U.S. patent application number 10/079185 was filed with the patent office on 2003-03-06 for human rnase iii and compositions and uses thereof.
Invention is credited to Crooke, Stanley T..
Application Number | 20030044941 10/079185 |
Document ID | / |
Family ID | 27752735 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044941 |
Kind Code |
A1 |
Crooke, Stanley T. |
March 6, 2003 |
Human RNase III and compositions and uses thereof
Abstract
The present invention provides polynucleotides encoding human
RNase III and polypeptides encoded thereby. Methods of using said
polynucleotides and polypeptides are also provided.
Inventors: |
Crooke, Stanley T.;
(Carlsbad, CA) |
Correspondence
Address: |
Woodcock Washburn LLP
46th Floor
One Liberty Place
Philadelphia
PA
19103
US
|
Family ID: |
27752735 |
Appl. No.: |
10/079185 |
Filed: |
February 20, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10079185 |
Feb 20, 2002 |
|
|
|
09900425 |
Jul 6, 2001 |
|
|
|
09900425 |
Jul 6, 2001 |
|
|
|
09479783 |
Jan 7, 2000 |
|
|
|
09479783 |
Jan 7, 2000 |
|
|
|
08870608 |
Jun 6, 1997 |
|
|
|
6107094 |
|
|
|
|
08870608 |
Jun 6, 1997 |
|
|
|
08659440 |
Jun 6, 1996 |
|
|
|
5898031 |
|
|
|
|
Current U.S.
Class: |
435/91.2 ;
435/455 |
Current CPC
Class: |
Y02A 50/30 20180101;
C12N 2310/316 20130101; C12N 2310/318 20130101; C12N 2310/311
20130101; C12N 2310/314 20130101; C12N 15/113 20130101; C12N
2310/322 20130101; C12Y 301/26003 20130101; C12N 2310/335 20130101;
C07H 21/00 20130101; C12N 2310/3181 20130101; C12N 15/1135
20130101; C12N 2310/321 20130101; C12N 2310/312 20130101; C12N
2310/341 20130101; C12N 9/22 20130101; C12N 2310/3341 20130101;
C12N 2310/346 20130101; Y02A 50/473 20180101; A61K 38/00 20130101;
C12N 2310/315 20130101; C12N 2310/321 20130101; C12N 2310/3525
20130101; C12N 2310/321 20130101; C12N 2310/3527 20130101; C12N
2310/321 20130101; C12N 2310/3521 20130101 |
Class at
Publication: |
435/91.2 ;
435/455 |
International
Class: |
C12P 019/34; C12N
015/87 |
Claims
What is claimed is:
1. A method for eliciting modification of a selected RNA target in
a cell comprising: (a) providing an RNA-like polynucleotide
hybridizable with said RNA target; (b) hybridizing the RNA-like
polynucleotide and the RNA to form a polynucleotide-target duplex;
and (c) contacting the duplex with a polypeptide comprising an
RNase III domain, under conditions selected to effect modification
of the duplex by the polypeptide, and modification of the RNA
target thereby.
2. The method of claim 1 wherein said modification of the RNA
target occurs in the cell's nucleus.
3. The method of claim 1 wherein the polypeptide comprising an
RNase III domain is an RNase III polypeptide.
4. The method of claim 1 wherein the RNase III polypeptide is a
human RNase III polypeptide.
5. The method of claim 1 wherein modification of the selected RNA
target is cleavage of the RNA target.
6. The method of claim 1 wherein the polypeptide comprising an
RNase III domain is present in enriched amounts.
7. The method of claim 6 wherein the polypeptide comprising an
RNase III domain present in enriched amounts is overexpressed or
exogenously added.
8. The method of claim 1 wherein the polypeptide comprising an
RNase III domain is a purified RNase III polypeptide.
9. The method of claim 1 wherein the RNA-like polynucleotide has a
modification at the 2' position of at least one sugar.
10. The method of claim 1 wherein step (c) is performed within a
cell.
11. The method of claim 1 wherein step (b) is performed within a
cell.
12. The method of claim 1 wherein step (b) is performed outside a
cell.
13. The method of claim 1 wherein at least one furanosyl moiety of
the RNA-like polynucleotide is a ribofuranosyl moiety.
14. The method of claim 13 wherein a majority of the furanosyl
moieties of the RNA-like polynucleotide are ribofuranosyl
moieties.
15. A method for promoting gene silencing in a cell comprising
providing to the cell, in an amount effective to promote said gene
silencing, a polypeptide comprising an RNase III domain.
16. The method of claim 15 wherein said promotion of gene silencing
occurs in the cell's nucleus.
17. The method of claim 15 wherein the polypeptide comprising an
RNase III domain is an RNase III polypeptide.
18. The method of claim 15 wherein the RNase III polypeptide is a
human RNase III polypeptide.
19. The method of claim 15 wherein the RNase III polypeptide is
exogenously added.
20. The method of claim 15 wherein the RNase III polypeptide is
provided through upregulation of endogenous production of the
polypeptide.
21. The method of claim 15 wherein said RNase III polypeptide is a
purified RNase III polypeptide.
22. The method of claim 15 wherein said RNase III polypeptide is
expressed by an exogenously added vector encoding said RNase III
polypeptide.
23. The method of claim 15 wherein said cell is a mammalian
cell.
24. The method of claim 15 wherein said cell is a human cell.
25. A method for promoting gene silencing in a cell comprising
enriching the amount or activity of RNase III polypeptide in said
cell to a level effective to promote said gene silencing.
26. The method of claim 25 wherein said promotion of gene silencing
occurs in the cell's nucleus.
27. The method of claim 25 wherein said enriching is by exogenous
addition of RNase III polypeptide.
28. The method of claim 27 wherein said exogenously added RNase III
polypeptide is a purified RNase III polypeptide.
29. The method of claim 25 wherein the RNase III polypeptide is
provided through upregulation of endogenous production of the
polypeptide.
30. The method of claim 25 wherein said enriching is by addition of
a vector encoding the RNase III polypeptide.
31. The method of claim 25 wherein said cell is a mammalian
cell.
32. The method of claim 25 wherein said cell is a human cell.
33. A method for promoting gene silencing of a gene in a cell
comprising: (a) providing to said cell a polynucleotide
hybridizable with a target RNA encoded by a selected gene whose
expression is to be silenced; (b) hybridizing said polynucleotide
and said target RNA to form a polynucleotide-target duplex; and (c)
contacting said duplex with a polypeptide comprising an RNase III
domain, under conditions selected to effect cleavage or
modification of the target RNA strand of the polynucleotide-target
RNA duplex by the polypeptide comprising an RNase III domain, and
silencing of the gene thereby.
