhibition. Thus, sequence identity may be optimized by alignment
algorithms known in the art and calculating the percent difference
between the nucleotide sequences. Alternatively, the duplex region
of the RNA may be defined functionally as a nucleotide sequence
that is capable of hybridizing with a portion of the target gene
transcript. In another embodiment, dsRNA constructs containing
nucleotide sequences identical to a non-coding portion of the
target gene are preferred for inhibition. Exemplary non-coding
regions include introns and the promoter region. Sequences with
insertions, deletions, and single point mutations relative to the
target non-coding sequence may also be used.
[0052] Yet another aspect of the invention pertains to transgenic
non-human mammals which include a transgene encoding a dsRNA
construct, wherein the dsRNA is identical or similar to either the
coding or non-coding sequence of the target gene, preferably which
is stably integrated into the genome of cells in which it occurs.
The animals can be derived by oocyte microinjection, for example,
in which case all of the nucleated cells of the animal will include
the transgene, or can be derived using embryonic stem (ES) cells
which have been transfected with the transgene, in which case the
animal is a chimera and only a portion of its nucleated cells will
include the transgene. In certain instances, the
sequence-independent dsRNA response, e.g., the PKR response, is
also inhibited in those cells including the transgene.
[0053] In still other embodiments, dsRNA itself can be introduced
into an ES cell in order to effect gene silencing, and that
phenotype will be carried for at least several rounds of division,
e.g., into the progeny of that cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1: RNAi in S2 cells. (a) Drosophila S2 cells were
transfected with a plasmid that directs lacZ expression from the
copia promoter in combination with dsRNAs corresponding to either
human CD8 or lacZ, or with no dsRNA, as indicated. (b) S2 cells
were co-transfected with a plasmid that directs expression of a
GFP-US9 fusion protein and dsRNAs of either lacZ or cyclin E, as
indicated. Upper panels show FACS profiles of the bulk population.
Lower panels show FACS profiles from GFP-positive cells. (c) Total
RNA was extracted from cells transfected with lacZ, cyclin E, fizzy
or cyclin A dsRNAs, as indicated. Northern blots were hybridized
with sequences not present in the transfected dsRNAs.
[0055] FIG. 2: RNAi in vitro. (a) Transcripts corresponding to
either the first 600 nucleotides of Drosophila cyclin E (E600) or
the first 800 nucleotides of lacZ (Z800) were incubated in lysates
derived from cells that had been transfected with either lacZ or
cyclin E (cycE) dsRNAs, as indicated. Time points were 0, 10, 20,
30, 40 and 60 min for cyclin E and 0, 10, 20, 30 and 60 min for
lacZ. (b) Transcripts were incubated in an extract of S2 cells that
had been transfected with cyclin E dsRNA (cross-hatched box,
below). Transcripts corresponded to the first 800 nucleotides of
lacZ or the first 600, 300, 220 or 100 nucleotides of cyclin E, as
indicated. Eout is a transcript derived from the portion of the
cyclin E cDNA not contained within the transfected dsRNA. E-ds is
identical to the dsRNA that had been transfected into S2 cells.
Time points were 0 and 30 min. (c) Synthetic transcripts
complementary to the complete cyclin E cDNA (Eas) or the final 600
nucleotides (Eas600) or 300 nucleotides (Eas300) were incubated in
extract for 0 or 30 min.
[0056] FIG. 3: Substrate requirements of the RISC. Extracts were
prepared from cells transfected with cyclin E dsRNA. Aliquots were
incubated for 30 min at 30.degree. C. before the addition of either
the cyclin E (E600) or lacZ (Z800) substrate. Individual 20 .mu.l
aliquots, as indicated, were pre-incubated with 1 mM CaCl.sub.2 and
5 mM EGTA, 1 mM CaCl.sub.2, 5 mM EGTA and 60 U of micrococcal
nuclease, 1 mM CaCl.sub.2 and 60 U of micrococcal nuclease or 10 U
of DNase I (Promega) and 5 mM EGTA. After the 30 min
pre-incubation, EGTA was added to those samples that lacked it.
Yeast tRNA (1 .mu.g) was added to all samples. Time points were at
0 and 30 min.
[0057] FIG. 4: The RISC contains a potential guide RNA. (a)
Northern blots of RNA from either a crude lysate or the S100
fraction (containing the soluble nuclease activity, see Methods)
were hybridized to a riboprobe derived from the sense strand of the
cyclin E mRNA. (b) Soluble cyclin-E-specific nuclease activity was
fractionated as described in Methods. Fractions from the
anion-exchange resin were incubated with the lacZ, control
substrate (upper panel) or the cyclin E substrate (centre panel).
Lower panel, RNA from each fraction was analysed by northern
blotting with a uniformly labelled transcript derived from sense
strand of the cyclin E cDNA. DNA oligonucleotides were used as size
markers.
[0058] FIG. 5: Generation of 22mers and degradation of mRNA are
carried out by distinct enzymatic complexes. (a) Extracts prepared
either from 0-12 hour Drosophila embryos or Drosophila S2 cells
(see Methods) were incubated for 0, 15, 30, or 60 minutes (left to
right) with a uniformly-labeled double-stranded RNA corresponding
to the first 500 nucleotides of the Drosophila cyclin E coding
region. M indicates a marker prepared by in vitro transcription of
a synthetic template. The template was designed to yield a 22
nucleotide transcript. The doublet most probably results from
improper initiation at the +1 position. (b) Whole-cell extracts
were prepared from S2 cells that had been transfected with a dsRNA
corresponding to the first 500 nt. of the luciferase coding region.
S10 extracts were spun at 30,000.times.g for 20 minutes which
represents our standard RISC extract. S100 extracts were prepared
by further centrifugation of S10 extracts for 60 minutes at
100,000.times.g. Assays for mRNA degradation were carried out as
described previously for 0, 30 or 60 minutes (left to right in each
set) with either a single-stranded luciferase mRNA or a
single-stranded cyclin E mRNA, as indicated. (c) S10 or S100
extracts were incubated with cyclin E dsRNAs for 0, 60 or 120
minutes (L to R).
[0059] FIG. 6: Production of 22mers by recombinant CG4792/Dicer.
(a) Drosophila S2 cells were transfected with plasmids that direct
the expression of T7-epitope tagged versions of Drosha,
CG4792/Dicer-1 and Homeless. Tagged proteins were purified from
cell lysates by immunoprecipitation and were incubated with cyclin
E dsRNA. For comparison, reactions were also performed in
Drosophila embryo and S2 cell extracts. As a negative control,
immunoprecipitates were prepared from cells transfected with a
.beta.-galactosidase expression vector. Pairs of lanes show
reactions performed for 0 or 60 minutes. The synthetic marker (M)
is as described in the legend to FIG. 1. (b) Diagrammatic
representations of the domain structures of CG4792/Dicer-1, Drosha
and Homeless are shown. (c) Immunoprecipitates were prepared from
detergent lysates of S2 cells using an antiserum raised against the
C-terminal 8 amino acids of Drosophila Dicer-1 (CG4792). As
controls, similar preparations were made with a pre-immune serum
and with an immune serum that had been pre-incubated with an excess
of antigenic peptide. Cleavage reactions in which each of these
precipitates was incubated with an .about.500 nt. fragment of
Drosophila cyclin E are shown. For comparsion, an incubation of the
substrate in Drosophila embryo extract was electrophoresed in
parallel. (d) Dicer immunoprecipitates were incubated with dsRNA
substrates in the presence or absence of ATP. For comparison, the
same substrate was incubated with S2 extracts that either contained
added ATP or that were depleted of ATP using glucose and hexokinase
(see methods). (e) Drosophila S2 cells were transfected with
uniformly, .sup.32P-labelled dsRNA corresponding to the first 500
nt. of GFP. RISC complex was affinity purified using a
histidine-tagged version of Drosophila Ago-2, a recently identified
component of the RISC complex (Hammond et al., in prep). RISC was
isolated either under conditions in which it remains ribosome
associated (ls, low salt) or under conditions that extract it from
the ribosome in a soluble form (hs, high salt). For comparison, the
spectrum of labelled RNAs in the total lysate is shown. (f) Guide
RNAs produced by incubation of dsRNA with a Dicer immunoprecipitate
are compared to guide RNAs present in an affinity-purified RISC
complex. These precisely comigrate on a gel that has
single-nucleotide resolution. The lane labelled control is an
affinity selection for RISC from a cell that had been transfected
with labeled dsRNA but not with the epitope-tagged Drosophila
Ago-2.
[0060] FIG. 7: Dicer participates in RNAi. (a) Drosophila S2 cells
were transfected with dsRNAs corresponding to the two Drosophila
Dicers (CG4792 and CG6493) or with a control dsRNA corresponding to
murine caspase 9. Cytoplasmic extracts of these cells were tested
for Dicer activity. Transfection with Dicer dsRNA reduced activity
in lysates by 7.4-fold. (b) The Dicer-1 antiserum (CG4792) was used
to prepare immunoprecipitates from S2 cells that had been treated
as described above. Dicer dsRNA reduced the activity of Dicer-1 in
this assay by 6.2-fold. (c) Cells that had been transfected two
days previously with either mouse caspase 9 dsRNA or with Dicer
dsRNA were cotransfected with a GFP expression plasmid and either
control, luciferase dsRNA or GFP dsRNA. Three independent
experiments were quantified by FACS. A comparison of the relative
percentage of GFP-positive cells is shown for control (GFP plasmid
plus luciferase dsRNA) or silenced (GFP plamsid plus GFP dsRNA)
populations in cells that had previously been transfected with
either control (caspase 9) or Dicer dsRNAs.
[0061] FIG. 8: Dicer is an evolutionarily conserved ribonuclease.
(a) A model for production of 22mers by Dicer. Based upon the
proposed mechanism of action of Ribonuclease III, we propose that
Dicer acts on its substrate as a dimer. The positioning of the two
ribonuclease domains (RIIIa and RIIIb) within the enzyme would thus
determine the size of the cleavage product. An equally plausible
alternative model could be derived in which the RIIIa and RIIIb
domains of each Dicer enzyme would cleave in concert at a single
position. In this model, the size of the cleavage product would be
determined by interaction between two neighboring Dicer enzymes.
(b) Comparison of the domain structures of potential Dicer homologs
in various organisms (Drosophila--CG4792, CG6493, C.
elegans--K12H4.8, Arabidopsis--CARPEL FACTORY, T25K16.4,
AC012328.sub.--1, human Helicase-MOI and S. pombe--YC9A_SCHPO). The
ZAP domains were identified both by analysis of individual
sequences with Pfam and by Psi-blast searches. The ZAP domain in
the putative S. pombe Dicer is not detected by PFAM but is
identified by Psi-Blast and is thus shown in a different color. For
comparison, a domain structure of the RDE1/QDE2/ARGONAUTE family is
shown. It should be noted that the ZAP domains are more similar
within each of the Dicer and ARGONAUTE families than they are
between the two groups. (c) An alignment of the ZAP domains in
selected Dicer and Argonaute family members is shown. The alignment
was produced using ClustalW.
[0062] FIG. 9: Purification strategy for RISC. (second step in RNAi
model).
[0063] FIG. 10: Fractionation of RISC activity over sizing column.
Activity fractionates as 500 KDa complex. Also, antibody to
Drosophila argonaute 2 cofractionates with activity.
[0064] FIGS. 11-13: Fractionation of RISC over monoS, monoQ,
Hydroxyapatite columns. Drosophila argonaute 2 protein also
cofactionates.
[0065] FIG. 14: Alignment of Drosophila argonaute 2 with other
family members.
[0066] FIG. 15: Confirmation of Drosophila argonaute 2. S2 cells
were transfected with labeled dsRNA and His tagged argonaute.
Argonaute was isolated on nickel agarose and RNA component was
identified on 15% acrylamide gel.
[0067] FIG. 16: S2 cell and embryo extracts were assayed for 22mer
generating activity.
[0068] FIG. 17: RISC can be separated from 22mer generating
activity (dicer). Spinning extracts (S100) can clear RISC activity
from supernatant (left panel) however, S100 spins still contain
dicer activity (right panel).
[0069] FIG. 18: Dicer is specific for dsRNA and prefers longer
substrates.
[0070] FIG. 19: Dicer was fractionated over several columns.
[0071] FIG. 20: Identification of dicer as enzyme which can process
dsRNA into 22mers. Various RNaseIII family members were expressed
with n terminal tags, immunoprecipitated, and assayed for 22mer
generating activity (left panel). In right panel, antibodies to
dicer could also precipitate 22mer generating activity.
[0072] FIG. 21: Dicer requires ATP.
[0073] FIG. 22: Dicer produces RNAs that are the same size as RNAs
present in RISC.
[0074] FIG. 23: Human dicer homolog when expressed and
immunoprecipitated has 22mer generating activity.
[0075] FIG. 24: Sequence of Drosophila argonaute 2. Peptides
identified by microsequencing are shown in underline.
[0076] FIG. 25: Molecular charaterization of Drosophila argonaute
2. The presence of an intron in coding sequence was determined by
northern blotting using intron probe. This results in a different
5' reading frame then the published genome seqeunce. Number of
polyglutamine repeats was determined by genomic PCR.
[0077] FIG. 26: Dicer activity can be created in human cells by
expression of human dicer gene. Host cell was 293. Crude extracts
had dicer activity, while activity was absent from untransfected
cells. Activity is not dissimilar to that seen in drosophila embryo
extracts.
[0078] FIG. 27: A .about.500 nt. fragment of the gene that is to be
silenced (X) is inserted into the modified vector as a stable
direct repeat using standard cloning procedures. Treatment with
commercially available cre recombinase reverses sequences within
the loxP sites (L) to create an inverted repeat. This can be stably
maintained and amplified in an sbc mutant bacterial strain (DL759).
Transcription in vivo from the promoter of choice (P) yields a
hairpin RNA that causes silencing. A zeocin resistance marker is
included to insure maintenance of the direct and inverted repeat
structures; however this is non-essential in vivo and could be
removed by pre-mRNA splicing if desired. (Smith et al. (2000)
Nature 407: 319-20).
[0079] FIG. 28: RNAi in P19 embryonal carcinoma cells.
Ten-centimeter plates of P19 cells were transfected by using 5
.mu.g of GFP plasmid and 40 .mu.g of the indicated dsRNA (or no
RNA). Cells were photographed by fluorescent (tope panel) and
phase-contrast microscopy (bottom panel) at 72 h after
transfection; silencing was also clearly evident at 48 h
posttransfection.
[0080] FIG. 29: RNAi of firefly and Renilla luciferase in P19
cells. (A and B) P19 cells were transfected with plasmids that
direct the expression of firefly and Renilla luciferase and dsRNA
500 mers (25 or 250 ng, as indicated in A and B, respectively),
that were either homologous to the firefly luciferase mRNA (dsFF)
or nonhomologous (dsGFP). Luciferase activities were assayed at
various times after transfection, as indicated. Ratios of firefly
to Renilla activity are normalized to dsGFP controls. (C and D) P19
cells in 12-well culture dishes (2 ml of media) were transfected
with 0.25 .mu.g of a 9:1 mix of pGL3-Control and pRL-SV40 as well
as 2 .mu.g of the indicated RNA. Extracts were prepared 9 h after
transfection. (C) Ratio of firefly to Renilla luciferase is shown.
(D) Ratio of Renilla to firefly luciferase is shown. Values are
normalized to dsGFP. The average of three independent experiments
is shown; error bars indicate standard deviation.
[0081] FIG. 30: The panels at the right show expression of either
RFP or GFP following transient transfection into wild type P19
cells. The panels at the left demonstrate the specific suppression
of GFP expression in P19 clones which stably express a 500 nt
double stranded GFP hairpin. P19 clones which stably express the
double stranded GFP hairpin were transiently transfected with RFP
or GFP, and expression of RFP or GFP was assessed by visual
inspection.
[0082] FIG. 31: Specific silencing of luciferase expression by
dsRNA in murine embryonic stem cells. Mouse embryonic stem cells in
12-well culture dishes (1 ml of media) were transfected with 1.5
.mu.g of dsRNA along with 0.25 .mu.g of a 10:1 mixture of the
reporter plasmids pGL3-Control and pRL-SV40. Extracts were prepared
and assayed 20 h after transfection. The ratio of firefly to
Renilla luciferase expression is shown for FF ds500; the ratio of
Renilla to firefly is shown for Ren ds500. Both are normalized to
ratios from the dsGFP transfection. The average of three
independent experiments is shown; error bars indicate standard
deviation.
[0083] FIG. 32: RNAi in C2C12 murine myoblast cells. (A) Mouse
C2C12-cells in 12-well culture dishes (1 ml of media) were
transfected with 1 .mu.g of the indicated dsRNA along with 0.250
.mu.g of the reporter plasmids pGL3-Control and pRL-SV40. Extracts
were prepared and assayed 24 h after transfection. The ratio of
firefly to Renilla luciferase expression is shown; values are
normalized to ratios from the no dsRNA control. The average of
three independent experiments is shown; error bars indicate
standard deviation. (B) C2C12 cells cotransfected with 1 .mu.g of
either plasmid alone or a plasmid containing a hyperactive mutant
of vaccinia virus K3L (Kawagishi-Kobayashi et al. 2000, Virology
276: 424-434). The absolute counts of Renilla and firefly
luciferase activity are shown. (C) The ratios of firefly/Renilla
activity from B, normalized to no dsRNA controls.
[0084] FIG. 33: Hela, Chinese hamster ovary, and P19 (pluripotent,
mouse embryonic carcinoma) cell lines transfected with plasmids
expressing Photinus pyralis (firefly) and Renilla reniformis (sea
pansy) luciferases and with dsRNA 500mers (400 ng), homologous to
either firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP).
Dual luciferase assays were carried out using an Analytical
Scientific Instruments model 3010 Luminometer. In this assay
Renilla luciferase serves as an internal control for dsRNA-specific
suppression of firefly luciferase activity. These data demonstrate
that 500mer dsRNA can specifically suppress cognate gene expression
in vivo.
[0085] FIG. 34: Expression of a hairpin RNA produces P19 EC cell
lines that stably silence GFP. (A) A cartoon of the FLIP cassette
used to construct the GFP hairpin. GFP represents the first 500
coding base pairs of EGFP. Zeo, zeocin resistance gene; L, Lox; P,
the cytomegalovirus promoter in the expression plasmid pcDNA3.
Homologous GFP fragments are first cloned as direct repeats into
the FLIP cassette. To create inverted repeats for hairpin
production, the second repeat is flipped by using Cre recombinase.
When transcribed, the inverted repeat forms a GFP dsRNA with a
hairpin loop. (B) P19 cell lines stably expressing the GFP hairpin
plasmid, GFPhp.1 (clone 10) and GFPhp.2 (clone 12), along with wt
P19 were transfected with 0.25 .mu.g each of GFP and RFP reporter
genes. Fluorescence micrographs were taken by using filters
appropriate for GFP and RFP. Magnification is 200.times.. (C) P19
GFPhp.1 cells were transfected with pEGFP and 0, 0.5, or 1 .mu.g of
Dicer or firefly dsRNA. Fluorescence micrographs were taken at 48 h
posttransfection and are superimposed with bright field images to
reveal non-GFP expressing cells. Magnification is 100.times.. (D)
In vitro and in vivo processing of dsRNA in P19 cells. In vitro
Dicer assays were performed on S2 cells and three independently
prepared P19 extracts by using .sup.32P-labeled dsRNA (30.degree.
C. for 30 min). A Northern blot of RNA extracted from control and
GFPhp.1 P19 cells shows the production of .apprxeq.22mer RNA
species in hairpin-expressing cells but not in control cells. Blots
were probed with a .sup.32P-labeled "sense" GFP transcript.
[0086] FIG. 35: dsRNA induces silencing at the posttranscriptional
level. P19 cell extracts were used for in vitro translation of
firefly and Renilla luciferase mRNA (100 ng each). Translation
reactions were programmed with various amounts of dsRNA 500mers,
either homologous to firefly luciferase mRNA (dsLUC) or
nonhomologous (dsGFP). Luciferase assays were carried out after a 1
h incubation at 30.degree. C. Ratios of firefly to Renilla activity
are normalized to no dsRNA controls. Standard deviations from the
mean are shown.
[0087] FIG. 36: S10 fractions from P19 cell lysates were used for
in vitro translations of mRNA coding for Photinus pyralis (firefly)
and Renilla reniformis (sea pansy) luciferases. Translation
reactions were programmed with dsRNA, ssRNA, or asRNA 500mers,
either complementary to firefly luciferase mRNA (dsFF, ssFF, or
asFF), complementary to Renilla luciferase (dsREN, ssREN, or asREN)
or non-complementary (dsGFP). Reactions were carried out at
30.degree. C. for 1 hour, after a 30 min preincubation with dsRNA,
ssRNA, or asRNA. Dual luciferase assays were carried out using an
Analytical Scientific Instruments model 3010 Luminometer. On the
left, Renilla luciferase serves as an internal control for
dsRNA-specific suppression of firefly luciferase activity. On the
right, firefly luciferase serves as an internal control for
dsRNA-specific suppression of Renilla luciferase activity. These
data demonstrate that 500mer double-stranded RNA (dsRNA) but not
single-stranded (ssRNA) or anti-sense RNA (asRNA) suppresses
cognate gene expression in vitro in a manner consistent with
post-transcriptional gene silencing.
[0088] FIG. 37: P19 cells were grown in 6-well tissue culture
plates to approximately 60% confluence. Various amounts of dsRNA,
either homologous to firefly luciferase mRNA (dsLUC) or
non-homologous (dsGFP), were added to each well and incubated for
12 hrs under normal tissue culture conditions. Cells were then
transfected with plasmids expressing Photinus pyralis (firefly) and
Renilla reniformis (sea pansy) luciferases and with dsRNA 500mers
(500 ng). Dual luciferase assays were carried out 12 hrs
post-transfection using an Analytical Scientific Instruments model
3010 Luminometer. In this assay Renilla luciferase serves as an
internal control for dsRNA-specific suppression of firefly
luciferase activity. These data show that 500mer dsRNA can
specifically suppress cognate gene expression in vivo without
transfection under normal tissue culture conditions.
[0089] FIG. 38: Previous methods for generating siRNAs required
costly chemical synthesis. The invention provides an in vitro
method for synthesizing siRNAs using standard RNA transcription
reactions.
[0090] FIG. 39: Short hairpins suppress gene expression in
Drosophila S2 cells. (A) Sequences and predicted secondary
structure of representative chemically synthesized RNAs. Sequences
correspond to positions 112-134 (siRNA) and 463-491 (shRNAs) of
Firefly luciferase carried on pGL3-Control. An siRNA targeted to
position 463-485 of the luciferase sequence was virtually identical
to the 112-134 siRNA in suppressing expression, but is not shown.
(B) Exogenously supplied short hairpins suppress expression of the
targeted Firefly luciferase gene in vivo. Six-well plates of S2
cells were transfected with 250 ng/well of plasmids that direct the
expression of firefly and Renilla luciferase and 500 ng/well of the
indicated RNA. Luciferase activities were assayed 48 h after
transfection. Ratios of firefly to Renilla luciferase activity were
normalized to a control transfected with an siRNA directed at the
green fluorescent protein (GFP). The average of three independent
experiments is shown; error bars indicate standard deviation. (C)
Short hairpins are processed by the Drosophila Dicer enzyme. T7
transcribed hairpins shFfL22, shFfL29, and shFfS29 were incubated
with (+) and without (-) 0-2-h Drosophila embryo extracts. Those
incubated with extract produced .about.22-nt siRNAs, consistent
with the ability of these hairpins to induce RNA interference. A
long dsRNA input (cyclin E 500-mer) was used as a control. Cleavage
reactions were performed as described in Bernstein et al., 2001,
Nature, 409:363-366.
[0091] FIG. 40: Short hairpins function in mammalian cells. HEK
293T, HeLa, COS-1, and NIH 3T3 cells were transfected with plasmids
and RNAs as in FIG. 1 and subjected to dual luciferase assays 48 h
posttransfection. The ratios of firefly to Renilla luciferase
activity are normalized to a control transfected with an siRNA
directed at the green fluorescent protein (GFP). The average of
three independent experiments is shown; error bars indicate
standard deviation.
[0092] FIG. 41: siRNAs and short hairpins transcribed in vitro
suppress gene expression in mammalian cells. (A) Sequences and
predicted secondary structure of representative in vitro
transcribed siRNAs. Sequences correspond to positions 112-134
(siRNA) and 463-491 (shRNAs) of firefly luciferase carried on
pGL3-Control. (B) In vitro transcribed siRNAs suppress expression
of the targeted firefly luciferase gene in vivo. HEK 293T cells
were transfected with plasmids as in FIG. 2. The presence of
non-base-paired guanosine residues at the 5' end of siRNAs
significantly alters the predicted end structure and abolishes
siRNA activity. (C) Sequences and predicted secondary structure of
representative in vitro transcribed shRNAs. Sequences correspond to
positions 112-141 of firefly luciferase carried on pGL3-Control.
(D) Short hairpins transcribed in vitro suppress expression of the
targeted firefly luciferase gene in vivo. HEK 293T cells were
transfected with plasmids as in FIG. 2.
[0093] FIG. 42: Transcription of functional shRNAs in vivo. (A)
Schematic of the pShh1 vector. Sequences encoding shRNAs with
between 19 and 29 bases of homology to the targeted gene are
synthesized as 60-75-bp double-stranded DNA oligonucleotides and
ligated into an EcoRV site immediately downstream of the U6
promoter. (B) Sequence and predicted secondary structure of the Ff1
hairpin. (C) An shRNA expressed from the pShh1 vector suppresses
luciferase expression in mammalian cells. HEK 293T, HeLa, COS-1,
and NIH 3T3 cells were transfected with reporter plasmids as in
FIG. 1, and pShh1 vector, firefly siRNA, or pshh1 firefly shRNA
constructs as indicated. The ratios of firefly to Renilla
luciferase activity were determined 48 h after transfection and
represent the average of three independent experiments; error bars
indicate standard deviation.
[0094] FIG. 43: Dicer is required for shRNA-mediated gene
silencing. HEK 293T cells were transfected with luciferase reporter
plasmids as well as pShh1-Ff1 and an siRNA targeting human Dicer
either alone or in combination, as indicated. The Dicer siRNA
sequence (TCA ACC AGC CAC TGC TGG A) corresponds to coordinates
3137-3155 of the human Dicer sequence. The ratios of firefly to
Renilla luciferase activity were determined 26 h after transfection
and represent the average of three independent experiments; error
bars indicate standard deviation.
[0095] FIG. 44: Stable shRNA-mediated gene silencing of an
endogenous gene. (A) Sequence and predicted secondary structure of
the p53 hairpin. The 5' shRNA stem contains a 27-nt sequence
derived from mouse p53 (nucleotides 166-192), whereas the 3' stem
harbors the complimentary antisense sequence. (B) Senescence bypass
in primary mouse embryo fibroblasts (MEFs) expressing an shRNA
targeted at p53. Wild-type MEFs, passage 5, were transfected with
pBabe-RasV12 with control plasmid or with p53hp (5 .mu.g each with
FuGENE; Roche). Two days after transfection, cells were
trypsinized, counted, and plated at a density of
1.times.10.sup.5/10-cm plate in media containing 2.0 .mu.g/mL of
puromycin. Control cells cease proliferation and show a senescent
morphology (left panel). Cells expressing the p53 hairpin continue
to grow (right panel). Photos were taken 14 d posttransfection.
[0096] FIG. 45: A mixture of two short hairpins, both corresponding
to firefly luciferase, does not result in a synergistic suppression
of gene expression. Suppression of firefly luciferase gene
expression resulting from transfection of a mixture of two
different short hairpins (HP #1 and HP #2) was examined. The
mixture of HP #1 and HP #2 did not have a more robust effect on the
suppression of firefly luciferase gene expression than expression
of HP #1 alone.
[0097] FIG. 46: Encoded short hairpins specifically suppress gene
expression in vivo. DNA oligonucleotides encoding 29 nucleotide
hairpins corresponding to firefly luciferase were inserted into a
vector containing the U6 promoter. Three independent constructs
were examined for their ability to specifically suppress firefly
luciferase gene expression in 293T cells. siOligo1-2, siOligo1-6,
and siOligo1-19 (construct in the correct orientation) each
suppressed gene expression as effectively as siRNA. In contrast,
siOligo1-10 (construct in the incorrect orientation) did not
suppress gene expression. An independent construct targeted to a
different portion of the firefly luciferase gene did not
effectively suppress gene expression in either orientation
(siOligo2-23, siOligo2-36).
[0098] FIGS. 47-49: Strategies for stable expression of short
dsRNAs.
[0099] FIG. 50: Dual luciferase assays were performed as described
in detail in FIGS. 28-35, however the cells used in these
experiments were PKR-/- murine embryonic fibroblasts (MEFs).
Briefly, RNAi using long dsRNAs typically envokes a non-specific
response in MEFs (due to PKR activity). To evaluate the effect of
long dsRNA constructs to specifically inhibit gene expression in
MEFs, RNAi was examined in PKR-/- MEFs. Such cells do not respond
to dsRNA with a non-specific response. The data summarized in this
figure demonstrates that in the absence of the non-specific PKR
response, long dsRNA constructs specifically suppress gene
expression in MEFs.
[0100] FIG. 51: Is a schematic representation of the mouse
tyrosinase promoter. Primers were used to amplify three separate
regions in the proximal promoter, or to amplify sequence
corresponding to an enhancer located approximately 12 kb
upstream.
[0101] FIG. 52: Reporter expression plasmids and siRNA sequences
used in Figures X and Y. PGL-3-Control and Pluc-NS5B are the
expression plasmids used for transfection into mouse liver. The
nucleotide sequences of the siRNAs used in the study are shown
underneath.
[0102] FIG. 53: RNA interference in adult mice using siRNAs. (a)
Representative images of light emitted from mice co-transfected
with the luciferase plasmid pGL3-control and either no siRNA,
luciferase siRNA or unrelated siRNA. A pseudocolour image
representing intensity of emitted light (red, most intense; blue,
least intense) superimposed on a greyscale reference image (for
orientation) shows that RNAi functions in adult mice. Annealed
21-nucleotide siRNAs (40 .mu.g; Dharmacon) were co-injected into
the livers of mice with 2 .mu.g pGL3-control DNA (Promega) and 800
units of RNasin (Promega) in 1.8 ml PBS buffer in 5-7 s. After 72
h, mice were anaesthetized and given 3 mg luciferin
intraperitoneally 15 min before imaging. (b) siRNA results (six
mice per group) from a representative experiment. Mice receiving
luciferase siRNA emitted significantly less light than
reporter-alone controls (one-way ANOVA with post hoc Fisher's
test). Results for reporter alone and unrelated siRNA were
statistically similar. Animals were treated according to the US
National Institutes of Health's guidelines for animal care and the
guidelines of Stanford University.
[0103] FIG. 54: RNA interference in adult mice using shRNAs. (a)
Representative images of light emitted from mice co-transfected
with the luciferase plasmid control, pShh1-Ff1, and pShh1-Ff1rev.
pShh1-Ff1, but not pShh1-Ff1rev, reduced luciferase expression in
mice relative to the reporter-alone control. pShh1-Ff1 or pShh1-rev
(10 .mu.g) were co-injected with 2 .mu.g pGL3-control in 1.8 ml PBS
buffer. (b) Average of three independent shRNA experiments (n=5).
Average values for the reporter-alone group are designated as 100%
in each of the three experiments. Animals were treated according to
the US National Institutes of Health's guidelines for animal care
and the guidelines of Stanford University.
[0104] FIG. 55: Heritable repression of Neil1 expression by RNAi in
several tissues. (a) Expression of Neil1 mRNA in the livers of
three mice containing the Neil1 shRNA transgene (shRNA-positive) or
three siblings lacking the transgene (shRNA-negative) was assayed
by RT-PCR (top row is Neil1). An RT-PCR of .beta.-actin was done to
ensure that equal quantities of mRNAs were tested for each mouse
(second row). Expression of the neomycin resistance gene (neo),
carried on the shRNA vector, was tested similarly (third row).
Finally, the mice were genotyped using genomic DNA that was
PCR-amplified with vector-specific primers (bottom row). (b)
Similar studies were performed in the heart. (c) Similar studies
were performed in the spleen. Animal procedures have been approved
by the SUNY, Stony Brook Institutional Animal Care and Use
Committee (IACUC).
[0105] FIG. 56: Reduction in Neil1 protein correlates with the
presence of siRNAs. (a) Expression of Neil1 protein was examined in
protein extracts from the livers of mice carrying the shRNA
transgene (shRNA-positive) or siblings lacking the transgene
(shRNA-negative) by western blotting with Neil1-specific antiserum.
A western blot for PCNA was used to standardize loading. (b) The
presence of siRNAs in RNA derived from the livers of transgenic
mice as assayed by northern blotting using a 300 nt probe, part of
which was complementary to the shRNA sequence. We note siRNAs only
in mice transgenic for the shRNA expression cassette.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0106] I. Overview
[0107] The present invention provides methods for attenuating gene
expression in a cell using gene-targeted double stranded RNA
(dsRNA). The dsRNA contains a nucleotide sequence that hybridizes
under physiologic conditions of the cell to the nucleotide sequence
of at least a portion of the gene to be inhibited (the "target"
gene). The nucleotide sequence can hybridize to either coding or
non-coding sequence of the target gene.
[0108] A significant aspect to certain embodiments of the present
invention relates to the demonstration in the present application
that RNAi can in fact be accomplished both in cultured mammalian
cells and in whole organisms. This had not been previously
described in the art.
[0109] Another salient feature of the present invention concerns
the ability to carry out RNAi in higher eukaryotes, particularly in
non-oocytic cells of mammals, e.g., cells from adult mammals as an
example.
[0110] Furthermore, in contrast to the teachings of the prior art,
we demonstrate that RNAi in mammalian systems can be mediated with
dsRNA identical or similar to non-coding sequence of a target gene.
It was previously believed that although dsRNA identical or similar
to non-coding sequences (i.e., promoter, enhancer, or intronic
sequences) did not inhibit RNAi, such dsRNAs were not thought to
mediate RNAi.
[0111] As described in further detail below, the present
invention(s) are based on the discovery that the RNAi phenomenum is
mediated by a set of enzyme activities, including an essential RNA
component, that are evolutionarily conserved in eukaryotes ranging
from plants to mammals.
[0112] One enzyme contains an essential RNA component. After
partial purification, a multi-component nuclease (herein "RISC
nuclease") co-fractionates with a discrete, 22-nucleotide RNA
species which may confer specificity to the nuclease through
homology to the substrate mRNAs. The short RNA molecules are
generated by a processing reaction from the longer input dsRNA.
Without wishing to be bound by any particular theory, these 22mer
guide RNAs may serve as guide sequences that instruct the RISC
nuclease to destroy specific mRNAs corresponding to the dsRNA
sequences.
[0113] As illustrated, double stranded forms of the 22-mer guide
RNA can be sufficient in length to induce sequence-dependent dsRNA
inhibition of gene expression. In the illustrated example, dsRNA
contructs are administered to cells having a recombinant luciferase
reporter gene. In the control cell, e.g., no exogeneously added
RNA, the level of expression of the luciferase reporter is
normalized to be the value of "1". As illustrated, both long
(500-mer) and short (22-mer) dsRNA constructs complementary to the
luciferase gene could inhibit expression of that gene product
relative to the control cell. On the other hand, similarly sized
dsRNA complementary to the coding sequence for another protein,
green fluorescence protein (GFP), did not significantly effect the
expression of luciferase--indicating that the inhibitory phenomena
was in each case sequence-dependent. Likewise, single stranded
22-mers of luciferase did not inhibit expression of that
gene--indicating that the inhibitory phenomena is double
stranded-dependent.
[0114] The appended examples also identify an enzyme, Dicer, that
can produce the putative guide RNAs. Dicer is a member of the RNAse
III family of nucleases that specifically cleave dsRNA and is
evolutionarily conserved in worms, flies, plants, fungi and, as
described herein, mammals. The enzyme has a distinctive structure
which includes a helicase domain and dual RNAse III motifs. Dicer
also contains a region of homology to the RDE1/QDE2/ARGONAUTE
family, which have been genetically linked to RNAi in lower
eukaryotes. Indeed, activation of, or overexpression of Dicer may
be sufficient in many cases to permit RNA interference in otherwise
non-receptive cells, such as cultured eukaryotic cells, or
mammalian (non-oocytic) cells in culture or in whole organisms.