34. The method of claim 33 wherein said promotion of gene silencing
occurs in the cell's nucleus.
35. The method of claim 33 wherein the polypeptide comprising an
RNase III domain is an RNase III polypeptide.
36. The method of claim 33 wherein the RNase III polypeptide is a
human RNase III polypeptide.
37. The method of claim 36 wherein the human RNase III polypeptide
comprises an amino acid sequence with at least 90% homology to SEQ
ID NO: 2.
38. The method of claim 33 wherein the polynucleotide is provided
as a single stranded polynucleotide.
39. The method of claim 33 wherein the polynucleotide is provided
as part of a double stranded nucleic acid structure.
40. The method of claim 33 wherein the polynucleotide is an
antisense oligonucleotide.
41. The method of claim 33 wherein the polynucleotide is an
RNA-like polynucleotide.
42. The method of claim 33 wherein at least one sugar moiety of the
polynucleotide is a ribofuranosyl sugar moiety.
43. The method of claim 42 wherein at least 50% of the sugar
moieties of the polynucleotide are ribofuranosyl sugar
moieties.
44. The method of claim 33 wherein the polynucleotide has at least
one modification of the base, sugar or internucleoside linkage.
45. The method of claim 44 wherein the polynucleotide has a
modification at the 2' position of at least one sugar.
46. The method of claim 33 wherein the RNase III polypeptide is
present in enriched amounts.
47. The method of claim 46 wherein the RNase III polypeptide
present in enriched amounts is overexpressed or exogenously
added.
48. The method of claim 46 wherein the RNase III polypeptide is a
purified RNase III polypeptide.
49. The method of claim 46 wherein said enriching is by addition of
a vector encoding said RNase III polypeptide.
50. The method of claim 46 wherein the RNase III polypeptide is
provided through upregulation of endogenous production of the
polypeptide.
51. The method of claim 33 wherein said cell is a mammalian
cell.
52. The method of claim 33 wherein said cell is a human cell.
53. The method of claim 33 wherein said polynucleotide-target RNA
duplex forms inside the cell.
54. The method of claim 33 wherein said polynucleotide-target RNA
duplex forms outside the cell.
55. A method for inhibiting the expression of a gene in a cell
comprising providing to said cell an agent effective to elicit
RNase III modification of double-stranded RNA in a cell.
56. The method of claim 55 wherein said inhibition of gene
expression occurs in the cell's nucleus.
57. The method of claim 55 wherein said agent is a nucleic acid
which is hybridizable with an RNA encoded by the gene whose
expression is to be inhibited.
58. The method of claim 55 wherein said RNase III modification is
RNase III cleavage.
59. The method of claim 55 wherein the polynucleotide is provided
as a single stranded polynucleotide.
60. The method of claim 55 wherein the polynucleotide is provided
as part of a double stranded nucleic acid structure.
61. The method of claim 55 wherein the polynucleotide is an
antisense oligonucleotide.
62. The method of claim 55 wherein the polynucleotide is an
RNA-like polynucleotide.
63. The method of claim 55 wherein at least one sugar moiety of the
polynucleotide is a ribofuranosyl sugar moiety.
64. The method of claim 63 wherein at least 50% of the sugar
moieties of the polynucleotide are ribofuranosyl sugar
moieties.
65. The method of claim 55 wherein the polynucleotide has at least
one modification of the base, sugar or internucleoside linkage.
66. The method of claim 65 wherein the polynucleotide has a
modification at the 2' position of at least one sugar.
67. A method for promoting inhibition of expression of a gene in a
cell comprising: (a) providing to said cell a polynucleotide
hybridizable with a target RNA encoded by the gene whose expression
is to be inhibited; (b) hybridizing the polynucleotide and the
target RNA to to form a polynucleotide-target duplex; and (c)
contacting the duplex with a polypeptide comprising an RNase III
domain, under conditions effective to effect cleavage or
modification of the target RNA strand of the polynucleotide-target
RNA duplex by the RNase III polypeptide, and inhibition of
expression of the gene thereby.
68. The method of claim 67 wherein said promotion of inhibition of
gene expression occurs in the cell's nucleus.
69. The method of claim 67 wherein the polypeptide comprising an
RNase III domain is an RNase III polypeptide.
70. The method of claim 69 wherein the RNase III polypeptide is a
human RNase III polypeptide.
71. The method of claim 70 wherein the human RNase III polypeptide
comprises an amino acid sequence with at least 90% sequence
identity to SEQ ID NO: 2.
72. The method of claim 67 wherein the polynucleotide is provided
as a single stranded polynucleotide.
73. The method of claim 67 wherein the polynucleotide is provided
as part of a double stranded nucleic acid structure.
74. The method of claim 67 wherein the polynucleotide is an
antisense oligonucleotide.
75. The method of claim 67 wherein the polynucleotide is an
RNA-like polynucleotide.
76. The method of claim 67 wherein at least one sugar moiety of the
polynucleotide is a ribofuranosyl sugar moiety.
77. The method of claim 76 wherein at least 50% of the sugar
moieties of the polynucleotide are ribofuranosyl sugar
moieties.
78. The method of claim 67 wherein the polynucleotide has at least
one modification of the base, sugar or internucleoside linkage.
79. The method of claim 78 wherein the polynucleotide has a
modification at the 2' position of at least one sugar.
80. The method of claim 67 wherein the polypeptide comprising an
RNase III domain is present in enriched amounts.
81. The method of claim 80 wherein the polypeptide comprising an
RNase III domain and present in enriched amounts is overexpressed
or exogenously added.
82. The method of claim 81 wherein the polypeptide comprising an
RNase III domain and present in enriched amounts is a purified
RNase III polypeptide.
83. The method of claim 81 wherein said enriching is by addition of
a vector encoding said polypeptide comprising an RNase III
domain.
84. The method of claim 67 wherein said cell is a human cell.
85. The method of claim 67 wherein step (c) is performed within a
cell.
86. The method of claim 67 wherein step (b) is performed within a
cell.
87. The method of claim 67 wherein step (b) is performed outside a
cell.
88. A cell having enhanced RNase III activity over an activity
exhibited by a second cell, said second cell not enriched with
respect to the amount or activity of RNase III polypeptide.
89. The cell of claim 88 wherein said enhanced RNase III activity
is detectable in the cell's nucleus.
90. The cell of claim 88 wherein said enhanced RNase III activity
is due to overexpression of RNase III.
91. The cell of claim 88 wherein the RNase III polypeptide is
provided through upregulation of endogenous production of the RNase
III polypeptide.
92. The cell of claim 88 wherein said enhanced RNase III activity
is due to exogenously added RNase III.
93. A method for eliciting modification of an RNA target in a cell
comprising: (a) providing an RNA-like polynucleotide hybridizable
with said RNA target; (b) hybridizing the RNA-like polynucleotide
and the RNA to form a polynucleotide-target duplex; and (c)
contacting the duplex with a polypeptide comprising an RNase III
domain, under conditions selected to effect modification of the
duplex by the polypeptide, and modification of the RNA target
thereby.
94. A hybrid RNase III comprising at least one domain from a human
RNase III and at least one domain from an RNase III of an organism
other than human.
95. The hybrid RNase III of claim 94 wherein the non-human RNase
III domain is derived from an organism selected from the group
consisting of E. coli, S. pombe, C. elegans and S. cerevisiae.
Description
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 09/900,425, filed Jul. 6, 2001, which is a
continuation-in-part of U.S. application Ser. No. 09/479,783, filed
Jan. 7, 2000, which is a divisional of U.S. application Ser. No.
08/870,608, filed Jun. 6, 1997, U.S. Pat. No. 6,107,094, which is a
continuation in part of U.S. application Ser. No. 08/659,440, filed
Jun. 6, 1996, U.S. Pat. No. 5,898,031. All of the above are
assigned to the assignee of the present invention and are
incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a human RNase III, the gene
for which has now been cloned and characterized, and compositions
and uses thereof. Antisense inhibitors of human RNase III are also
described.
BACKGROUND OF THE INVENTION
[0003] Ribonuclease III (RNase III) is an endoribonuclease that
cleaves double stranded RNA. The enzyme is expressed in many
organisms and is highly conserved. I. S. Mian, Nucleic Acids Res.,
1997, 25, 3187-95. All RNase III species cloned to date contain an
RNase III signature sequence and vary in size from 25 to 50 kDa.
Multiple functions have been ascribed to RNase III. In both E. coli
and S. cerevisiae, RNase III has been reported to be involved in
the processing of pre-ribosomal RNA (pre-rRNA). Elela et al., Cell,
1996, 85, 115-24. RNase III has also been reported to be involved
in the processing of small molecular weight nuclear RNAs (snRNAs)
and small molecular weight nucleolar RNAs (snoRNAs) in S.
cerevisiae. Chanfreau et al., Genes Dev. 1996, 11, 2741-51; Qu et
al., Mol. Cell. Biol. 1996, 19, 1144-58. In E. coli, RNase III has
also been reported to be involved in the degradation of some mRNA
species. D. Court, in Control of messenger RNA stability, 1993,
Academic Press, Inc, pp. 71-116.
[0004] A human double strand RNase (dsRNase) activity has been
described. Wu et al., J. Biol. Chem., 1998, 273, 2532-2542; Crooke,
U.S. Pat. No. 5,898,031; U.S. Pat. No. 6,017,094. By the rational
design and testing of chemically modified antisense
oligonucleotides that contained oligoribonucleotide stretches of
varying length, a dsRNase activity was demonstrated in human T24
bladder carcinoma cells which produced 5'-phosphate and 3'-hydroxyl
termini upon cleavage of the complementary cellular RNA target.
This pattern of cleavage products is a feature of E. coli RNase
III. The cleavage activity in human cells required the formation of
a dsRNA region in the oligonucleotide. This human dsRNase activity
is believed to be useful as an alternative terminating mechanism to
RNase H for antisense therapeutics. Because it relies on "RNA-like"
oligonucleotides, which generally have higher potency than the
"DNA-like" oligonucleotides required for RNase H activity, it may
prove an attractive alternative to RNase H-based antisense
approaches.
[0005] RNA interference (RNAi) is a form of sequence-specific,
post-transcriptional gene silencing in animals and plants, elicited
by double-stranded RNA (dsRNA) that is homologous in sequence to
the silenced gene. Elbashir et al., Nature, 2001, 411, 494-498.
dsRNA triggers the specific degradation of homologous RNAs, only
within the region of homology. The dsRNA is processed to 21- to
23-nucleotide fragments, sometimes called short interfering RNAs
(siRNAs) which are believed to be the guide fragments for
sequence-specific mRNA degradation. The processing of longer dsRNA
to these short siRNA fragments is believed to be accomplished by
RNase III. Elbashir et al., ibid., Elbashir et al., Genes and
Devel., 2001, 15, 188-200. Thus it is believed that the human RNase
III of the present invention may be useful in further understanding
and exploiting the gene silencing mechanism, particularly in human
cells.
[0006] Despite the substantial information about members of the
RNase III family and the cloning of genes encoding proteins with
RNase III activity from a number of lower organisms (E. coli, yeast
and others), no human RNase III has previously been cloned. This
has hampered efforts to understand the structure of the enzyme(s),
its distribution and the functions it may serve. The present
application describes the cloning and characterization of a cDNA
that expresses a human RNase III. Cloning and sequencing of the
cDNA encoding human RNase III allowed characterization of this
nucleic acid as well as of the location and function of the RNase
III protein itself.
SUMMARY OF THE INVENTION
[0007] The present invention provides a polynucleotide sequence
(set forth herein as SEQ ID NO: 1) which has been identified as
encoding human RNase III by the homology of the calculated
expressed polypeptide (provided herein as SEQ ID NO: 2) with known
amino acid sequences of yeast and worm RNase III as well as by
functional analysis.
[0008] The present invention provides polynucleotides that encode
human RNase III, the human RNase III polypeptide, vectors
comprising nucleic acids encoding human RNase III, host cells
containing such vectors, antibodies targeted to human RNase III,
nucleic acid probes capable of hybridizing to a nucleic acid
encoding a human RNase III polypeptide, and antisense inhibitors of
RNase III expression. Methods of inhibiting RNase III expression or
activity are also provided, as are pharmaceutical compositions
which include a human RNase III polypeptide, an antisense inhibitor
of RNase III expression, or a vector containing a nucleic acid
encoding human RNase III.
[0009] Methods for identifying agents which modulate activity
and/or levels of human RNase III are also provided. Methods of
promoting inhibition of expression of a selected protein via
antisense, methods of screening oligonucleotides to identify active
antisense oligonucleotides against a particular target, methods of
prognosticating efficacy of antisense therapy, methods of promoting
RNA interference (RNAi) or other forms of gene silencing in a cell
and methods of eliciting cleavage or modification of a selected
cellular RNA target are also provided. All of these methods exploit
the RNA-binding and cleaving activity of RNase III polypeptides. In
preferred embodiments the polynucleotides used in these methods are
RNA-like oligonucleotides. Also provided are methods of identifying
agents which increase or decrease activity or levels of human RNase
III.