[0115] In certain embodiments, the cells can be treated with an
agent(s) that inhibits the general double-stranded RNA response(s)
by the host cells, such as may give rise to sequence-independent
apoptosis. For instance, the cells can be treated with agents that
inhibit the dsRNA-dependent protein kinase known as PKR (protein
kinase RNA-activated). Double stranded RNAs in mammalian cells
typically activate protein kinase PKR and lead to apoptosis. The
mechanism of action of PKR includes phosphorylation and
inactivation of eIF2.alpha. (Fire (1999) Trends Genet 15: 358). It
has also been reported that induction of NF-.kappa.B by PKR is
involved in apoptosis commitment and this process is mediated
through activation of the IKK complex. This sequence-independent
response may reflect a form of primitive immune response, since the
presence of dsRNA is a common feature of many viral lifecycles.
[0116] As described herein, Applicants have demonstrated that the
PKR response can be overcome in favor of the sequence-specific RNAi
response. However, in certain instances, it may be desirable to
treat the cells with agents which inhibit expression of PKR, cause
its destruction, and/or inhibit the kinase activity of PKR, and
such methods are specifically contemplated for use in the present
invention. Likewise, overexpression of agents which ectopically
activate eIF2.alpha. can be used. Other agents which can be used to
suppress the PKR response include inhibitors of I.kappa.B
phosphorylation of I.kappa.B, inhibitors of I.kappa.B
ubiquitination, inhibitors of I.kappa.B degradation, inhibitors of
NF-.kappa.B nuclear translocation, and inhibitors of NF-.kappa.B
interaction with .kappa.B response elements.
[0117] Other inhibitors of sequence-independent dsRNA response in
cells include the gene product of the vaccinia virus E3L. The E3L
gene product contains two distinct domains. A conserved
carboxy-terminal domain has been shown to bind double-stranded RNA
(dsRNA) and inhibit the antiviral dsRNA response by cells.
Expression of at least that portion of the E3L gene in the host
cell, or the use of polypeptide or peptidomimetics thereof, can be
used to suppress the general dsRNA response. Caspase inhibitors
sensitize cells to killing by double-stranded RNA. Accordingly,
ectopic expression or activation of caspases in the host cell can
be used to suppress the general dsRNA response.
[0118] In other embodiments, the subject method is carried out in
cells which have little or no general response to double stranded
RNA, e.g., have no PKR-dependent dsRNA response, at least under the
culture conditions. As illustrated in FIGS. 28-32, CHO and P19
cells can be used without having to inhibit PKR or other general
dsRNA responses.
[0119] Thus, the present invention provides a process and
compositions for inhibiting expression of a target gene in a cell,
especially a mammalian cell. In certain embodiments, the process
comprises introduction of RNA (the "dsRNA construct") with partial
or fully double-stranded character into the cell or into the
extracellular environment. Inhibition is specific in that a
nucleotide sequence from a portion of the target gene is chosen to
produce the dsRNA construct. The dsRNA may be identical or similar
to coding or non-coding sequence of the target gene. In preferred
embodiments, the method utilizes a cell in which Dicer and/or
Argonaute activities are recombinantly expressed or otherwise
ectopically activated. This process can be (1) effective in
attenuating gene expression, (2) specific to the targeted gene, and
(3) general in allowing inhibition of many different types of
target gene.
[0120] II. Definitions
[0121] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0122] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a genomic integrated vector,
or "integrated vector", which can become integrated into the
chromsomal DNA of the host cell. Another type of vector is an
episomal vector, i.e., a nucleic acid capable of extra-chromosomal
replication. Vectors capable of directing the expression of genes
to which they are operatively linked are referred to herein as
"expression vectors". In the present specification, "plasmid" and
"vector" are used interchangeably unless otherwise clear from the
context.
[0123] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as applicable to the embodiment being
described, single-stranded (such as sense or antisense) and
double-stranded polynucleotides.
[0124] As used herein, the term "gene" or "recombinant gene" refers
to a nucleic acid comprising an open reading frame encoding a
polypeptide of the present invention, including both exon and
(optionally) intron sequences. The nucleic acid may also optionally
include non-coding sequences such as promoter or enhancer
sequences. A "recombinant gene" refers to nucleic acid encoding
such regulatory polypeptides, that may optionally include intron
sequences that are derived from chromosomal DNA. The term "intron"
refers to a DNA sequence present in a given gene that is not
translated into protein and is generally found between exons.
[0125] A "protein coding sequence" or a sequence that "encodes" a
particular polypeptide or peptide, is a nucleic acid sequence that
is transcribed (in the case of DNA) and is translated (in the case
of mRNA) into a polypeptide in vitro or in vivo when placed under
the control of appropriate regulatory sequences. The boundaries of
the coding sequence are determined by a start codon at the 5'
(amino) terminus and a translation stop codon at the 3' (carboxy)
terminus. A coding sequence can include, but is not limited to,
cDNA from procaryotic or eukaryotic mRNA, genomic DNA sequences
from procaryotic or eukaryotic DNA, and even synthetic DNA
sequences. A transcription termination sequence will usually be
located 3' to the coding sequence.
[0126] Likewise, "encodes", unless evident from its context, will
be meant to include DNA sequences that encode a polypeptide, as the
term is typically used, as well as DNA sequences that are
transcribed into inhibitory antisense molecules.
[0127] The term "loss-of-function", as it refers to genes inhibited
by the subject RNAi method, refers to a diminishment in the level
of expression of a gene(s) in the presence of one or more dsRNA
construct(s) when compared to the level in the absense of such
dsRNA construct(s).
[0128] The term "expression" with respect to a gene sequence refers
to transcription of the gene and, as appropriate, translation of
the resulting mRNA transcript to a protein. Thus, as will be clear
from the context, expression of a protein coding sequence results
from transcription and translation of the coding sequence.
[0129] "Cells," "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0130] The term "cultured cells" refers to cells suspended in
culture, e.g., dispersed in culture or in the form tissue. It does
not, however, include oocytes or whole embryos (including
blastocysts and the like) which may be provided in culture. In
certain embodiments, the cultured cells are adults cells, e.g.,
non-embryonic.
[0131] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle.
[0132] As used herein, the terms "transduction" and "transfection"
are art recognized and mean the introduction of a nucleic acid,
e.g., an expression vector, into a recipient cell by nucleic
acid-mediated gene transfer. "Transformation", as used herein,
refers to a process in which a cell's genotype is changed as a
result of the cellular uptake of exogenous DNA or RNA, and, for
example, the transformed cell expresses a dsRNA contruct.
[0133] "Transient transfection" refers to cases where exogenous DNA
does not integrate into the genome of a transfected cell, e.g.,
where episomal DNA is transcribed into mRNA and translated into
protein.
[0134] A cell has been "stably transfected" with a nucleic acid
construct when the nucleic acid construct is capable of being
inherited by daughter cells.
[0135] As used herein, a "reporter gene construct" is a nucleic
acid that includes a "reporter gene" operatively linked to at least
one transcriptional regulatory sequence. Transcription of the
reporter gene is controlled by these sequences to which they are
linked. The activity of at least one or more of these control
sequences can be directly or indirectly regulated by the target
receptor protein. Exemplary transcriptional control sequences are
promoter sequences. A reporter gene is meant to include a
promoter-reporter gene construct that is heterologously expressed
in a cell.
[0136] As used herein, "transformed cells" refers to cells that
have spontaneously converted to a state of unrestrained growth,
i.e., they have acquired the ability to grow through an indefinite
number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or
hyperplastic, with respect to their loss of growth control. For
purposes of this invention, the terms "transformed phenotype of
malignant mammalian cells" and "transformed phenotype" are intended
to encompass, but not be limited to, any of the following
phenotypic traits associated with cellular transformation of
mammalian cells: immortalization, morphological or growth
transformation, and tumorigenicity, as detected by prolonged growth
in cell culture, growth in semi-solid media, or tumorigenic growth
in immuno-incompetent or syngeneic animals.
[0137] As used herein, "proliferating" and "proliferation" refer to
cells undergoing mitosis.
[0138] As used herein, "immortalized cells" refers to cells that
have been altered via chemical, genetic, and/or recombinant means
such that the cells have the ability to grow through an indefinite
number of divisions in culture.
[0139] The "growth state" of a cell refers to the rate of
proliferation of the cell and the state of differentiation of the
cell.
[0140] "MHC antigen", as used herein, refers to a protein product
of one or more MHC genes; the term includes fragments or analogs of
products of MHC genes which can evoke an immune response in a
recipient organism. Examples of MHC antigens include the products
(and fragments or analogs thereof) of the human MHC genes, i.e.,
the HLA genes.
[0141] The term "histocompatibility" refers to the similarity of
tissue between different individuals. The level of
histocompatibility describes how well matched the patient and donor
are. The major histocompatibility determinants are the human
leukocyte antigens (HLA). HLA typing is performed between the
potential marrow donor and the potential transplant recipient to
determine how close a HLA match the two are. The closer the match
the less the donated marrow and the patient's body will react
against each other.
[0142] The term "human leukocyte antigens" or "HLA", refers to
proteins (antigens) found on the surface of white blood cells and
other tissues that are used to match donor and patient. For
instances, a patient and potential donor may have their white blood
cells tested for such HLA antigens as, HLA-A, B and DR. Each
individual has two sets of these antigens, one set inherited from
each parent. For this reason, it is much more likely for a brother
or sister to match the patient than an unrelated individual, and
much more likely for persons of the same racial and ethnic
backgrounds to match each other.
[0143] III. Exemplary Embodiments of Isolation Method
[0144] One aspect of the invention provides a method for
potentiating RNAi by induction or ectopic activation of an RNAi
enzyme in a cell (in vivo or in vitro) or cell-free mixtures. In
preferred embodiments, the RNAi activity is activated or added to a
mammalian cell, e.g., a human cell, which cell may be provided in
vitro or as part of a whole organism. In other embodiments, the
subject method is carried out using eukaryotic cells generally
(except for oocytes) in culture. For instance, the Dicer enzyme may
be activated by virtue of being recombinantly expressed or it may
be activated by use of an agent which (i) induces expression of the
endogenous gene, (ii) stabilizes the protein from degradation,
and/or (iii) allosterically modifies the enzyme to increase its
activity (by altering its Kcat, Km or both).
[0145] A. Dicer and Argonaut Activities
[0146] In certain embodiments, at least one of the activated RNAi
enzymes is Dicer, or a homolog thereof. In certain preferred
embodiments, the present method provides for ectopic activation of
Dicer. As used herein, the term "Dicer" refers to a protein which
(a) mediates an RNAi response and (b) has an amino acid sequence at
least 50 percent identical, and more preferably at least 75, 85, 90
or 95 percent identical to SEQ ID No. 2 or 4, and/or which can be
encoded by a nucleic acid which hybridizes under wash conditions of
2.times.SSC at 22.degree. C., and more preferably 0.2.times.SSC at
65.degree. C., to a nucleotide represented by SEQ ID No. 1 or 3.
Accordingly, the method may comprise introducing a dsRNA contruct
into a cell in which Dicer has been recombinantly expressed or
otherwise ectopically activated.
[0147] In certain embodiment, at least one of the activated RNAi
enzymes is Argonaut, or a homolog thereof. In certain preferred
embodiments, the present method provides for ectopic activation of
Argonaut. As used herein, the term "Argonaut" refers to a protein
which (a) mediates an RNAi response and (b) has an amino acid
sequence at least 50 percent identical, and more preferably at
least 75, 85, 90 or 95 percent identical to the amino acid sequence
shown in FIG. 24. Accordingly, the method may comprise introducing
a dsRNA contruct into a cell in which Argonaut has been
recombinantly expressed or otherwise ectopically activated.
[0148] This invention also provides expression vectors containing a
nucleic acid encoding a Dicer or Argonaut polypeptide, operably
linked to at least one transcriptional regulatory sequence.
Operably linked is intended to mean that the nucleotide sequence is
linked to a regulatory sequence in a manner which allows expression
of the nucleotide sequence. Regulatory sequences are art-recognized
and are selected to direct expression of the subject Dicer or
Argonaut proteins. Accordingly, the term transcriptional regulatory
sequence includes promoters, enhancers and other expression control
elements. Such regulatory sequences are described in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). For instance, any of a wide variety of
expression control sequences, sequences that control the expression
of a DNA sequence when operatively linked to it, may be used in
these vectors to express DNA sequences encoding Dicer or Argonaut
polypeptides of this invention. Such useful expression control
sequences, include, for example, a viral LTR, such as the LTR of
the Moloney murine leukemia virus, the early and late promoters of
SV40, adenovirus or cytomegalovirus immediate early promoter, the
lac system, the trp system, the TAC or TRC system, T7 promoter
whose expression is directed by T7 RNA polymerase, the major
operator and promoter regions of phage .lambda., the control
regions for fd coat protein, the promoter for 3-phosphoglycerate
kinase or other glycolytic enzymes, the promoters of acid
phosphatase, e.g., Pho5, the promoters of the yeast .alpha.-mating
factors, the polyhedron promoter of the baculovirus system and
other sequences known to control the expression of genes of
prokaryotic or eukaryotic cells or their viruses, and various
combinations thereof. It should be understood that the design of
the expression vector may depend on such factors as the choice of
the host cell to be transformed and/or the type of protein desired
to be expressed.
[0149] Moreover, the vector's copy number, the ability to control
that copy number and the expression of any other proteins encoded
by the vector, such as antibiotic markers, should also be
considered.
[0150] The recombinant Dicer or Argonaut genes can be produced by
ligating a nucleic acid encoding a Dicer or Argonaut polypeptide
into a vector suitable for expression in either prokaryotic cells,
eukaryotic cells, or both. Expression vectors for production of
recombinant forms of the subject Dicer or Argonaut polypeptides
include plasmids and other vectors. For instance, suitable vectors
for the expression of a Dicer or Argonaut polypeptide include
plasmids of the types: pBR322-derived plasmids, pEMBL-derived
plasmids, pEX-derived plasmids, pBTac-derived plasmids and
pUC-derived plasmids for expression in prokaryotic cells, such as
E. coli.
[0151] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2,
and YRP17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used. In an
illustrative embodiment, a Dicer or Argonaut polypeptide is
produced recombinantly utilizing an expression vector generated by
sub-cloning the coding sequence of a Dicer or Argonaut gene.
[0152] The preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989) Chapters 16 and 17.
[0153] In yet another embodiment, the subject invention provides a
"gene activation" construct which, by homologous recombination with
a genomic DNA, alters the transcriptional regulatory sequences of
an endogenous Dicer or Argonaut gene. For instance, the gene
activation construct can replace the endogenous promoter of a Dicer
or Argonaut gene with a heterologous promoter, e.g., one which
causes constitutive expression of the Dicer or Argonaut gene or
which causes inducible expression of the gene under conditions
different from the normal expression pattern of Dicer or Argonaut.
A variety of different formats for the gene activation constructs
are available. See, for example, the Transkaryotic Therapies, Inc
PCT publications WO93/09222, WO95/31560, WO96/29411, WO95/31560 and
WO94/12650.
[0154] In preferred embodiments, the nucleotide sequence used as
the gene activation construct can be comprised of (1) DNA from some
portion of the endogenous Dicer or Argonaut gene (exon sequence,
intron sequence, promoter sequences, etc.) which direct
recombination and (2) heterologous transcriptional regulatory
sequence(s) which is to be operably linked to the coding sequence
for the genomic Dicer or Argonaut gene upon recombination of the
gene activation construct. For use in generating cultures of Dicer
or Argonaut producing cells, the construct may further include a
reporter gene to detect the presence of the knockout construct in
the cell.
[0155] The gene activation construct is inserted into a cell, and
integrates with the genomic DNA of the cell in such a position so
as to provide the heterologous regulatory sequences in operative
association with the native Dicer or Argonaut gene. Such insertion
occurs by homologous recombination, i.e., recombination regions of
the activation construct that are homologous to the endogenous
Dicer or Argonaut gene sequence hybridize to the genomic DNA and
recombine with the genomic sequences so that the construct is
incorporated into the corresponding position of the genomic
DNA.
[0156] The terms "recombination region" or "targeting sequence"
refer to a segment (i.e., a portion) of a gene activation construct
having a sequence that is substantially identical to or
substantially complementary to a genomic gene sequence, e.g.,
including 5' flanking sequences of the genomic gene, and can
facilitate homologous recombination between the genomic sequence
and the targeting transgene construct.
[0157] As used herein, the term "replacement region" refers to a
portion of a activation construct which becomes integrated into an
endogenous chromosomal location following homologous recombination
between a recombination region and a genomic sequence.
[0158] The heterologous regulatory sequences, e.g., which are
provided in the replacement region, can include one or more of a
variety of elements, including: promoters (such as constitutive or
inducible promoters), enhancers, negative regulatory elements,
locus control regions, transcription factor binding sites, or
combinations thereof.
[0159] Promoters/enhancers which may be used to control the
expression of the targeted gene in vivo include, but are not
limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama
et al. (1989) J. Exp. Med 169: 13), the human .beta.-actin promoter
(Gunning et al. (1987) PNAS 84: 4831-4835), the
glucocorticoid-inducible promoter present in the mouse mammary
tumor virus long terminal repeat (MMTV LTR) (Klessig et al. (1984)
Mol. Cell Biol. 4: 1354-1362), the long terminal repeat sequences
of Moloney murine leukemia virus (MuLV LTR) (Weiss et al. (1985)
RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.), the SV40 early or late region promoter (Bemoist et
al. (1981) Nature 290: 304-310; Templeton et al. (1984) Mol. Cell
Biol. 4: 817; and Sprague et al. (1983) J. Virol. 45: 773), the
promoter contained in the 3' long terminal repeat of Rous sarcoma
virus (RSV) (Yamamoto et al. (1980) Cell 22: 787-797), the herpes
simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner et
al. (1981) PNAS 82: 3567-71), and the herpes simplex virus LAT
promoter (Wolfe et al. (1992) Nature Genetics 1: 379-384).
[0160] In still other embodiments, the replacement region merely
deletes a negative transcriptional control element of the native
gene, e.g., to activate expression, or ablates a positive control
element, e.g., to inhibit expression of the targeted gene.
[0161] B. Cell/Organism
[0162] The cell with the target gene may be derived from or
contained in any organism (e.g., plant, animal, protozoan, virus,
bacterium, or fungus). The dsRNA construct may be synthesized
either in vivo or in vitro. Endogenous RNA polymerase of the cell
may mediate transcription in vivo, or cloned RNA polymerase can be
used for transcription in vivo or in vitro. For generating double
stranded transcripts from a transgene in vivo, a regulatory region
may be used to transcribe the RNA strand (or strands). Furthermore,
dsRNA can be generated by transcribing an RNA strand which forms a
hairpin, thus producing a dsRNA.
[0163] Genetic manipulation becomes possible in organisms that are
not classical genetic models. Breeding and screening programs may
be accelerated by the ability to rapidly assay the consequences of
a specific, targeted gene disruption. Gene disruptions may be used
to discover the function of the target gene, to produce disease
models in which the target gene are involved in causing or
preventing a pathological condition, and to produce organisms with
improved economic properties.
[0164] The cell with the target gene may be derived from or
contained in any organism. The organism may be a plant, animal,
protozoan, bacterium, virus, or fungus. The plant may be a monocot,
dicot or gymnosperm; the animal may be a vertebrate or
invertebrate. Preferred microbes are those used in agriculture or
by industry, and those that are pathogenic for plants or animals.
Fungi include organisms in both the mold and yeast
morphologies.
[0165] Plants include arabidopsis; field crops (e.g., alfalfa,
barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower,
sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops
(e.g., asparagus, beet, broccoli, cabbage, carrot, cauliflower,
celery, cucumber, eggplant, lettuce, onion, pepper, potato,
pumpkin, radish, spinach, squash, taro, tomato, and zucchini);
fruit and nut crops (e.g., almond, apple, apricot, banana,
blackberry, blueberry, cacao, cherry, coconut, cranberry, date,
fajoa, filbert, grape, grapefruit, guava, kiwi, lemon, lime, mango,
melon, nectarine, orange, papaya, passion fruit, peach, peanut,
pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine,
walnut, and watermelon); and ornamentals (e.g., alder, ash, aspen,
azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm,
fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,
rhododendron, rose, and rubber).
[0166] Examples of vertebrate animals include fish, mammal, cattle,
goat, pig, sheep, rodent, hamster, mouse, rat, primate, and
human.
[0167] Invertebrate animals include nematodes, other worms,
drosophila, and other insects. Representative generae of nematodes
include those that infect animals (e.g., Ancylostoma, Ascaridia,
Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia,
Cooperia, Dictyocaulus, Haernonchus, Heterakis, Nematodirus,
Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus,
Toxascaris, Trichuris, Trichostrongylus, Tflichonema, Toxocara,
Uncinaria) and those that infect plants (e.g., Bursaphalenchus,
Criconerriella, Diiylenchus, Ditylenchus, Globodera,
Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus,
Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus,
and Xiphinerna). Representative orders of insects include
Coleoptera, Diptera, Lepidoptera, and Homoptera.
[0168] The cell having the target gene may be from the germ line or
somatic, totipotent or pluripotent, dividing or non-dividing,
parenchyma or epithelium, immortalized or transformed, or the like.
The cell may be a stem cell or a differentiated cell. Cell types
that are differentiated include adipocytes, fibroblasts, myocytes,
cardiomyocytes, endothelium, neurons, glia, blood cells,
megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils,
basophils, mast cells, leukocytes, granulocytes, keratinocytes,
chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of
the endocrine or exocrine glands.
[0169] C. Targeted Genes
[0170] The target gene may be a gene derived from the cell, an
endogenous gene, a transgene, or a gene of a pathogen which is
present in the cell after infection thereof. Depending on the
particular target gene and the dose of double stranded RNA material
delivered, the procedure may provide partial or complete loss of
function for the target gene. Lower doses of injected material and
longer times after administration of dsRNA may result in inhibition
in a smaller fraction of cells. Quantitation of gene expression in
a cell may show similar amounts of inhibition at the level of
accumulation of target mRNA or translation of target protein.
[0171] "Inhibition of gene expression" refers to the absence (or
observable decrease) in the level of protein and/or mRNA product
from a target gene. "Specificity" refers to the ability to inhibit
the target gene without manifest effects on other genes of the
cell. The consequences of inhibition can be confirmed by
examination of the outward properties of the cell or organism (as
presented below in the examples) or by biochemical techniques such
as RNA solution hybridization, nuclease protection, Northern
hybridization, reverse transcription, gene expression monitoring
with a microarray, antibody binding, enzyme linked immunosorbent
assay (ELISA), Western blotting, radioimmunoassay (RIA), other
immunoassays, and fluorescence activated cell analysis (FACS). For
RNA-mediated inhibition in a cell line or whole organism, gene
expression is conveniently assayed by use of a reporter or drug
resistance gene whose protein product is easily assayed. Such
reporter genes include acetohydroxyacid synthase (AHAS), alkaline
phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase
(GUS), chloramphenicol acetyltransferase (CAT), green fluorescent
protein (GFP), horseradish peroxidase (HRP), luciferase (Luc),
nopaline synthase (NOS), octopine synthase (OCS), and derivatives
thereof. Multiple selectable markers are available that confer
resistance to ampicillin, bleomycin, chloramphenicol, gentamycin,
hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,
puromycin, and tetracyclin.
[0172] Depending on the assay, quantitation of the amount of gene
expression allows one to determine a degree of inhibition which is
greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell
not treated according to the present invention. Lower doses of
injected material and longer times after administration of dsRNA
may result in inhibition in a smaller fraction of cells (e.g., at
least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells).
Quantitation of gene expression in a cell may show similar amounts
of inhibition at the level of accumulation of target mRNA or
translation of target protein. As an example, the efficiency of
inhibition may be determined by assessing the amount of gene
product in the cell: mRNA may be detected with a hybridization
probe having a nucleotide sequence outside the region used for the
inhibitory double-stranded RNA, or translated polypeptide may be
detected with an antibody raised against the polypeptide sequence
of that region.
[0173] As disclosed herein, the present invention is not limited to
any type of target gene or nucleotide sequence. But the following
classes of possible target genes are listed for illustrative
purposes: developmental genes (e.g., adhesion molecules, cyclin
kinase inhibitors, Writ family members, Pax family members, Winged
helix family members, Hox family members, cytokines/lymphokines and
their receptors, growth/differentiation factors and their
receptors, neurotransmitters and their receptors); oncogenes (e.g.,
ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI,
ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2,
MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI, TCL3,
and YES); tumor suppressor genes (e.g., APC, BRCA 1, BRCA2, MADH4,
MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC
synthases and oxidases, ACP desaturases and hydroxylases,
ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases,
amylases, amyloglucosidases, catalases, cellulases, chalcone
synthases, chitinases, cyclooxygenases, decarboxylases,
dextrinases, DNA and RNA polymerases, galactosidases, glucanases,
glucose oxidases, granule-bound starch synthases, GTPases,
helicases, hemicellulases, integrases, inulinases, invertases,
isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,
nopaline synthases, octopine synthases, pectinesterases,
peroxidases, phosphatases, phospholipases, phosphorylases,
phytases, plant growth regulator synthases, polygalacturonases,
proteinases and peptidases, pullanases, recombinases, reverse
transcriptases, RUBISCOs, topoisomerases, and xylanases).
[0174] D. dsRNA Constructs
[0175] The dsRNA construct may comprise one or more strands of
polymerized ribonucleotide. It may include modifications to either
the phosphate-sugar backbone or the nucleoside. For example, the
phosphodiester linkages of natural RNA may be modified to include
at least one of a nitrogen or sulfur heteroatom. Modifications in
RNA structure may be tailored to allow specific genetic inhibition
while avoiding a general panic response in some organisms which is
generated by dsRNA. Likewise, bases may be modified to block the
activity of adenosine deaminase. The dsRNA construct may be
produced enzymatically or by partial/total organic synthesis, any
modified ribonucleotide can be introduced by in vitro enzymatic or
organic synthesis.
[0176] The dsRNA construct may be directly introduced into the cell
(i.e., intracellularly); or introduced extracellularly into a
cavity, interstitial space, into the circulation of an organism,
introduced orally, or may be introduced by bathing an organism in a
solution containing RNA. Methods for oral introduction include
direct mixing of RNA with food of the organism, as well as
engineered approaches in which a species that is used as food is
engineered to express an RNA, then fed to the organism to be
affected. Physical methods of introducing nucleic acids include
injection of an RNA solution directly into the cell or
extracellular injection into the organism.
[0177] The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the
cell. The RNA may be introduced in an amount which allows delivery
of at least one copy per cell. Higher doses (e.g., at least 5, 10,
100, 500 or 1000 copies per cell) of double-stranded material may
yield more effective inhibition; lower doses may also be useful for
specific applications. Inhibition is sequence-specific in that
nucleotide sequences corresponding to the duplex region of the RNA
are targeted for genetic inhibition.
[0178] dsRNA constructs containing a nucleotide sequences identical
to a portion, of either coding or non-coding sequence, of the
target gene are preferred for inhibition. RNA sequences with
insertions, deletions, and single point mutations relative to the
target sequence (ds RNA similar to the target gene) have also been
found to be effective for inhibition. Thus, sequence identity may
be optimized by sequence comparison and alignment algorithms known
in the art (see Gribskov and Devereux, Sequence Analysis Primer,
Stockton Press, 1991, and references cited therein) and calculating
the percent difference between the nucleotide sequences by, for
example, the Smith-Waterman algorithm as implemented in the BESTFIT
software program using default parameters (e.g., University of
Wisconsin Genetic Computing Group). Greater than 90% sequence
identity, or even 100% sequence identity, between the inhibitory
RNA and the portion of the target gene is preferred. Alternatively,
the duplex region of the RNA may be defined functionally as a
nucleotide sequence that is capable of hybridizing with a portion
of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH
6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. hybridization for
12-16 hours; followed by washing). In certain preferred
embodiments, the length of the dsRNA is at least 20, 21 or 22
nucleotides in length, e.g., corresponding in size to RNA products
produced by Dicer-dependent cleavage. In certain embodiments, the
dsRNA construct is at least 25, 50, 100, 200, 300 or 400 bases. In
certain embodiments, the dsRNA construct is 400-800 bases in
length.
[0179] 100% sequence identity between the RNA and the target gene
is not required to practice the present invention. Thus the
invention has the advantage of being able to tolerate sequence
variations that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence.
[0180] The dsRNA construct may be synthesized either in vivo or in
vitro. Endogenous RNA polymerase of the cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vivo or in vitro. For transcription from a
transgene in vivo or an expression construct, a regulatory region
(e.g., promoter, enhancer, silencer, splice donor and acceptor,
polyadenylation) may be used to transcribe the dsRNA strand (or
strands). Inhibition may be targeted by specific transcription in
an organ, tissue, or cell type; stimulation of an environmental
condition (e.g., infection, stress, temperature, chemical
inducers); and/or engineering transcription at a developmental
stage or age. The RNA strands may or may not be polyadenylated; the
RNA strands may or may not be capable of being translated into a
polypeptide by a cell's translational apparatus. The dsRNA
construct may be chemically or enzymatically synthesized by manual
or automated reactions. The dsRNA construct may be synthesized by a
cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,
T3, T7, SP6). The use and production of an expression construct are
known in the art (see also WO 97/32016; U.S. Pat. Nos. 5,593,874,
5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the references
cited therein). If synthesized chemically or by in vitro enzymatic
synthesis, the RNA may be purified prior to introduction into the
cell. For example, RNA can be purified from a mixture by extraction
with a solvent or resin, precipitation, electrophoresis,
chromatography or a combination thereof. Alternatively, the dsRNA
construct may be used with no or a minimum of purification to avoid
losses due to sample processing. The dsRNA construct may be dried
for storage or dissolved in an aqueous solution. The solution may
contain buffers or salts to promote annealing, and/or stabilization
of the duplex strands.
[0181] Physical methods of introducing nucleic acids include
injection of a solution containing the dsRNA construct, bombardment
by particles covered by the dsRNA construct, soaking the cell or
organism in a solution of the RNA, or electroporation of cell
membranes in the presence of the dsRNA construct. A viral construct
packaged into a viral particle would accomplish both efficient
introduction of an expression construct into the cell and
transcription of dsRNA construct encoded by the expression
construct. Other methods known in the art for introducing nucleic
acids to cells may be used, such as lipid-mediated carrier
transport, chemical mediated transport, such as calcium phosphate,
and the like. Thus the dsRNA construct may be introduced along with
components that perform one or more of the following activities:
enhance RNA uptake by the cell, promote annealing of the duplex
strands, stabilize the annealed strands, or other-wise increase
inhibition of the target gene.
[0182] E. Illustrative Uses
[0183] One utility of the present invention is as a method of
identifying gene function in an organism, especially higher
eukaryotes, comprising the use of double-stranded RNA to inhibit
the activity of a target gene of previously unknown function.
Instead of the time consuming and laborious isolation of mutants by
traditional genetic screening, functional genomics would envision
determining the function of uncharacterized genes by employing the
invention to reduce the amount and/or alter the timing of target
gene activity. The invention could be used in determining potential
targets for pharmaceuticals, understanding normal and pathological
events associated with development, determining signaling pathways
responsible for postnatal development/aging, and the like. The
increasing speed of acquiring nucleotide sequence information from
genomic and expressed gene sources, including total sequences for
mammalian genomes, can be coupled with the invention to determine
gene function in a cell or in a whole organism. The preference of
different organisms to use particular codons, searching sequence
databases for related gene products, correlating the linkage map of
genetic traits with the physical map from which the nucleotide
sequences are derived, and artificial intelligence methods may be
used to define putative open reading frames from the nucleotide
sequences acquired in such sequencing projects.
[0184] A simple assay would be to inhibit gene expression according
to the partial sequence available from an expressed sequence tag
(EST). Functional alterations in growth, development, metabolism,
disease resistance, or other biological processes would be
indicative of the normal role of the EST's gene product.
[0185] The ease with which the dsRNA construct can be introduced
into an intact cell/organism containing the target gene allows the
present invention to be used in high throughput screening (HTS).
For example, duplex RNA can be produced by an amplification
reaction using primers flanking the inserts of any gene library
derived from the target cell or organism. Inserts may be derived
from genomic DNA or mRNA (e.g., cDNA and cRNA). Individual clones
from the library can be replicated and then isolated in separate
reactions, but preferably the library is maintained in individual
reaction vessels (e.g., a 96 well microtiter plate) to minimize the
number of steps required to practice the invention and to allow
automation of the process.
[0186] In an exemplary embodiment, the subject invention provides
an arrayed library of RNAi constructs. The array may be in the form
of solutions, such as multi-well plates, or may be "printed" on
solid substrates upon which cells can be grown. To illustrate,
solutions containing duplex RNAs that are capable of inhibiting the
different expressed genes can be placed into individual wells
positioned on a microtiter plate as an ordered array, and intact
cells/organisms in each well can be assayed for any changes or
modifications in behavior or development due to inhibition of
target gene activity.
[0187] In one embodiment, the subject method uses an arrayed
library of RNAi constructs to screen for combinations of RNAi that
are lethal to host cells. Synthetic lethality is a bedrock
principle of experimental genetics. A synthetic lethality describes
the properties of two mutations which, individually, are tolerated
by the organism but which, in combination, are lethal. The subject
arrays can be used to identify loss-of-function mutations that are
lethal in combination with alterations in other genes, such as
activated oncogenes or loss-of-function mutations to tumor
suppressors. To achieve this, one can create "phenotype arrays"
using cultured cells. Expression of each of a set of genes, such as
the host cell's genome, can be individually systematically
disrupted using RNA interference. Combination with alterations in
oncogene and tumor suppressor pathways can be used to identify
synthetic lethal interactions that may identify novel therapeutic
targets.
[0188] In certain embodiments, the RNAi constructs can be fed
directly to, or injected into, the cell/organism containing the
target gene. Alternatively, the duplex RNA can be produced by in
vivo or in vitro transcription from an expression construct used to
produce the library. The construct can be replicated as individual
clones of the library and transcribed to produce the RNA; each
clone can then be fed to, injected into, or delivered by another
method known in the art to the cell/organism containing the target
gene. The function of the target gene can be assayed from the
effects it has on the cell/organism when gene activity is
inhibited. This screening could be amenable to small subjects that
can be processed in large number, for example, tissue culture cells
derived from mammals, especially primates, and most preferably
humans.
[0189] If a characteristic of an organism is determined to be
genetically linked to a polymorphism through RFLP or QTL analysis,
the present invention can be used to gain insight regarding whether
that genetic polymorphism might be directly responsible for the
characteristic. For example, a fragment defining the genetic
polymorphism or sequences in the vicinity of such a genetic
polymorphism can be amplified to produce an RNA, the duplex RNA can
be introduced to the organism or cell, and whether an alteration in
the characteristic is correlated with inhibition can be determined.
Of course, there may be trivial explanations for negative results
with this type of assay, for example: inhibition of the target gene
causes lethality, inhibition of the target gene may not result in
any observable alteration, the fragment contains nucleotide
sequences that are not capable of inhibiting the target gene, or
the target gene's activity is redundant.
[0190] The present invention may be useful in allowing the
inhibition of essential genes. Such genes may be required for cell
or organism viability at only particular stages of development or
only in specific cellular compartments or tissues. The functional
equivalent of conditional mutations may be produced by inhibiting
activity of the target gene when or where it is not required for
viability. The invention allows addition of RNA at specific times
of development and locations in the organism without introducing
permanent mutations into the target genome.
[0191] The present invention may be useful in allowing the
inhibition of genes that have been difficult to inhibit using other
methods due to gene redundancy. Since the present methods may be
used to deliver more than one dsRNA to a cell or organism, dsRNA
identical or similar to more than one gene, wherein the genes have
a redundant function during normal development, may be
delivered.
[0192] If alternative splicing produced a family of transcripts
that were distinguished by usage of characteristic exons, the
present invention can target inhibition through the appropriate
exons to specifically inhibit or to distinguish among the functions
of family members. For example, a protein factor that contained an
alternatively spliced transmembrane domain may be expressed in both
membrane bound and secreted forms. Instead of isolating a nonsense
mutation that terminates translation before the transmembrane
domain, the functional consequences of having only secreted hormone
can be determined according to the invention by targeting the exon
containing the transmembrane domain and thereby inhibiting
expression of membrane-bound hormone. That is, the subject method
can be used for selected ablation of splicing variants.
[0193] The present invention may be used alone or as a component of
a kit having at least one of the reagents necessary to carry out
the in vitro or in vivo introduction of RNA to test samples or
subjects. Preferred components are the dsRNA and a vehicle that
promotes introduction of the dsRNA. Such a kit may also include
instructions to allow a user of the kit to practice the
invention.
[0194] Alternatively, an organism may be engineered to produce
dsRNA which produces commercially or medically beneficial results,
for example, resistance to a pathogen or its pathogenic effects,
improved growth, or novel developmental patterns.
IV. Exemplification
[0195] The invention, now being generally described, will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention and are not intended to
limit the invention.
EXAMPLE 1
An RNA-Directed Nuclease Mediates RNAi Gene Silencing
[0196] In a diverse group of organisms that includes Caenorhabditis
elegans, Drosophila, planaria, hydra, trypanosomes, fungi and
plants, the introduction of double-stranded RNAs inhibits gene
expression in a sequence-specific manner (Sharp (1999) Genes and
Development 13: 139-141; Sanchez-Alvarado and Newmark (1999) PNAS
96: 5049-5054; Lohman et al. (1999) Developmental Biology 214:
211-214; Cogoni and Macino (1999) Nature 399: 166-169; Waterhouse
et al. (1998) PNAS 95: 13959-13964; Montgomery and Fire (1998)
Trends Genet. 14: 225-228; Ngo et al. (1998) PNAS 95: 14687-14692).
These responses, called RNA interference or post-transcriptional
gene silencing, may provide anti-viral defence, modulate
transposition or regulate gene expression (Sharp (1999) Genes and
Development 13: 139-141; Montgomery and Fire (1998) Trends Genet.
14: 225-228; Tabara et al. (1999) Cell 99: 123-132; Ketting et al.
(1999) Cell 99: 133-141; Ratcliff et al. (1997) Science 276:
1558-1560). We have taken a biochemical approach towards
elucidating the mechanisms underlying this genetic phenomenon. Here
we show that `loss-of-function` phenotypes can be created in
cultured Drosophila cells by transfection with specific
double-stranded RNAs. This coincides with a marked reduction in the
level of cognate cellular messenger RNAs. Extracts of transfected
cells contain a nuclease activity that specifically degrades
exogenous transcripts homologous to transfected double-stranded
RNA. This enzyme contains an essential RNA component. After partial
purification, the sequence-specific nuclease co-fractionates with a
discrete, .about.25-nucleotide RNA species which may confer
specificity to the enzyme through homology to the substrate
mRNAs.
[0197] Although double-stranded RNAs (dsRNAs) can provoke gene
silencing in numerous biological contexts including Drosophila
(Kennerdell et al. (1998) Cell 95: 1017-1026; Misquitta and
Paterson (1999) PNAS 96: 1451-1456), the mechanisms underlying this
phenomenon have remained mostly unknown. We therefore wanted to
establish a biochemically tractable model in which such mechanisms
could be investigated.
[0198] Transient transfection of cultured, Drosophila S2 cells with
a lacZ expression vector resulted in .beta.-galactosidase activity
that was easily detectable by an in situ assay (FIG. 1a). This
activity was greatly reduced by co-transfection with a dsRNA
corresponding to the first 300 nucleotides of the lacZ sequence,
whereas co-transfection with a control dsRNA (CD8) (FIG. 1a) or
with single-stranded RNAs of either sense or antisense orientation
(data not shown) had little or no effect. This indicated that
dsRNAs could interfere, in a sequence-specific fashion, with gene
expression in cultured cells.
[0199] To determine whether RNA interference (RNAi) could be used
to target endogenous genes, we transfected S2 cells with a dsRNA
corresponding to the first 540 nucleotides of Drosophila cyclin E,
a gene that is essential for progression into S phase of the cell
cycle. During log-phase growth, untreated S2 cells reside primarily
in G2/M (FIG. 1b). Transfection with lacZ dsRNA had no effect on
cell-cycle distribution, but transfection with the cyclin E dsRNA
caused a G1-phase cell-cycle arrest (FIG. 1b). The ability of
cyclin E dsRNA to provoke this response was length-dependent.
Double-stranded RNAs of 540 and 400 nucleotides were quite
effective, whereas dsRNAs of 200 and 300 nucleotides were less
potent. Double-stranded cyclin E RNAs of 50 or 100 nucleotides were
inert in our assay, and transfection with a single-stranded,
antisense cyclin E RNA had virtually no effect.
[0200] One hallmark of RNAi is a reduction in the level of mRNAs
that are homologous to the dsRNA. Cells transfected with the cyclin
E dsRNA (bulk population) showed diminished endogenous cyclin E
mRNA as compared with control cells (FIG. 1c). Similarly,
transfection of cells with dsRNAs homologous to fizzy, a component
of the anaphase-promoting complex (APC) or cyclin A, a cyclin that
acts in S, G2 and M, also caused reduction of their cognate mRNAs
(FIG. 1c). The modest reduction in fizzy mRNA levels in cells
transfected with cyclin A dsRNA probably resulted from arrest at a
point in the division cycle at which fizzy transcription is low
(Wolf and Jackson (1998) Current Biology 8: R637-R639; Kramer et
al. (1998) Current Biology 8: 1207-1210). These results indicate
that RNAi may be a generally applicable method for probing gene
function in cultured Drosophila cells.
[0201] The decrease in mRNA levels observed upon transfection of
specific dsRNAs into Drosophila cells could be explained by effects
at transcriptional or post-transcriptional levels. Data from other
systems have indicated that some elements of the dsRNA response may
affect mRNA directly (reviewed in Sharp (1999) Genes and
Development 13: 139-141; Montgomery and Fire (1998) Trends Genet.
14: 225-228). We therefore sought to develop a cell-free assay that
reflected, at least in part, RNAi.
[0202] S2 cells were transfected with dsRNAs corresponding to
either cyclin E or lacZ. Cellular extracts were incubated with
synthetic mRNAs of lacZ or cyclin E. Extracts prepared from cells
transfected with the 540-nucleotide cyclin E dsRNA efficiently
degraded the cyclin E transcript; however, the lacZ transcript was
stable in these lysates (FIG. 2a). Conversely, lysates from cells
transfected with the lacZ dsRNA degraded the lacZ transcript but
left the cyclin E mRNA intact. These results indicate that RNAi
ablates target mRNAs through the generation of a sequence-specific
nuclease activity. We have termed this enzyme RISC (RNA-induced
silencing complex). Although we occasionally observed possible
intermediates in the degradation process (see FIG. 2), the absence
of stable cleavage end-products indicates an exonuclease (perhaps
coupled to an endonuclease). However, it is possible that the RNAi
nuclease makes an initial endonucleolytic cut and that non-specific
exonucleases in the extract complete the degradation process
(Shuttleworth and Colman (1988) EMBO J. 7: 427-434). In addition,
our ability to create an extract that targets lacZ in vitro
indicates that the presence of an endogenous gene is not required
for the RNAi response.
[0203] To examine the substrate requirements for the dsRNA-induced,
sequence-specific nuclease activity, we incubated a variety of
cyclin-E-derived transcripts with an extract derived from cells
that had been transfected with the 540-nucleotide cyclin E dsRNA
(FIG. 2b, c). Just as a length requirement was observed for the
transfected dsRNA, the RNAi nuclease activity showed a dependence
on the size of the RNA substrate. Both a 600-nucleotide transcript
that extends slightly beyond the targeted region (FIG. 2b) and an
.about.1-kilobase (kb) transcript that contains the entire coding
sequence (data not shown) were completely destroyed by the extract.
Surprisingly, shorter substrates were not degraded as efficiently.
Reduced activity was observed against either a 300- or a
220-nucleotide transcript, and a .about.100-nucleotide transcript
was resistant to nuclease in our assay. This was not due solely to
position effects because .about.100-nucleotide transcripts derived
from other portions of the transfected dsRNA behaved similarly
(data not shown). As expected, the nuclease activity (or
activities) present in the extract could also recognize the
antisense strand of the cyclin E mRNA. Again, substrates that
contained a substantial portion of the targeted region were
degraded efficiently whereas those that contained a shorter stretch
of homologous sequence (.about.130 nucleotides) were recognized
inefficiently (FIG. 2c, as600). For both the sense and antisense
strands, transcripts that had no homology with the transfected
dsRNA (FIG. 2b, Eout; FIG. 2c, as300) were not degraded. Although
we cannot exclude the possibility that nuclease specificity could
have migrated beyond the targeted region, the resistance of
transcripts that do not contain homology to the dsRNA is consistent
with data from C. elegans. Double-stranded RNAs homologous to an
upstream cistron have little or no effect on a linked downstream
cistron, despite the fact that unprocessed, polycistronic mRNAs can
be readily detected (Tabara et al. (1998) Science 282: 430-432;
Bosher et al. (1999) Genetics 153: 1245-1256). Furthermore, the
nuclease was inactive against a dsRNA identical to that used to
provoke the RNAi response in vivo (FIG. 2b). In the in vitro
system, neither a 5' cap nor a poly(A) tail was required, as such
transcripts were degraded as efficiently as uncapped and
non-polyadenylated RNAs.
[0204] Gene silencing provoked by dsRNA is sequence specific. A
plausible mechanism for determining specificity would be
incorporation of nucleic-acid guide sequences into the complexes
that accomplish silencing (Hamilton and Baulcombe (1999) Science
286: 950-952). In accord with this idea, pre-treatment of extracts
with a Ca.sup.2+-dependent nuclease (micrococcal nuclease)
abolished the ability of these extracts to degrade cognate mRNAs
(FIG. 3). Activity could not be rescued by addition of non-specific
RNAs such as yeast transfer RNA. Although micrococcal nuclease can
degrade both DNA and RNA, treatment of the extract with DNAse I had
no effect (FIG. 3). Sequence-specific nuclease activity, however,
did require protein (data not shown). Together, our results support
the possibility that the RNAi nuclease is a ribonucleoprotein,
requiring both RNA and protein components. Biochemical
fractionation (see below) is consistent with these components being
associated in extract rather than being assembled on the target
mRNA after its addition.
[0205] In plants, the phenomenon of co-suppression has been
associated with the existence of small (.about.25-nucleotide) RNAs
that correspond to the gene that is being silenced (Hamilton and
Baulcombe (1999) Science 286: 950-952). To address the possibility
that a similar RNA might exist in Drosophila and guide the
sequence-specific nuclease in the choice of substrate, we partially
purified our activity through several fractionation steps. Crude
extracts contained both sequence-specific nuclease activity and
abundant, heterogeneous RNAs homologous to the transfected dsRNA
(FIGS. 2 and 4a). The RNAi nuclease fractionated with ribosomes in
a high-speed centrifugation step. Activity could be extracted by
treatment with high salt, and ribosomes could be removed by an
additional centrifugation step. Chromatography of soluble nuclease
over an anion-exchange column resulted in a discrete peak of
activity (FIG. 4b, cyclin E). This retained specificity as it was
inactive against a heterologous mRNA (FIG. 4b, lacZ). Active
fractions also contained an RNA species of 25 nucleotides that is
homologous to the cyclin E target (FIG. 4b, northern). The band
observed on northern blots may represent a family of discrete RNAs
because it could be detected with probes specific for both the
sense and antisense cyclin E sequences and with probes derived from
distinct segments of the dsRNA (data not shown). At present, we
cannot determine whether the 25-nucleotide RNA is present in the
nuclease complex in a double-stranded or single-stranded form.
[0206] RNA interference allows an adaptive defence against both
exogenous and endogenous dsRNAs, providing something akin to a
dsRNA immune response. Our data, and that of others (Hamilton and
Baulcombe (1999) Science 286: 950-952), is consistent with a model
in which dsRNAs present in a cell are converted, either through
processing or replication, into small specificity determinants of
discrete size in a manner analogous to antigen processing. Our
results suggest that the post-transcriptional component of
dsRNA-dependent gene silencing is accomplished by a
sequence-specific nuclease that incorporates these small RNAs as
guides that target specific messages based upon sequence
recognition. The identical size of putative specificity
determinants in plants (Hamilton and Baulcombe (1999) Science 286:
950-952) and animals predicts a conservation of both the mechanisms
and the components of dsRNA-induced, post-transcriptional gene
silencing in diverse organisms. In plants, dsRNAs provoke not only
post-transcriptional gene silencing but also chromatin remodelling
and transcriptional repression (Jones et al. (1998) EMBO J. 17:
6385-6393; Jones et al. (1999) Plant Cell 11: 2291-2301). It is now
critical to determine whether conservation of gene-silencing
mechanisms also exists at the transcriptional level and whether
chromatin remodelling can be directed in a sequence-specific
fashion by these same dsRNA-derived guide sequences.
[0207] Methods:
[0208] Cell culture and RNA methods S2 cells (Schneider (1972) J.
Embryol Exp Morpho 27: 353-365) were cultured at 27.degree. C. in
90% Schneider's insect media (Sigma), 10% heat inactivated fetal
bovine serum (FBS). Cells were transfected with dsRNA and plasmid
DNA by calcium phosphate co-precipitation (DiNocera and Dawid
(1983) PNAS 80: 7095-7098). Identical results were observed when
cells were transfected using lipid reagents (for example,
Superfect, Qiagen). For FACS analysis, cells were additionally
transfected with a vector that directs expression of a green
fluorescent protein (GFP)-US9 fusion protein (Kalejta et al. (1999)
Exp Cell Res. 248: 322-328). These cells were fixed in 90% ice-cold
ethanol and stained with propidium iodide at 25 .mu.g/ml. FACS was
performed on an Elite flow cytometer (Coulter). For northern
blotting, equal loading was ensured by over-probing blots with a
control complementary DNA (RP49). For the production of dsRNA,
transcription templates were generated by polymerase chain reaction
such that they contained T7 promoter sequences on each end of the
template. RNA was prepared using the RiboMax kit (Promega).
Confirmation that RNAs were double stranded came from their
complete sensitivity to RNAse III (a gift from A. Nicholson).
Target mRNA transcripts were synthesized using the Riboprobe kit
(Promega) and were gel purified before use.
[0209] Extract preparation Log-phase S2 cells were plated on 15-cm
tissue culture dishes and transfected with 30 .mu.g dsRNA and 30
.mu.g carrier plasmid DNA. Seventy-two hours after transfection,
cells were harvested in PBS containing 5 mM EGTA, washed twice in
PBS and once in hypotonic buffer (10 mM HEPES pH 7.3, 6 mM
.beta.-mercaptoethanol). Cells were suspended in 0.7 packed-cell
volumes of hypotonic buffer containing Complete protease inhibitors
(Boehringer) and 0.5 units/ml of RNasin (Promega). Cells were
disrupted in a dounce homogenizer with a type B pestle, and lysates
were centrifuged at 30,000 g for 20 min. Supernatants were used in
an in vitro assay containing 20 mM HEPES pH 7.3, 110 mM KOAc, 1 mM
Mg(OAc).sub.2, 3 mM EGTA, 2 mM CaCl.sub.2, 1 mM DTT. Typically, 5
.mu.l extract was used in a 10 .mu.l assay that contained also
10,000 c.p.m. synthetic mRNA substrate.
[0210] Extract fractionation Extracts were centrifuged at 200,000 g
for 3 h and the resulting pellet (containing ribosomes) was
extracted in hypotonic buffer containing also 1 mM MgCl.sub.2 and
300 mM KOAc. The extracted material was spun at 100,000 g for 1 h
and the resulting supernatant was fractionated on Source 15Q column
(Pharmacia) using a KCl gradient in buffer A (20 mM HEPES pH 7.0, 1
mM dithiothreitol, 1 mM MgCl.sub.2). Fractions were assayed for
nuclease activity as described above. For northern blotting,
fractions were proteinase K/SDS treated, phenol extracted, and
resolved on 15% acrylamide 8M urea gels. RNA was electroblotted
onto Hybond N+ and probed with strand-specific riboprobes derived
from cyclin E mRNA. Hybridization was carried out in 500 mM
NaPO.sub.4 pH 7.0, 15% formamide, 7% SDS, 1% BSA. Blots were washed
in 1.times.SSC at 37-45.degree. C.
EXAMPLE 2
Role for a Bidentate Ribonuclease in the Initiation Step of RNA
Interference
[0211] Genetic approaches in worms, fungi and plants have
identified a group of proteins that are essential for
double-stranded RNA-induced gene silencing. Among these are
ARGONAUTE family members (e.g. RDE1, QDE2) (Tabara et al. (1999)
Cell 99: 123-132; Catalanotto et al. (2000) Nature 404: 245; Fagard
et al. (2000) PNAS 97: 11650-11654), recQ-family helicases (MUT-7,
QDE3) (Ketting et al. (1999) Cell 99: 133-141; Cogoni and Macino.
(1999) Science 286: 2342-2344), and RNA-dependent RNA polymerases
(e.g. EGO-1, QDE1, SGS2/SDE1) (Cogoni and Macino (1999) Nature 399:
166-169; Smardon et al. (2000) Current Biology 10: 169-178;
Mourrain et al. (2000) Cell 101: 533-542; Dalmay et al. (2000) Cell
101: 543-553). While potential roles have been proposed, none of
these genes has been assigned a definitive function in the
silencing process. Biochemical studies have suggested that PTGS is
accomplished by a multicomponent nuclease that targets mRNAs for
degradation (Hammond et al. (2000) Nature 404: 293-296; Zamore et
al. (2000) Cell 101 25-33; Tuschl et al. (1999) Genes and
Development 13: 3191-3197). We have shown that the specificity of
this complex may derive from the incorporation of a small guide
sequence that is homologous to the mRNA substrate (Hammond et al.
(2000) Nature 404: 293-296). Originally identified in plants that
were actively silencing transgenes (Hamilton and Baulcombe (1999)
Science 286: 950-952), these .about.22 nt. RNAs have been produced
during RNAi in vitro using an extract prepared from Drosophila
embryos (Zamore et al. (2000) Cell 101 25-33). Putative guide RNAs
can also be produced in extracts from Drosophila S2 cells (FIG.
5a). With the goal of understanding the mechanism of
post-transcriptional gene silencing, we have undertaken both
biochemical fractionation and candidate gene approaches to identify
the enzymes that execute each step of RNAi.
[0212] Our previous studies resulted in the partial purification of
a nuclease, RISC, that is an effector of RNA interference. See
Example 1. This enzyme was isolated from Drosophila S2 cells in
which RNAi had been initiated in vivo by transfection with dsRNA.
We first sought to determine whether the RISC enzyme and the enzyme
that initiates RNAi via processing of dsRNA into 22mers are
distinct activities. RISC activity could be largely cleared from
extracts by high-speed centrifugation (100,000.times.g for 60 min.)
while the activity that produces 22mers remained in the supernatant
(FIG. 5b,c). This simple fractionation indicated that RISC and the
22mer-generating activity are separable and thus distinct enzymes.
However, it seems likely that they might interact at some point
during the silencing process.
[0213] RNAse III family members are among the few nucleases that
show specificity for double-stranded RNA (Nicholson (1999) FEMS
Microbiol Rev 23: 371-390). Analysis of the Drosophila and C.
elegans genomes reveals several types of RNAse III enzymes. First
is the canonical RNAse III which contains a single RNAse III
signature motif and a double-stranded RNA binding domain (dsRBD;
e.g. RNC_CAEEL). Second is a class represented by Drosha (Filippov
et al. (2000) Gene 245: 213-221), a Drosophila enzyme that contains
two RNAse III motifs and a dsRBD (CeDrosha in C. elegans). A third
class contains two RNAse III signatures and an amino terminal
helicase domain (e.g. Drosophila CG4792, CG6493, C. elegans
K12H4.8), and these had previously been proposed by Bass as
candidate RNAi nucleases (Bass (2000) Cell 101: 235-238).
Representatives of all three classes were tested for the ability to
produce discrete, .about.22 nt. RNAs from dsRNA substrates.
[0214] Partial digestion of a 500 nt. cyclin E dsRNA with purified,
bacterial RNAse III produced a smear of products while nearly
complete digestion produced a heterogeneous group of 11-17
nucleotide RNAs (not shown). In order to test the dual-RNAse III
enzymes, we prepared T7 epitope-tagged versions of Drosha and
CG4792. These were expressed in transfected S2 cells and isolated
by immunoprecipitation using antibody-agarose conjugates. Treatment
of the dsRNA with the CG4792 immunoprecipitate yielded .about.22
nt. fragments similar to those produced in either S2 or embryo
extracts (FIG. 6a). Neither activity in extract nor activity in
immunoprecipitates depended on the sequence of the RNA substrate
since dsRNAs derived from several genes were processed equivalently
(see Supplement 1). Negative results were obtained with Drosha and
with immunoprecipitates of a DExH box helicase (Homeless (Gillespie
et al. (1995) Genes and Development 9: 2495-2508); see FIG. 6a,b).
Western blotting confirmed that each of the tagged proteins was
expressed and immunoprecipitated similarly (see Supplement 2).
Thus, we conclude that CG4792 may carry out the initiation step of
RNA interference by producing .about.22 nt. guide sequences from
dsRNAs. Because of its ability to digest dsRNA into uniformly
sized, small RNAs, we have named this enzyme Dicer (Dcr). Dicer
mRNA is expressed in embryos, in S2 cells, and in adult flies,
consistent with the presence of functional RNAi machinery in all of
these contexts (see Supplement 3).
[0215] The possibility that Dicer might be the nuclease responsible
for the production of guide RNAs from dsRNAs prompted us to raise
an antiserum directed against the carboxy-terminus of the Dicer
protein (Dicer-1, CG4792). This antiserum could immunoprecipitate a
nuclease activity from either Drosophila embryo extracts or from S2
cell lysates that produced .about.22 nt. RNAs from dsRNA substrates
(FIG. 6C). The putative guide RNAs that are produced by the Dicer-1
enzyme precisely comigrate with 22mers that are produced in extract
and with 22mers that are associated with the RISC enzyme (FIG.
6D,F). It had previously been shown that the enzyme that produced
guide RNAs in Drosophila embryo extracts was ATP-dependent (Zamore
et al. (2000) Cell 101 25-33). Depletion of this cofactor resulted
in an .about.6-fold lower rate of dsRNA cleavage and in the
production of RNAs with a slightly lower mobility. Of interest was
the fact that both Dicer-I immunoprecipitates and extracts from S2
cells require ATP for the production of 22mers (FIG. 6D). We do not
observe the accumulation of lower mobility products in these cases,
although we do routinely observe these in ATP-depleted embryo
extracts. The requirement of this nuclease for ATP is a quite
unusual property. We hypothesize that this requirement could
indicate that the enzyme may act processively on the dsRNA, with
the helicase domain harnessing the energy of ATP hydrolysis both
for unwinding guide RNAs and for translocation along the
substrate.
[0216] Efficient induction of RNA interference in C. elegans and in
Drosophila has several requirements. For example, the initiating
RNA must be double-stranded, and it must be several hundred
nucleotides in length. To determine whether these requirements are
dictated by Dicer, we characterized the ability of extracts and of
immunoprecipitated enzyme to digest various RNA substrates. Dicer
was inactive against single stranded RNAs regardless of length (see
Supplement 4). The enyzme could digest both 200 and 500 nucleotide
dsRNAs but was significantly less active with shorter substrates
(see Supplement 4). Double-stranded RNAs as short as 35 nucleotides
could be cut by the enzyme, albeit very inefficiently (data not
shown). In contrast, E. coli RNAse III could digest to completion
dsRNAs of 35 or 22 nucleotides (not shown). This suggests that the
substrate preferences of the Dicer enzyme may contribute to but not
wholly determine the size dependence of RNAi.
[0217] To determine whether the Dicer enzyme indeed played a role
in RNAi in vivo, we sought to deplete Dicer activity from S2 cells
and test the effect on dsRNA-induced gene silencing. Transfection
of S2 cells with a mixture of dsRNAs homologous to the two
Drosophila Dicer genes (CG4792 and CG6493) resulted in an
.about.6-7 fold reduction of Dicer activity either in whole cell
lysates or in Dicer-1 immunoprecipitates (FIG. 7A,B). Transfection
with a control dsRNA (murine caspase 9) had no effect.
Qualitatively similar results were seen if Dicer was examined by
Northern blotting (not shown). Depletion of Dicer in this manner
substantially compromised the ability of cells to silence
subsequently an exogenous, GFP transgene by RNAi (FIG. 7C). These
results indicate that Dicer is involved in RNAi in vivo. The lack
of complete inhibition of silencing could result from an incomplete
suppression of Dicer (which is itself required for RNAi) or could
indicate that in vivo, guide RNAs can be produced by more than one
mechanism (e.g. through the action of RNA-dependent RNA
polymerases).
[0218] Our results indicate that the process of RNA interference
can be divided into at least two distinct steps. According to this
model, initiation of PTGS would occur upon processing of a
double-stranded RNA by Dicer into .about.22 nucleotide guide
sequences, although we cannot formally exclude the possibility that
another, Dicer-associated nuclease may participate in this process.
These guide RNAs would be incorporated into a distinct nuclease
complex (RISC) that targets single-stranded mRNAs for degradation.
An implication of this model is that guide sequences are themselves
derived directly from the dsRNA that triggers the response. In
accord with this model, we have demonstrated that .sup.32P-labeled,
exogenous dsRNAs that have been introduced into S2 cells by
transfection are incorporated into the RISC enzyme as 22 mers (FIG.
7E). However, we cannot exclude the possibility that RNA-dependent
RNA polymerases might amplify 22mers once they have been generated
or provide an alternative method for producing guide RNAs.
[0219] The structure of the Dicer enzyme provokes speculation on
the mechanism by which the enzyme might produce discretely sized
fragments irrespective of the sequence of the dsRNA (see Supplement
1, FIG. 8a). It has been established that bacterial RNAse III acts
on its substrate as a dimer (Nicholson (1999) FEMS Microbiol Rev
23: 371-390; Robertson et al. (1968) J Biol Chem 243: 82-91; Dunn
(1976) J Biol Chem 251: 3807-3814). Similarly, a dimer of Dicer
enzymes may be required for cleavage of dsRNAs into .about.22 nt.
pieces. According to one model, the cleavage interval would be
determined by the physical arrangement of the two RNAse III domains
within Dicer enzyme (FIG. 8a). A plausible alternative model would
dictate that cleavage was directed at a single position by the two
RIII domains in a single Dicer protein. The 22 nucleotide interval
could be dictated by interaction of neighboring Dicer enzymes or by
translocation along the mRNA substrate. The presence of an integral
helicase domain suggests that the products of Dicer cleavage might
be single-stranded 22 mers that are incorporated into the RISC
enzyme as such.
[0220] A notable feature of the Dicer family is its evolutionary
conservation. Homologs are found in C. elegans (K12H4.8),
Arabidopsis (e.g., CARPEL FACTORY (Jacobson et al. (1999)
Development 126: 5231-5243), T25K16.4, AC012328.sub.--1), mammals
(Helicase-MOI (Matsuda et al. (2000) Biochim Biophys Acta 1490:
163-169) and S. pombe (YC9A_SCHPO) (FIG. 8b, see Supplements 6,7
for sequence comparisons). In fact, the human Dicer family member
is capable of generating .about.22 nt. RNAs from dsRNA substrates
(Supplement 5) suggesting that these structurally similar proteins
may all share similar biochemical functions. It has been
demonstrated that exogenous dsRNAs can affect gene function in
early mouse embryos (Wianny et al. (2000) Nature Cell Biology 2:
70-75), and our results suggest that this regulation may be
accomplished by an evolutionarily conserved RNAi machinery.
[0221] In addition to RNAseIII and helicase motifs, searches of the
PFAM database indicate that each Dicer family member also contains
a ZAP domain (FIG. 8c) (Sonnhammer et al. (1997) Proteins 28:
405-420). This sequence was defined based solely upon its
conservation in the Zwille/ARGONAUTE/Piwi family that has been
implicated in RNAi by mutations in C. elegans (Rde-1) and
Neurospora (Qde-2) (Tabara et al. (1999) Cell 99: 123-132;
Catalanotto et al (2000) Nature 404: 245). Although the function of
this domain is unknown, it is intriguing that this region of
homology is restricted to two gene families that participate in
dsRNA-dependent silencing. Both the ARGONAUTE and Dicer families
have also been implicated in common biological processes, namely
the determination of stem-cell fates. A hypomorphic allele of
carpel factory, a member of the Dicer family in Arabidopsis, is
characterized by increased proliferation in floral meristems
(Jacobsen et al. (1999) Development 126 5231-5243). This phenotype
and a number of other characteristic features are also shared by
Arabidopsis ARGONAUTE (ago1-1) mutants (Bohmert et al. (1998) EMBO
J 17: 170-180; C. Kidner and R. Martiennsen, pers. comm.). These
genetic analyses begin to provide evidence that RNAi may be more
than a defensive response to unusual RNAs but may also play
important roles in the regulation of endogenous genes.
[0222] With the identification of Dicer as a catalyst of the
initiation step of RNAi, we have begun to unravel the biochemical
basis of this unusual mechanism of gene regulation. It will be of
critical importance to determine whether the conserved family
members from other organisms, particularly mammals, also play a
role in dsRNA-mediated gene regulation.
[0223] Methods:
[0224] Plasmid constructs. A full-length cDNA encoding Drosha was
obtained by PCR from an EST sequenced by the Berkeley Drosophila
genome project. The Homeless clone was a gift from Gillespie and
Berg (Univ. Washington). The T7 epitope-tag was added to the amino
terminus of each by PCR, and the tagged cDNAs were cloned into
pRIP, a retroviral vector designed specifically for expression in
insect cells (E. Bernstein, unpublished). In this vector,
expression is driven by the Orgyia pseudotsugata IE2 promoter
(Invitrogen). Since no cDNA was available for CG4792/Dicer, a
genomic clone was amplified from a bacmid (BACR23F10; obtained from
the BACPAC Resource Center in the Dept. of Human Genetics at the
Roswell Park Cancer Institute). Again, during amplification, a T7
epitope tag was added at the amino terminus of the coding sequence.
The human Dicer gene was isolated from a cDNA library prepared from
HaCaT cells (GJH, unpublished). A T7-tagged version of the complete
coding sequence was cloned into pCDNA3 (Invitrogen) for expression
in human cells (LinX-A).
[0225] Cell culture and extract preparation. S2 and embryo culture.
S2 cells were cultured at 27.degree. C. in 5% CO.sub.2 in
Schneider's insect media supplemented with 10% heat inactivated
fetal bovine serum (Gemini) and 1% antibiotic-antimycotic solution
(Gibco BRL). Cells were harvested for extract preparation at
10.times.10.sup.6 cells/ml. The cells were washed 1.times. in PBS
and were resuspended in a hypotonic buffer (10 mM Hepes pH 7.0, 2
mM MgCl.sub.2, 6 mM PME) and dounced. Cell lysates were spun
20,000.times.g for 20 minutes. Extracts were stored at -80.degree.
C. Drosophila embryos were reared in fly cages by standard
methodologies and were collected every 12 hours. The embryos were
dechorionated in 50% chlorox bleach and washed thoroughly with
distilled water. Lysis buffer (10 mM Hepes, 10 mM KCl, 1.5 mM
MgCl.sub.2, 0.5 mM EGTA, 10 mM .beta.-glycerophosphate, 1 mM DTT,
0.2 mM PMSF) was added to the embryos, and extracts were prepared
by homogenization in a tissue grinder. Lysates were spun for two
hours at 200,000.times.g and were frozen at -80.degree. C. LinX-A
cells, a highly-transfectable derivative of human 293 cells, (Lin
Xie and GJH, unpublished) were maintained in DMEM/10% FCS.
[0226] Transfections and immunoprecipitations. S2 cells were
transfected using a calcium phosphate procedure essentially as
previously described (Hammond et al. (2000) Nature 404: 293-296).
Transfection rates were .about.90% as monitored in controls using
an in situ .beta.-galactosidase assay. LinX-A cells were also
transfected by calcium phosphate co-precipitation. For
immunoprecipitations, cells (.about.5.times.10.sup.- 6 per IP) were
transfected with various clones and lysed three days later in IP
buffer (125 mM KOAc, 1 mM MgOAc, 1 mM CaCl.sub.2, 5 mM EGTA, 20 mM
Hepes pH 7.0, 1 mM DTT, 1% NP-40 plus Complete protease inhibitors
(Roche)). Lysates were spun for 10 minutes at 14,000.times.g and
supernatants were added to T7 antibody-agarose beads (Novagen).
Antibody binding proceeded for 4 hours at 4.degree. C. Beads were
centrifuged and washed in lysis buffer three times, and once in
reaction buffer. The Dicer antiserum was raised in rabbits using a
KLH-conjugated peptide corresponding to the C-terminal 8 amino
acids of Drosophila Dicer-1 (CG4792).
[0227] Cleavage reactions. RNA preparation. Templates to be
transcribed into dsRNA were generated by PCR with forward and
reverse primers, each containing a T7 promoter sequence. RNAs were
produced using Riboprobe (Promega) kits and were uniformly labeling
during the transcription reaction with .sup.32P-UTP.
Single-stranded RNAs were purified from 1% agarose gels. dsRNA
cleavage. Five microliters of embryo or S2 extracts were incubated
for one hour at 30.degree. C. with dsRNA in a reaction containing
20 mM Hepes pH 7.0, 2 mM MgOAc, 2 mM DTT, 1 mM ATP and 5% Superasin
(Ambion). Immunoprecipitates were treated similarly except that a
minimal volume of reaction buffer (including ATP and Superasin) and
dsRNA were added to beads that had been washed in reaction buffer
(see above). For ATP depletion, Drosophila embryo extracts were
incubated for 20 minutes at 30.degree. C. with 2 mM glucose and
0.375 U of hexokinase (Roche) prior to the addition of dsRNA.
[0228] Northern and Western analysis. Total RNA was prepared from
Drosophila embryos (0-12 hour), from adult flies, and from S2 cells
using Trizol (Lifetech). Messenger RNA was isolated by affinity
selection using magnetic oligo-dT beads (Dynal). RNAs were
electrophoresed on denaturing formaldehyde/agarose gels, blotted
and probed with randomly primed DNAs corresponding to Dicer. For
Western analysis, T7-tagged proteins were immunoprecipitated from
whole cell lysates in IP buffer using anti-T7-antibody-agarose
conjugates. Proteins were released from the beads by boiling in
Laemmli buffer and were separated by electrophoresis on 8% SDS
PAGE. Following transfer to nitrocellulose, proteins were
visualized using an HRP-conjugated anti-T7 antibody (Novagen) and
chemiluminescent detection (Supersignal, Pierce).
[0229] RNAi of Dicer. Drosophila S2 cells were transfected either
with a dsRNA corresponding to mouse caspase 9 or with a mixture of
two dsRNAs corresponding to Drosophila Dicer-1 and Dicer-2 (CG4792
and CG6493). Two days after the initial transfection, cells were
again transfected with a mixture containing a GFP expression
plasmid and either luciferase dsRNA or GFP dsRNA as previously
described (Hammond et al. (2000) Nature 404: 293-296). Cells were
assayed for Dicer activity or fluorescence three days after the
second transfection. Quantification of fluorescent cells was done
on a Coulter EPICS cell sorter after fixation. Control
transfections indicated that Dicer activity was not affected by the
introduction of caspase 9 dsRNA.
EXAMPLE 3
A Simplified Method for the Creation of Hairpin Constructs for RNA
Interference
[0230] In numerous model organisms, double stranded RNAs have been
shown to cause effective and specific suppression of gene function
(Bosher and Labouesse (2000) Nature Cell Biology 2: E31-E36). This
response, termed RNA interference or post-transcriptional gene
silencing, has evolved into a highly effective reverse genetic tool
in C. elegans, Drosophila, plants and numerous other systems. In
these cases, double-stranded RNAs can be introduced by injection,
transfection or feeding; however, in all cases, the response is
both transient and systemic. Recently, stable interference with
gene expression has been achieved by expression of RNAs that form
snap-back or hairpin structures (Fortier and Belote (2000) Genesis
26: 240-244; Kennerdell and Carthew (2000) Nature Biotechnology 18:
896-898; Lam and Thummel (2000) Current Biology 10: 957-963; Shi et
al. (2000) RNA 6: 1069-1076; Smith et al. (2000) Nature 407:
319-320; Tavemarakis et al. (2000) Nature Genetics 24: 180-183).
This has the potential not only to allow stable silencing of gene
expression but also inducible silencing as has been observed in
trypanosomes and adult Drosophila (Fortier and Belote (2000)
Genesis 26: 240-244; Lam and Thummel (2000) Current Biology 10:
957-963; Shi et al. (2000) RNA 6: 1069-1076). The utility of this
approach is somewhat hampered by the difficulties that arise in the
construction of bacterial plasmids containing the long inverted
repeats that are necessary to provoke silencing. In a recent
report, it was stated that more than 1,000 putative clones were
screened to identify the desired construct (Tavemarakis et al.