[0010] The compositions and methods of the present invention are
useful for research, biological and clinical purposes. For example,
the methods, polynucleotides and antisense oligonucleotides are
useful in defining the roles of RNase III and the interaction of
human RNase III and cellular RNA (including pre-mRNA or
pre-rRNA).
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the amino acid sequence of human RNase III (SEQ
ID NO: 2) and a comparison of the sequence of the RNase III domain
of the human RNase III to RNase III domains of C. elegans (Worm;
SEQ ID NO: 3), S. pombe (PAC; SEQ ID NO: 4) and S. cerevisiae (RNT;
SEQ ID NO: 5) and E. coli (RNC; SEQ ID NO: 6). Bold letters:
identical amino acids of human RNase III to other species. @@@:
putative catalytic center. HHH: alpha helix; BBB: beta sheet (dsRNA
binding region at C-terminus). Amino acid identity of human RNase
III to Worm (41%), PAC (17%), RNT (15%) and RNC (16%). *: Potential
phosphorylation sites analyzed using OMIGA (Oxford Molecular
Ltd.).
DETAILED DESCRIPTION OF THE INVENTION
[0012] A cDNA encoding human RNase III has now been cloned and
characterized. The cloned sequence is provided herein as SEQ ID NO:
1. This cDNA encodes a protein of 160 kDa which is ubiquitously
expressed in human cell and tissue types, and is involved in
processing of preribosomal RNA (pre-rRNA).
[0013] Thus, in accordance with one aspect of the present
invention, there are provided isolated polynucleotides which encode
human RNase III polypeptides. By "polynucleotides" it is meant to
include any form of RNA or DNA such as mRNA, pre-mRNA or cDNA or
genomic DNA, respectively, obtained by cloning or produced
synthetically by well known chemical techniques. The term
"polynucleotide" is also meant to include oligonucleotides, e.g.
synthetic antisense oligonucleotides. DNA or RNA polynucleotides
may be double- or single-stranded. Single-stranded DNA or RNA
polynucleotides may comprise the coding or sense strand or the
non-coding or antisense strand.
[0014] Methods of isolating a polynucleotide of the present
invention via cloning techniques are well known. For example, to
obtain the polynucleotide sequence of SEQ ID NO: 1, a similarity
search of the yeast RNT1 gene (RNase III, Genbank accession no.
AAB04172; SEQ ID NO: 5) and the Caenorhabditis elegans RNase III
gene (Genbank accession no. 001326; SEQ ID NO: 3) with the XREF
database (National Center for Biotechnology Information, NIH,
Rockville Md.) was performed. A 393 base pair (bp) human EST clone
(GenBank AA083888) was identified.
[0015] Using primers based on this EST sequence, a clone (U4)
corresponding to the COOH-terminal portion of the protein
(nucleotides 3569-4764 of full length cDNA) was cloned by 3' RACE.
Eight positive clones were isolated by screening a liver cDNA
library with this clone. With primers based on one of these clones,
5' RACE was performed to clone a cDNA of approximately 1 kb, which
corresponds to the middle part of the full length cDNA. In the same
way, a cDNA of the NH.sub.2-terminal portion was cloned. Primers
based on the NH.sub.2-terminal-most clone were used to perform
additional 5'-RACE to obtain the NH.sub.2-terminal portion of the
cDNA. The overlapping clones were sequenced and assembled to a full
length human RNase III cDNA with a total of 4764 nucleotides. This
human RNase III polynucleotide sequence is provided herein as SEQ
ID NO: 1 and has been deposited as GenBank accession no. AF189011.
The cDNA contained a coding sequence of 4125 nucleotides (from
246-4370 of SEQ ID NO:1) that was calculated to encode a 1374 amino
acid protein. This polypeptide sequence is provided herein as SEQ
ID NO: 2, shown in FIG. 1. The calculated molecular weight of the
protein is 160 kDa based on the prediction of the first translated
methionine as the translation initiation site. Northern
hybridization analyses demonstrated that the human RNase III mRNA
was approximately 5 kb in size. It was found to be ubiquitously
expressed in human tissues and cell lines. Compared to C. elegans,
yeast and bacterial RNase III, human RNase III is substantially
larger and contains multiple domains. The RNase III domain (amino
acids 949-1374) is located at the carboxy terminus of the protein
and is homologous to C. elegans, yeast and bacterial RNase III. The
human RNase also contains proline rich (amino acids 1-220) and
serine-arginine rich (amino acids 221-470) domains near the amino
terminus. The SR and RNase III domains are separated by 478 amino
acids.
[0016] The RNase III domain of human RNase III is conserved with
other species and is most homologous with C. elegans RNase III (41%
identity). Both the human RNase III domain and C. elegans RNase III
contain two RNase III signature sequences (HNERLEFLGDS; SEQ ID NO
7). Sequence identity was also compared with the yeasts S. pombe
(PAC gene)(17% homology) and S. cerevisiae (RNT gene) (15%
homology) and with E. coli RNase III (RNC gene) (16% homology).
Human RNase III also contains multiple phosphorylation sites. The
SR domain is usually present in SR or SR related proteins that play
crucial roles in mRNA splicing. The fusion of SR and RNase III
domains into a single protein suggests that human RNase III may be
involved in a number of RNA metabolic events. The presence of
multiple potential phosphorylation sites suggests that the enzyme
is regulated by phosphorylation.
[0017] As used herein, the phrase "homologous nucleotide sequence,"
or "homologous amino acid sequence," or variations thereof, refers
to sequences characterized by a homology, at the nucleotide level
or amino acid level, of at least the specified percentage.
Homologous nucleotide sequences include those sequences coding for
isoforms of proteins. Such isoforms can be expressed in different
tissues of the same organism as a result of, for example,
alternative splicing of RNA. Alternatively, isoforms can be encoded
by different genes. Homologous nucleotide sequences include
nucleotide sequences encoding for a protein of a species other than
humans, including, but not limited to, mammals. Homologous
nucleotide sequences also include, but are not limited to,
naturally occurring allelic variations and mutations of the
nucleotide sequences set forth herein. A homologous nucleotide
sequence does not, however, include the nucleotide sequence
encoding other known RNase IIIs. Homologous amino acid sequences
include those amino acid sequences which contain conservative amino
acid substitutions and which polypeptides have the same binding
and/or activity. A homologous amino acid sequence does not,
however, include the amino acid sequence encoding other known RNase
IIIs. Percent homology can be determined by, for example, the Gap
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, Madison Wis.),
using the default settings, which uses the algorithm of Smith and
Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated
herein by reference in its entirety).