(2000) Nature Genetics 24: 180-183).
[0231] The presence of hairpin structures often induces plasmid
rearrangement, in part due to the E. coli sbc proteins that
recognize and cleave cruciform DNA structures (Connelly et al.
(1996) Genes Cell 1: 285-291). We have developed a method for the
construction of hairpins that does not require cloning of inverted
repeats, per se. Instead, the fragment of the gene that is to be
silenced is cloned as a direct repeat, and the inversion is
accomplished by treatment with a site-specific recombinase, either
in vitro (or potentially in vivo) (see FIG. 27). Following
recombination, the inverted repeat structure is stable in a
bacterial strain that lacks an intact SBC system (DL759). We have
successfully used this strategy to construct numerous hairpin
expression constructs that have been successfully used to provoke
gene silencing in Drosophila cells.
[0232] In the following examples, we use this method to express
long dsRNAs in a variety of mammalian cell types. We show that such
long dsRNAs mediate RNAi in a variety of cell types. Additionally,
since the vector described in FIG. 27 contains a selectable marker,
dsRNAs produced in this manner can be stably expressed in cells.
Accordingly, this method allows not only the examination of
transient effects of RNA suppression in a cell, but also the
effects of stable and prolonged RNA suppression.
[0233] Methods:
[0234] Plasmids expressing hairpin RNAs were constructed by cloning
the first 500 basepairs of the GFP coding region into the FLIP
cassette of pRIP-FLIP as a direct repeat. The FLIP cassette
contains two directional cloning sites, the second of which is
flanked by LoxP sites. The Zeocin gene, present between the cloning
sites, maintains selection and stability. To create an inverted
repeat for hairpin production, the direct repeat clones were
exposed to Cre recombinase (Stratagene) in vitro and, afterwards,
transformed into DL759 E. Coli. These bacteria permit the
replication of DNA containing cruciform structures, which tend to
form inverted repeats.
EXAMPLE 4
Long dsRNAs Suppress Gene Expression in Mammalian Cells
[0235] Previous experiments have demonstrated that dsRNA, produced
using a variety of methods including via the construction of
hairpins, can suppress gene expression in Drosophila cells. We now
demonstrate that dsRNA can also suppress gene expression in
mammalian cells in culture. Additionally, the power of RNAi as a
genetic tool would be greatly enhanced by the ability to engineer
stable silencing of gene expression. We therefore undertook an
effort to identify mammalian cells in which long dsRNAs could be
used as RNAi triggers in the hope that these same cell lines would
provide a platform upon which to develop stable silencing
strategies. We demonstrate that RNA suppression can be mediated by
stably expressing a long hairpin in a mammalian cell line. The
ability to engineer stable silencing of gene expression in cultured
mammalian cells, in addition to the ability to transiently silence
gene expression, has many important applications.
[0236] A. RNAi in Pluripotent Murine P19 Cells.
[0237] We first sought to determine whether long dsRNA triggers
could induce sequence-specific silencing in cultured murine cells,
both to develop this approach as a tool for probing gene function
and to allow mechanistic studies of dsRNA-induced silencing to be
propagated to mammalian systems. We, therefore, attempted to extend
previous studies in mouse embryos (Wianny et al. 2000, Nat. Cell
Biol. 2: 70-75; Svoboda et al. 2000, Development 127: 4147-4156) by
searching for RNAi-like mechanisms in pluripotent, embryonic cell
types. We surveyed a number of cell lines of embryonic origin for
the degree to which generalized suppression of gene expression
occurred upon introduction of dsRNA. As an assay, we tested the
effects of dsRNA on the expression of GFP as measured in situ by
counting fluorescent cells. As expected, in both human embryonic
kidney cells (293) and mouse embryo fibroblasts, GFP expression was
virtually eliminated irrespective of the sequence of the
cotransfected dsRNA. In some pluripotent teratocarcinoma and
teratoma cell lines (e.g., N-Teral, F9), the PKR response was
attenuated but still evident; however, in contrast, transfection of
nonhomologous dsRNAs had no effect on the expression of reporter
genes (e.g., GFP or luciferase) either in mouse embryonic stem
cells or in p19 embryonal carcinoma cells (FIG. 28).
[0238] Transfection of P19 embryonal carcinoma cells with GFP in
the presence of cognate dsRNA corresponding to the first
.apprxeq.500 nts of the GFP coding sequence had a strikingly
different effect. GFP expression was eliminated in the vast
majority of cotransfected cells (FIG. 28), suggesting that these
cultured murine cells might respond to dsRNA in a manner similar to
that which we had previously demonstrated in cultured, Drosophila
S2 cells (Hammond et al. 2000, Nature 404: 293-296).
[0239] To quantify the extent to which dsRNA could induce
sequence-specific gene silencing, we used a dual luciferase
reporter assay similar to that which had first been used to
demonstrate RNAi in Drosophila embryo extracts (Tuschl et al. 1999,
Genes Dev. 13: 3191-3197). P19 EC cells were transfected with a
mixture of two plasmids that individually direct the expression of
firefly luciferase and Renilla luciferase. These were cotransfected
with no dsRNA, with dsRNA that corresponds to the first
.apprxeq.500 nts of the firefly luciferase, or with dsRNA
corresponding to the first .apprxeq.500 nts of GFP as a control.
Cotransfection with GFP dsRNA gave luciferase activities that were
similar to the no-dsRNA control, both in the firefly/Renilla
activity ratio and in the absolute values of both activities. In
contrast, in cells that received the firefly luciferase dsRNA, the
ratio of firefly to Renilla luciferase activity was reduced by up
to 30-fold (250 ng, FIG. 29B). For comparison, we carried out an
identical set of experiments in Drosophila S2 cells. Although
qualitatively similar results were obtained, the silencing response
was more potent. At equivalent levels of dsRNA, S2 cells suppressed
firefly luciferase activity to virtually background levels.
[0240] The complementary experiment, in which dsRNA was homologous
to Renilla luciferase, was also performed. Again, in this case,
suppression of the expression of the Renilla enzyme was
.apprxeq.10-fold (FIG. 29D). Thus, the dsRNA response in P19 cells
was flexible, and the silencing machinery was able to adapt to
dsRNAs directed against any of the reporters that were tested.
[0241] We took two approaches to test whether this response was
specific for dsRNA. Pretreatment of the trigger with purified RNase
III, a dsRNA-specific ribonuclease, before transfection greatly
reduced its ability to provoke silencing. Furthermore, transfection
of cells with single-stranded antisense RNAs directed against
either firefly or Renilla luciferase had little or no effect on
expression of the reporters (FIGS. 29C and 29D). Considered
together, these results provided a strong indication that
double-stranded RNAs provoke a potent and specific silencing
response in P19 embryonal carcinoma cells. Efficient silencing
could be provoked with relatively low concentrations of dsRNA (25
ng/ml culture media; see FIG. 29A). The response was
concentration-dependent, with maximal suppression of
.apprxeq.20-fold being achieved at a dose of 1.5 .mu.g/ml culture
media. Silencing was established rapidly and was evident by 9 h
posttransfection (the earliest time point examined). Furthermore,
the response persisted without significant changes in the degree of
suppression for up to 72 h following a single dose of dsRNA.
[0242] FIG. 30 further shows wildtype P19 cells which have been
co-transfected with either RFP or GFP (right panel). Note the
robust expression of RFP or GFR respectively approximately 42 hours
post-transfection. We isolated P19 clones which stably express a
500 nt. GFP hairpin. Such clones were then transfected with either
RFP or GFP, and expression of RFP or GFP was assessed by visual
inspection of the cells. The left panel demonstrates that a 500 nt
GFP hairpin specifically suppresses expression of GFP in P19
cells.
[0243] B. RNAi in Embryonic Stem Cells.
[0244] To assess whether the presence of a sequence-specific
response to dsRNA was a peculiarity of P19 cells or whether it also
extended to normal murine embryonic cells, we performed similar
silencing assays in mouse embryonic stem cells. Cotransfection of
embryonic stem cells with noncognate dsRNAs (e.g., GFP), again, had
no dramatic effect on either the absolute values or the ratios of
Renilla and firefly luciferase activity (FIG. 31). However,
transfection with either firefly or Renilla luciferase dsRNA
dramatically and specifically reduced the activity of the targeted
enzyme (FIG. 31).
[0245] This result suggests that RNAi can operate in multiple
murine cell types of embryonic origin, including normal embryonic
stem cells. The ability to provoke silencing in a cell type that is
normally used for the generation of genetic, mosaic animals
suggests the possibility of eventually testing the biological
effects of silencing both in culture and in reconstituted animal
models. Our ability to successfully manipulate ES cell via RNAi
allows the use of RNAi in the generation of transgenic and
knock-out mice.
[0246] C. RNAI in Murine Somatic Cells.
[0247] RNAi effector pathways are likely to be present in mammalian
somatic cells, based on the ability of siRNAs to induce transient
silencing (Elbashir et al. 2001, Nature 411: 494-498). Furthermore,
we have shown that RNAi initiator and effector pathways clearly
exist in embryonic cells that can enforce silencing in response to
long dsRNA triggers. We therefore sought to test whether the RNAi
machinery might exist intact in some somatic cell lines.
[0248] Transfection of HeLa cells with luciferase reporters in
combination with long dsRNA triggers caused a nearly complete
suppression of activity, irrespective of the RNA sequence. In a
murine myoblast cell line, C2C12, we noted a mixture of two
responses. dsRNAs homologous to firefly luciferase provoked a
sequence-specific effect, producing a degree of suppression that
was slightly more potent than was observed upon transfection with
cognate .apprxeq.21-nt siRNA (Elbashir et al. 2001, Nature 411:
494-498) (see FIG. 32A). However, with long dsRNA triggers, the
specific effect was superimposed upon a generalized suppression of
reporter gene expression that was presumably because of PKR
activation (FIG. 32B).
[0249] Numerous mammalian viruses have evolved the ability to block
PKR as an aid to efficient infection. For example, adenoviruses
express VA RNAs, which mimic dsRNA with respect to binding but not
to activation of PKR (Clarke et al. 1995, RNA 1: 7-20). Vaccinia
virus uses two strategies to evade PKR. The first is expression of
E3L, which binds and masks dsRNAs (Kawagishi-Kobayashi et al. 2000,
Virology 276: 424-434). The second is expression of K3L, which
binds and inhibits PKR via its ability to mimic the natural
substrate of this enzyme, eIF2.alpha. (Kawagishi-Kobayashi et al.
2000, supra).
[0250] Transfection of C2C12 cells with a vector that directs K3L
expression attenuates the generalized repression of reporter genes
in response to dsRNA. However, this protein had no effect on the
magnitude of specific inhibition by RNAi (FIG. 32C).
[0251] FIG. 33 further shows the results of a transient
co-transfection assay performed in Hela cells, CHO cells, and P19
cells. The cell lines were each transfected with plasmids
expressing Photinus pyralis (firefly) and Renila reniformis (sea
pansy) luciferases. The cells lines were additionally transfected
with 400 ng of 500nt dsRNAs corresponding to either firefly
luciferase (dsLUC) or dsGFP. The results demonstrate that dsRNA can
specifically mediate suppression in a multiple mammalian cells
types in culture.
[0252] These results raise the possibility that, at least in some
cell lines and/or cell types, blocking nonspecific responses to
dsRNA will enable the use of long dsRNAs for the study of gene
function. This might be accomplished through the use of viral
inhibitors, as described here, or through the use of cells isolated
from animals that are genetically modified to lack undesirable
responses.
[0253] D. Stable Suppression of Gene Expression Using RNAi.
[0254] To date, dsRNAs have been used to induce sequence-specific
gene silencing in either cultured mammalian cells or in embryos
only in a transient fashion. However, the most powerful
applications of genetic manipulation are realized only with the
creation of stable mutants. The ability to induce silencing by
using long dsRNAs offers the opportunity to translate into
mammalian cells work from model systems such as Drosophila, plants,
and C. elegans wherein stable silencing has been achieved by
enforced expression of hairpin RNAs (Kennerdell et al. 2000, Nat.
Biotechnol. 18: 896-898; Smith et al. 2000, Nature 407: 319-320;
Tavemarakis et al. 2000, Nat. Genet. 24: 180-183).
[0255] P19 EC cells were transfected with a control vector or with
an expression vector that directs expression of a .apprxeq.500-nt
GFP hairpin RNA from an RNA polymerase II promoter
(cytomegalovirus). Colonies arising from cells that had stably
integrated either construct were selected and expanded into clonal
cell lines. Each cell line was assayed for persistent RNAi by
transient cotransfection with a mixture of two reporter genes,
dsRED to mark transfected cells and GFP to test for stable
silencing.
[0256] Transfection of clonal P19 EC cells that had stably
integrated the control vector produced equal numbers of red and
green cells, as would be expected in the absence of any specific
silencing response (FIG. 34B), whereas cells that express the GFP
hairpin RNA gave a very different result. These cells expressed the
dsRED protein with an efficiency comparable to that observed in
cells containing the control vector. However, the cells failed to
express the cotransfected GFP reporter (FIG. 34B). These data
provide a strong indication that continuous expression of a hairpin
dsRNA can provoke stable, sequence-specific silencing of a target
gene.
[0257] In Drosophila S2 cells and C. elegans, RNAi is initiated by
the Dicer enzyme, which processes dsRNA into .apprxeq.22-nt siRNAs
(Bernstein et al. 2001, Nature 409: 363-366; Grishok et al. 2001,
Cell 106: 23-34; Hutvagner et al. 2001, Science 293: 834-838;
Ketting et al. 2001, Genes Dev. 15: 2654-2659; Knight et al. 2001,
Science 293: 2269-2271). In both, S2 cells and C. elegans
experiments by using dsRNA to target Dicer suppress the RNAi
response. Whether Dicer plays a central role in hairpin-induced
gene silencing in P19 cells was tested by transfecting P19 cells
stably transfected with GFP hairpin constructs with mouse Dicer
dsRNA. Treatment with Dicer dsRNA, but not control dsRNA, resulted
in derepression of GFP (FIG. 34C).
[0258] E. dsRNA Induces Posttranscriptional Silencing.
[0259] A key feature of RNAi is that it exerts its effect at the
posttranscriptional level by destruction of targeted mRNAs (Hammond
et al. 2001, Nat. Rev. Genet. 2: 110-119). To test whether dsRNAs
induced silencing in mouse cells via posttranscriptional
mechanisms, we used an assay identical to that, used initially to
characterize RNAi responses in Drosophila embryo extracts (Tuschl
et al. 1999, Genes Dev. 13: 3191-3197). We prepared lysates from
P19 EC cells that were competent for in vitro translation of capped
mRNAs corresponding to Renilla and firefly luciferase. Addition of
nonspecific dsRNAs to these extracts had no substantial effect on
either the absolute amount of luciferase expression or on the ratio
of firefly to Renilla luciferase (FIG. 35). In contrast, addition
of dsRNA homologous to the firefly luciferase induced a dramatic
and dose-dependent suppression of activity. Addition of RNA
corresponding to only the antisense strand of the dsRNA had little
effect, comparable to a nonspecific dsRNA control, and pretreatment
of the dsRNA silencing trigger with RNase III greatly reduced its
potential to induce silencing in vitro. A second hallmark of RNAi
is the production of small, .apprxeq.22-nt siRNAs, which determine
the specificity of silencing. We found that such RNA species were
generated from dsRNA in P19 cell extracts (FIG. 34D, in vitro),
indicative of the presence of a mouse Dicer activity. These species
were also produced in cells that stably express GFP hairpin RNAs
(FIG. 34D, in vivo). Considered together, the posttranscriptional
nature of dsRNA-induced silencing, the association of silencing
with the production of .apprxeq.22-nt siRNAs, and the dependence of
this response on Dicer, a key player in the RNAi pathway, strongly
suggests that dsRNA suppresses gene expression in murine cells via
a conventional RNAi mechanism.
[0260] F RNAi-Mediated Gene Silencing is Specific and Requires
dsRNAs.
[0261] We carried out experiments to verify that the suppressive
effects observed in the in vitro system were specific to double
stranded RNA. Briefly, experiments were performed in accordance
with the methods outlined above. Either dsRNA (ds), single-stranded
RNA (ss), or antisense-RNA (as) corresponding to firefly (FF) or
Renilla (Ren) luciferase was added to the translation reaction.
Following reactions performed at 30.degree. C. for 1 hour, dual
luciferase assays were performed using an Analytical Scientific
Instruments model 3010 Luminometer.
[0262] FIG. 36 summarizes the results of these experiments which
demonstrate that the suppression of gene expression observed in
this in vitro assay is specific for dsRNA. These results further
support the conclusion that dsRNA suppresses gene expression in
this mammalian in vitro system in a manner consistent with
post-transcriptional silencing.
[0263] G. Mammalian Cells Soaked with dsRNAs Results in Gene
Silencing.
[0264] Studies of post-transcriptional silencing in invertebrates
have demonstrated that transfection or injection of the dsRNA is
not necessary to achieve the suppressive affects. For example,
dsRNA suppression in C. elegans can be observed by either soaking
the worms in dsRNA, or by feeding the worms bacteria expressing the
dsRNA of interest. We addressed whether dsRNA suppression in
mammalian cells could be observed without transfection of the
dsRNA. Such a result would present additional potential for easily
using dsRNA suppression in mammalian cells, and would also allow
the use of dsRNA to suppress gene expression in cell types which
have been difficult to transfect (i.e., cell types with a low
transfection efficiency, or cell types which have proven difficult
to transfect at all).
[0265] P19 cells were grown in 6-well tissue culture plates to
approximately 60% confluency in growth media (.alpha.MEM/10% FBS).
Varying concentrations of firefly dsRNA were added to the cultures,
and cells were cultured for 12 hours in growth media+dsRNA. Cells
were then transfected with plasmids expressing firefly or sea pansy
luciferase, as described in detail above. Dual luciferase assays
were carried out 12 hours post-transfection using an Analytical
Scientific Instruments model 3010 Luminometer.
[0266] FIG. 37 summarizes these results which demonstrate that
dsRNA can suppress gene expression in mammalian cells without
transfection. Culturing cells in the presence of dsRNA resulted in
a dose dependent suppression of firefly luciferase gene
expression.
[0267] Methods:
[0268] Cell Culture. P19 mouse embryonic carcinoma cells (American
Type Culture Collection, CRL-1825) were cultured in .alpha.-MEM
(GIBCO/BRL) supplemented with 10% heat-inactivated FBS and 1%
antibiotic/antimycotic solution (GIBCO/BRL). Mouse embryo stem
cells (J1, provided by S. Kim, Cold Spring Harbor Laboratory) were
cultured in DMEM containing ESgro (Chemicon) according to the
manufacturer's instructions. C2C12 murine myoblast cells (gift of
N. Tonks, Cold Spring Harbor Laboratory) were cultured in DMEM
(GIBCO/BRL) supplemented with 10% heat-inactivated FBS and 1%
antibiotic/antimycotic solution (GIBCO/BRL).
[0269] RNA Preparation. For the production of dsRNA, transcription
templates were generated by PCR; they contained T7 promoter
sequences on each end of the template (see Hammond et al. 2000,
Nature 404: 293-296). dsRNAs were prepared by using the RiboMax kit
(Ambion, Austin, Tex.). Firefly and Renilla luciferase mRNA
transcripts were synthesized by using the Riboprobe kit (Promega)
and were gel purified before use.
[0270] Transfection and Gene Silencing Assays. Cells were
transfected with indicated amounts of dsRNA and plasmid DNA by
using FuGENE6 (Roche Biochemicals) according to the manufacturer's
instructions. Cells were transfected at 50-70% confluence in
12-well plates containing either 1 or 2 ml of medium per well. Dual
luciferase assays (Promega) were carried out by cotransfecting
cells with plasmids contain firefly luciferase under the control of
SV40 promoter (pGL3-Control, Promega) and Renilla luciferase under
the control of the SV40 early enhancer/promoter region (pSV40,
Promega). These plasmids were cotransfected by using a 1:1 or 10:1
ratio of pGL3-control (250 ng/well) to pRL-SV40. Both ratios
yielded similar results. For some experiments, cells were
transfected with vectors that direct expression of enhanced green
fluorescent protein (EGFP)-US9 fusion protein (Kalejta et al. 1999,
Exp. Cell Res. 248: 322-328) or red fluorescent protein (RFP)
(pDsRed N1, CLONTECH). RNAi in S2 cells was performed as described
(Hammond et al. 2000, Nature 404: 293-296).
[0271] Plasmids expressing hairpin RNAs (RNAs with a
self-complimentary stem loop) were constructed by cloning the first
500 bp of the EGFP coding region (CLONTECH) into the FLIP cassette
of pRIP-FLIP as a direct repeat. The FLIP cassette contains two
directional cloning sites, the second of which sports flanking LoxP
sites (see FIG. 35A). The Zeocin gene (Stratagene), present between
the cloning sites, maintains selection and, thus, stability of the
FLIP cassette. The FLIP cassette containing EGFP direct repeats was
subcloned into pcDNA3 (Invitrogen). To create an inverted repeat
for hairpin production, EGFP direct repeat clones were exposed to
Cre recombinase (Stratagene) in vitro and, afterward, transformed
into DL759 Escherichia coli (Connelly et al. 1996, Genes Cells 1:
285-291). These bacteria permit the replication of DNA containing
cruciform structures, which tend to form from inverted repeats.
DL759 transformants were screened for plasmids containing inverted
repeats (.apprxeq.50%).
[0272] Silencing of Dicer was accomplished by using a dsRNA
comprising exon 25 of the mouse Dicer gene and corresponding to
nucleotides 5284-5552 of the human Dicer cDNA.
[0273] In Vitro Translation and in Vitro Dicer Assays.
Logarithmically growing cells were harvested in PBS containing 5 mM
EGTA washed twice in PBS and once in hypotonic buffer (10 mM Hepes,
pH 7.3/6 mM .beta.-mercaptoethanol). Cells were suspended in 0.7
packed-cell volumes of hypotonic buffer containing Complete
protease inhibitors (Roche Molecular Biochemicals) and 0.5 units/ml
of RNasin (Promega). Cells were disrupted in a Dounce homogenizer
with a type B pestle, and lysates were centrifuged at
30,000.times.g for 20 min. Supernatants were used in an in vitro
translation assay containing capped m7G(5')pppG firefly and Renilla
luciferase mRNA or in in vitro Dicer assays containing
.sup.32P-labeled dsRNA. For in vitro translation assays, 5 .mu.l of
extract were mixed with 100 ng of firefly and Renilla mRNA along
with 1 .mu.g of dsRNA (or buffer)/10 mM DTT/0.5 mM spermidine/200
mM Hepes, 3.3 mM MgOAc/800 mM KOAc/1 mM ATP/1 mM GTP/4 units of
Rnasin/215 .mu.g of creatine phosphate/1 .mu.g of creatine
phosphate kinase/1 mM amino acids (Promega). Reactions were carried
out for 1 h at 30.degree. C. and quenched by adding 1.times.
passive lysis buffer (Promega). Extracts were then assayed for
luciferase activity. In vitro assays for Dicer activity were
performed as described (Bernstein et al. 2001, Nature 409:
363-366).
[0274] Construction of Stable Silencing Lines. Ten-centimeter
plates of P19 cells were transfected with 5 .mu.g of GFP hairpin
expression plasmid and selected for stable integrants by using
G-418 (300 ng/ml) for 14 days. Clones were selected and screened
for silencing of GFP.
EXAMPLE 5
Compositions and Methods for Synthesizing siRNAs
[0275] Previous results have indicated that short synthetic RNAs
(siRNAs) can efficiently induce RNA suppression. Since short RNAs
do not activate the non-specific PKR response, they offer a means
for efficiently silencing gene expression in a range of cell types.
However, the current state of the art with respect to siRNAs has
several limitations. Firstly, siRNAs are currently chemically
synthesized at great cost (approx. $400/siRNA). Such high costs
make siRNAs impractical for either small laboratories or for use in
large scale screening efforts. Accordingly, there is a need in the
art for methods for generating siRNAs at reduced cost.
[0276] We provide compositions and methods for synthesizing siRNAs
by T7 polymerase. This approach allows for the efficient sythesis
of siRNAs at a cost consistent with standard RNA transcription
reactions (approx. $16/siRNA). This greatly reduced cost makes the
use of siRNA a reasonable approach for small laboratories, and also
will facilitate their use in large-scale screening projects.
[0277] FIG. 38 shows the method for producing siRNAs using T7
polymerase. Briefly, T7 polymerase is used to transcribe both a
sense and antisense transcript. The transcripts are then annealed
to provide an siRNA. One of skill in the art will recognize that
any one of the available RNA polymerases can be readily substituted
for T7 to practice the invention (i.e., T3, Sp6, etc.).
[0278] This approach is amenable to the generation of a single
siRNA species, as well as to the generation of a library of siRNAs.
Such a library of siRNAs can be used in any number of
high-throughput screens including cell based phenotypic screens and
gene array based screens.
EXAMPLE 6
Generation of Short Hairpin dsRNA and Suppression of Gene
Expression Using Such Short Hairpins
[0279] Since the realization that small, endogenously encoded
hairpin RNAs could regulate gene expression via elements of the
RNAi machinery, we have sought to exploit this biological mechanism
for the regulation of desired target genes. Here we show that short
hairpin RNAs (shRNAs) can induce sequence-specific gene silencing
in mammalian cells. As is normally done with siRNAs, silencing can
be provoked by transfecting exogenously synthesized hairpins into
cells. However, silencing can also be triggered by endogenous
expression of shRNAs. This observation opens the door to the
production of continuous cells lines in which RNAi is used to
stably suppress gene expression in mammalian cells. Furthermore,
similar approaches should prove efficacious in the creation of
transgenic animals and potentially in therapeutic strategies in
which long-term suppression of gene function is essential to
produce a desired effect.
[0280] Several groups (Grishok et al. 2001, Cell 106: 23-34;
Ketting et al. 2001, Genes & Dev. 15: 2654-2659; Knight et al.
2001, Science 293: 2269-2271; Hutvagner et al. 2001, Science 293:
834-838) have shown that endogenous triggers of gene silencing,
specifically small temporal RNAs (stRNAs) let-7 and lin-4, function
at least in part through RNAi pathways. Specifically, these small
RNAs are encoded by hairpin precursors that are processed by Dicer
into mature, .about.21-nt forms. Moreover, genetic studies in C.
elegans have shown a requirement for Argonaute-family proteins in
stRNA function. Specifically, alg-1 and alg-2, members of the EIF2c
subfamily, are implicated both in stRNA processing and in their
downstream effector functions (Grishok et al., 2001, supra). We
have recently shown that a component of RISC, the effector nuclease
of RNAi, is a member of the Argonaute family, prompting a model in
which stRNAs may function through RISC-like complexes, which
regulate mRNA translation rather than mRNA stability (Hammond et
al. 2001, Science 293: 1146-1150).
[0281] A. Short Hairpin RNAs Triggeedr Gene Silencing in Drosophila
Cells.
[0282] We wished to test the possibility that we might retarget
these small, endogenously encoded hairpin RNAs to regulate genes of
choice with the ultimate goal of subverting this regulatory system
for manipulating gene expression stably in mammalian cell lines and
in transgenic animals. Whether triggered by long dsRNAs or by
siRNAs, RNAi is generally more potent in the suppression of gene
expression in Drosophila S2 cells than in mammalian cells. We
therefore chose this model system in which to test the efficacy of
short hairpin RNAs (shRNAs) as inducers of gene silencing.
[0283] Neither stRNAs nor the broader group of miRNAs that has
recently been discovered form perfect hairpin structures. Indeed,
each of these RNAs is predicted to contain several bulged
nucleotides within their rather short (.about.30-nt) stem
structures. Because the position and character of these bulged
nucleotides have been conserved throughout evolution and among at
least a subset of miRNAs, we sought to design retargeted miRNA
mimics to conserve these predicted structural features. Only the
let-7 and lin-4 miRNAs have known miRNA targets (Wightman et al.
1993, Cell 75: 855-862; Slack et al. 2000, Mol. Cell 5: 659-669).
In both cases, pairing to binding sites within the regulated
transcripts is imperfect, and in the case of lin-4, the presence of
a bulged nucleotide is critical to suppression (Ha et al. 1996,
Genes & Dev. 10: 3041-3050). We therefore also designed shRNAs
that paired imperfectly with their target substrates. A subset of
these shRNAs is depicted in FIG. 39A.
[0284] To permit rapid testing of large numbers of shRNA variants
and quantitative comparison of the efficacy of suppression, we
chose to use a dual-luciferase reporter system, as previously
described for assays of RNAi in both Drosophila extracts (Tuschl et
al. 1999, Genes & Dev. 13: 3191-3197) and mammalian cells
(Caplen et al. 2001, Proc. Natl. Acad. Sci. 98: 9742-9747; Elbashir
et al., 2001, Nature 411: 494-498). Cotransfection of firefly and
Renilla luciferase reporter plasmids with either long dsRNAs or
with siRNAs homologous to the firefly luciferase gene yielded an
.about.95% suppression of firefly luciferase without effect on
Renilla luciferase (FIG. 39B; data not shown). Firefly luciferase
could also be specifically silenced by cotransfection with
homologous shRNAs. The most potent inhibitors were those composed
of simple hairpin structures with complete homology to the
substrate. Introduction of G-U basepairs either within the stem or
within the substrate recognition sequence had little or no effect
(FIGS. 39A and 39B; data not shown).
[0285] These results show that short hairpin RNAs can induce gene
silencing in Drosophila S2 cells with potency similar to that of
siRNAs (FIG. 39B). However, in our initial observation of RNA
interference in Drosophila S2 cells, we noted a profound dependence
of the efficiency of silencing on the length of the dsRNA trigger
(Hammond et al. 2000, Nature 404: 293-296). Indeed, dsRNAs of fewer
than .about.200 nt triggered silencing very inefficiently.
Silencing is initiated by an RNase III family nuclease, Dicer, that
processes long dsRNAs into .about.22-nt siRNAs. In accord with
their varying potency as initiators of silencing, long dsRNAs are
processed much more readily than short RNAs by the Dicer enzyme
(Bernstein et al. 2001, Nature 409: 363-366). We therefore tested
whether shRNAs were substrates for the Dicer enzyme.
[0286] We had noted previously that let-7 (Ketting et al. 2001,
Genes & Dev. 15: 2654-2659) and other miRNAs (E. Bernstein,
unpublished data) are processed by Dicer with an unexpectedly high
efficiency as compared with short, nonhairpin dsRNAs. Similarly,
Dicer efficiently processed shRNAs that targeted firefly
luciferase, irrespective of whether they were designed to mimic a
natural Dicer substrate (let-7) or whether they were simple hairpin
structures (FIG. 39C). These data suggest that recombinant shRNAs
can be processed by Dicer into siRNAs and are consistent with the
idea that these short hairpins trigger gene silencing via an RNAi
pathway.
[0287] B. Short Hairpin RNAs Activated Gene Silencing in Mammalian
Cells.
[0288] Mammalian cells contain several endogenous systems that were
predicted to hamper the application of RNAi. Chief among these is a
dsRNA-activated protein kinase, PKR, which effects a general
suppression of translation via phosphorylation of EIF-2.alpha.
(Williams 1997, Biochem. Soc. Trans. 25: 509-513; Gil et al. 2000,
Apoptosis 5: 107-114). Activation of these, and other
dsRNA-responsive pathways, generally requires duplexes exceeding 30
bp in length, possibly to permit dimerization of the enzyme on its
allosteric activator (e.g., Clarke et al. 1995, RNA 1: 7-20). Small
RNAs that mimic Dicer products, siRNAs, presumably escape this
limit and trigger specific silencing, in part because of their
size. However, short duplex RNAs that lack signature features of
siRNAs can efficiently induce silencing in Drosophila S2 cells but
not in mammalian cells (A. A. Caudy, unpublished data).
Endogenously encoded miRNAs may also escape PKR surveillance
because of their size but perhaps also because of the discontinuity
of their duplex structure. Given that shRNAs of <30 bp were
effective inducers of RNAi in Drosophila S2 cells, we tested
whether these RNAs could also induce sequence-specific silencing in
mammalian cells.
[0289] Human embryonic kidney (HEK293T) cells were cotransfected
with chemically synthesized shRNAs and with a mixture of firefly
and Renilla luciferase reporter plasmids. As had been observed in
S2 cells, shRNAs were effective inducers of gene silencing. Once
again, hairpins designed to mimic let-7 were consistently less
effective than were simple hairpin RNAs, and the introduction of
mismatches between the antisense strand of the shRNA and the mRNA
target abolished silencing (FIG. 40A; data not shown). Overall,
shRNAs were somewhat less potent silencing triggers than were
siRNAs. Whereas siRNAs homologous to firefly luciferase routinely
yielded .about.90%-95% suppression of gene expression, suppression
levels achieved with shRNAs ranged from 80%-90% on average. As we
also observe with siRNAs, the most important determinant of the
potency of the silencing trigger is its sequence. We find that
roughly 50% of both siRNAs and shRNAs are competent for suppressing
gene expression. However, neither analysis of the predicted
structures of the target mRNA nor analysis of alternative
structures in siRNA duplexes or shRNA hairpins has proved of
predictive value for choosing effective inhibitors of gene
expression.
[0290] We have adopted as a standard, shRNA duplexes containing 29
bp. However, the size of the helix can be reduced to .about.25 nt
without significant loss of potency. Duplexes as short as 22 bp can
still provoke detectable silencing, but do so less efficiently than
do longer duplexes. In no case did we observe a reduction in the
internal control reporter (Renilla luciferase) that would be
consistent with an induction of nonspecific dsRNA responses.
[0291] The ability of shRNAs to induce gene silencing was not
confined to 293T cells. Similar results were also obtained in a
variety of other mammalian cell lines, including human cancer cells
(HeLa), transformed monkey epithelial cells (COS-1), murine
fibroblasts (NIH 3T3), and diploid human fibroblasts (IMR90; FIG.
40; data not shown).
[0292] C. Synthesis of Effective Inhibitors of Gene Expression
Using T7 RNA Polymerse.
[0293] The use of siRNAs to provoke gene silencing is developing
into a standard methodology for investigating gene function in
mammalian cells. To date, siRNAs have been produced exclusively by
chemical synthesis (e.g., Caplen et al. 2001, Proc. Natl. Acad.
Sci. 98: 9742-9747; Elbashir et al. 2001, Nature 411: 494-498).
However, the costs associated with this approach are significant,
limiting its potential utility as a tool for investigating in
parallel the functions of large numbers of genes. Short hairpin
RNAs are presumably processed into active siRNAs in vivo by Dicer.
Thus, these may be more tolerant of terminal structures, both with
respect to nucleotide overhangs and with respect to phosphate
termini. We therefore tested whether shRNAs could be prepared by in
vitro transcription with T7 RNA polymerase.
[0294] Transcription templates that were predicted to generate
siRNAs and shRNAs similar to those prepared by chemical RNA
synthesis were prepared by DNA synthesis (FIG. 41A,C). These were
tested for efficacy both in S2 cells (data not shown) and in human
293 cells (FIG. 41B,D). Overall, the performance of the
T7-synthesized hairpin or siRNAs closely matched the performance of
either produced by chemical synthesis, both with respect to the
magnitude of inhibition and with respect to the relative efficiency
of differing sequences. Because T7 polymerase prefers to initiate
at twin guanosine residues, however, it was critical to consider
initiation context when designing in vitro transcribed siRNAs (FIG.
41B). In contrast, shRNAs, which are processed by Dicer (see FIG.
39C), tolerate the addition of these bases at the 5' end of the
transcript.
[0295] Studies in Drosophila embryo extracts indicate that siRNAs
possess 5' phosphorylated termini, consistent with their production
by an RNase III family nuclease. In vitro, this terminus is
critical to the induction of RNAi by synthetic RNA oligonucleotides
(Elbashir et al. 2001, EMBO J. 20: 6877-6888; Nykanen et al. 2001,
Cell 107: 309-321). Chemically synthesized siRNAs are
nonphosphorylated, and enzymatic addition of a 5' phosphate group
in vitro prior to transfection does not increase the potency of the
silencing effect (A. A. Caudy, unpublished data). This suggests
either that the requirement for phosphorylated termini is less
stringent in mammalian cells or that a kinase efficiently
phosphorylates siRNAs in vivo. RNAs synthesized with T7 RNA
polymerase, however, possess 5' triphosphate termini. We therefore
explored the possibility of synthesizing siRNAs with T7 polymerase
followed by treatment in vitro with pyrophosphatase to modify the
termini to resemble those of siRNAs. Surprisingly,
monophosphorylated siRNAs (data not shown) were as potent in
inducing gene silencing as transcription products bearing
triphosphate termini (FIG. 41B). This may suggest either that the
requirement for monophosphorylated termini is less stringent in
mammalian cells or that siRNAs are modified in vivo to achieve an
appropriate terminal structure.
[0296] Considered together, our data suggest that both shRNAs and
siRNA duplexes can be prepared by synthesis with T7 RNA polymerase
in vitro. This significantly reduces the cost of RNAi in mammalian
cells and paves the way for application of RNAi on a whole-genome
scale.
[0297] D. Transcription of Small Hairpin RNAs In Vivo by RNA
Polymerase III.
[0298] Although siRNAs are an undeniably effective tool for probing
gene function in mammalian cells, their suppressive effects are by
definition of limited duration. Delivery of siRNAs can be
accomplished by any of a number of transient transfection
methodologies, and both the timing of peak suppression and the
recovery of protein levels as silencing decays can vary with both
the cell-type and the target gene. Therefore, one limitation on
siRNAs is the development of continuous cell lines in which the
expression of a desired target is stably silenced.
[0299] Hairpin RNAs, consisting of long duplex structures, have
been proved as effective triggers of stable gene silencing in
plants, in C. elegans, and in Drosophila (Kennerdell et al. 2000,
Nat. Biotechnol. 18: 896-898; Smith et al. 2000, Nature 407:
319-320; Tavernarakis et al. 2000, Nat. Genet. 24: 180-183). We
have recently shown stable suppression of gene expression in
cultured mammalian cells by continuous expression of a long hairpin
RNA (Paddison et al. 2002, Proc. Natl. Acad. Sci. 99: 1443-1448).
However, the scope of this approach was limited by the necessity of
expressing such hairpins only in cells that lack a detectable PKR
response. In principle, shRNAs could bypass such limitations and
provide a tool for evoking stable suppression by RNA in mammalian
somatic cells.
[0300] To test this possibility, we initially cloned sequences
encoding a firefly luciferase shRNA into a CMV-based expression
plasmid. This was predicted to generate a capped, polyadenylated
RNA polymerase II transcript in which the hairpin was extended on
both the 5' and 3' ends by vector sequences and poly(A). This
construct was completely inert in silencing assays in 293T
cells.
[0301] During our studies on chemically and T7-synthesized shRNAs,
we noted that the presence of significant single-stranded
extensions (either 5' or 3' of the duplex) reduced the efficacy of
shRNAs. We therefore explored the use of alternative promoter
strategies in an effort to produce more defined hairpin RNAs. In
particular, RNA polymerase III promoters have well-defined
initiation and termination sites and naturally produce a variety of
small, stable RNA species. Although many Pol III promoters contain
essential elements within the transcribed region, limiting their
utility for our purposes; class III promoters use exclusively
nontranscribed promoter sequences. Of these, the U6 snRNA promoter
and the H1 RNA promoter have been well studied (Lobo et al. 1990,
Nucleic Acids Res. 18: 2891-2899; Hannon et al. 1991, J. Biol.
Chem. 266: 22796-22799; Chong et al. 2001, J. Biol. Chem. 276:
20727-20734).
[0302] By placing a convenient cloning site immediately behind the
U6 snRNA promoter, we have constructed pShh-1, an expression vector
in which short hairpins are harnessed for gene silencing. Into this
vector either of two shRNA sequences derived from firefly
luciferase were cloned from synthetic oligonucleotides. These were
cotransfected with firefly and Renilla luciferase expression
plasmids into 293T cells. One of the two encoded shRNAs provoked
effective silencing of firefly luciferase without altering the
expression of the internal control (FIG. 42C). The second encoded
shRNA also produced detectable, albeit weak, repression. In both
cases, silencing was dependent on insertion of the shRNA in the
correct orientation with respect to the promoter (FIG. 42C; data
not shown). Although the shRNA itself is bilaterally symmetric,
insertion in the incorrect orientation would affect Pol III
termination and is predicted to produce a hairpin with both 5' and
3' single-stranded extensions. Similar results were also obtained
in a number of other mammalian cell lines including HeLa, COS-1,
NIH 3T3, and IMR90 (FIG. 42; data not shown). pShh1-Ff1 was,
however, incapable of effecting suppression of the luciferase
reporter in Drosophila cells, in which the human U6 promoter is
inactive.
[0303] E. Dicer is Required for shRNA-Mediated Gene Silencing.
[0304] As a definitive test of whether the plasmid-encoded shRNAs
brought about gene silencing via the mammalian RNAi pathway, we
assessed the dependence of suppression on an essential component of
the RNAi pathway. We transfected pShh1-Ff1 along with an siRNA
homologous to human Dicer. FIG. 43 shows that treatment of cells
with Dicer siRNAs is able to completely depress the silencing
induced by pShh1-Ff1. Addition of an unrelated siRNA had no effect
on the magnitude of suppression by pShh1-Ff1. Importantly, Dicer
siRNAs had no effect on siRNA-induced silencing of firefly
luciferase. These results are consistent with shRNAs operating via
an RNAi pathway similar to those provoked by stRNAs and long
dsRNAs. Furthermore, it suggests that siRNA-mediated silencing is
less sensitive to depletion of the Dicer enzyme.
[0305] F. Stable shRNA-Mediated Gene Silencing of an Endogenous
Gene.
[0306] The ultimate utility of encoded short hairpins will be in
the creation of stable mutants that permit the study of the
resulting phenotypes. We therefore tested whether we could create a
cellular phenotype through stable suppression. Expression of
activated alleles of the ras oncogene in primary mouse embryo
fibroblasts (MEFs) induces a stable growth arrest that resembles,
as a terminal phenotype, replicative senescence (Serrano et al.
1997, Cell 88: 593-602). Cells cease dividing and assume a typical
large, flattened morphology. Senescence can be countered by
mutations that inactivate the p53 tumor suppressor pathway (Serrano
et al. 1997, supra). As a test of the ability of vector-encoded
shRNAs to stably suppress an endogenous cellular gene, we generated
a hairpin that was targeted to the mouse p53 gene. As shown in FIG.
44, MEFs transfected with pBabe-RasV12 fail to proliferate and show
a senescent morphology when cotransfected with an empty control
vector. As noted previously by Serrano et al., the terminally
arrested state is achieved in 100% of drug-selected cells in
culture by 8 d posttransfection. However, upon cotransfection of an
activated ras expression construct with the pShh-p53, cells emerged
from drug selection that not only fail to adopt a senescent
morphology but also maintain the ability to proliferate for a
minimum of several weeks in culture (FIG. 44). These data strongly
suggest that shRNA expression constructs can be used for the
creation of continuous mammalian cell lines in which selected
target genes are stably suppressed.
[0307] G. Simultaneous Introduction of Multiple Hairpin RNAs Does
Not Produce Synergy.
[0308] In an attempt to further understand the mechanisms by which
short hairpins suppress gene expression, we examined the effects of
transfecting cells with a mixture of two different short hairpins
corresponding to firefly luciferase. FIG. 45 summarizes the results
of experiments which suggest that there is no synergistic affects
on suppression of firefly luciferase gene expression obtained when
cells are exposed to a mixture of such short hairpins.
[0309] Methods:
[0310] Cell culture. HEK 293T, HeLa, COS-1, MEF, and IMR90 cells
were cultured in DMEM (GIBCO BRL) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) and 1%
antibiotic/antimycotic solution (GIBCO BRL). NIH 3T3 cells were
cultured in DMEM supplemented with 10% heat-inactivated calf serum
and 1% antibiotic/antimycotic solution.
[0311] RNA preparation. Both shRNAs and siRNAs were produced in
vitro using chemically synthesized DNA oligonucleotide templates
(Sigma) and the T7 Megashortscript kit (Ambion). Transcription
templates were designed such that they contained T7 promoter
sequences at the 5' end. shRNA transcripts subjected to in vitro
Dicer processing were synthesized using a Riboprobe kit (Promega).
Chemically synthesized RNAs were obtained from Dharmacon, Inc.
[0312] Transfection and gene silencing assays. Cells were
transfected with indicated amounts of siRNA, shRNA, and plasmid DNA
using standard calcium phosphate procedures at 50%-70% confluence
in 6-well plates. Dual luciferase assays (Promega) were carried out
by cotransfecting cells with plasmids containing firefly luciferase
under the control of the SV40 promoter (pGL3-Control, Promega) and
Renilla luciferase under the control of the SV40 early
enhancer/promoter region (pSV40, Promega). Plasmids were
cotransfected using a 1:1 ratio of pGL3-Control (250 ng/well) to
pRL-SV40. RNAi in S2 cells was performed as previously described
(Hammond et al., 2000, Nature 404: 293-296). For stable silencing,
primary MEFs (a gift from S. Lowe, Cold Spring Harbor Laboratory,
NY) were cotransfected using Fugene 6 with pBabe-Ha-rasV12 and
pShh-p53 (no resistance marker), according to the manufacturer's
recommendations. Selection was for the presence of the activated
Ha-rasV12 plasmid, which carries a puromycin-resistance marker. The
pShh-p53 plasmid was present in excess, as is standard in a
cotransfection experiment. We have now generated a version of the
U6 promoter vector (pSHAG-1) that is compatible with the GATEWAY
system (Invitrogen), and this can be used to transport the shRNA
expression cassette into a variety of recipient vectors that carry
cis-linked selectable markers. Furthermore, we have validated
delivery of shRNAs using retroviral vectors. Updated plasmid
information can be obtained at
http://www.cshl.org/public/science/hannon.html.
[0313] Plasmids expressing hairpin RNAs. The U6 promoter region
from -265 to +1 was amplified by PCR, adding 5' KpnI and 3' EcoRV
sites for cloning into pBSSK+. A linker/terminator oligonucleotide
set bearing the U6 terminator sequence and linker ends of 5' EcoRV
and 3' NotI was cloned into the promoter construct, resulting in a
U6 cassette with an EcoRV site for insertion of new sequences. This
vector has been named pShh1. Blunt-ended, double-stranded DNA
oligonucleotides encoding shRNAs with between 19 and 29 bases of
homology to the targeted gene were ligated into the EcoRV site to
produce expression constructs. The oligonucleotide sequence used to
construct Ff1 was: TCCAATTCAGCGGGAGCCACCTGATGAAGCTTGATCG-
GGTGGCTCTCGCTGAG TTGGAATCCATTTTTTTT. This sequence is preceded by
the sequence GGAT, which is supplied by the vector, and contains a
tract of more than five Ts as a Pol III terminator.
[0314] In vitro Dicer assays. In vitro assays for Dicer activity
were performed as described (Bernstein et al., 2001, Nature 409:
363-366).
EXAMPLE 7
Encoded Short Hairpins Function in Vivo
[0315] An object of the present invention is to improve methods for
generating siRNAs and short hairpins for use in specifically
suppressing gene expression. Example 6 demonstrates that siRNAs and
short hairpins are highly effective in specifically suppressing
gene expression. Accordingly, it would be advantageous to combine
the efficient suppression of gene expression attainable using short
hairpins and siRNAs with a method to encode such RNA on a plasmid
and express it either transiently or stably.
[0316] FIG. 46 demonstrates that short hairpins encoded on a
plasmid are effective in suppressing gene expression. DNA
oligonucleotides encoding 29 nucleotide hairpins corresponding to
firefly luciferase were inserted into a vector containing the U6
promoter. Three independent constructs were examined for their
ability to specifically suppress firefly luciferase gene expression
in 293T cells. siOligo1-2, siOligo1-6, and siOligo1-19 (construct
in the correct orientation) each suppressed gene expression as
effectively as siRNA. In contrast, siOligo1-10 (construct in the
incorrect orientation) did not suppress gene expression.
Additionally, an independent construct targeted to a different
portion of the firefly luciferase gene did not effectively suppress
gene expression in either orientation (siOligo2-23,
siOligo2-36).
[0317] The results summarized in FIG. 46 demonstrate that transient
expression of siRNAs and short hairpins encoded on a plasmid can
efficiently suppress gene expression. One of skill can choose from
amongst a range of vectors to either transiently or stably express
an siRNA or short hairpin. Non-limiting examples of vectors and
strategies to stably express short dsRNAs are presented in FIGS.
47-49.
EXAMPLE 8
dsRNA Suppression in the Absence of a PKR Response
[0318] One potential impediment to the use of RNAi to suppress gene
expression in some cell types, is the non-specific PKR response
that can be triggered by long dsRNAs. Numerous mammalian viruses
have evolvd the ability to block PKR inorder to aid in the
infection of potential host cells. For example, adenoviruses
express RNAs which mimic dsRNA but do not activate the PKR
response. Vaccinia virus uses two strategies to evade PKR: the
expression of E3L which binds and masks dsRNA; the expression of
K3L to mimic the natural PKR substrate eIF2.alpha..
[0319] Our understanding of the mechanisms by which viruses avoid
the PKR response allows us to design approaches to circumvent the
PKR response in cell types in which in might be advantageous to
suppression gene expression with long dsRNAs. Possible approaches
include treating cells with an agent that inhibits protein kinase
RNA-activated (PKR) apoptosis, such as by treatment with agents
which inhibit expression of PKR, cause its destruction, and/or
inhibit the kinase activity of PKR. Accordingly, RNAi suppression
of gene expression in such cell types could involve first
inhibiting the PKR response, and then delivering a dsRNA identical
or similar to a target gene.
[0320] A. In a murine myoblast cell line, C2C12, we noted that the
cells responded to long dsRNAs with a mixture of specific and
non-specific (presumably PKR) responses. In order to attenuate the
non-specific PKR response while maintaining the robust and specific
suppression due to the long dsRNA, C2C12 cells were transfected
with a vector that directs K3L expression. This additional step
successfully attenuated the PKR response, however expression of K3L
protein had no effect on the magnitude of specific inhibition.
[0321] B. However, since the efficacy of such a two step approach
had not been previously demonstrated, it was formerly possible that
dsRNA suppression would not be possible in cells with a PKR
response. FIG. 50 summarizes results which demonstrate that such a
two step approach is possible, and that robust and specific dsRNA
mediated suppression is possible in cells which had formerly
possessed a robust PKR response.
[0322] Briefly, dual luciferase assay were carried out as described
in detail above. The experiments were carried out using PKR-/- MEFs
harvested from E13.5 PKR-/- mosue embryos. MEFs typically have a
robust PKR response, and thus treatment with long dsRNAs typically
results in non-specific suppression of gene expression and
apoptosis. However, in PKR-/- cells examined 12, 42, and 82 hours
after transfection, expression of dsRenilla luciferase RNA
specifically suppresses expression Renilla reniformis (sea pansy)
luciferase. This suppression is stable over time.
[0323] These results demonstrate that the non-specific PKR response
can be blocked without affecting specific suppression of gene
expression mediated by dsRNA. This allows the use of long dsRNAs to
suppress gene expression in a diverse range of cell types,
including those that would be previously intractable due to the
confounding influences of the non-specific PKR response to long
dsRNA.
EXAMPLE 9
Suppression of Gene Expression Using dsRNA which Corresponds to
Non-Coding Sequence
[0324] Current models for the mechanisms which drive RNAi have
suggested that the dsRNA construct must contain coding sequence
corresponding to the gene of interest. Although evidence has
demonstrated that such coding sequence need not be a perfect match
to the endogenous coding sequence (i.e., it may be similar), it has
been widely held that the dsRNA construct must correspond to coding
sequence. We present evidence that contradicts the teachings of the
prior art, and demonstrate that dsRNA corresponding to non-coding
regions of a gene can suppress gene function in vivo. These results
are significant not only because they demonstrate that dsRNA
identical or similar to non-coding sequences (i.e., promoter
sequences, enhancer sequences, or intronic sequences) can mediate
suppression, but also because we demonstrate the in vivo
suppression of gene expression using dsRNA technology in a mouse
model.
[0325] We generated doubled stranded RNA corresponding to four
segments of the mouse tyrosinase gene promoter. Three of these
segments correspond to the proximal promoter and one corresponds to
an enhancer (FIG. 51). The tyrosinase gene encodes the rate
limiting enzyme involved in the melanin biosynthetic pathway
(Bilodeau et al. (2001) Pigment Cell Research 14: 328-336).
Accordingly, suppression of the tyrosinase gene is expected to
inhibit pigmentation.
[0326] Double stranded RNA corresponding to each of the above
promoter segments was injected into the pronuclei of fertilized
eggs. Pups were born after 19 days. In total 42/136 (31%) of the
embryos were carried to term. This number is within the expected
range for transgenesis (30-40%). Two pups out of 42 (5%) appear
totally unpigmented at birth, consistent with suppression of
tyrosinase function.
[0327] Methods:
[0328] dsRNA from non-coding promoter region of tyrosinase gene.
Four segments of the mouse tyrosinase gene promoter were amplified
by PCR using primers which incorporated T7 RNA polymerase promoters
into the PCR products (shown in bold--FIG. 51). Sequences of the
mouse tyrosinase gene 5' flanking regions were obtained from
GenBank (accession number D00439 and X51743). The sequence of the
tyrosinase enhancer, located approximately 12 kb upstream of the
transcriptional start site, was also obtained from GenBank
(accession number X76647).
[0329] The sequences of the primers used were as follows: note the
sequence of the T7 RNA polymerase promoter is shown in bold
1 (a) Tyrosinase enhancer (.about.12 kb upstream):
5'TAATACGACTCACTATAGGGCAAGGTCATAGTTCCTGCCAGCTG 3'
5'TAATACGACTCACTATAGGGCAGATATTTTCTTACCACCCACCC 3' (b) -1404 to
-1007: 5'TAATACGACTCACTATAGGGTTAAGTTTAACAGGAGAAGCTGGA 3'
5'TAATACGACTCACTATAGGGAAATCATTGCTTTCCTGATAATGC 3' (c) -1003 to
-506: 5'TAATACGACTCACTATAGGGTAGATTTCCGCAGCCCCAGTGTTC 3'
5'TAATACGACTCACTATAGGGGTTGCCTCTCATTTTTCCTTGATT 3' (d) -505 to -85:
5'TAATACGACTCACTATAGGGTATTTTAGACTGATTACTTTTATAA 3'
5'TAATACGACTCACTATAGGGTCACATGTTTTGGCTAAGACCTAT 3'
[0330] PCR products were gel purified from 1% TAE agarose gels
using QiaExII Gel Extraction Kit (Qiagen). Double stranded RNA was
produced from these templates using T7-Megashortscript Kit
(Ambion). Enzymes and unincorporated nucleotides were removed using
Qiaquick MinElute PCR Purification Kit. RNA was phenol/chloroform
extracted twice, and ethanol precipitated. Pellets were resuspended
in injection buffer ((10 mM Tris (pH 7.5), 0.15 nM EDTA (pH 8.0))
at a concentration of 20 ng/ul and run on a 1% TAE agarose gel to
confirm integrity.
[0331] Generation of mice: An equal mixture of double stranded RNA
from each of the above primer sets was injected into the pronuclei
of fertilized eggs from C57BL6J mice. A total of 136 injections was
performed, and 34 embryos were implanted into each of 4
pseudopregnant CD-1 females. Pups were born after 19 days. In
total, 42/136 (31%) of the embryos were carried to term. 2/42 pups
(5%) appear totally unpigmented at birth.
[0332] It is not clear whether the RNAi mediated by dsRNA identical
or similar to non-coding sequence works via the same mechanism as
PTGS observed in the presence of dsRNA identical or similar to
coding sequence. However, whether these results ultimately reveal
similar or differing mechanisms does not diminish the tremendous
utility of the compositions and methods of the present invention to
suppress expression of one or more genes in vitro or in vivo.
[0333] The present invention demonstrates that dsRNA ranging in
length from 20-500 nt can readily suppress expression of target
genes both in vitro and in vivo. Furthermore, the present invention
demonstrates that the dsRNAs can be generated using a variety of
methods including the formation of hairpins, and that these dsRNAs
can be expressed either stably or transiently. Finally, the present
invention demonstrates that dsRNA identical or similar to
non-coding sequences can suppress target gene expression.
EXAMPLE 10
RNA Interference in Adult Mice
[0334] RNA interference is an evolutionarily conserved surveillance
mechanism that responds to double-stranded RNA by sequence-specific
silencing of homologous genes. Here we show that transgene
expression can be suppressed in adult mice by synthetic small
interfering RNAs and by small-hairpin RNAs transcribed in vivo from
DNA templates. We also show the therapeutic potential of this
technique by demonstrating effective targeting of a sequence from
hepatitis C virus by RNA interference in vivo.
[0335] Small interfering RNAs (siRNAs) mimic intermediates in the
RNA-interference (RNAi) pathway and can silence genes in somatic
cells without activating non-specific suppression by
double-stranded RNA-dependent protein kinase (Elbashir et al. 2001,
Nature 411: 494-498). To investigate whether siRNAs also inhibit
gene expression in vivo, we used a modification of hydrodynamic
transfection methods (Zhang et al. 1999, Hum. Gene Therapy 10:
1735-1737; Liu et al. 1999, Gene Therapy 6: 1258-1266; Chang et al.
2001, J. Virol. 75: 3469-3473) to deliver naked siRNAs to the
livers of adult mice. Either an siRNA derived from firefly
luciferase or an unrelated siRNA was co-injected with a
luciferase-expression plasmid (for construct description and
sequences, see FIG. 52). We monitored luciferase expression in
living animals using quantitative whole-body imaging (Contag, et
al. 1997, Photochem. Photobiol. 66: 523-531) (see FIGS. 53a, 54a),
and found that it was dependent on reporter-plasmid dose.
[0336] In each experiment, serum measurements of a co-injected
human .chi.-1 antitrypsin (hAAT) plasmid (Yant et al. 2000, Nature
Genet. 25: 35-41) served to normalize transfection efficiency and
to monitor non-specific translational inhibition. Average serum
concentrations of HAAT after 74 h were similar in all groups.
[0337] Our results indicate that there was specific, siRNA-mediated
inhibition of luciferase expression in adult mice (P<0.0115) and
that unrelated siRNAs had no effect (P<0.864; FIGS. 53a, 53b).
In 11 independent experiments, luciferase siRNAs reduced luciferase
expression (as judged by emitted light) by an average of 81%
(.+-.2.2%). These findings indicate that RNAi can downregulate gene
expression in adult mice.
[0338] As RNAi degrades respiratory syncitial virus RNAs in culture
(Bitko et al. 2001, BMC Microbiol. 1: 34), we investigated whether
RNAi could be directed against a human pathogenic RNA expressed in
a mouse, namely that of hepatitis C virus (HCV). Infection by HCV
(an RNA virus that infects 1 in 40 people worldwide) is the most
common reason for liver transplantation in the United States and
Europe. We fused the NS5B region (non-structural protein 5B,
viral-polymerase-encoding region) of this virus with luciferase RNA
and monitored RNAi by co-transfection in vivo. An siRNA targeting
the NS5B region reduced luciferase expression from the chimaeric
HCV NS5B protein-luciferase fusion by 75% (.+-.6.8%; 6 animals per
group). This result suggests that it may be feasible to use RNAi as
a therapy against other important human pathogens.
[0339] Although our results show that siRNAs are functional in
mice, delivery remains a major obstacle. Unlike siRNAs, functional
small-hairpin RNAs (shRNAs) can be expressed in vivo from DNA
templates using RNA polymerase III promoters (Paddison et al. 2002,
Genes Dev. 16: 948-958; Tuschl 2002, Nature Biotechnol. 20:
446-448); they are as effective as siRNAs in inducing gene
suppression. Expression of a cognate shRNA (pShh1-Ff1) inhibited
luciferase expression by up to 98% (.+-.0.6%), with an average
suppression of 92.8% (.+-.3.39%) in three independent experiments
(see FIGS. 54a, 54b). An empty shRNA-expression vector had no
effect; reversing the orientation of the shRNA (pShh1-Ff1rev)
insert prevents gene silencing because it alters the termination by
RNA polymerase III and generates an improperly structured shRNA.
These findings indicate that plasmid-encoded shRNAs can induce a
potent and specific RNAi response in adult mice.
[0340] RNAi may find application in functional genomics or in
identifying targets for designer drugs. It is a more promising
system than gene-knockout mice because groups of genes can be
simultaneously rendered ineffective without the need for
time-consuming crosses. Gene therapy currently depends on the
ectopic expression of exogenous proteins; however, RNAi may
eventually complement this gain-of-function approach by silencing
disease-related genes with DNA constructs that direct the
expression of shRNAs. Our method of RNAi delivery could also be
tailored to take advantage of developing viral and non-viral
gene-transfer vectors in a clinical context.
EXAMPLE 11
Germline Transmission of RNAi in Mice
[0341] MicroRNA molecules (miRNAs) are small, noncoding RNA
molecules that have been found in a diverse array of eukaryotes,
including mammals. miRNA precursors share a characteristic
secondary structure, forming short `hairpin` RNAs. Genetic and
biochemical studies have indicated that miRNAs are processed to
their mature forms by Dicer, an RNAse III family nuclease, and
function through RNA-mediated interference (RNAi) and related
pathways to regulate the expression of target genes (Hannon 2002,
Nature 418: 244-251; Pasquinelli et al. 2002, Annu. Rev. Cell. Dev.
Biol. 18: 495-513). Recently, we and others have remodeled miRNAs
to permit experimental manipulation of gene expression in mammalian
cells and have dubbed these synthetic silencing triggers `short
hairpin RNAs` (shRNAs) (Paddison et al. 2002, Cancer Cell 2:
17-23). Silencing by shRNAs requires the RNAi machinery and
correlates with the production of small interfering RNAs (siRNAs),
which are a signature of RNAi.
[0342] Expression of shRNAs can elicit either transient or stable
silencing, depending upon whether the expression cassette is
integrated into the genome of the recipient cultured cell (Paddison
et al. 2002, Cancer Cell 2: 17-23). shRNA expression vectors also
induce gene silencing in adult mice following transient delivery
(Lewis et al. 2002, Nat. Genet. 32: 107-108; McCaffrey et al. 2002,
Nature 418: 38-39). However, for shRNAs to be a viable genetic tool
in mice, stable manipulation of gene expression is essential.
Hemann and colleagues have demonstrated long-term suppression of
gene expression in vivo following retroviral delivery of
shRNA-expression cassettes to hematopoietic stem cells (Hemann et
al. 2003, Nat. Genet. in the press). Here we sought to test whether
shRNA-expression cassettes that were passed through the mouse
germline could enforce heritable gene silencing.
[0343] We began by taking standard transgenesis approaches (Gordon
et al. 1993, Methods Enzymol. 225: 747-771) using shRNAs directed
against a variety of targets with expected phenotypes, including
the genes encoding tyrosinase (albino), myosin VIIa (shaker), Bmp-5
(crinkled ears), Hox a-10 (limb defects), homogentisate
1,2,-dioxygenase (urine turns black upon exposure to air), Hairless
(hair loss) and melanocortin 1 receptor (yellow). Three constructs
per gene were linearized and injected into pronuclei to produce
transgenic founder animals. Although we noted the presence of the
transgene in some animals, virtually none showed a distinct or
reproducible phenotype that was expected for a hypomorphic allele
of the targeted gene.
[0344] Therefore, we decided to take another approach: verifying
the presence of the shRNA and its activity toward a target gene in
cultured embryonic stem (ES) cells and then asking whether those
cells retained suppression in a chimeric animal in vivo. We also
planned to test whether such cells could pass a functional
RNAi-inducing construct through the mouse germline. For these
studies, we chose to examine a novel gene, Neil1, which is proposed
to have a role in DNA repair. Oxidative damage accounts for 10,000
DNA lesions per cell per day in humans and is thought to contribute
to carcinogenesis, aging and tissue damage following ischemia (Ames
et al. 1993, Proc. Natl. Acad. Sci. USA 90: 7915-7922; Jackson et
al. 2001, Mutat. Res. 477: 7-21). Oxidative DNA damage includes
abasic sites, strand breaks and at least 20 oxidized bases, many of
which are cytotoxic or pro-mutagenic (Dizdaroglu et al. 2002, Free
Radic. Biol. Med. 32: 1102-1115). DNA N-glycosylases initiate the
base excision repair pathway by recognizing specific bases in DNA
and cleaving the sugar base bond to release the damaged base (David
et al. 1998, Chem. Rev. 98: 1221-1262).
[0345] The Neil genes are a newly discovered family of mammalian
DNA N-glycosylases related to the Fpg/Nei family of proteins from
Escherichia coli (Hazra et al. 2002, Proc. Natl. Acad. Sci. USA 99:
3523-3528; Bandaru et al. 2002, DNA Repair 1: 517-529). Neil1
recognizes and removes a wide spectrum of oxidized pyrimidines and
ring-opened purines from DNA, including thymine glycol (Tg),
2,6-diamino-4-hydroxy-5-formamidopyrimidin- e (FapyG) and
4,6-diamino-5-formidopyrimidine (FapyA). Tg, FapyG and FapyA are
among the most prevalent oxidized bases produced by ionizing
radiation (Dizdaroglu et al. 2002, Free Radic. Biol. Med. 32:
1102-1115) and can block replicative DNA polymerases, which can, in
turn, cause cell death (Asagoshi et al. 2002, J. Biol. Chem. 277:
14589-14597; Clark et al. 1989, Biochemistry 28: 775-779).
[0346] The Nth1 and Ogg1 glycosylases each remove subsets of
oxidized DNA bases that overlap with substrates of Neil1 (Nishimura
2002, Free Radic. Biol. Med. 32: 813-821; Asagoshi et al. 2000,
Biochemistry 39: 11389-11398; Dizdaroglu et al. 1999, Biochemistry
38: 243-246). However, mice with null mutations in either Nth1
(Ocampo et al. 2002, Mol. Cell. Biol. 22: 6111-6121; Takao et al.
2002, EMBO J. 21: 3486-3493) or Ogg1 (Klungland et al. 1999, Proc.
Natl. Acad. Sci. USA 96: 13300-13305; Minowa et al. 2000, Proc.
Natl. Acad. Sci. USA 97: 4156-4161) are viable, raising the
possibility that Neil1 activity tempers the loss of Nth1 or Ogg1.
Recently, a residual Tg-DNA glycosylase activity in Nth1.sup.-/-
mice has been identified as Neil1 (Takao et al. 2002, J. Biol.
Chem. 277: 42205-42213).
[0347] We constructed a single shRNA expression vector targeting a
sequence near the 5' end of the Neil1 coding region. This vector
was introduced into mouse embryonic stem cells by electroporation,
and individual stable integrants were tested for expression of the
Neil1 protein (see the weblink:
http://www.cshl.edu/public/SCIENCE/hannon.html for detailed
procedures). The majority of cell lines showed an .about.80%
reduction in Neil1 protein, which correlated with a similar change
in levels of Neil1 mRNA. These cells showed an approximately
two-fold increase in their sensitivity to ionizing radiation,
consistent with a role for Neil1 in DNA repair. Two independent ES
cell lines were injected into BL/6 blastocysts, and several
high-percentage chimeras were obtained. These chimeras were
out-crossed, and germline transmission of the shRNA-expression
construct was noted in numerous F.sub.1 progeny (13/27 for one line
and 12/26 for the other).
[0348] To determine whether the silencing of Neil1 that had been
observed in ES cells was transmitted faithfully, we examined Neil1
mRNA and protein levels. Both were reduced by approximately the
same extent that had been observed in the engineered ES cells
(FIGS. 55, 56). Consistent with this having occurred through the
RNAi pathway, we detected the presence of siRNAs corresponding to
the shRNA sequence in F.sub.1 animals that carry the shRNA
expression vector but not in those that lack the vector (FIG.
56b).
[0349] The aforementioned data demonstrate that shRNAs can be used
to create germline transgenic mice in which RNAi has silenced a
target gene. These observations open the door to using of RNAi as a
complement to standard knock-out methodologies and provide a means
to rapidly assess the consequences of suppressing a gene of
interest in a living animal. Coupled with activator-dependent U6
promoters, the use of shRNAs will ultimately provide methods for
tissue-specific, inducible and reversible suppression of gene
expression in mice.
[0350] V. Equivalents
[0351] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0352] All of the above-cited references and publications are
hereby incorporated by reference.