[0018] In a preferred embodiment, the polynucleotide of the present
invention comprises the nucleic acid sequence of SEQ ID NO: 1.
However, as will be obvious to those of skill in the art upon this
disclosure, due to the degeneracy of the genetic code,
polynucleotides of the present invention may comprise other nucleic
acid sequences encoding the polypeptide of SEQ ID NO: 2 and
derivatives, variants or active fragments thereof.
[0019] The invention further provides homologs of the human RNase
III DNA. Such homologs, in general, share at least 50%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 98%, or at least
99% homology with the human RNase III DNA of the invention. Species
homologs, sometimes referred to as "orthologs," in general, share
at least 50%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, or at least 99% homology with the human RNase III DNA of
the invention. Generally, percent sequence "homology" with respect
to polynucleotides of the invention can be calculated as the
percentage of nucleotide bases in the candidate sequence that are
identical to nucleotides in the human RNase III sequence set forth
in the appended Sequence Listing, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity.
[0020] Another aspect of the present invention relates to the
polypeptides encoded by the polynucleotides of the present
invention. In a preferred embodiment, a polypeptide of the present
invention comprises the deduced amino acid sequence of human RNase
III provided in SEQ ID NO: 2. However, by "polypeptide" it is also
meant to include fragments, derivatives and analogs of SEQ ID NO: 2
which retain essentially the same biological activity and/or
function as human RNase III. Alternatively, polypeptides of the
present invention may retain their ability to bind to double
stranded RNA even though they do not function as active RNase III
enzymes in other capacities. Thus an enzyme may "modify" its RNA
substrate, e.g., bind and interfere with the function of the RNA
but not cleave it, or may bind and cleave. In some embodiments
cleavage is a preferred form of modification. In another
embodiment, polypeptides of the present invention may retain
nuclease activity but without specificity for an RNA/RNA duplex.
Polypeptides of the present invention include recombinant
polypeptides, isolated natural polypeptides and synthetic
polypeptides, and fragments thereof which retain one or more of the
activities described above.
[0021] The invention further provides homologs of the human RNase
III polypeptide. Such homologs, in general, share at least 50%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, or at least
99% homology with the RNase III polypeptides of the invention.
Generally, percent sequence "homology" with respect to polypeptides
of the invention can be calculated as the percentage of amino acid
residues in the candidate sequence that are identical to amino acid
residues in the RNAse III sequences set forth in the appended
Sequence Listing, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity.
[0022] In some embodiments the present invention provides
recombinant polypeptides that comprise RNase III domains from one
or more organisms. Domains of RNase III that exhibit certain
functions can be replaced with RNase III domains from other
organisms that exhibit similar functions, while maintaining the
overall function of the polypeptide. As a non-limiting example, a
hybrid RNase III may comprise one or more E. coli RNase III domain,
one or more C. elegans RNase III domain, and one or more human
RNase III domain. As a non-limiting example, such hybrid RNase III
polypeptides can be produced by first designing and producing
recombinant DNA molecules encoding such polypeptides. Such
recombinant DNA molecules are produced, for example, by replacing
DNA sequences that encode individual domains in a polynucleotide
encoding RNase III with DNA sequences from other organisms that
encode RNase III domains that exhibit similar functions. The
recombinant DNA construct thus produced can then be expressed and
purified using means familiar to one of ordinary skill in the
art.
[0023] To confirm the expression of the human RNase III protein,
two anti-peptide antibodies were produced. The "anti-III" peptide
antibody was derived from a peptide corresponding to amino acids
1356-1374 within the RNase III domain present in the C-terminal
portion of the putative protein. The "anti-SR" peptide antibody was
derived from a peptide corresponding to amino acids 266-284 within
the SR-domain of the putative protein. Using these antibodies,
Western blot analyses were performed to determine the size and
localization of human RNase III. The anti-SR peptide antibody
recognized a band in HeLa whole cell lysate with a molecular weight
of approximately 160 kDa which is near the calculated protein size
confirming that the full coding region is expressed in HeLa cells.
Similar experiments were performed using different human cell lines
e.g. A549, T24 and HL60 with equivalent results. To determine the
localization of the protein, nuclear and non-nuclear fractions from
HeLa cells and other human cell lines were prepared and equal
amounts of proteins were analyzed by Western blots. RNase III was
present primarily in the nuclear fractions. Non-nuclear fractions
contained only trace amounts of protein, possibly due to the
contamination during sample preparation. The anti-III peptide
antibody gave results equivalent to those obtained with the anti-SR
peptide antibody. To better understand the localization of human
RNase III, the protein was identified in cells by indirect
immunofluorescence microscopy. The nuclei of HeLa cells were
stained by both anti-SR and anti-III antibodies, confirming that
human RNase III is present in the nucleus. RNase III is localized
extensively in nucleus and occasionally observed in nucleoli. This
localization suggests possible involvement in both pre-mRNA and
pre-rRNA processing. In E. coli, RNase III is associated with
ribosomes in the cytoplasm. Robertson et al., J. Biol. Chem, 1968,
243, 82-91. Eukaryotic RNase III has not previously been shown to
be localized in the nucleus.
[0024] The localization of human RNase III to nucleoli was found to
be cell cycle regulated. Double thymidine treatment was used to
synchronize HeLa cells to early-S phase. Two to four hours after
releasing the thymidine block, HeLa cells entered S phase as
determined by fluorescence activated cell sorting (FACS). Six to
eight hours after release, HeLa cells entered the G2/M phase. There
were no significant changes in the MRNA or protein levels of the
RNase III during pre-S, S or G2/M phases. However, the subcellular
localization of the protein changed during the cell cycle. When the
cells were treated with thymidine and synchronized in early S
phase, RNase III protein was present only in the nucleus and not
the nucleoli, as determined by immunofluorescent labeling. After
releasing from thymidine block, RNase III was translocated to
nucleoli, reaching a peak at 4 hours when cells were in S phase. At
that time, RNase III was present both in the nucleoli and the
nucleus. The protein was present in the nucleoli for approximately
8 hours, and then disappeared from nucleoli as cells entered M
phase. Localization of RNase III in the nucleoli was confirmed by
double staining with an anti-nucleolin monoclonal antibody (MBL,
Watertown, Mass.).
[0025] In human cells, nucleoli undergo phases of condensation and
dissociation as a function of the cell cycle. Nucleoli dissociate
upon entering prophase and disappear entirely during the late
prophase and metaphase periods of mitosis, then begin to reappear
during telophase and form dense organelles during the G1 phase.
Human RNase III was only translocated to and remained in the
nucleoli during S phase suggesting that RNase III may serve one or
more specific functions in nucleoli during S phase.