Sequence CWU 1
1
45 1 5775 DNA Homo sapiens CDS (1)..(5775) 1 atg aaa agc cct gct
ttg caa ccc ctc agc atg gca ggc ctg cag ctc 48 Met Lys Ser Pro Ala
Leu Gln Pro Leu Ser Met Ala Gly Leu Gln Leu 1 5 10 15 atg acc cct
gct tcc tca cca atg ggt cct ttc ttt gga ctg cca tgg 96 Met Thr Pro
Ala Ser Ser Pro Met Gly Pro Phe Phe Gly Leu Pro Trp 20 25 30 caa
caa gaa gca att cat gat aac att tat acg cca aga aaa tat cag 144 Gln
Gln Glu Ala Ile His Asp Asn Ile Tyr Thr Pro Arg Lys Tyr Gln 35 40
45 gtt gaa ctg ctt gaa gca gct ctg gat cat aat acc atc gtc tgt tta
192 Val Glu Leu Leu Glu Ala Ala Leu Asp His Asn Thr Ile Val Cys Leu
50 55 60 aac act ggc tca ggg aag aca ttt att gct agt act act cta
cta aag 240 Asn Thr Gly Ser Gly Lys Thr Phe Ile Ala Ser Thr Thr Leu
Leu Lys 65 70 75 80 agc tgt ctc tat cta gat cta ggg gag act tca gct
aga aat gga aaa 288 Ser Cys Leu Tyr Leu Asp Leu Gly Glu Thr Ser Ala
Arg Asn Gly Lys 85 90 95 agg acg gtg ttc ttg gtc aac tct gca aac
cag gtt gct caa caa gtg 336 Arg Thr Val Phe Leu Val Asn Ser Ala Asn
Gln Val Ala Gln Gln Val 100 105 110 tca gct gtc aga act cat tca gat
ctc aag gtt ggg gaa tac tca aac 384 Ser Ala Val Arg Thr His Ser Asp
Leu Lys Val Gly Glu Tyr Ser Asn 115 120 125 cta gaa gta aat gca tct
tgg aca aaa gag aga tgg aac caa gag ttt 432 Leu Glu Val Asn Ala Ser
Trp Thr Lys Glu Arg Trp Asn Gln Glu Phe 130 135 140 act aag cac cag
gtt ctc att atg act tgc tat gtc gcc ttg aat gtt 480 Thr Lys His Gln
Val Leu Ile Met Thr Cys Tyr Val Ala Leu Asn Val 145 150 155 160 ttg
aaa aat ggt tac tta tca ctg tca gac att aac ctt ttg gtg ttt 528 Leu
Lys Asn Gly Tyr Leu Ser Leu Ser Asp Ile Asn Leu Leu Val Phe 165 170
175 gat gag tgt cat ctt gca atc cta gac cac ccc tat cga gaa ttt atg
576 Asp Glu Cys His Leu Ala Ile Leu Asp His Pro Tyr Arg Glu Phe Met
180 185 190 aag ctc tgt gaa att tgt cca tca tgt cct cgc att ttg gga
cta act 624 Lys Leu Cys Glu Ile Cys Pro Ser Cys Pro Arg Ile Leu Gly
Leu Thr 195 200 205 gct tcc att tta aat ggg aaa tgg gat cca gag gat
ttg gaa gaa aag 672 Ala Ser Ile Leu Asn Gly Lys Trp Asp Pro Glu Asp
Leu Glu Glu Lys 210 215 220 ttt cag aaa cta gag aaa att ctt aag agt
aat gct gaa act gca act 720 Phe Gln Lys Leu Glu Lys Ile Leu Lys Ser
Asn Ala Glu Thr Ala Thr 225 230 235 240 gac ctg gtg gtc tta gac agg
tat act tct cag cca tgt gag att gtg 768 Asp Leu Val Val Leu Asp Arg
Tyr Thr Ser Gln Pro Cys Glu Ile Val 245 250 255 gtg gat tgt gga cca
ttt act gac aga agt ggg ctt tat gaa aga ctg 816 Val Asp Cys Gly Pro
Phe Thr Asp Arg Ser Gly Leu Tyr Glu Arg Leu 260 265 270 ctg atg gaa
tta gaa gaa gca ctt aat ttt atc aat gat tgt aat ata 864 Leu Met Glu
Leu Glu Glu Ala Leu Asn Phe Ile Asn Asp Cys Asn Ile 275 280 285 tct
gta cat tca aaa gaa aga gat tct act tta att tcg aaa cag ata 912 Ser
Val His Ser Lys Glu Arg Asp Ser Thr Leu Ile Ser Lys Gln Ile 290 295
300 cta tca gac tgt cgt gcc gta ttg gta gtt ctg gga ccc tgg tgt gca
960 Leu Ser Asp Cys Arg Ala Val Leu Val Val Leu Gly Pro Trp Cys Ala
305 310 315 320 gat aaa gta gct gga atg atg gta aga gaa cta cag aaa
tac atc aaa 1008 Asp Lys Val Ala Gly Met Met Val Arg Glu Leu Gln
Lys Tyr Ile Lys 325 330 335 cat gag caa gag gag ctg cac agg aaa ttt
tta ttg ttt aca gac act 1056 His Glu Gln Glu Glu Leu His Arg Lys
Phe Leu Leu Phe Thr Asp Thr 340 345 350 ttc cta agg aaa ata cat gca
cta tgt gaa gag cac ttc tca cct gcc 1104 Phe Leu Arg Lys Ile His
Ala Leu Cys Glu Glu His Phe Ser Pro Ala 355 360 365 tca ctt gac ctg
aaa ttt gta act cct aaa gta atc aaa ctg ctc gaa 1152 Ser Leu Asp
Leu Lys Phe Val Thr Pro Lys Val Ile Lys Leu Leu Glu 370 375 380 atc
tta cgc aaa tat aaa cca tat gag cga cac agt ttt gaa agc gtt 1200
Ile Leu Arg Lys Tyr Lys Pro Tyr Glu Arg His Ser Phe Glu Ser Val 385
390 395 400 gag tgg tat aat aat aga aat cag gat aat tat gtg tca tgg
agt gat 1248 Glu Trp Tyr Asn Asn Arg Asn Gln Asp Asn Tyr Val Ser
Trp Ser Asp 405 410 415 tct gag gat gat gat gag gat gaa gaa att gaa
gaa aaa gag aag cca 1296 Ser Glu Asp Asp Asp Glu Asp Glu Glu Ile
Glu Glu Lys Glu Lys Pro 420 425 430 gag aca aat ttt cct tct cct ttt
acc aac att ttg tgc gga att att 1344 Glu Thr Asn Phe Pro Ser Pro
Phe Thr Asn Ile Leu Cys Gly Ile Ile 435 440 445 ttt gtg gaa aga aga
tac aca gca gtt gtc tta aac aga ttg ata aag 1392 Phe Val Glu Arg
Arg Tyr Thr Ala Val Val Leu Asn Arg Leu Ile Lys 450 455 460 gaa gct
ggc aaa caa gat cca gag ctg gct tat atc agt agc aat ttc 1440 Glu
Ala Gly Lys Gln Asp Pro Glu Leu Ala Tyr Ile Ser Ser Asn Phe 465 470
475 480 ata act gga cat ggc att ggg aag aat cag cct cgc aac aac acg
atg 1488 Ile Thr Gly His Gly Ile Gly Lys Asn Gln Pro Arg Asn Asn
Thr Met 485 490 495 gaa gca gaa ttc aga aaa cag gaa gag gta ctt agg
aaa ttt cga gca 1536 Glu Ala Glu Phe Arg Lys Gln Glu Glu Val Leu
Arg Lys Phe Arg Ala 500 505 510 cat gag acc aac ctg ctt att gca aca
agt att gta gaa gag ggt gtt 1584 His Glu Thr Asn Leu Leu Ile Ala
Thr Ser Ile Val Glu Glu Gly Val 515 520 525 gat ata cca aaa tgc aac
ttg gtg gtt cgt ttt gat ttg ccc aca gaa 1632 Asp Ile Pro Lys Cys
Asn Leu Val Val Arg Phe Asp Leu Pro Thr Glu 530 535 540 tat cga tcc
tat gtt caa tct aaa gga aga gca agg gca ccc atc tct 1680 Tyr Arg
Ser Tyr Val Gln Ser Lys Gly Arg Ala Arg Ala Pro Ile Ser 545 550 555
560 aat tat ata atg tta gcg gat aca gac aaa ata aaa agt ttt gaa gaa
1728 Asn Tyr Ile Met Leu Ala Asp Thr Asp Lys Ile Lys Ser Phe Glu
Glu 565 570 575 gac ctt aaa acc tac aaa gct att gaa aag atc ttg aga
aac aag tgt 1776 Asp Leu Lys Thr Tyr Lys Ala Ile Glu Lys Ile Leu
Arg Asn Lys Cys 580 585 590 tcc aag tcg gtt gat act ggt gag act gac
att gat cct gtc atg gat 1824 Ser Lys Ser Val Asp Thr Gly Glu Thr
Asp Ile Asp Pro Val Met Asp 595 600 605 gat gat cac gtt ttc cca cca
tat gtg ttg agg cct gac gat ggt ggt 1872 Asp Asp His Val Phe Pro
Pro Tyr Val Leu Arg Pro Asp Asp Gly Gly 610 615 620 cca cga gtc aca
atc aac acg gcc att gga cac atc aat aga tac tgt 1920 Pro Arg Val
Thr Ile Asn Thr Ala Ile Gly His Ile Asn Arg Tyr Cys 625 630 635 640
gct aga tta cca agt gat ccg ttt act cat cta gct cct aaa tgc aga
1968 Ala Arg Leu Pro Ser Asp Pro Phe Thr His Leu Ala Pro Lys Cys
Arg 645 650 655 acc cga gag ttg cct gat ggt aca ttt tat tca act ctt
tat ctg cca 2016 Thr Arg Glu Leu Pro Asp Gly Thr Phe Tyr Ser Thr
Leu Tyr Leu Pro 660 665 670 att aac tca cct ctt cga gcc tcc att gtt
ggt cca cca atg agc tgt 2064 Ile Asn Ser Pro Leu Arg Ala Ser Ile
Val Gly Pro Pro Met Ser Cys 675 680 685 gta cga ttg gct gaa aga gtt
gtc gct ctc att tgc tgt gag aaa ctg 2112 Val Arg Leu Ala Glu Arg
Val Val Ala Leu Ile Cys Cys Glu Lys Leu 690 695 700 cac aaa att ggc
gaa ctg gat gac cat ttg atg cca gtt ggg aaa gag 2160 His Lys Ile
Gly Glu Leu Asp Asp His Leu Met Pro Val Gly Lys Glu 705 710 715 720
act gtt aaa tat gaa gag gag ctt gat ttg cat gat gaa gaa gag acc
2208 Thr Val Lys Tyr Glu Glu Glu Leu Asp Leu His Asp Glu Glu Glu
Thr 725 730 735 agt gtt cca gga aga cca ggt tcc acg aaa cga agg cag
tgc tac cca 2256 Ser Val Pro Gly Arg Pro Gly Ser Thr Lys Arg Arg
Gln Cys Tyr Pro 740 745 750 aaa gca att cca gag tgt ttg agg gat agt
tat ccc aga cct gat cag 2304 Lys Ala Ile Pro Glu Cys Leu Arg Asp
Ser Tyr Pro Arg Pro Asp Gln 755 760 765 ccc tgt tac ctg tat gtg ata
gga atg gtt tta act aca cct tta cct 2352 Pro Cys Tyr Leu Tyr Val
Ile Gly Met Val Leu Thr Thr Pro Leu Pro 770 775 780 gat gaa ctc aac
ttt aga agg cgg aag ctc tat cct cct gaa gat acc 2400 Asp Glu Leu
Asn Phe Arg Arg Arg Lys Leu Tyr Pro Pro Glu Asp Thr 785 790 795 800
aca aga tgc ttt gga ata ctg acg gcc aaa ccc ata cct cag att cca
2448 Thr Arg Cys Phe Gly Ile Leu Thr Ala Lys Pro Ile Pro Gln Ile
Pro 805 810 815 cac ttt cct gtg tac aca cgc tct gga gag gtt acc ata
tcc att gag 2496 His Phe Pro Val Tyr Thr Arg Ser Gly Glu Val Thr
Ile Ser Ile Glu 820 825 830 ttg aag aag tct ggt ttc atg ttg tct cta
caa atg ctt gag ttg att 2544 Leu Lys Lys Ser Gly Phe Met Leu Ser
Leu Gln Met Leu Glu Leu Ile 835 840 845 aca aga ctt cac cag tat ata
ttc tca cat att ctt cgg ctt gaa aaa 2592 Thr Arg Leu His Gln Tyr
Ile Phe Ser His Ile Leu Arg Leu Glu Lys 850 855 860 cct gca cta gaa
ttt aaa cct aca gac gct gat tca gca tac tgt gtt 2640 Pro Ala Leu
Glu Phe Lys Pro Thr Asp Ala Asp Ser Ala Tyr Cys Val 865 870 875 880
cta cct ctt aat gtt gtt aat gac tcc agc act ttg gat att gac ttt
2688 Leu Pro Leu Asn Val Val Asn Asp Ser Ser Thr Leu Asp Ile Asp
Phe 885 890 895 aaa ttc atg gaa gat att gag aag tct gaa gct cgc ata
ggc att ccc 2736 Lys Phe Met Glu Asp Ile Glu Lys Ser Glu Ala Arg
Ile Gly Ile Pro 900 905 910 agt aca aag tat aca aaa gaa aca ccc ttt
gtt ttt aaa tta gaa gat 2784 Ser Thr Lys Tyr Thr Lys Glu Thr Pro
Phe Val Phe Lys Leu Glu Asp 915 920 925 tac caa gat gcc gtt atc att
cca aga tat cgc aat ttt gat cag cct 2832 Tyr Gln Asp Ala Val Ile
Ile Pro Arg Tyr Arg Asn Phe Asp Gln Pro 930 935 940 cat cga ttt tat
gta gct gat gtg tac act gat ctt acc cca ctc agt 2880 His Arg Phe
Tyr Val Ala Asp Val Tyr Thr Asp Leu Thr Pro Leu Ser 945 950 955 960
aaa ttt cct tcc cct gag tat gaa act ttt gca gaa tat tat aaa aca
2928 Lys Phe Pro Ser Pro Glu Tyr Glu Thr Phe Ala Glu Tyr Tyr Lys
Thr 965 970 975 aag tac aac ctt gac cta acc aat ctc aac cag cca ctg
ctg gat gtg 2976 Lys Tyr Asn Leu Asp Leu Thr Asn Leu Asn Gln Pro
Leu Leu Asp Val 980 985 990 gac cac aca tct tca aga ctt aat ctt ttg
aca cct cga cat ttg aat 3024 Asp His Thr Ser Ser Arg Leu Asn Leu
Leu Thr Pro Arg His Leu Asn 995 1000 1005 cag aag ggg aaa gcg ctt
cct tta agc agt gct gag aag agg aaa 3069 Gln Lys Gly Lys Ala Leu
Pro Leu Ser Ser Ala Glu Lys Arg Lys 1010 1015 1020 gcc aaa tgg gaa
agt ctg cag aat aaa cag ata ctg gtt cca gaa 3114 Ala Lys Trp Glu
Ser Leu Gln Asn Lys Gln Ile Leu Val Pro Glu 1025 1030 1035 ctc tgt
gct ata cat cca att cca gca tca ctg tgg aga aaa gct 3159 Leu Cys
Ala Ile His Pro Ile Pro Ala Ser Leu Trp Arg Lys Ala 1040 1045 1050
gtt tgt ctc ccc agc ata ctt tat cgc ctt cac tgc ctt ttg act 3204
Val Cys Leu Pro Ser Ile Leu Tyr Arg Leu His Cys Leu Leu Thr 1055
1060 1065 gca gag gag cta aga gcc cag act gcc agc gat gct ggc gtg
gga 3249 Ala Glu Glu Leu Arg Ala Gln Thr Ala Ser Asp Ala Gly Val
Gly 1070 1075 1080 gtc aga tca ctt cct gcg gat ttt aga tac cct aac
tta gac ttc 3294 Val Arg Ser Leu Pro Ala Asp Phe Arg Tyr Pro Asn
Leu Asp Phe 1085 1090 1095 ggg tgg aaa aaa tct att gac agc aaa tct
ttc atc tca att tct 3339 Gly Trp Lys Lys Ser Ile Asp Ser Lys Ser
Phe Ile Ser Ile Ser 1100 1105 1110 aac tcc tct tca gct gaa aat gat
aat tac tgt aag cac agc aca 3384 Asn Ser Ser Ser Ala Glu Asn Asp
Asn Tyr Cys Lys His Ser Thr 1115 1120 1125 att gtc cct gaa aat gct
gca cat caa ggt gct aat aga acc tcc 3429 Ile Val Pro Glu Asn Ala
Ala His Gln Gly Ala Asn Arg Thr Ser 1130 1135 1140 tct cta gaa aat
cat gac caa atg tct gtg aac tgc aga acg ttg 3474 Ser Leu Glu Asn
His Asp Gln Met Ser Val Asn Cys Arg Thr Leu 1145 1150 1155 ctc agc
gag tcc cct ggt aag ctc cac gtt gaa gtt tca gca gat 3519 Leu Ser
Glu Ser Pro Gly Lys Leu His Val Glu Val Ser Ala Asp 1160 1165 1170
ctt aca gca att aat ggt ctt tct tac aat caa aat ctc gcc aat 3564
Leu Thr Ala Ile Asn Gly Leu Ser Tyr Asn Gln Asn Leu Ala Asn 1175
1180 1185 ggc agt tat gat tta gct aac aga gac ttt tgc caa gga aat
cag 3609 Gly Ser Tyr Asp Leu Ala Asn Arg Asp Phe Cys Gln Gly Asn
Gln 1190 1195 1200 cta aat tac tac aag cag gaa ata ccc gtg caa cca
act acc tca 3654 Leu Asn Tyr Tyr Lys Gln Glu Ile Pro Val Gln Pro
Thr Thr Ser 1205 1210 1215 tat tcc att cag aat tta tac agt tac gag
aac cag ccc cag ccc 3699 Tyr Ser Ile Gln Asn Leu Tyr Ser Tyr Glu
Asn Gln Pro Gln Pro 1220 1225 1230 agc gat gaa tgt act ctc ctg agt
aat aaa tac ctt gat gga aat 3744 Ser Asp Glu Cys Thr Leu Leu Ser
Asn Lys Tyr Leu Asp Gly Asn 1235 1240 1245 gct aac aaa tct acc tca
gat gga agt cct gtg atg gcc gta atg 3789 Ala Asn Lys Ser Thr Ser
Asp Gly Ser Pro Val Met Ala Val Met 1250 1255 1260 cct ggt acg aca
gac act att caa gtg ctc aag ggc agg atg gat 3834 Pro Gly Thr Thr
Asp Thr Ile Gln Val Leu Lys Gly Arg Met Asp 1265 1270 1275 tct gag
cag agc cct tct att ggg tac tcc tca agg act ctt ggc 3879 Ser Glu
Gln Ser Pro Ser Ile Gly Tyr Ser Ser Arg Thr Leu Gly 1280 1285 1290
ccc aat cct gga ctt att ctt cag gct ttg act ctg tca aac gct 3924
Pro Asn Pro Gly Leu Ile Leu Gln Ala Leu Thr Leu Ser Asn Ala 1295
1300 1305 agt gat gga ttt aac ctg gag cgg ctt gaa atg ctt ggc gac
tcc 3969 Ser Asp Gly Phe Asn Leu Glu Arg Leu Glu Met Leu Gly Asp
Ser 1310 1315 1320 ttt tta aag cat gcc atc acc aca tat cta ttt tgc
act tac cct 4014 Phe Leu Lys His Ala Ile Thr Thr Tyr Leu Phe Cys
Thr Tyr Pro 1325 1330 1335 gat gcg cat gag ggc cgc ctt tca tat atg
aga agc aaa aag gtc 4059 Asp Ala His Glu Gly Arg Leu Ser Tyr Met
Arg Ser Lys Lys Val 1340 1345 1350 agc aac tgt aat ctg tat cgc ctt
gga aaa aag aag gga cta ccc 4104 Ser Asn Cys Asn Leu Tyr Arg Leu
Gly Lys Lys Lys Gly Leu Pro 1355 1360 1365 agc cgc atg gtg gtg tca
ata ttt gat ccc cct gtg aat tgg ctt 4149 Ser Arg Met Val Val Ser
Ile Phe Asp Pro Pro Val Asn Trp Leu 1370 1375 1380 cct cct ggt tat
gta gta aat caa gac aaa agc aac aca gat aaa 4194 Pro Pro Gly Tyr
Val Val Asn Gln Asp Lys Ser Asn Thr Asp Lys 1385 1390 1395 tgg gaa
aaa gat gaa atg aca aaa gac tgc atg ctg gcg aat ggc 4239 Trp Glu
Lys Asp Glu Met Thr Lys Asp Cys Met Leu Ala Asn Gly 1400 1405 1410
aaa ctg gat gag gat tac gag gag gag gat gag gag gag gag agc 4284
Lys Leu Asp Glu Asp Tyr Glu Glu Glu Asp Glu Glu Glu Glu Ser 1415
1420 1425 ctg atg tgg agg gct ccg aag gaa gag gct gac tat gaa gat
gat 4329 Leu Met Trp Arg Ala Pro Lys Glu Glu Ala Asp Tyr Glu Asp
Asp 1430 1435 1440 ttc ctg gag tat gat cag gaa cat atc aga ttt ata
gat aat atg 4374 Phe Leu Glu Tyr Asp Gln Glu His Ile Arg Phe Ile
Asp Asn Met 1445 1450 1455 tta atg ggg tca gga gct ttt gta aag aaa
atc tct ctt tct cct 4419 Leu Met Gly Ser Gly Ala Phe Val Lys Lys
Ile Ser Leu Ser Pro 1460 1465 1470 ttt tca acc act gat tct gca tat
gaa tgg aaa atg ccc aaa aaa 4464 Phe Ser Thr Thr Asp Ser Ala Tyr
Glu Trp Lys Met Pro Lys Lys 1475 1480 1485 tcc tcc tta ggt agt atg
cca ttt tca tca gat ttt gag gat ttt 4509 Ser Ser Leu Gly Ser Met
Pro Phe Ser Ser Asp
Phe Glu Asp Phe 1490 1495 1500 gac tac agc tct tgg gat gca atg tgc
tat ctg gat cct agc aaa 4554 Asp Tyr Ser Ser Trp Asp Ala Met Cys
Tyr Leu Asp Pro Ser Lys 1505 1510 1515 gct gtt gaa gaa gat gac ttt
gtg gtg ggg ttc tgg aat cca tca 4599 Ala Val Glu Glu Asp Asp Phe
Val Val Gly Phe Trp Asn Pro Ser 1520 1525 1530 gaa gaa aac tgt ggt
gtt gac acg gga aag cag tcc att tct tac 4644 Glu Glu Asn Cys Gly
Val Asp Thr Gly Lys Gln Ser Ile Ser Tyr 1535 1540 1545 gac ttg cac
act gag cag tgt att gct gac aaa agc ata gcg gac 4689 Asp Leu His
Thr Glu Gln Cys Ile Ala Asp Lys Ser Ile Ala Asp 1550 1555 1560 tgt
gtg gaa gcc ctg ctg ggc tgc tat tta acc agc tgt ggg gag 4734 Cys
Val Glu Ala Leu Leu Gly Cys Tyr Leu Thr Ser Cys Gly Glu 1565 1570
1575 agg gct gct cag ctt ttc ctc tgt tca ctg ggg ctg aag gtg ctc
4779 Arg Ala Ala Gln Leu Phe Leu Cys Ser Leu Gly Leu Lys Val Leu
1580 1585 1590 ccg gta att aaa agg act gat cgg gaa aag gcc ctg tgc
cct act 4824 Pro Val Ile Lys Arg Thr Asp Arg Glu Lys Ala Leu Cys
Pro Thr 1595 1600 1605 cgg gag aat ttc aac agc caa caa aag aac ctt
tca gtg agc tgt 4869 Arg Glu Asn Phe Asn Ser Gln Gln Lys Asn Leu
Ser Val Ser Cys 1610 1615 1620 gct gct gct tct gtg gcc agt tca cgc
tct tct gta ttg aaa gac 4914 Ala Ala Ala Ser Val Ala Ser Ser Arg
Ser Ser Val Leu Lys Asp 1625 1630 1635 tcg gaa tat ggt tgt ttg aag
att cca cca aga tgt atg ttt gat 4959 Ser Glu Tyr Gly Cys Leu Lys
Ile Pro Pro Arg Cys Met Phe Asp 1640 1645 1650 cat cca gat gca gat
aaa aca ctg aat cac ctt ata tcg ggg ttt 5004 His Pro Asp Ala Asp
Lys Thr Leu Asn His Leu Ile Ser Gly Phe 1655 1660 1665 gaa aat ttt
gaa aag aaa atc aac tac aga ttc aag aat aag gct 5049 Glu Asn Phe
Glu Lys Lys Ile Asn Tyr Arg Phe Lys Asn Lys Ala 1670 1675 1680 tac
ctt ctc cag gct ttt aca cat gcc tcc tac cac tac aat act 5094 Tyr
Leu Leu Gln Ala Phe Thr His Ala Ser Tyr His Tyr Asn Thr 1685 1690
1695 atc act gat tgt tac cag cgc tta gaa ttc ctg gga gat gcg att
5139 Ile Thr Asp Cys Tyr Gln Arg Leu Glu Phe Leu Gly Asp Ala Ile
1700 1705 1710 ttg gac tac ctc ata acc aag cac ctt tat gaa gac ccg
cgg cag 5184 Leu Asp Tyr Leu Ile Thr Lys His Leu Tyr Glu Asp Pro
Arg Gln 1715 1720 1725 cac tcc ccg ggg gtc ctg aca gac ctg cgg tct
gcc ctg gtc aac 5229 His Ser Pro Gly Val Leu Thr Asp Leu Arg Ser
Ala Leu Val Asn 1730 1735 1740 aac acc atc ttt gca tcg ctg gct gta
aag tac gac tac cac aag 5274 Asn Thr Ile Phe Ala Ser Leu Ala Val
Lys Tyr Asp Tyr His Lys 1745 1750 1755 tac ttc aaa gct gtc tct cct
gag ctc ttc cat gtc att gat gac 5319 Tyr Phe Lys Ala Val Ser Pro
Glu Leu Phe His Val Ile Asp Asp 1760 1765 1770 ttt gtg cag ttt cag
ctt gag aag aat gaa atg caa gga atg gat 5364 Phe Val Gln Phe Gln
Leu Glu Lys Asn Glu Met Gln Gly Met Asp 1775 1780 1785 tct gag ctt
agg aga tct gag gag gat gaa gag aaa gaa gag gat 5409 Ser Glu Leu
Arg Arg Ser Glu Glu Asp Glu Glu Lys Glu Glu Asp 1790 1795 1800 att
gaa gtt cca aag gcc atg ggg gat att ttt gag tcg ctt gct 5454 Ile
Glu Val Pro Lys Ala Met Gly Asp Ile Phe Glu Ser Leu Ala 1805 1810
1815 ggt gcc att tac atg gat agt ggg atg tca ctg gag aca gtc tgg
5499 Gly Ala Ile Tyr Met Asp Ser Gly Met Ser Leu Glu Thr Val Trp
1820 1825 1830 cag gtg tac tat ccc atg atg cgg cca cta ata gaa aag
ttt tct 5544 Gln Val Tyr Tyr Pro Met Met Arg Pro Leu Ile Glu Lys
Phe Ser 1835 1840 1845 gca aat gta ccc cgt tcc cct gtg cga gaa ttg
ctt gaa atg gaa 5589 Ala Asn Val Pro Arg Ser Pro Val Arg Glu Leu
Leu Glu Met Glu 1850 1855 1860 cca gaa act gcc aaa ttt agc ccg gct
gag aga act tac gac ggg 5634 Pro Glu Thr Ala Lys Phe Ser Pro Ala
Glu Arg Thr Tyr Asp Gly 1865 1870 1875 aag gtc aga gtc act gtg gaa
gta gta gga aag ggg aaa ttt aaa 5679 Lys Val Arg Val Thr Val Glu
Val Val Gly Lys Gly Lys Phe Lys 1880 1885 1890 ggt gtt ggt cga agt
tac agg att gcc aaa tct gca gca gca aga 5724 Gly Val Gly Arg Ser
Tyr Arg Ile Ala Lys Ser Ala Ala Ala Arg 1895 1900 1905 aga gcc ctc
cga agc ctc aaa gct aat caa cct cag gtt ccc aat 5769 Arg Ala Leu
Arg Ser Leu Lys Ala Asn Gln Pro Gln Val Pro Asn 1910 1915 1920 agc
tga 5775 Ser 2 1924 PRT Homo sapiens 2 Met Lys Ser Pro Ala Leu Gln
Pro Leu Ser Met Ala Gly Leu Gln Leu 1 5 10 15 Met Thr Pro Ala Ser
Ser Pro Met Gly Pro Phe Phe Gly Leu Pro Trp 20 25 30 Gln Gln Glu
Ala Ile His Asp Asn Ile Tyr Thr Pro Arg Lys Tyr Gln 35 40 45 Val
Glu Leu Leu Glu Ala Ala Leu Asp His Asn Thr Ile Val Cys Leu 50 55
60 Asn Thr Gly Ser Gly Lys Thr Phe Ile Ala Ser Thr Thr Leu Leu Lys
65 70 75 80 Ser Cys Leu Tyr Leu Asp Leu Gly Glu Thr Ser Ala Arg Asn
Gly Lys 85 90 95 Arg Thr Val Phe Leu Val Asn Ser Ala Asn Gln Val
Ala Gln Gln Val 100 105 110 Ser Ala Val Arg Thr His Ser Asp Leu Lys
Val Gly Glu Tyr Ser Asn 115 120 125 Leu Glu Val Asn Ala Ser Trp Thr
Lys Glu Arg Trp Asn Gln Glu Phe 130 135 140 Thr Lys His Gln Val Leu
Ile Met Thr Cys Tyr Val Ala Leu Asn Val 145 150 155 160 Leu Lys Asn
Gly Tyr Leu Ser Leu Ser Asp Ile Asn Leu Leu Val Phe 165 170 175 Asp
Glu Cys His Leu Ala Ile Leu Asp His Pro Tyr Arg Glu Phe Met 180 185
190 Lys Leu Cys Glu Ile Cys Pro Ser Cys Pro Arg Ile Leu Gly Leu Thr
195 200 205 Ala Ser Ile Leu Asn Gly Lys Trp Asp Pro Glu Asp Leu Glu
Glu Lys 210 215 220 Phe Gln Lys Leu Glu Lys Ile Leu Lys Ser Asn Ala
Glu Thr Ala Thr 225 230 235 240 Asp Leu Val Val Leu Asp Arg Tyr Thr
Ser Gln Pro Cys Glu Ile Val 245 250 255 Val Asp Cys Gly Pro Phe Thr
Asp Arg Ser Gly Leu Tyr Glu Arg Leu 260 265 270 Leu Met Glu Leu Glu
Glu Ala Leu Asn Phe Ile Asn Asp Cys Asn Ile 275 280 285 Ser Val His
Ser Lys Glu Arg Asp Ser Thr Leu Ile Ser Lys Gln Ile 290 295 300 Leu
Ser Asp Cys Arg Ala Val Leu Val Val Leu Gly Pro Trp Cys Ala 305 310
315 320 Asp Lys Val Ala Gly Met Met Val Arg Glu Leu Gln Lys Tyr Ile
Lys 325 330 335 His Glu Gln Glu Glu Leu His Arg Lys Phe Leu Leu Phe
Thr Asp Thr 340 345 350 Phe Leu Arg Lys Ile His Ala Leu Cys Glu Glu
His Phe Ser Pro Ala 355 360 365 Ser Leu Asp Leu Lys Phe Val Thr Pro
Lys Val Ile Lys Leu Leu Glu 370 375 380 Ile Leu Arg Lys Tyr Lys Pro
Tyr Glu Arg His Ser Phe Glu Ser Val 385 390 395 400 Glu Trp Tyr Asn
Asn Arg Asn Gln Asp Asn Tyr Val Ser Trp Ser Asp 405 410 415 Ser Glu
Asp Asp Asp Glu Asp Glu Glu Ile Glu Glu Lys Glu Lys Pro 420 425 430
Glu Thr Asn Phe Pro Ser Pro Phe Thr Asn Ile Leu Cys Gly Ile Ile 435
440 445 Phe Val Glu Arg Arg Tyr Thr Ala Val Val Leu Asn Arg Leu Ile
Lys 450 455 460 Glu Ala Gly Lys Gln Asp Pro Glu Leu Ala Tyr Ile Ser
Ser Asn Phe 465 470 475 480 Ile Thr Gly His Gly Ile Gly Lys Asn Gln
Pro Arg Asn Asn Thr Met 485 490 495 Glu Ala Glu Phe Arg Lys Gln Glu
Glu Val Leu Arg Lys Phe Arg Ala 500 505 510 His Glu Thr Asn Leu Leu
Ile Ala Thr Ser Ile Val Glu Glu Gly Val 515 520 525 Asp Ile Pro Lys
Cys Asn Leu Val Val Arg Phe Asp Leu Pro Thr Glu 530 535 540 Tyr Arg
Ser Tyr Val Gln Ser Lys Gly Arg Ala Arg Ala Pro Ile Ser 545 550 555
560 Asn Tyr Ile Met Leu Ala Asp Thr Asp Lys Ile Lys Ser Phe Glu Glu
565 570 575 Asp Leu Lys Thr Tyr Lys Ala Ile Glu Lys Ile Leu Arg Asn
Lys Cys 580 585 590 Ser Lys Ser Val Asp Thr Gly Glu Thr Asp Ile Asp
Pro Val Met Asp 595 600 605 Asp Asp His Val Phe Pro Pro Tyr Val Leu
Arg Pro Asp Asp Gly Gly 610 615 620 Pro Arg Val Thr Ile Asn Thr Ala
Ile Gly His Ile Asn Arg Tyr Cys 625 630 635 640 Ala Arg Leu Pro Ser
Asp Pro Phe Thr His Leu Ala Pro Lys Cys Arg 645 650 655 Thr Arg Glu
Leu Pro Asp Gly Thr Phe Tyr Ser Thr Leu Tyr Leu Pro 660 665 670 Ile
Asn Ser Pro Leu Arg Ala Ser Ile Val Gly Pro Pro Met Ser Cys 675 680
685 Val Arg Leu Ala Glu Arg Val Val Ala Leu Ile Cys Cys Glu Lys Leu
690 695 700 His Lys Ile Gly Glu Leu Asp Asp His Leu Met Pro Val Gly
Lys Glu 705 710 715 720 Thr Val Lys Tyr Glu Glu Glu Leu Asp Leu His
Asp Glu Glu Glu Thr 725 730 735 Ser Val Pro Gly Arg Pro Gly Ser Thr
Lys Arg Arg Gln Cys Tyr Pro 740 745 750 Lys Ala Ile Pro Glu Cys Leu
Arg Asp Ser Tyr Pro Arg Pro Asp Gln 755 760 765 Pro Cys Tyr Leu Tyr
Val Ile Gly Met Val Leu Thr Thr Pro Leu Pro 770 775 780 Asp Glu Leu
Asn Phe Arg Arg Arg Lys Leu Tyr Pro Pro Glu Asp Thr 785 790 795 800
Thr Arg Cys Phe Gly Ile Leu Thr Ala Lys Pro Ile Pro Gln Ile Pro 805
810 815 His Phe Pro Val Tyr Thr Arg Ser Gly Glu Val Thr Ile Ser Ile
Glu 820 825 830 Leu Lys Lys Ser Gly Phe Met Leu Ser Leu Gln Met Leu
Glu Leu Ile 835 840 845 Thr Arg Leu His Gln Tyr Ile Phe Ser His Ile
Leu Arg Leu Glu Lys 850 855 860 Pro Ala Leu Glu Phe Lys Pro Thr Asp
Ala Asp Ser Ala Tyr Cys Val 865 870 875 880 Leu Pro Leu Asn Val Val
Asn Asp Ser Ser Thr Leu Asp Ile Asp Phe 885 890 895 Lys Phe Met Glu
Asp Ile Glu Lys Ser Glu Ala Arg Ile Gly Ile Pro 900 905 910 Ser Thr
Lys Tyr Thr Lys Glu Thr Pro Phe Val Phe Lys Leu Glu Asp 915 920 925
Tyr Gln Asp Ala Val Ile Ile Pro Arg Tyr Arg Asn Phe Asp Gln Pro 930
935 940 His Arg Phe Tyr Val Ala Asp Val Tyr Thr Asp Leu Thr Pro Leu
Ser 945 950 955 960 Lys Phe Pro Ser Pro Glu Tyr Glu Thr Phe Ala Glu
Tyr Tyr Lys Thr 965 970 975 Lys Tyr Asn Leu Asp Leu Thr Asn Leu Asn
Gln Pro Leu Leu Asp Val 980 985 990 Asp His Thr Ser Ser Arg Leu Asn
Leu Leu Thr Pro Arg His Leu Asn 995 1000 1005 Gln Lys Gly Lys Ala
Leu Pro Leu Ser Ser Ala Glu Lys Arg Lys 1010 1015 1020 Ala Lys Trp
Glu Ser Leu Gln Asn Lys Gln Ile Leu Val Pro Glu 1025 1030 1035 Leu
Cys Ala Ile His Pro Ile Pro Ala Ser Leu Trp Arg Lys Ala 1040 1045
1050 Val Cys Leu Pro Ser Ile Leu Tyr Arg Leu His Cys Leu Leu Thr
1055 1060 1065 Ala Glu Glu Leu Arg Ala Gln Thr Ala Ser Asp Ala Gly
Val Gly 1070 1075 1080 Val Arg Ser Leu Pro Ala Asp Phe Arg Tyr Pro
Asn Leu Asp Phe 1085 1090 1095 Gly Trp Lys Lys Ser Ile Asp Ser Lys
Ser Phe Ile Ser Ile Ser 1100 1105 1110 Asn Ser Ser Ser Ala Glu Asn
Asp Asn Tyr Cys Lys His Ser Thr 1115 1120 1125 Ile Val Pro Glu Asn
Ala Ala His Gln Gly Ala Asn Arg Thr Ser 1130 1135 1140 Ser Leu Glu
Asn His Asp Gln Met Ser Val Asn Cys Arg Thr Leu 1145 1150 1155 Leu
Ser Glu Ser Pro Gly Lys Leu His Val Glu Val Ser Ala Asp 1160 1165
1170 Leu Thr Ala Ile Asn Gly Leu Ser Tyr Asn Gln Asn Leu Ala Asn
1175 1180 1185 Gly Ser Tyr Asp Leu Ala Asn Arg Asp Phe Cys Gln Gly
Asn Gln 1190 1195 1200 Leu Asn Tyr Tyr Lys Gln Glu Ile Pro Val Gln
Pro Thr Thr Ser 1205 1210 1215 Tyr Ser Ile Gln Asn Leu Tyr Ser Tyr
Glu Asn Gln Pro Gln Pro 1220 1225 1230 Ser Asp Glu Cys Thr Leu Leu
Ser Asn Lys Tyr Leu Asp Gly Asn 1235 1240 1245 Ala Asn Lys Ser Thr
Ser Asp Gly Ser Pro Val Met Ala Val Met 1250 1255 1260 Pro Gly Thr
Thr Asp Thr Ile Gln Val Leu Lys Gly Arg Met Asp 1265 1270 1275 Ser
Glu Gln Ser Pro Ser Ile Gly Tyr Ser Ser Arg Thr Leu Gly 1280 1285
1290 Pro Asn Pro Gly Leu Ile Leu Gln Ala Leu Thr Leu Ser Asn Ala
1295 1300 1305 Ser Asp Gly Phe Asn Leu Glu Arg Leu Glu Met Leu Gly
Asp Ser 1310 1315 1320 Phe Leu Lys His Ala Ile Thr Thr Tyr Leu Phe
Cys Thr Tyr Pro 1325 1330 1335 Asp Ala His Glu Gly Arg Leu Ser Tyr
Met Arg Ser Lys Lys Val 1340 1345 1350 Ser Asn Cys Asn Leu Tyr Arg
Leu Gly Lys Lys Lys Gly Leu Pro 1355 1360 1365 Ser Arg Met Val Val
Ser Ile Phe Asp Pro Pro Val Asn Trp Leu 1370 1375 1380 Pro Pro Gly
Tyr Val Val Asn Gln Asp Lys Ser Asn Thr Asp Lys 1385 1390 1395 Trp
Glu Lys Asp Glu Met Thr Lys Asp Cys Met Leu Ala Asn Gly 1400 1405
1410 Lys Leu Asp Glu Asp Tyr Glu Glu Glu Asp Glu Glu Glu Glu Ser
1415 1420 1425 Leu Met Trp Arg Ala Pro Lys Glu Glu Ala Asp Tyr Glu
Asp Asp 1430 1435 1440 Phe Leu Glu Tyr Asp Gln Glu His Ile Arg Phe
Ile Asp Asn Met 1445 1450 1455 Leu Met Gly Ser Gly Ala Phe Val Lys
Lys Ile Ser Leu Ser Pro 1460 1465 1470 Phe Ser Thr Thr Asp Ser Ala
Tyr Glu Trp Lys Met Pro Lys Lys 1475 1480 1485 Ser Ser Leu Gly Ser
Met Pro Phe Ser Ser Asp Phe Glu Asp Phe 1490 1495 1500 Asp Tyr Ser
Ser Trp Asp Ala Met Cys Tyr Leu Asp Pro Ser Lys 1505 1510 1515 Ala
Val Glu Glu Asp Asp Phe Val Val Gly Phe Trp Asn Pro Ser 1520 1525
1530 Glu Glu Asn Cys Gly Val Asp Thr Gly Lys Gln Ser Ile Ser Tyr
1535 1540 1545 Asp Leu His Thr Glu Gln Cys Ile Ala Asp Lys Ser Ile
Ala Asp 1550 1555 1560 Cys Val Glu Ala Leu Leu Gly Cys Tyr Leu Thr
Ser Cys Gly Glu 1565 1570 1575 Arg Ala Ala Gln Leu Phe Leu Cys Ser
Leu Gly Leu Lys Val Leu 1580 1585 1590 Pro Val Ile Lys Arg Thr Asp
Arg Glu Lys Ala Leu Cys Pro Thr 1595 1600 1605 Arg Glu Asn Phe Asn
Ser Gln Gln Lys Asn Leu Ser Val Ser Cys 1610 1615 1620 Ala Ala Ala
Ser Val Ala Ser Ser Arg Ser Ser Val Leu Lys Asp 1625 1630 1635 Ser
Glu Tyr Gly Cys Leu Lys Ile Pro Pro Arg Cys Met Phe Asp 1640 1645
1650 His Pro Asp Ala Asp Lys Thr Leu Asn His Leu Ile Ser Gly Phe
1655 1660 1665 Glu Asn Phe Glu Lys Lys Ile Asn Tyr Arg Phe Lys Asn
Lys Ala 1670 1675 1680 Tyr Leu Leu Gln Ala Phe Thr His Ala Ser Tyr
His Tyr Asn Thr 1685 1690 1695 Ile Thr Asp Cys Tyr Gln Arg Leu Glu
Phe Leu Gly Asp Ala Ile 1700
1705 1710 Leu Asp Tyr Leu Ile Thr Lys His Leu Tyr Glu Asp Pro Arg
Gln 1715 1720 1725 His Ser Pro Gly Val Leu Thr Asp Leu Arg Ser Ala
Leu Val Asn 1730 1735 1740 Asn Thr Ile Phe Ala Ser Leu Ala Val Lys
Tyr Asp Tyr His Lys 1745 1750 1755 Tyr Phe Lys Ala Val Ser Pro Glu
Leu Phe His Val Ile Asp Asp 1760 1765 1770 Phe Val Gln Phe Gln Leu
Glu Lys Asn Glu Met Gln Gly Met Asp 1775 1780 1785 Ser Glu Leu Arg
Arg Ser Glu Glu Asp Glu Glu Lys Glu Glu Asp 1790 1795 1800 Ile Glu
Val Pro Lys Ala Met Gly Asp Ile Phe Glu Ser Leu Ala 1805 1810 1815
Gly Ala Ile Tyr Met Asp Ser Gly Met Ser Leu Glu Thr Val Trp 1820
1825 1830 Gln Val Tyr Tyr Pro Met Met Arg Pro Leu Ile Glu Lys Phe
Ser 1835 1840 1845 Ala Asn Val Pro Arg Ser Pro Val Arg Glu Leu Leu
Glu Met Glu 1850 1855 1860 Pro Glu Thr Ala Lys Phe Ser Pro Ala Glu
Arg Thr Tyr Asp Gly 1865 1870 1875 Lys Val Arg Val Thr Val Glu Val
Val Gly Lys Gly Lys Phe Lys 1880 1885 1890 Gly Val Gly Arg Ser Tyr
Arg Ile Ala Lys Ser Ala Ala Ala Arg 1895 1900 1905 Arg Ala Leu Arg
Ser Leu Lys Ala Asn Gln Pro Gln Val Pro Asn 1910 1915 1920 Ser 3
6750 DNA Drosophila melanogaster CDS (1)..(6750) 3 atg gcg ttc cac
tgg tgc gac aac aat ctg cac acc acc gtg ttc acg 48 Met Ala Phe His
Trp Cys Asp Asn Asn Leu His Thr Thr Val Phe Thr 1 5 10 15 ccg cgc
gac ttt cag gtg gag cta ctg gcc acc gcc tac gag cgg aac 96 Pro Arg
Asp Phe Gln Val Glu Leu Leu Ala Thr Ala Tyr Glu Arg Asn 20 25 30
acg att att tgc ctg ggc cat cga agt tcc aag gag ttt ata gcc ctc 144
Thr Ile Ile Cys Leu Gly His Arg Ser Ser Lys Glu Phe Ile Ala Leu 35
40 45 aag ctg ctc cag gag ctg tcg cgt cga gca cgc cga cat ggt cgt
gtc 192 Lys Leu Leu Gln Glu Leu Ser Arg Arg Ala Arg Arg His Gly Arg
Val 50 55 60 agt gtc tat ctc agt tgc gag gtt ggc acc agc acg gaa
cca tgc tcc 240 Ser Val Tyr Leu Ser Cys Glu Val Gly Thr Ser Thr Glu
Pro Cys Ser 65 70 75 80 atc tac acg atg ctc acc cac ttg act gac ctg
cgg gtg tgg cag gag 288 Ile Tyr Thr Met Leu Thr His Leu Thr Asp Leu
Arg Val Trp Gln Glu 85 90 95 cag ccg gat atg caa att ccc ttt gat
cat tgc tgg acg gac tat cac 336 Gln Pro Asp Met Gln Ile Pro Phe Asp
His Cys Trp Thr Asp Tyr His 100 105 110 gtt tcc atc cta cgg cca gag
gga ttt ctt tat ctg ctc gaa act cgc 384 Val Ser Ile Leu Arg Pro Glu
Gly Phe Leu Tyr Leu Leu Glu Thr Arg 115 120 125 gag ctg ctg ctg agc
agc gtc gaa ctg atc gtg ctg gaa gat tgt cat 432 Glu Leu Leu Leu Ser
Ser Val Glu Leu Ile Val Leu Glu Asp Cys His 130 135 140 gac agc gcc
gtt tat cag agg ata agg cct ctg ttc gag aat cac att 480 Asp Ser Ala
Val Tyr Gln Arg Ile Arg Pro Leu Phe Glu Asn His Ile 145 150 155 160
atg cca gcg cca ccg gcg gac agg cca cgg att ctc gga ctc gct gga 528
Met Pro Ala Pro Pro Ala Asp Arg Pro Arg Ile Leu Gly Leu Ala Gly 165
170 175 ccg ctg cac agc gcc gga tgt gag ctg cag caa ctg agc gcc atg
ctg 576 Pro Leu His Ser Ala Gly Cys Glu Leu Gln Gln Leu Ser Ala Met
Leu 180 185 190 gcc acc ctg gag cag agt gtg ctt tgc cag atc gag acg
gcc agt gat 624 Ala Thr Leu Glu Gln Ser Val Leu Cys Gln Ile Glu Thr
Ala Ser Asp 195 200 205 att gtc acc gtg ttg cgt tac tgt tcc cga ccg
cac gaa tac atc gta 672 Ile Val Thr Val Leu Arg Tyr Cys Ser Arg Pro
His Glu Tyr Ile Val 210 215 220 cag tgc gcc ccc ttc gag atg gac gaa
ctg tcc ctg gtg ctt gcc gat 720 Gln Cys Ala Pro Phe Glu Met Asp Glu
Leu Ser Leu Val Leu Ala Asp 225 230 235 240 gtg ctc aac aca cac aag
tcc ttt tta ttg gac cac cgc tac gat ccc 768 Val Leu Asn Thr His Lys
Ser Phe Leu Leu Asp His Arg Tyr Asp Pro 245 250 255 tac gaa atc tac
ggc aca gac cag ttt atg gac gaa ctg aaa gac ata 816 Tyr Glu Ile Tyr
Gly Thr Asp Gln Phe Met Asp Glu Leu Lys Asp Ile 260 265 270 ccc gat
ccc aag gtg gac ccc ctg aac gtc atc aac tca cta ctg gtc 864 Pro Asp
Pro Lys Val Asp Pro Leu Asn Val Ile Asn Ser Leu Leu Val 275 280 285
gtg ctg cac gag atg ggt cct tgg tgc acg cag cgg gct gca cat cac 912
Val Leu His Glu Met Gly Pro Trp Cys Thr Gln Arg Ala Ala His His 290
295 300 ttt tac caa tgc aat gag aag tta aag gtg aag acg ccg cac gaa
cgt 960 Phe Tyr Gln Cys Asn Glu Lys Leu Lys Val Lys Thr Pro His Glu
Arg 305 310 315 320 cac tac ttg ctg tac tgc cta gtg agc acg gcc ctt
atc caa ctg tac 1008 His Tyr Leu Leu Tyr Cys Leu Val Ser Thr Ala
Leu Ile Gln Leu Tyr 325 330 335 tcc ctc tgc gaa cac gca ttc cat cga
cat tta gga agt ggc agc gat 1056 Ser Leu Cys Glu His Ala Phe His
Arg His Leu Gly Ser Gly Ser Asp 340 345 350 tca cgc cag acc atc gaa
cgc tat tcc agc ccc aag gtg cga cgt ctg 1104 Ser Arg Gln Thr Ile
Glu Arg Tyr Ser Ser Pro Lys Val Arg Arg Leu 355 360 365 ttg cag aca
ctg agg tgc ttc aag ccg gaa gag gtg cac acc caa gcg 1152 Leu Gln
Thr Leu Arg Cys Phe Lys Pro Glu Glu Val His Thr Gln Ala 370 375 380
gac gga ctg cgc aga atg cgg cat cag gtg gat cag gcg gac ttc aat
1200 Asp Gly Leu Arg Arg Met Arg His Gln Val Asp Gln Ala Asp Phe
Asn 385 390 395 400 cgg tta tct cat acg ctg gaa agc aag tgc cga atg
gtg gat caa atg 1248 Arg Leu Ser His Thr Leu Glu Ser Lys Cys Arg
Met Val Asp Gln Met 405 410 415 gac caa ccg ccg acg gag aca cga gcc
ctg gtg gcc act ctt gag cag 1296 Asp Gln Pro Pro Thr Glu Thr Arg
Ala Leu Val Ala Thr Leu Glu Gln 420 425 430 att ctg cac acg aca gag
gac agg cag acg aac aga agc gcc gct cgg 1344 Ile Leu His Thr Thr
Glu Asp Arg Gln Thr Asn Arg Ser Ala Ala Arg 435 440 445 gtg act cct
act cct act ccc gct cat gcg aag ccg aaa cct agc tct 1392 Val Thr
Pro Thr Pro Thr Pro Ala His Ala Lys Pro Lys Pro Ser Ser 450 455 460
ggt gcc aac act gca caa cca cga act cgt aga cgt gtg tac acc agg
1440 Gly Ala Asn Thr Ala Gln Pro Arg Thr Arg Arg Arg Val Tyr Thr
Arg 465 470 475 480 cgc cac cac cgg gat cac aat gat ggc agc gac acg
ctc tgc gca ctg 1488 Arg His His Arg Asp His Asn Asp Gly Ser Asp
Thr Leu Cys Ala Leu 485 490 495 att tac tgc aac cag aac cac acg gct
cgc gtg ctc ttt gag ctt cta 1536 Ile Tyr Cys Asn Gln Asn His Thr
Ala Arg Val Leu Phe Glu Leu Leu 500 505 510 gcg gag att agc aga cgt
gat ccc gat ctc aag ttc cta cgc tgc cag 1584 Ala Glu Ile Ser Arg
Arg Asp Pro Asp Leu Lys Phe Leu Arg Cys Gln 515 520 525 tac acc acg
gac cgg gtg gca gat ccc acc acg gag ccc aaa gag gct 1632 Tyr Thr
Thr Asp Arg Val Ala Asp Pro Thr Thr Glu Pro Lys Glu Ala 530 535 540
gag ttg gag cac cgg cgg cag gaa gag gtg cta aag cgc ttc cgc atg
1680 Glu Leu Glu His Arg Arg Gln Glu Glu Val Leu Lys Arg Phe Arg
Met 545 550 555 560 cat gac tgc aat gtc ctg atc ggt act tcg gtg ctg
gaa gag ggc atc 1728 His Asp Cys Asn Val Leu Ile Gly Thr Ser Val
Leu Glu Glu Gly Ile 565 570 575 gat gtg ccc aag tgc aat ttg gtt gtg
cgc tgg gat ccg cca acc aca 1776 Asp Val Pro Lys Cys Asn Leu Val
Val Arg Trp Asp Pro Pro Thr Thr 580 585 590 tat cgc agt tac gtt cag
tgc aaa ggt cga gcc cgt gct gct cca gcc 1824 Tyr Arg Ser Tyr Val
Gln Cys Lys Gly Arg Ala Arg Ala Ala Pro Ala 595 600 605 tat cat gtc
att ctc gtc gct ccg agt tat aaa agc cca act gtg ggg 1872 Tyr His
Val Ile Leu Val Ala Pro Ser Tyr Lys Ser Pro Thr Val Gly 610 615 620
tca gtg cag ctg acc gat cgg agt cat cgg tat att tgc gcg act ggt
1920 Ser Val Gln Leu Thr Asp Arg Ser His Arg Tyr Ile Cys Ala Thr
Gly 625 630 635 640 gat act aca gag gcg gac agc gac tct gat gat tca
gcg atg cca aac 1968 Asp Thr Thr Glu Ala Asp Ser Asp Ser Asp Asp
Ser Ala Met Pro Asn 645 650 655 tcg tcc ggc tcg gat ccc tat act ttt
ggc acg gca cgc gga acc gtg 2016 Ser Ser Gly Ser Asp Pro Tyr Thr
Phe Gly Thr Ala Arg Gly Thr Val 660 665 670 aag atc ctc aac ccc gaa
gtg ttc agt aaa caa cca ccg aca gcg tgc 2064 Lys Ile Leu Asn Pro
Glu Val Phe Ser Lys Gln Pro Pro Thr Ala Cys 675 680 685 gac att aag
ctg cag gag atc cag gac gaa ttg cca gcc gca gcg cag 2112 Asp Ile
Lys Leu Gln Glu Ile Gln Asp Glu Leu Pro Ala Ala Ala Gln 690 695 700
ctg gat acg agc aac tcc agc gac gaa gcc gtc agc atg agt aac acg
2160 Leu Asp Thr Ser Asn Ser Ser Asp Glu Ala Val Ser Met Ser Asn
Thr 705 710 715 720 tct cca agc gag agc agt aca gaa caa aaa tcc aga
cgc ttc cag tgc 2208 Ser Pro Ser Glu Ser Ser Thr Glu Gln Lys Ser
Arg Arg Phe Gln Cys 725 730 735 gag ctg agc tct tta acg gag cca gaa
gac aca agt gat act aca gcc 2256 Glu Leu Ser Ser Leu Thr Glu Pro
Glu Asp Thr Ser Asp Thr Thr Ala 740 745 750 gaa atc gat act gct cat
agt tta gcc agc acc acg aag gac ttg gtg 2304 Glu Ile Asp Thr Ala
His Ser Leu Ala Ser Thr Thr Lys Asp Leu Val 755 760 765 cat caa atg
gca cag tat cgc gaa atc gag cag atg ctg cta tcc aag 2352 His Gln
Met Ala Gln Tyr Arg Glu Ile Glu Gln Met Leu Leu Ser Lys 770 775 780
tgc gcc aac aca gag ccg ccg gag cag gag cag agt gag gcg gaa cgt
2400 Cys Ala Asn Thr Glu Pro Pro Glu Gln Glu Gln Ser Glu Ala Glu
Arg 785 790 795 800 ttt agt gcc tgc ctg gcc gca tac cga ccc aag ccg
cac ctg cta aca 2448 Phe Ser Ala Cys Leu Ala Ala Tyr Arg Pro Lys
Pro His Leu Leu Thr 805 810 815 ggc gcc tcc gtg gat ctg ggt tct gct
ata gct ttg gtc aac aag tac 2496 Gly Ala Ser Val Asp Leu Gly Ser
Ala Ile Ala Leu Val Asn Lys Tyr 820 825 830 tgc gcc cga ctg cca agc
gac acg ttc acc aag ttg acg gcg ttg tgg 2544 Cys Ala Arg Leu Pro
Ser Asp Thr Phe Thr Lys Leu Thr Ala Leu Trp 835 840 845 cgc tgc acc
cga aac gaa agg gct gga gtg acc ctg ttt cag tac aca 2592 Arg Cys
Thr Arg Asn Glu Arg Ala Gly Val Thr Leu Phe Gln Tyr Thr 850 855 860
ctc cgt ctg ccc atc aac tcg cca ttg aag cat gac att gtg ggt ctt
2640 Leu Arg Leu Pro Ile Asn Ser Pro Leu Lys His Asp Ile Val Gly
Leu 865 870 875 880 ccg atg cca act caa aca ttg gcc cgc cga ctg gct
gcc ttg cag gct 2688 Pro Met Pro Thr Gln Thr Leu Ala Arg Arg Leu
Ala Ala Leu Gln Ala 885 890 895 tgc gtg gaa ctg cac agg atc ggt gag
tta gac gat cag ttg cag cct 2736 Cys Val Glu Leu His Arg Ile Gly
Glu Leu Asp Asp Gln Leu Gln Pro 900 905 910 atc ggc aag gag gga ttt
cgt gcc ctg gag ccg gac tgg gag tgc ttt 2784 Ile Gly Lys Glu Gly
Phe Arg Ala Leu Glu Pro Asp Trp Glu Cys Phe 915 920 925 gaa ctg gag
cca gag gac gaa cag att gtg cag cta agc gat gaa cca 2832 Glu Leu
Glu Pro Glu Asp Glu Gln Ile Val Gln Leu Ser Asp Glu Pro 930 935 940
cgt ccg gga aca acg aag cgt cgt cag tac tat tac aaa cgc att gca
2880 Arg Pro Gly Thr Thr Lys Arg Arg Gln Tyr Tyr Tyr Lys Arg Ile
Ala 945 950 955 960 tcc gaa ttt tgc gat tgc cgt ccc gtt gcc gga gcg
cca tgc tat ttg 2928 Ser Glu Phe Cys Asp Cys Arg Pro Val Ala Gly
Ala Pro Cys Tyr Leu 965 970 975 tac ttt atc caa ctg acg ctc caa tgt
ccg att ccc gaa gag caa aac 2976 Tyr Phe Ile Gln Leu Thr Leu Gln
Cys Pro Ile Pro Glu Glu Gln Asn 980 985 990 acg cgg gga cgc aag att
tat ccg ccc gaa gat gcg cag cag gga ttc 3024 Thr Arg Gly Arg Lys
Ile Tyr Pro Pro Glu Asp Ala Gln Gln Gly Phe 995 1000 1005 ggc att
cta acc acc aaa cgg ata ccc aag ctg agt gct ttc tcg 3069 Gly Ile
Leu Thr Thr Lys Arg Ile Pro Lys Leu Ser Ala Phe Ser 1010 1015 1020
ata ttc acg cgt tcc ggt gag gtg aag gtt tcc ctg gag tta gct 3114
Ile Phe Thr Arg Ser Gly Glu Val Lys Val Ser Leu Glu Leu Ala 1025
1030 1035 aag gaa cgc gtg att cta act agc gaa caa ata gtc tgc atc
aac 3159 Lys Glu Arg Val Ile Leu Thr Ser Glu Gln Ile Val Cys Ile
Asn 1040 1045 1050 gga ttt tta aac tac acg ttc acc aat gta ctg cgt
ttg caa aag 3204 Gly Phe Leu Asn Tyr Thr Phe Thr Asn Val Leu Arg
Leu Gln Lys 1055 1060 1065 ttt ctg atg ctc ttc gat ccg gac tcc acg
gaa aat tgt gta ttc 3249 Phe Leu Met Leu Phe Asp Pro Asp Ser Thr
Glu Asn Cys Val Phe 1070 1075 1080 att gtg ccc acc gtg aag gca cca
gct ggc ggc aag cac atc gac 3294 Ile Val Pro Thr Val Lys Ala Pro
Ala Gly Gly Lys His Ile Asp 1085 1090 1095 tgg cag ttt ctg gag ctg
atc caa gcg aat gga aat aca atg cca 3339 Trp Gln Phe Leu Glu Leu
Ile Gln Ala Asn Gly Asn Thr Met Pro 1100 1105 1110 cgg gca gtg ccc
gat gag gag cgc cag gcg cag ccg ttt gat ccg 3384 Arg Ala Val Pro
Asp Glu Glu Arg Gln Ala Gln Pro Phe Asp Pro 1115 1120 1125 caa cgc
ttc cag gat gcc gtc gtt atg ccg tgg tat cgc aac cag 3429 Gln Arg
Phe Gln Asp Ala Val Val Met Pro Trp Tyr Arg Asn Gln 1130 1135 1140
gat caa ccg cag tat ttc tat gtg gcg gag ata tgt cca cat cta 3474
Asp Gln Pro Gln Tyr Phe Tyr Val Ala Glu Ile Cys Pro His Leu 1145
1150 1155 tcc cca ctc agc tgc ttc cct ggt gac aac tac cgc acg ttc
aag 3519 Ser Pro Leu Ser Cys Phe Pro Gly Asp Asn Tyr Arg Thr Phe
Lys 1160 1165 1170 cac tac tac ctc gtc aag tat ggt ctg acc ata cag
aat acc tcg 3564 His Tyr Tyr Leu Val Lys Tyr Gly Leu Thr Ile Gln
Asn Thr Ser 1175 1180 1185 cag ccg cta ttg gac gtg gat cac acc agt
gcg cgg tta aac ttc 3609 Gln Pro Leu Leu Asp Val Asp His Thr Ser
Ala Arg Leu Asn Phe 1190 1195 1200 ctc acg cca cga tac gtt aat cgc
aag ggc gtt gct ctg ccc act 3654 Leu Thr Pro Arg Tyr Val Asn Arg
Lys Gly Val Ala Leu Pro Thr 1205 1210 1215 agt tcg gag gag aca aag
cgg gca aag cgc gag aat ctc gaa cag 3699 Ser Ser Glu Glu Thr Lys
Arg Ala Lys Arg Glu Asn Leu Glu Gln 1220 1225 1230 aag cag atc ctt
gtg cca gag ctc tgc act gtg cat cca ttc ccc 3744 Lys Gln Ile Leu
Val Pro Glu Leu Cys Thr Val His Pro Phe Pro 1235 1240 1245 gcc tcc
ttg tgg cga act gcc gtg tgc ctg ccc tgc atc ctg tac 3789 Ala Ser
Leu Trp Arg Thr Ala Val Cys Leu Pro Cys Ile Leu Tyr 1250 1255 1260
cgc ata aat ggt ctt cta ttg gcc gac gat att cgg aaa cag gtt 3834
Arg Ile Asn Gly Leu Leu Leu Ala Asp Asp Ile Arg Lys Gln Val 1265
1270 1275 tct gcg gat ctg ggg ctg gga agg caa cag atc gaa gat gag
gat 3879 Ser Ala Asp Leu Gly Leu Gly Arg Gln Gln Ile Glu Asp Glu
Asp 1280 1285 1290 ttc gag tgg ccc atg ctg gac ttt ggg tgg agt cta
tcg gag gtg 3924 Phe Glu Trp Pro Met Leu Asp Phe Gly Trp Ser Leu
Ser Glu Val 1295 1300 1305 ctc aag aaa tcg cgg gag tcc aaa caa aag
gag tcc ctt aag gat 3969 Leu Lys Lys Ser Arg Glu Ser Lys Gln Lys
Glu Ser Leu Lys Asp 1310 1315 1320 gat act att aat ggc aaa gac tta
gct gat gtt gaa aag aaa ccg 4014 Asp Thr Ile Asn Gly Lys Asp Leu
Ala Asp Val Glu Lys Lys Pro 1325 1330 1335 act agc gag gag acc caa
cta gat aag gat tca aaa gac gat aag 4059 Thr Ser Glu Glu Thr Gln
Leu Asp Lys Asp Ser Lys Asp Asp Lys 1340 1345 1350 gtt gag aaa agt
gct att gaa cta atc att gag gga gag gag aag 4104 Val Glu Lys Ser
Ala Ile Glu Leu Ile Ile Glu Gly Glu Glu Lys 1355
1360 1365 ctg caa gag gct gat gac ttc att gag ata ggc act tgg tca
aac 4149 Leu Gln Glu Ala Asp Asp Phe Ile Glu Ile Gly Thr Trp Ser
Asn 1370 1375 1380 gat atg gcc gac gat ata gct agt ttt aac caa gaa
gac gac gac 4194 Asp Met Ala Asp Asp Ile Ala Ser Phe Asn Gln Glu
Asp Asp Asp 1385 1390 1395 gag gat gac gcc ttc cat ctc cca gtt tta
ccg gca aac gtt aag 4239 Glu Asp Asp Ala Phe His Leu Pro Val Leu
Pro Ala Asn Val Lys 1400 1405 1410 ttc tgt gat cag caa acg cgc tac
ggt tcg ccc aca ttt tgg gat 4284 Phe Cys Asp Gln Gln Thr Arg Tyr
Gly Ser Pro Thr Phe Trp Asp 1415 1420 1425 gtg agc aat ggc gaa agc
ggc ttc aag ggt cca aag agc agt cag 4329 Val Ser Asn Gly Glu Ser
Gly Phe Lys Gly Pro Lys Ser Ser Gln 1430 1435 1440 aat aag cag ggt
ggc aag ggc aaa gca aag ggt ccg gca aag ccc 4374 Asn Lys Gln Gly
Gly Lys Gly Lys Ala Lys Gly Pro Ala Lys Pro 1445 1450 1455 aca ttt
aac tat tat gac tcg gac aat tcg ctg ggt tcc agc tac 4419 Thr Phe
Asn Tyr Tyr Asp Ser Asp Asn Ser Leu Gly Ser Ser Tyr 1460 1465 1470
gat gac gac gat aac gca ggt ccg ctc aat tac atg cat cac aac 4464
Asp Asp Asp Asp Asn Ala Gly Pro Leu Asn Tyr Met His His Asn 1475
1480 1485 tac agt tcg gat gac gac gat gtg gca gat gat atc gat gcg
gga 4509 Tyr Ser Ser Asp Asp Asp Asp Val Ala Asp Asp Ile Asp Ala
Gly 1490 1495 1500 cgc att gcg ttc acc tcc aag aat gaa gcg gag act
att gaa acc 4554 Arg Ile Ala Phe Thr Ser Lys Asn Glu Ala Glu Thr
Ile Glu Thr 1505 1510 1515 gca cag gaa gtg gaa aag cgc cag aag cag
ctg tcc atc atc cag 4599 Ala Gln Glu Val Glu Lys Arg Gln Lys Gln
Leu Ser Ile Ile Gln 1520 1525 1530 gcg acc aat gct aac gag cgg cag
tat cag cag aca aag aac ctg 4644 Ala Thr Asn Ala Asn Glu Arg Gln
Tyr Gln Gln Thr Lys Asn Leu 1535 1540 1545 ctc att gga ttc aat ttt
aag cat gag gac cag aag gaa cct gcc 4689 Leu Ile Gly Phe Asn Phe
Lys His Glu Asp Gln Lys Glu Pro Ala 1550 1555 1560 act ata aga tat
gaa gaa tcc ata gct aag ctc aaa acg gaa ata 4734 Thr Ile Arg Tyr
Glu Glu Ser Ile Ala Lys Leu Lys Thr Glu Ile 1565 1570 1575 gaa tcc
ggc ggc atg ttg gtg ccg cac gac cag cag ttg gtt cta 4779 Glu Ser
Gly Gly Met Leu Val Pro His Asp Gln Gln Leu Val Leu 1580 1585 1590
aaa aga agt gat gcc gct gag gct cag gtt gca aag gta tcg atg 4824
Lys Arg Ser Asp Ala Ala Glu Ala Gln Val Ala Lys Val Ser Met 1595
1600 1605 atg gag cta ttg aag cag ctg ctg ccg tat gta aat gaa gat
gtg 4869 Met Glu Leu Leu Lys Gln Leu Leu Pro Tyr Val Asn Glu Asp
Val 1610 1615 1620 ctg gcc aaa aag ctg ggt gat agg cgc gag ctt ctg
ctg tcg gat 4914 Leu Ala Lys Lys Leu Gly Asp Arg Arg Glu Leu Leu
Leu Ser Asp 1625 1630 1635 ttg gta gag cta aat gca gat tgg gta gcg
cga cat gag cag gag 4959 Leu Val Glu Leu Asn Ala Asp Trp Val Ala
Arg His Glu Gln Glu 1640 1645 1650 acc tac aat gta atg gga tgc gga
gat agt ttt gac aac tat aac 5004 Thr Tyr Asn Val Met Gly Cys Gly
Asp Ser Phe Asp Asn Tyr Asn 1655 1660 1665 gat cat cat cgg ctg aac
ttg gat gaa aag caa ctg aaa ctg caa 5049 Asp His His Arg Leu Asn
Leu Asp Glu Lys Gln Leu Lys Leu Gln 1670 1675 1680 tac gaa cga att
gaa att gag cca cct act tcc acg aag gcc ata 5094 Tyr Glu Arg Ile
Glu Ile Glu Pro Pro Thr Ser Thr Lys Ala Ile 1685 1690 1695 acc tca
gcc ata tta cca gct ggc ttc agt ttc gat cga caa ccg 5139 Thr Ser
Ala Ile Leu Pro Ala Gly Phe Ser Phe Asp Arg Gln Pro 1700 1705 1710
gat cta gtg ggc cat cca gga ccc agt ccc agc atc att ttg caa 5184
Asp Leu Val Gly His Pro Gly Pro Ser Pro Ser Ile Ile Leu Gln 1715
1720 1725 gcc ctc aca atg tcc aat gct aac gat ggc atc aat ctg gag
cga 5229 Ala Leu Thr Met Ser Asn Ala Asn Asp Gly Ile Asn Leu Glu
Arg 1730 1735 1740 ctg gag aca att gga gat tcc ttt cta aag tat gcc
att acc acc 5274 Leu Glu Thr Ile Gly Asp Ser Phe Leu Lys Tyr Ala
Ile Thr Thr 1745 1750 1755 tac ttg tac atc acc tac gag aat gtg cac
gag gga aaa cta agt 5319 Tyr Leu Tyr Ile Thr Tyr Glu Asn Val His
Glu Gly Lys Leu Ser 1760 1765 1770 cac ctg cgc tcc aag cag gtt gcc
aat ctc aat ctc tat cgt ctg 5364 His Leu Arg Ser Lys Gln Val Ala
Asn Leu Asn Leu Tyr Arg Leu 1775 1780 1785 ggc aga cgt aag aga ctg
ggt gaa tat atg ata gcc act aaa ttc 5409 Gly Arg Arg Lys Arg Leu
Gly Glu Tyr Met Ile Ala Thr Lys Phe 1790 1795 1800 gag ccg cac gac
aat tgg ctg cca ccc tgc tac tac gtg cca aag 5454 Glu Pro His Asp
Asn Trp Leu Pro Pro Cys Tyr Tyr Val Pro Lys 1805 1810 1815 gag cta
gag aag gcg ctc atc gag gcg aag atc ccc act cac cat 5499 Glu Leu
Glu Lys Ala Leu Ile Glu Ala Lys Ile Pro Thr His His 1820 1825 1830
tgg aag ctg gcc gat ctg cta gac att aag aac cta agc agt gtg 5544
Trp Lys Leu Ala Asp Leu Leu Asp Ile Lys Asn Leu Ser Ser Val 1835
1840 1845 caa atc tgc gag atg gtt cgc gaa aaa gcc gat gcc ctg ggc
ttg 5589 Gln Ile Cys Glu Met Val Arg Glu Lys Ala Asp Ala Leu Gly
Leu 1850 1855 1860 gag cag aat ggg ggt gcc caa aat gga caa ctt gac
gac tcc aat 5634 Glu Gln Asn Gly Gly Ala Gln Asn Gly Gln Leu Asp
Asp Ser Asn 1865 1870 1875 gat agc tgc aat gat ttt agc tgt ttt att
ccc tac aac ctt gtt 5679 Asp Ser Cys Asn Asp Phe Ser Cys Phe Ile
Pro Tyr Asn Leu Val 1880 1885 1890 tcg caa cac agc att ccg gat aag
tct att gcc gat tgc gtc gaa 5724 Ser Gln His Ser Ile Pro Asp Lys
Ser Ile Ala Asp Cys Val Glu 1895 1900 1905 gcc ctc att gga gcc tat
ctc att gag tgc gga ccc cga ggg gct 5769 Ala Leu Ile Gly Ala Tyr
Leu Ile Glu Cys Gly Pro Arg Gly Ala 1910 1915 1920 tta ctc ttt atg
gcc tgg ctg ggc gtg aga gtg ctc cct atc aca 5814 Leu Leu Phe Met
Ala Trp Leu Gly Val Arg Val Leu Pro Ile Thr 1925 1930 1935 agg cag
ttg gac ggg ggt aac cag gag caa cga ata ccc ggt agc 5859 Arg Gln
Leu Asp Gly Gly Asn Gln Glu Gln Arg Ile Pro Gly Ser 1940 1945 1950
aca aaa ccg aat gcc gaa aat gtg gtc acc gtt tac ggt gca tgg 5904