[0026] The present invention also provides antisense inhibitors of
RNase III expression, which may be used, for example,
therapeutically, prophylactically or as research reagents. The
modulation of function of a target nucleic acid (in this case a
nucleic acid encoding RNase III) by compounds which specifically
hybridize to it is generally referred to as "antisense". The
functions of DNA to be interfered with include replication and
transcription. The functions of RNA to be interfered with include
all vital functions such as, for example, translocation of the RNA
to the site of protein translation, translation of protein from the
RNA, splicing of the RNA to yield one or more mRNA species, and
catalytic activity which may be engaged in or facilitated by the
RNA. The overall effect of such interference with target nucleic
acid function is modulation of the expression of the target. In the
context of the present invention, "modulation" means either an
increase (stimulation) or a decrease (inhibition) in the expression
of a gene. In the context of the present invention, inhibition is
the preferred form of modulation of gene expression and mRNA is a
preferred target. In some embodiments gene silencing is a preferred
form of inhibition of gene expression and refers to a decrease in
gene expression mediated by a double-stranded RNA polynucleotide,
one strand of which is homologous to the RNA to be silenced.
[0027] It is preferred to target specific nucleic acids for
antisense. "Targeting" an antisense compound to a particular
nucleic acid, in the context of this invention, is a multistep
process. The process usually begins with the identification of a
nucleic acid sequence whose function is to be modulated. This may
be, for example, a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious agent.
The targeting process also includes determination of a site or
sites within this gene for the antisense interaction to occur such
that the desired effect, e.g., detection or modulation of
expression of the protein, will result. Within the context of the
present invention, a preferred intragenic site is the region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of the gene. Since, as is known in the
art, the translation initiation codon is typically 5'-AUG (in
transcribed mRNA molecules; 5'-ATG in the corresponding DNA
molecule), the translation initiation codon is also referred to as
the "AUG codon," the "start codon" or the "AUG start codon". A
minority of genes have a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and
5'-CUG have been shown to function in vivo. Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of the target, regardless of the sequence(s) of such
codons.
[0028] It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences,
i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences
are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start
codon region" and "translation initiation codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation termination codon.
[0029] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Other target regions
include the 5' untranslated region (5'UTR), known in the art to
refer to the portion of an mRNA in the 5' direction from the
translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an
mRNA or corresponding nucleotides on the gene, and the 3'
untranslated region (3'UTR), known in the art to refer to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap of an MRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap. The
5' cap region may also be a preferred target region.
[0030] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence. mRNA
splice sites, i.e., intron-exon junctions, may also be preferred
target regions, and are particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular mRNA splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred targets. It has also been found that
introns can also be effective, and therefore preferred, target
regions for antisense compounds targeted, for example, to DNA or
pre-mRNA.
[0031] Once one or more target sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect.
[0032] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases which pair through the formation of
hydrogen bonds. "Complementary," as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of an oligonucleotide is
capable of hydrogen bonding with a nucleotide at the same position
of a DNA or RNA molecule, then the oligonucleotide and the DNA or
RNA are considered to be complementary to each other at that
position. The oligonucleotide and the DNA or RNA are complementary
to each other when a sufficient number of corresponding positions
in each molecule are occupied by nucleotides which can hydrogen
bond with each other. Thus, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity or precise pairing such that stable and
specific binding occurs between the oligonucleotide and the DNA or
RNA target. It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and in the case of in vitro assays, under
conditions in which the assays are performed.
[0033] Antisense and other compounds of the invention which
hybridize to the target and inhibit expression of the target are
identified through experimentation, and the sequences of these
compounds are hereinbelow identified as preferred embodiments of
the invention. The target sites to which these preferred sequences
are complementary are hereinbelow referred to as "active sites" and
are therefore preferred sites for targeting. Therefore another
embodiment of the invention encompasses compounds, including
primers, probes, siRNAs, other double stranded RNAs including RNAi
or gene silencing agents, ribozymes, external guide sequence (EGS)
oligonucleotides (oligozymes), and other short catalytic RNAs or
catalytic oligonucleotides which hybridize to these active
sites.
[0034] Antisense compounds are commonly used as research reagents
and diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
[0035] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense oligonucleotides have been employed as therapeutic
moieties in the treatment of disease states in animals and man.
Antisense oligonucleotide drugs, including ribozymes, have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
oligonucleotides can be useful therapeutic modalities that can be
configured to be useful in treatment regimes for treatment of
cells, tissues and animals, especially humans.
[0036] In the context of this invention, the term "polynucleotide",
which includes oligonucleotides, refers to an oligomer or polymer
of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or
mimetics thereof. This term includes oligonucleotides composed of
naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as oligonucleotides
having non-naturally-occurring portions which function similarly.
Such modified or substituted oligonucleotides are often preferred
over native forms because of desirable properties such as, for
example, enhanced cellular uptake, enhanced affinity for nucleic
acid target and increased stability in the presence of
nucleases.
[0037] In general, nucleic acids or polynucleotides (including
oligonucleotides) may be described as "DNA-like" (i.e., having
2'-deoxy sugars and, generally, T rather than U bases) or
"RNA-like" (i.e., having 2'-hydroxyl or 2'-modified sugars and,
generally U rather than T bases). Nucleic acid helices can adopt
more than one type of structure, most commonly the A- and B-forms.
It is believed that, in general, oligonucleotides which have
B-form-like structure are "DNA-like" and those which have
A-form-like structure are "RNA-like".
[0038] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics such as are described below. The antisense
compounds in accordance with this invention preferably comprise
from about 8 to about 50 nucleobases (i.e. from about 8 to about 50
linked nucleosides). Particularly preferred antisense compounds are
antisense oligonucleotides, even more preferably those comprising
from about 12 to about 30 nucleobases. Antisense compounds include
ribozymes, external guide sequence (EGS) oligonucleotides
(oligozymes), and other short catalytic RNAs or catalytic
oligonucleotides which hybridize to the target nucleic acid and
modulate its expression.
[0039] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn, the respective ends of this
linear polymeric structure can be further joined to form a circular
structure, however, open linear structures are generally preferred.
Within the oligonucleotide structure, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0040] Specific examples of preferred antisense compounds useful in
this invention include oligonucleotides containing modified
backbones or non-natural internucleoside linkages. As defined in
this specification, oligonucleotides having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified oligonucleotides that do not have a phosphorus atom
in their internucleoside backbone can also be considered to be
oligonucleosides.
[0041] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest- ers,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0042] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S.: U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference.
[0043] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0044] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S.: U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439, certain of which are commonly owned with
this application, and each of which is herein incorporated by
reference.
[0045] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S.: U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each
of which is herein incorporated by reference. Further teaching of
PNA compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0046] Most preferred embodiments of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0047] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and O
(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.s- ub.3)].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2,
N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2O
CH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et
al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy
group. A further preferred modification includes
2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'O-dimethylaminoethoxyethoxy (also known in the art as
2'--O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0048] A further prefered modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 3' or 4' carbon atom
wherein n is 1 or 2. LNAs and preparation thereof are described in
WO 98/39352 and WO 99/14226.
[0049] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub- .2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S.: U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0050] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.dbd.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazi- n-2(3H)-one),
phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin--
2(3H)-one), G-clamps such as a substituted phenoxazine cytidine
(e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , ed.,
CRC Press, 1993. Certain of these nucleobases are particularly
useful for increasing the binding affinity of the oligomeric
compounds of the invention. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0051] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S.: U.S. Pat.
Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;
5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;
5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985;
5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which
are commonly owned with the instant application, and each of which
is herein incorporated by reference, and U.S. Pat. No. 5,750,692,
which is commonly owned with the instant application and also
herein incorporated by reference.
[0052] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. The
compounds of the invention can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen sequence-specific hybridization with RNA. Groups that
enhance the pharmaco-kinetic properties, in the context of this
invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Representative conjugate
groups are disclosed in International Patent Application
PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which
is incorporated herein by reference. Conjugate moieties include but
are not limited to lipid moieties such as a cholesterol moiety
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,
6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,
1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309;
Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a
thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,
533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues
(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et
al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,
1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol
or triethylammonium 1,2-di-O-hexadecyl-rac-glyc-
ero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36,
3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a
polyamine or a polyethylene glycol chain (Manoharan et al.,
Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane
acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,
3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys.
Acta, 1995, 1264, 229-237), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the
invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0053] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S.: U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference.
[0054] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention preferably includes antisense compounds which
are chimeric compounds. "Chimeric" antisense compounds or
"chimeras," in the context of this invention, are antisense
compounds, particularly oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids.
[0055] By way of example, RNase H cleaves the RNA strand of an
RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of oligonucleotide inhibition of gene expression.
Consequently, comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Oligonucleotides, particularly chimeric
oligonucleotides, designed to elicit target cleavage by RNase H,
thus are generally more potent than oligonucleotides of the same
base sequence which are not so optimized. Cleavage of the RNA
target can be routinely detected by, for example, gel
electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0056] Chimeric oligonucleotides may have one or more modifications
of the internucleoside (backbone) linkage, the sugar or the base.
In a preferred embodiment, the oligonucleotide is a chimeric
oligonucleotide having a modification at the 2' position of at
least one sugar moiety. Presently believed to be particularly
preferred are chimeric oligonucleotides which have approximately
four or more deoxynucleotides in a row, which provide an RNase H
cleavage site, flanked on one or both sides by a region of
2'-modified oligonucleotides.
[0057] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S.: U.S. Pat. Nos. 5,013,830; 5,149,797;
5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;
5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which
are commonly owned with the instant application, and each of which
is herein incorporated by reference in its entirety.
[0058] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0059] Antisense inhibition of human RNase III expression was used
to further evaluate the role(s) of RNase III. To identify optimal
sites in RNase III mRNA for antisense effects, 2'-O-methoxyethyl
chimeric antisense oligonucleotides targeted to 10 sites in the
MRNA were designed and screened for inhibition of RNase III. These
are shown in Table 1. These chimeric or "gapped" oligonucleotides
are designed to serve as substrates for RNase H when bound to RNA
resulting in degradation of the target RNA and oligonucleotides of
this type have been shown to be highly specific when used under the
described conditions.
1TABLE 1 Antisense inhibition of human RNase III ISIS # Sequence
(5'--> 3') Target sites % Inhib'n SEQ ID NO: 25690
ATCCCTTTCTTCCGCATGTG 3051-3070 79 8 25691 GCCAAGGCGTGACATGATAT
3085-4004 96 9 25692 CGGATCATTAAAGAGCAAGC 3442-3461 78 10 25693
TATTCACCAAAGAGCTTCGC 3776-3795 49 11 25694 CAATCGTGGAAAGAAGCAGA
3973-3992 50 12 25695 GCTCCCATTTCCGCTTGCTG 4197-4216 81 13 25696
ATGCTCTCTTTCCCACCTCA 4308-4327 70 14 25697 AAATACTCCACACTTGCATG
4378-4397 79 15 25698 TGCACATTCACCAAAGTCAA 4420-4439 44 16 25699
AGTCTAGGGTCACAATCTGG 4688-4707 31 17 27110 TTCAGTTGTAGTGGTCCGAC
3-mismatch of N/D 18 25691
[0060] All oligonucleotides in Table 1 have phosphorothioate
(P.dbd.S or PS) backbones and 2'-methoxyethoxy (2'MOE) "wings "
flanking a 2'deoxy gap. 2'MOE nucleotides are shown in bold. All
cytosines are 5-methyl cytosines (5meC). Target site refers to
nucleotide numbers on the cloned RNase III cDNA (SEQ ID NO: 1) to
which the oligonucleotide binds. Oligonucleotide concentration was
200 nM. Table 1 shows that ISIS 25690, 25691, 25692, 25693, 25694,
25695, 25696 and 25697 (SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 and 15)
inhibited human RNase III expression by about 50% or more. These
compounds are therefore preferred. The most effective agent was
ISIS 25691 (SEQ ID NO: 9), targeted to nucleotides 3085-4004 in the
coding region of the mRNA. This compound was selected for further
studies.
[0061] Increasing concentrations of ISIS 25691 caused increasing
loss of RNase III mRNA, with 300 nM resulting in loss of more than
90% of the RNase III mRNA. The mismatch control oligonucleotide,
ISIS 27110 (SEQ ID NO: 18), at 300 nM had no effect on the RNase
III mRNA level. ISIS 25691 at 300 nM suppressed RNase III mRNA
levels in HeLa cells from 2 to 72 hours after a single treatment.