Thr Lys Pro Asn Ala Glu Asn Val Val Thr Val Tyr Gly Ala Trp 1955
1960 1965 ccc acg ccg cgt agt cca ctg ctg cac ttt gct cca aat gct
acg 5949 Pro Thr Pro Arg Ser Pro Leu Leu His Phe Ala Pro Asn Ala
Thr 1970 1975 1980 gag gag ctg gac cag tta cta agc ggc ttt gag gag
ttt gag gag 5994 Glu Glu Leu Asp Gln Leu Leu Ser Gly Phe Glu Glu
Phe Glu Glu 1985 1990 1995 agt ttg gga tac aag ttc cgg gat cgg tcg
tac ctg ttg caa gcc 6039 Ser Leu Gly Tyr Lys Phe Arg Asp Arg Ser
Tyr Leu Leu Gln Ala 2000 2005 2010 atg aca cat gcc agt tac acg ccc
aat cga ttg acg gat tgc tat 6084 Met Thr His Ala Ser Tyr Thr Pro
Asn Arg Leu Thr Asp Cys Tyr 2015 2020 2025 cag cgt ctg gag ttc ctg
ggc gat gct gtt cta gat tac ctc att 6129 Gln Arg Leu Glu Phe Leu
Gly Asp Ala Val Leu Asp Tyr Leu Ile 2030 2035 2040 acg cgg cat tta
tac gaa gat ccc cgc cag cat tct cca ggc gca 6174 Thr Arg His Leu
Tyr Glu Asp Pro Arg Gln His Ser Pro Gly Ala 2045 2050 2055 tta acg
gat ttg cgg tca gca ctg gtg aat aat aca ata ttc gcc 6219 Leu Thr
Asp Leu Arg Ser Ala Leu Val Asn Asn Thr Ile Phe Ala 2060 2065 2070
tcc ctg gct gtt cgc cat ggc ttc cac aag ttc ttc cgg cac ctc 6264
Ser Leu Ala Val Arg His Gly Phe His Lys Phe Phe Arg His Leu 2075
2080 2085 tcg ccg ggc ctt aac gat gtg att gac cgt ttt gtg cgg atc
cag 6309 Ser Pro Gly Leu Asn Asp Val Ile Asp Arg Phe Val Arg Ile
Gln 2090 2095 2100 cag gag aat gga cac tgc atc agt gag gag tac tac
tta ttg tcc 6354 Gln Glu Asn Gly His Cys Ile Ser Glu Glu Tyr Tyr
Leu Leu Ser 2105 2110 2115 gag gag gag tgc gat gac gcc gag gac gtt
gag gtg ccc aag gca 6399 Glu Glu Glu Cys Asp Asp Ala Glu Asp Val
Glu Val Pro Lys Ala 2120 2125 2130 ttg ggc gac gtt ttc gag tcg atc
gca ggt gcc att ttt ctc gac 6444 Leu Gly Asp Val Phe Glu Ser Ile
Ala Gly Ala Ile Phe Leu Asp 2135 2140 2145 tca aac atg tcg ctg gac
gtg gtt tgg cac gta tat agc aac atg 6489 Ser Asn Met Ser Leu Asp
Val Val Trp His Val Tyr Ser Asn Met 2150 2155 2160 atg agc ccg gag
atc gag cag ttc agc aac tca gtg cca aaa tcg 6534 Met Ser Pro Glu
Ile Glu Gln Phe Ser Asn Ser Val Pro Lys Ser 2165 2170 2175 ccc att
cgg gag ctc ctc gag ctg gag ccg gaa acc gcc aag ttc 6579 Pro Ile
Arg Glu Leu Leu Glu Leu Glu Pro Glu Thr Ala Lys Phe 2180 2185 2190
ggc aag ccc gag aag ctg gcg gat ggg cga cgg gtg cgc gtt acc 6624
Gly Lys Pro Glu Lys Leu Ala Asp Gly Arg Arg Val Arg Val Thr 2195
2200 2205 gtg gat gtc ttc tgc aaa gga acc ttc cgt ggc atc gga cgc
aac 6669 Val Asp Val Phe Cys Lys Gly Thr Phe Arg Gly Ile Gly Arg
Asn 2210 2215 2220 tat cgc att gcc aag tgc acg gcg gcc aaa tgc gca
ttg cgc caa 6714 Tyr Arg Ile Ala Lys Cys Thr Ala Ala Lys Cys Ala
Leu Arg Gln 2225 2230 2235 ctc aaa aag cag ggc ttg ata gcc aaa aaa
gac taa 6750 Leu Lys Lys Gln Gly Leu Ile Ala Lys Lys Asp 2240 2245
4 2249 PRT Drosophila melanogaster 4 Met Ala Phe His Trp Cys Asp
Asn Asn Leu His Thr Thr Val Phe Thr 1 5 10 15 Pro Arg Asp Phe Gln
Val Glu Leu Leu Ala Thr Ala Tyr Glu Arg Asn 20 25 30 Thr Ile Ile
Cys Leu Gly His Arg Ser Ser Lys Glu Phe Ile Ala Leu 35 40 45 Lys
Leu Leu Gln Glu Leu Ser Arg Arg Ala Arg Arg His Gly Arg Val 50 55
60 Ser Val Tyr Leu Ser Cys Glu Val Gly Thr Ser Thr Glu Pro Cys Ser
65 70 75 80 Ile Tyr Thr Met Leu Thr His Leu Thr Asp Leu Arg Val Trp
Gln Glu 85 90 95 Gln Pro Asp Met Gln Ile Pro Phe Asp His Cys Trp
Thr Asp Tyr His 100 105 110 Val Ser Ile Leu Arg Pro Glu Gly Phe Leu
Tyr Leu Leu Glu Thr Arg 115 120 125 Glu Leu Leu Leu Ser Ser Val Glu
Leu Ile Val Leu Glu Asp Cys His 130 135 140 Asp Ser Ala Val Tyr Gln
Arg Ile Arg Pro Leu Phe Glu Asn His Ile 145 150 155 160 Met Pro Ala
Pro Pro Ala Asp Arg Pro Arg Ile Leu Gly Leu Ala Gly 165 170 175 Pro
Leu His Ser Ala Gly Cys Glu Leu Gln Gln Leu Ser Ala Met Leu 180 185
190 Ala Thr Leu Glu Gln Ser Val Leu Cys Gln Ile Glu Thr Ala Ser Asp
195 200 205 Ile Val Thr Val Leu Arg Tyr Cys Ser Arg Pro His Glu Tyr
Ile Val 210 215 220 Gln Cys Ala Pro Phe Glu Met Asp Glu Leu Ser Leu
Val Leu Ala Asp 225 230 235 240 Val Leu Asn Thr His Lys Ser Phe Leu
Leu Asp His Arg Tyr Asp Pro 245 250 255 Tyr Glu Ile Tyr Gly Thr Asp
Gln Phe Met Asp Glu Leu Lys Asp Ile 260 265 270 Pro Asp Pro Lys Val
Asp Pro Leu Asn Val Ile Asn Ser Leu Leu Val 275 280 285 Val Leu His
Glu Met Gly Pro Trp Cys Thr Gln Arg Ala Ala His His 290 295 300 Phe
Tyr Gln Cys Asn Glu Lys Leu Lys Val Lys Thr Pro His Glu Arg 305 310
315 320 His Tyr Leu Leu Tyr Cys Leu Val Ser Thr Ala Leu Ile Gln Leu
Tyr 325 330 335 Ser Leu Cys Glu His Ala Phe His Arg His Leu Gly Ser
Gly Ser Asp 340 345 350 Ser Arg Gln Thr Ile Glu Arg Tyr Ser Ser Pro
Lys Val Arg Arg Leu 355 360 365 Leu Gln Thr Leu Arg Cys Phe Lys Pro
Glu Glu Val His Thr Gln Ala 370 375 380 Asp Gly Leu Arg Arg Met Arg
His Gln Val Asp Gln Ala Asp Phe Asn 385 390 395 400 Arg Leu Ser His
Thr Leu Glu Ser Lys Cys Arg Met Val Asp Gln Met 405 410 415 Asp Gln
Pro Pro Thr Glu Thr Arg Ala Leu Val Ala Thr Leu Glu Gln 420 425 430
Ile Leu His Thr Thr Glu Asp Arg Gln Thr Asn Arg Ser Ala Ala Arg 435
440 445 Val Thr Pro Thr Pro Thr Pro Ala His Ala Lys Pro Lys Pro Ser
Ser 450 455 460 Gly Ala Asn Thr Ala Gln Pro Arg Thr Arg Arg Arg Val
Tyr Thr Arg 465 470 475 480 Arg His His Arg Asp His Asn Asp Gly Ser
Asp Thr Leu Cys Ala Leu 485 490 495 Ile Tyr Cys Asn Gln Asn His Thr
Ala Arg Val Leu Phe Glu Leu Leu 500 505 510 Ala Glu Ile Ser Arg Arg
Asp Pro Asp Leu Lys Phe Leu Arg Cys Gln 515 520 525 Tyr Thr Thr Asp
Arg Val Ala Asp Pro Thr Thr Glu Pro Lys Glu Ala 530 535 540 Glu Leu
Glu His Arg Arg Gln Glu Glu Val Leu Lys Arg Phe Arg Met 545 550 555
560 His Asp Cys Asn Val Leu Ile Gly Thr Ser Val Leu Glu Glu Gly Ile
565 570 575 Asp Val Pro Lys Cys Asn Leu Val Val Arg Trp Asp Pro Pro
Thr Thr 580 585 590 Tyr Arg Ser Tyr Val Gln Cys Lys Gly Arg Ala Arg
Ala Ala Pro Ala 595 600 605 Tyr His Val Ile Leu Val Ala Pro Ser Tyr
Lys Ser Pro Thr Val Gly 610 615 620 Ser Val Gln Leu Thr Asp Arg Ser
His Arg Tyr Ile Cys Ala Thr Gly 625 630 635 640 Asp Thr Thr Glu Ala
Asp Ser Asp Ser Asp Asp Ser Ala Met Pro Asn 645 650 655 Ser Ser Gly
Ser Asp Pro Tyr Thr Phe Gly Thr Ala Arg Gly Thr Val 660 665 670 Lys
Ile Leu Asn Pro Glu Val Phe Ser Lys Gln Pro Pro Thr Ala Cys 675 680
685 Asp Ile Lys Leu Gln Glu Ile Gln Asp Glu Leu Pro Ala Ala Ala Gln
690 695 700 Leu Asp Thr Ser Asn Ser Ser Asp Glu Ala Val Ser Met Ser
Asn Thr 705 710 715 720 Ser Pro Ser Glu Ser Ser Thr Glu Gln Lys Ser
Arg Arg Phe Gln Cys 725 730 735 Glu Leu Ser Ser Leu Thr Glu Pro Glu
Asp Thr Ser Asp Thr Thr Ala 740 745 750 Glu Ile Asp Thr Ala His Ser
Leu Ala Ser Thr Thr Lys Asp Leu Val 755 760 765 His Gln Met Ala Gln
Tyr Arg Glu Ile Glu Gln Met Leu Leu Ser Lys 770 775 780 Cys Ala Asn
Thr Glu Pro Pro Glu Gln Glu Gln Ser Glu Ala Glu Arg 785 790 795 800
Phe Ser Ala Cys Leu Ala Ala Tyr Arg Pro Lys Pro His Leu Leu Thr 805
810 815 Gly Ala Ser Val Asp Leu Gly Ser Ala Ile Ala Leu Val Asn Lys
Tyr 820 825 830 Cys Ala Arg Leu Pro Ser Asp Thr Phe Thr Lys Leu Thr
Ala Leu Trp 835 840 845 Arg Cys Thr Arg Asn Glu Arg Ala Gly Val Thr
Leu Phe Gln Tyr Thr 850 855 860 Leu Arg Leu Pro Ile Asn Ser Pro Leu
Lys His Asp Ile Val Gly Leu 865 870 875 880 Pro Met Pro Thr Gln Thr
Leu Ala Arg Arg Leu Ala Ala Leu Gln Ala 885 890 895 Cys Val Glu Leu
His Arg Ile Gly Glu Leu Asp Asp Gln Leu Gln Pro 900 905 910 Ile Gly
Lys Glu Gly Phe Arg Ala Leu Glu Pro Asp Trp Glu Cys Phe 915 920 925
Glu Leu Glu Pro Glu Asp Glu Gln Ile Val Gln Leu Ser Asp Glu Pro 930
935 940 Arg Pro Gly Thr Thr Lys Arg Arg Gln Tyr Tyr Tyr Lys Arg Ile
Ala 945 950 955 960 Ser Glu Phe Cys Asp Cys Arg
Pro Val Ala Gly Ala Pro Cys Tyr Leu 965 970 975 Tyr Phe Ile Gln Leu
Thr Leu Gln Cys Pro Ile Pro Glu Glu Gln Asn 980 985 990 Thr Arg Gly
Arg Lys Ile Tyr Pro Pro Glu Asp Ala Gln Gln Gly Phe 995 1000 1005
Gly Ile Leu Thr Thr Lys Arg Ile Pro Lys Leu Ser Ala Phe Ser 1010
1015 1020 Ile Phe Thr Arg Ser Gly Glu Val Lys Val Ser Leu Glu Leu
Ala 1025 1030 1035 Lys Glu Arg Val Ile Leu Thr Ser Glu Gln Ile Val
Cys Ile Asn 1040 1045 1050 Gly Phe Leu Asn Tyr Thr Phe Thr Asn Val
Leu Arg Leu Gln Lys 1055 1060 1065 Phe Leu Met Leu Phe Asp Pro Asp
Ser Thr Glu Asn Cys Val Phe 1070 1075 1080 Ile Val Pro Thr Val Lys
Ala Pro Ala Gly Gly Lys His Ile Asp 1085 1090 1095 Trp Gln Phe Leu
Glu Leu Ile Gln Ala Asn Gly Asn Thr Met Pro 1100 1105 1110 Arg Ala
Val Pro Asp Glu Glu Arg Gln Ala Gln Pro Phe Asp Pro 1115 1120 1125
Gln Arg Phe Gln Asp Ala Val Val Met Pro Trp Tyr Arg Asn Gln 1130
1135 1140 Asp Gln Pro Gln Tyr Phe Tyr Val Ala Glu Ile Cys Pro His
Leu 1145 1150 1155 Ser Pro Leu Ser Cys Phe Pro Gly Asp Asn Tyr Arg
Thr Phe Lys 1160 1165 1170 His Tyr Tyr Leu Val Lys Tyr Gly Leu Thr
Ile Gln Asn Thr Ser 1175 1180 1185 Gln Pro Leu Leu Asp Val Asp His
Thr Ser Ala Arg Leu Asn Phe 1190 1195 1200 Leu Thr Pro Arg Tyr Val
Asn Arg Lys Gly Val Ala Leu Pro Thr 1205 1210 1215 Ser Ser Glu Glu
Thr Lys Arg Ala Lys Arg Glu Asn Leu Glu Gln 1220 1225 1230 Lys Gln
Ile Leu Val Pro Glu Leu Cys Thr Val His Pro Phe Pro 1235 1240 1245
Ala Ser Leu Trp Arg Thr Ala Val Cys Leu Pro Cys Ile Leu Tyr 1250
1255 1260 Arg Ile Asn Gly Leu Leu Leu Ala Asp Asp Ile Arg Lys Gln
Val 1265 1270 1275 Ser Ala Asp Leu Gly Leu Gly Arg Gln Gln Ile Glu
Asp Glu Asp 1280 1285 1290 Phe Glu Trp Pro Met Leu Asp Phe Gly Trp
Ser Leu Ser Glu Val 1295 1300 1305 Leu Lys Lys Ser Arg Glu Ser Lys
Gln Lys Glu Ser Leu Lys Asp 1310 1315 1320 Asp Thr Ile Asn Gly Lys
Asp Leu Ala Asp Val Glu Lys Lys Pro 1325 1330 1335 Thr Ser Glu Glu
Thr Gln Leu Asp Lys Asp Ser Lys Asp Asp Lys 1340 1345 1350 Val Glu
Lys Ser Ala Ile Glu Leu Ile Ile Glu Gly Glu Glu Lys 1355 1360 1365
Leu Gln Glu Ala Asp Asp Phe Ile Glu Ile Gly Thr Trp Ser Asn 1370
1375 1380 Asp Met Ala Asp Asp Ile Ala Ser Phe Asn Gln Glu Asp Asp
Asp 1385 1390 1395 Glu Asp Asp Ala Phe His Leu Pro Val Leu Pro Ala
Asn Val Lys 1400 1405 1410 Phe Cys Asp Gln Gln Thr Arg Tyr Gly Ser
Pro Thr Phe Trp Asp 1415 1420 1425 Val Ser Asn Gly Glu Ser Gly Phe
Lys Gly Pro Lys Ser Ser Gln 1430 1435 1440 Asn Lys Gln Gly Gly Lys
Gly Lys Ala Lys Gly Pro Ala Lys Pro 1445 1450 1455 Thr Phe Asn Tyr
Tyr Asp Ser Asp Asn Ser Leu Gly Ser Ser Tyr 1460 1465 1470 Asp Asp
Asp Asp Asn Ala Gly Pro Leu Asn Tyr Met His His Asn 1475 1480 1485
Tyr Ser Ser Asp Asp Asp Asp Val Ala Asp Asp Ile Asp Ala Gly 1490
1495 1500 Arg Ile Ala Phe Thr Ser Lys Asn Glu Ala Glu Thr Ile Glu
Thr 1505 1510 1515 Ala Gln Glu Val Glu Lys Arg Gln Lys Gln Leu Ser
Ile Ile Gln 1520 1525 1530 Ala Thr Asn Ala Asn Glu Arg Gln Tyr Gln
Gln Thr Lys Asn Leu 1535 1540 1545 Leu Ile Gly Phe Asn Phe Lys His
Glu Asp Gln Lys Glu Pro Ala 1550 1555 1560 Thr Ile Arg Tyr Glu Glu
Ser Ile Ala Lys Leu Lys Thr Glu Ile 1565 1570 1575 Glu Ser Gly Gly
Met Leu Val Pro His Asp Gln Gln Leu Val Leu 1580 1585 1590 Lys Arg
Ser Asp Ala Ala Glu Ala Gln Val Ala Lys Val Ser Met 1595 1600 1605
Met Glu Leu Leu Lys Gln Leu Leu Pro Tyr Val Asn Glu Asp Val 1610
1615 1620 Leu Ala Lys Lys Leu Gly Asp Arg Arg Glu Leu Leu Leu Ser
Asp 1625 1630 1635 Leu Val Glu Leu Asn Ala Asp Trp Val Ala Arg His
Glu Gln Glu 1640 1645 1650 Thr Tyr Asn Val Met Gly Cys Gly Asp Ser
Phe Asp Asn Tyr Asn 1655 1660 1665 Asp His His Arg Leu Asn Leu Asp
Glu Lys Gln Leu Lys Leu Gln 1670 1675 1680 Tyr Glu Arg Ile Glu Ile
Glu Pro Pro Thr Ser Thr Lys Ala Ile 1685 1690 1695 Thr Ser Ala Ile
Leu Pro Ala Gly Phe Ser Phe Asp Arg Gln Pro 1700 1705 1710 Asp Leu
Val Gly His Pro Gly Pro Ser Pro Ser Ile Ile Leu Gln 1715 1720 1725
Ala Leu Thr Met Ser Asn Ala Asn Asp Gly Ile Asn Leu Glu Arg 1730
1735 1740 Leu Glu Thr Ile Gly Asp Ser Phe Leu Lys Tyr Ala Ile Thr
Thr 1745 1750 1755 Tyr Leu Tyr Ile Thr Tyr Glu Asn Val His Glu Gly
Lys Leu Ser 1760 1765 1770 His Leu Arg Ser Lys Gln Val Ala Asn Leu
Asn Leu Tyr Arg Leu 1775 1780 1785 Gly Arg Arg Lys Arg Leu Gly Glu
Tyr Met Ile Ala Thr Lys Phe 1790 1795 1800 Glu Pro His Asp Asn Trp
Leu Pro Pro Cys Tyr Tyr Val Pro Lys 1805 1810 1815 Glu Leu Glu Lys
Ala Leu Ile Glu Ala Lys Ile Pro Thr His His 1820 1825 1830 Trp Lys
Leu Ala Asp Leu Leu Asp Ile Lys Asn Leu Ser Ser Val 1835 1840 1845
Gln Ile Cys Glu Met Val Arg Glu Lys Ala Asp Ala Leu Gly Leu 1850
1855 1860 Glu Gln Asn Gly Gly Ala Gln Asn Gly Gln Leu Asp Asp Ser
Asn 1865 1870 1875 Asp Ser Cys Asn Asp Phe Ser Cys Phe Ile Pro Tyr
Asn Leu Val 1880 1885 1890 Ser Gln His Ser Ile Pro Asp Lys Ser Ile
Ala Asp Cys Val Glu 1895 1900 1905 Ala Leu Ile Gly Ala Tyr Leu Ile
Glu Cys Gly Pro Arg Gly Ala 1910 1915 1920 Leu Leu Phe Met Ala Trp
Leu Gly Val Arg Val Leu Pro Ile Thr 1925 1930 1935 Arg Gln Leu Asp
Gly Gly Asn Gln Glu Gln Arg Ile Pro Gly Ser 1940 1945 1950 Thr Lys
Pro Asn Ala Glu Asn Val Val Thr Val Tyr Gly Ala Trp 1955 1960 1965
Pro Thr Pro Arg Ser Pro Leu Leu His Phe Ala Pro Asn Ala Thr 1970
1975 1980 Glu Glu Leu Asp Gln Leu Leu Ser Gly Phe Glu Glu Phe Glu
Glu 1985 1990 1995 Ser Leu Gly Tyr Lys Phe Arg Asp Arg Ser Tyr Leu
Leu Gln Ala 2000 2005 2010 Met Thr His Ala Ser Tyr Thr Pro Asn Arg
Leu Thr Asp Cys Tyr 2015 2020 2025 Gln Arg Leu Glu Phe Leu Gly Asp
Ala Val Leu Asp Tyr Leu Ile 2030 2035 2040 Thr Arg His Leu Tyr Glu
Asp Pro Arg Gln His Ser Pro Gly Ala 2045 2050 2055 Leu Thr Asp Leu
Arg Ser Ala Leu Val Asn Asn Thr Ile Phe Ala 2060 2065 2070 Ser Leu
Ala Val Arg His Gly Phe His Lys Phe Phe Arg His Leu 2075 2080 2085
Ser Pro Gly Leu Asn Asp Val Ile Asp Arg Phe Val Arg Ile Gln 2090
2095 2100 Gln Glu Asn Gly His Cys Ile Ser Glu Glu Tyr Tyr Leu Leu
Ser 2105 2110 2115 Glu Glu Glu Cys Asp Asp Ala Glu Asp Val Glu Val
Pro Lys Ala 2120 2125 2130 Leu Gly Asp Val Phe Glu Ser Ile Ala Gly
Ala Ile Phe Leu Asp 2135 2140 2145 Ser Asn Met Ser Leu Asp Val Val
Trp His Val Tyr Ser Asn Met 2150 2155 2160 Met Ser Pro Glu Ile Glu
Gln Phe Ser Asn Ser Val Pro Lys Ser 2165 2170 2175 Pro Ile Arg Glu
Leu Leu Glu Leu Glu Pro Glu Thr Ala Lys Phe 2180 2185 2190 Gly Lys
Pro Glu Lys Leu Ala Asp Gly Arg Arg Val Arg Val Thr 2195 2200 2205
Val Asp Val Phe Cys Lys Gly Thr Phe Arg Gly Ile Gly Arg Asn 2210
2215 2220 Tyr Arg Ile Ala Lys Cys Thr Ala Ala Lys Cys Ala Leu Arg
Gln 2225 2230 2235 Leu Lys Lys Gln Gly Leu Ile Ala Lys Lys Asp 2240
2245 5 1145 PRT Drosophila melanogaster 5 Met Gly Lys Lys Asp Lys
Asn Lys Lys Gly Gly Gln Asp Ser Ala Ala 1 5 10 15 Ala Pro Gln Pro
Gln Gln Gln Gln Lys Gln Gln Gln Gln Arg Gln Gln 20 25 30 Gln Pro
Gln Gln Leu Gln Gln Pro Gln Gln Leu Gln Gln Pro Gln Gln 35 40 45
Leu Gln Gln Pro Gln Gln Gln Gln Gln Gln Gln Pro His Gln Gln Gln 50
55 60 Gln Gln Ser Ser Arg Gln Gln Pro Ser Thr Ser Ser Gly Gly Ser
Arg 65 70 75 80 Ala Ser Gly Phe Gln Gln Gly Gly Gln Gln Gln Lys Ser
Gln Asp Ala 85 90 95 Glu Gly Trp Thr Ala Gln Lys Lys Gln Gly Lys
Gln Gln Val Gln Gly 100 105 110 Trp Thr Lys Gln Gly Gln Gln Gly Gly
His Gln Gln Gly Arg Gln Gly 115 120 125 Gln Asp Gly Gly Tyr Gln Gln
Arg Pro Pro Gly Gln Gln Gln Gly Gly 130 135 140 His Gln Gln Gly Arg
Gln Gly Gln Glu Gly Gly Tyr Gln Gln Arg Pro 145 150 155 160 Pro Gly
Gln Gln Gln Gly Gly His Gln Gln Gly Arg Gln Gly Gln Glu 165 170 175
Gly Gly Tyr Gln Gln Arg Pro Ser Gly Gln Gln Gln Gly Gly His Gln 180
185 190 Gln Gly Arg Gln Gly Gln Glu Gly Gly Tyr Gln Gln Arg Pro Pro
Gly 195 200 205 Gln Gln Gln Gly Gly His Gln Gln Gly Arg Gln Gly Gln
Glu Gly Gly 210 215 220 Tyr Gln Gln Arg Pro Ser Gly Gln Gln Gln Gly
Gly His Gln Gln Gly 225 230 235 240 Arg Gln Gly Gln Glu Gly Gly Tyr
Gln Gln Arg Pro Ser Gly Gln Gln 245 250 255 Gln Gly Gly His Gln Gln
Gly Arg Gln Gly Gln Glu Gly Gly Tyr Gln 260 265 270 Gln Arg Pro Ser
Gly Gln Gln Gln Gly Gly His Gln Gln Gly Arg Gln 275 280 285 Gly Gln
Glu Gly Gly Tyr Gln Gln Arg Pro Pro Gly Gln Gln Pro Asn 290 295 300
Gln Thr Gln Ser Gln Gly Gln Tyr Gln Ser Arg Gly Pro Pro Gln Gln 305
310 315 320 Gln Gln Ala Ala Pro Leu Pro Leu Pro Pro Gln Pro Ala Gly
Ser Ile 325 330 335 Lys Arg Gly Thr Ile Gly Lys Pro Gly Gln Val Gly
Ile Asn Tyr Leu 340 345 350 Asp Leu Asp Leu Ser Lys Met Pro Ser Val
Ala Tyr His Tyr Asp Val 355 360 365 Lys Ile Met Pro Glu Arg Pro Lys
Lys Phe Tyr Arg Gln Ala Phe Glu 370 375 380 Gln Phe Arg Val Asp Gln
Leu Gly Gly Ala Val Leu Ala Tyr Asp Gly 385 390 395 400 Lys Ala Ser
Cys Tyr Ser Val Asp Lys Leu Pro Leu Asn Ser Gln Asn 405 410 415 Pro
Glu Val Thr Val Thr Asp Arg Asn Gly Arg Thr Leu Arg Tyr Thr 420 425
430 Ile Glu Ile Lys Glu Thr Gly Asp Ser Thr Ile Asp Leu Lys Ser Leu
435 440 445 Thr Thr Tyr Met Asn Asp Arg Ile Phe Asp Lys Pro Met Arg
Ala Met 450 455 460 Gln Cys Val Glu Val Val Leu Ala Ser Pro Cys His
Asn Lys Ala Ile 465 470 475 480 Arg Val Gly Arg Ser Phe Phe Lys Met
Ser Asp Pro Asn Asn Arg His 485 490 495 Glu Leu Asp Asp Gly Tyr Glu
Ala Leu Val Gly Leu Tyr Gln Ala Phe 500 505 510 Met Leu Gly Asp Arg
Pro Phe Leu Asn Val Asp Ile Ser His Lys Ser 515 520 525 Phe Pro Ile
Ser Met Pro Met Ile Glu Tyr Leu Glu Arg Phe Ser Leu 530 535 540 Lys
Ala Lys Ile Asn Asn Thr Thr Asn Leu Asp Tyr Ser Arg Arg Phe 545 550
555 560 Leu Glu Pro Phe Leu Arg Gly Ile Asn Val Val Tyr Thr Pro Pro
Gln 565 570 575 Ser Phe Gln Ser Ala Pro Arg Val Tyr Arg Val Asn Gly
Leu Ser Arg 580 585 590 Ala Pro Ala Ser Ser Glu Thr Phe Glu His Asp
Gly Lys Lys Val Thr 595 600 605 Ile Ala Ser Tyr Phe His Ser Arg Asn
Tyr Pro Leu Lys Phe Pro Gln 610 615 620 Leu His Cys Leu Asn Val Gly
Ser Ser Ile Lys Ser Ile Leu Leu Pro 625 630 635 640 Ile Glu Leu Cys
Ser Ile Glu Glu Gly Gln Ala Leu Asn Arg Lys Asp 645 650 655 Gly Ala
Thr Gln Val Ala Asn Met Ile Lys Tyr Ala Ala Thr Ser Thr 660 665 670
Asn Val Arg Lys Arg Lys Ile Met Asn Leu Leu Gln Tyr Phe Gln His 675
680 685 Asn Leu Asp Pro Thr Ile Ser Arg Phe Gly Ile Arg Ile Ala Asn
Asp 690 695 700 Phe Ile Val Val Ser Thr Arg Val Leu Ser Pro Pro Gln
Val Glu Tyr 705 710 715 720 His Ser Lys Arg Phe Thr Met Val Lys Asn
Gly Ser Trp Arg Met Asp 725 730 735 Gly Met Lys Phe Leu Glu Pro Lys
Pro Lys Ala His Lys Cys Ala Val 740 745 750 Leu Tyr Cys Asp Pro Arg
Ser Gly Arg Lys Met Asn Tyr Thr Gln Leu 755 760 765 Asn Asp Phe Gly
Asn Leu Ile Ile Ser Gln Gly Lys Ala Val Asn Ile 770 775 780 Ser Leu
Asp Ser Asp Val Thr Tyr Arg Pro Phe Thr Asp Asp Glu Arg 785 790 795
800 Ser Leu Asp Thr Ile Phe Ala Asp Leu Lys Arg Ser Gln His Asp Leu
805 810 815 Ala Ile Val Ile Ile Pro Gln Phe Arg Ile Ser Tyr Asp Thr
Ile Lys 820 825 830 Gln Lys Ala Glu Leu Gln His Gly Ile Leu Thr Gln
Cys Ile Lys Gln 835 840 845 Phe Thr Val Glu Arg Lys Cys Asn Asn Gln
Thr Ile Gly Asn Ile Leu 850 855 860 Leu Lys Ile Asn Ser Lys Leu Asn
Gly Ile Asn His Lys Ile Lys Asp 865 870 875 880 Asp Pro Arg Leu Pro
Met Met Lys Asn Thr Met Tyr Ile Gly Ala Asp 885 890 895 Val Thr His
Pro Ser Pro Asp Gln Arg Glu Ile Pro Ser Val Val Gly 900 905 910 Val
Ala Ala Ser His Asp Pro Tyr Gly Ala Ser Tyr Asn Met Gln Tyr 915 920
925 Arg Leu Gln Arg Gly Ala Leu Glu Glu Ile Glu Asp Met Phe Ser Ile
930 935 940 Thr Leu Glu His Leu Arg Val Tyr Lys Glu Tyr Arg Asn Ala
Tyr Pro 945 950 955 960 Asp His Ile Ile Tyr Tyr Arg Asp Gly Val Ser
Asp Gly Gln Phe Pro 965 970 975 Lys Ile Lys Asn Glu Glu Leu Arg Cys
Ile Lys Gln Ala Cys Asp Lys 980 985 990 Val Gly Cys Lys Pro Lys Ile
Cys Cys Val Ile Val Val Lys Arg His 995 1000 1005 His Thr Arg Phe
Phe Pro Ser Gly Asp Val Thr Thr Ser Asn Lys 1010 1015 1020 Phe Asn
Asn Val Asp Pro Gly Thr Val Val Asp Arg Thr Ile Val 1025 1030 1035
His Pro Asn Glu Met Gln Phe Phe Met Val Ser Gly Gln Ala Ile 1040
1045 1050 Gln Gly Thr Ala Lys Pro Thr Arg Tyr Asn Val Ile Glu Asn
Thr 1055 1060 1065 Gly Asn Leu Asp Ile Asp Leu Leu Gln Gln Leu Thr
Tyr Asn Leu 1070 1075 1080 Cys His Met Phe Pro Arg Cys Asn Arg Ser
Val Ser Tyr Pro Ala 1085 1090 1095 Pro Ala Tyr Leu Ala His Leu Val
Ala Ala Arg Gly Arg Val Tyr 1100 1105 1110 Leu Thr Gly Thr Asn Arg
Phe Leu Asp Leu Lys Lys Glu Tyr Ala 1115 1120 1125 Lys Arg Thr
Ile
Val Pro Glu Phe Met Lys Lys Asn Pro Met Tyr 1130 1135 1140 Phe Val
1145 6 22 RNA photinus pyralis 6 ucgaaguacu cagcguaagu ga 22 7 22
RNA photinus pyralis 7 uaaagcuuca ugagucgcau uc 22 8 83 RNA
photinus pyralis 8 caucgacuga aaucccuccc ugguaauccg uuugggcucu
gcccugcuau gggauaaaug 60 guuaucaggg auuuuagucg auc 83 9 62 RNA
photinus pyralis 9 caucgacuga aaucccuggu aauccguugu uaacaacgga
uuaccaggga uuucagucga 60 ug 62 10 44 DNA Artificial Sequence primer
10 taatacgact cactataggg caaggtcata gttcctgcca gctg 44 11 44 DNA
Artificial Sequence primer 11 taatacgact cactataggg cagatatttt
cttaccaccc accc 44 12 44 DNA Artificial Sequence primer 12
taatacgact cactataggg ttaagtttaa caggagaagc tgga 44 13 44 DNA
Artificial Sequence primer 13 taatacgact cactataggg aaatcattgc
tttcctgata atgc 44 14 44 DNA Artificial Sequence primer 14
taatacgact cactataggg tagatttccg cagccccagt gttc 44 15 44 DNA
Artificial Sequence primer 15 taatacgact cactataggg gttgcctctc
atttttcctt gatt 44 16 45 DNA Artificial Sequence primer 16
taatacgact cactataggg tattttagac tgattacttt tataa 45 17 44 DNA
Artificial Sequence primer 17 taatacgact cactataggg tcacatgttt
tggctaagac ctat 44 18 19 DNA Dicer siRNA 18 tcaaccagcc actgctgga 19
19 71 DNA Artificial Sequence Oligonucleotide Primer 19 tccaattcag
cgggagccac ctgatgaagc ttgatcgggt ggctctcgct gagttggaat 60
ccattttttt t 71 20 21 RNA photinus pyralis 20 ucgaaguacu cagcguaagu
g 21 21 21 RNA Photinus Pyralis 21 aaagcuucau gagucgcauu c 21 22 81
RNA Photinus Pyralis 22 caucgacuga aaucccuggu aauccguuug gggcucugcc
cugcuauggg auaaauggau 60 uaucagggau uuuagucgau g 81 23 82 RNA
Photinus Pyralis 23 caucgacuga aaucccgccg uaauccguuu ggggcucugc
ccugcuaugg gauaaaugga 60 uuaucaggga uuuuagucga ug 82 24 23 RNA
Photinus Pyralis 24 ggucgaagua cucagcguaa gaa 23 25 23 DNA Photinus
Pyralis 25 aaagcuucat gagucgcauu cgg 23 26 21 RNA Photinus Pyralis
26 gguuguggau cuggauaccg g 21 27 21 RNA Photinus Pyralis 27
uuccaacacc uagaccuaug g 21 28 21 RNA Photinus Pyralis 28 ggugccaacc
cuauucuccu u 21 29 21 RNA Photinus Pyralis 29 gaccacgguu gggauaagag
g 21 30 22 RNA Photinus Pyralis 30 ggcuaugaag agaguacgcc cu 22 31
22 RNA Photinus Pyralis 31 uuccgauacu ucucucaugc gg 22 32 64 RNA
Photinus Pyralis 32 ggucgaagua cucagcguaa gugaugucca cuuaaguggg
uguuguuugu guuggguguu 60 uugg 64 33 60 RNA Photinus Pyralis 33
ggucgaagua cucagcguaa gugauguccu uaaggguguu guuuguguug gguguuuugg
60 34 56 RNA Photinus Pyralis 34 gggcgaagua cucagcguaa gugauguuua
aguguuguuu guguugggug uuuugg 56 35 50 RNA Photinus Pyralis 35
ggucgaagua cucagcguaa gugauuaauu guuuguguug gguguuuugg 50 36 69 RNA
Photinus Pyralis 36 ggcucgaguc gaaguacuca gcguaaguga uguccauuaa
guggguguug uuuguguugg 60 guguuuugg 69 37 70 RNA Photinus Pyralis 37
ggucgaagua cucagcguaa gugaugucca cuuaaguggg uguuguuugu guuggguguu
60 uuggcucgag 70 38 69 RNA Photinus Pyralis 38 ggauuccaau
ucagcgggag ccaccugaug aagcuugauc ggguggcucu cgcugaguug 60 gaauccauu
69 39 64 RNA Photinus Pyralis 39 ggucuaagug gagcccuucg aguguuagaa
gcuugugaca cucggagggc uucacuuggg 60 ccuu 64 40 21 DNA Photinus
Pyralis 40 cuuacgcuga guacuucgat t 21 41 21 DNA Photinus Pyralis 41
ucgaaguacu cagcguaagt t 21 42 21 DNA Photinus Pyralis 42 agcuucauaa
ggcgcaugct t 21 43 21 DNA Photinus Pyralis 43 gcaugcgccu uaugaagcut
t 21 44 23 DNA Photinus Pyralis 44 cugugagauc uacggagccu gtt 23 45
23 DNA Photinus Pyralis 45 caggcuccgu agaucucaca gtt 23
* * * * *
References