After treatment with ISIS 25691 at 100, 150 or 200 nM for 24 hours,
RNase III protein was reduced to 67%, 44% or 19% of control
respectively. The level of RNase II protein was slightly reduced at
5 hours after treatment and reached a maximum reduction of about
70% at 18 hours. Immunofluorescence staining showed that after
treatment with ISIS 25691 (150 nM, 24 hours), RNase III was
dramatically reduced or absent in the nucleus and nucleoli. After
treatment of HeLa cells with ISIS 25691 at 300 nM for 18 hours, the
morphology of HeLa cells changed from fusiform to oval. After 24
hours of treatment, approximately 5-10% of the cells detached from
the plate and could be stained with trypan blue indicating cell
death. The cells that remained attached to the solid substrate grew
much more slowly than untreated cells and appeared unable to enter
mitosis (data not shown). After 48 hours, 40-50% of the cells
treated with 300 nM ISIS 25691 were dead. These results were highly
reproducible and indicate that RNase III is required for HeLa cell
survival. The control oligonucleotide had no effect at any time or
at any concentration on cell morphology, RNase III mRNA or protein
levels demonstrating the antisense effect was highly specific.
[0062] One function that has been attributed to RNase III in lower
species is pre-ribosomal RNA (pre-rRNA) processing. Human pre-rRNA
processing is thought to involve cleavage of 45S pre-rRNA into 30S
and 32S fragments. The 32S RNA product of the cleavage of 45S
pre-rRNA contains 5.8S rRNA, ITS2 and 28S rRNA. Cleavage of the 32S
RNA results in 12S pre-rRNA and 28S rRNA products. The 12S pre-rRNA
is further cleaved to 5.8S rRNA. Because ribosomes are made in the
nucleolus, and the human RNase III protein appeared to be
translocated to and from the nucleolus during the cell cycle, its
potential role(s) in human pre-rRNA processing was evaluated. Two
hybridization probes for human pre-rRNA were synthesized, 5'ETS-1
(5'-CAA GGC ACG CCT CTC AGA TCG CTA GAG AAG GCT TTT CTC A-3'; SEQ
ID NO: 19), designed to bind to the 5' external transcribed spacer
(5'ETS) of human pre-rRNA and 5.8S-1 (5'-CAT TAA TTC TCG CAG CTA
GCG CTG CGT TCT TCA TCG ACG C-3'; SEQ ID NO: 20), designed to bind
to 5.8S rRNA. When total cellular RNA (15 .mu.g) from untreated
HeLa cells was fractionated by agarose gel electrophoresis,
transferred to a nylon membrane and probed with .sup.32P-5'ETS-1, a
band corresponding to 45S pre-rRNA and a very faint band
corresponding in mobility to 30S (5'ETS-18S-ITS1) pre-rRNA were
observed. When .sup.32P-5.8S-1 was used, bands corresponding to
45S, 32S (5.8S-ITS2-28S) and 12S (5.8S-ITS2) pre-rRNA and 5.8S rRNA
were observed. At concentrations at which the antisense
oligonucleotide ISIS 25691 dramatically reduced the RNase III
level, no effect on the 45S pre-rRNA level was observed. In
contrast, the 5.8S-1 probe demonstrated that antisense inhibition
of RNase III increased the levels of 32S and 12S pre-rRNAs.
[0063] To provide further confirmation that human RNase III is
involved in preribosomal RNA processing, the effects of ten
antisense oligonucleotides on RNase III mRNA levels were compared
to the effects of these oligonucleotides on accumulation of the two
pre-rRNA species (32S and 12S) that accumulated after treatment
with the most potent of the antisense inhibitors, ISIS 25691. The
potency of antisense inhibitors designed to bind to different sites
in RNase III mRNA varied. The correlation between the reduction of
RNase III RNA levels and the accumulation of both 32S and 12S
pre-rRNAs was excellent, thus confirming the conclusion derived
from the Northern blot analysis.
[0064] Antisense inhibition of RNase III resulted in substantial
accumulation of 12S pre-rRNA, less pronounced accumulation of 32S
pre-rRNA and no accumulation of 45S pre-rRNA. Thus this human RNase
III appears to be required for the processing of 12S pre-rRNA. It
may also be involved in the processing of 32S pre-rRNA. The
principal site of cleavage induced by human RNase III described
here is in the 5.8S-ITS2 region of pre-RNA.
[0065] RNase III enzymes are double-strand RNA (dsRNA)
endoribonucleases. To test whether the human RNase III domain can
specifically cleave dsRNA, the RNase III domain-coding region was
subcloned into a glutathione S-transferase (GST) expression vector.
The GST-RNase III fusion protein and GST alone were expressed,
purified using glutathione agarose and analyzed by coomassie blue
staining of the SDS-PAGE and Western Blot analysis with anti-human
RNase III peptide antibody. These studies showed that the human
RNase III domain was greater than 85% pure, though there was
evidence of slight degradation during expression and purification.
When incubated with labeled dsRNA and ssRNA, the GST-RNase III
fusion protein preferentially digested the dsRNA without
significant cleavage of ssRNA, while GST alone cleaved neither
dsRNA nor ssDNA substrate. Thus, the cleavage observed was not due
to contamination with ssRNases or dsRNases from E. coli.
Ribonucleases V.sub.1 (dsRNase), and T.sub.1 and A (ssRNases) were
used as controls to confirm that the cleavage observed was dsRNA
cleavage.
[0066] RNase III is a double-strand RNA endonuclease, specifically
cleaving double-helical structures in cellular and viral RNAs. It
is believed that this cleavage can be exploited to promote cleavage
of a cellular RNA target, by providing "RNA-like" antisense
oligonucleotides which hybridize to the cellular RNA target to form
an RNA duplex, thus eliciting RNase III cleavage. Methods of
promoting inhibition of expression by antisense oligonucleotides,
and methods for screening oligonucleotides are thus provided. In
the context of this invention, "promoting antisense inhibition" or
"promoting inhibition of expression" of a selected RNA target, or
of its protein product, means inhibiting expression of the target
or enhancing the inhibition of expression of the target. In some
embodiments of these methods, the RNase III is present in an
enriched amount. In the context of this invention, "enriched" means
an amount greater than would naturally be found. RNase III may be
present in an enriched amount through, for example, addition of
exogenous RNase III, through selection of cells which overexpress
RNase III or through manipulation of cells to cause overexpression
of RNase III. The exogenously added RNase III may be added in the
form of, for example, a cellular or tissue extract, a biochemically
purified or partially purified preparation of RNase III, or a
cloned and expressed RNase III polypeptide.
[0067] The expression of large quantities of a cloned human RNase
III of the present invention has been shown to be useful in
characterizing the activities of this enzyme. In addition, the
polynucleotides and polypeptides of the present invention provide a
means for identifying agents, such as the antisense compounds
described herein, which modulate the function of this enzyme in
human cells and tissues. For example, a host cell can be
genetically engineered to incorporate polynucleotides and express
polypeptides of the present invention. Polynucleotides can be
introduced into a host cell using any number of well known
techniques such as infection, transduction, transfection or
transformation. The polynucleotide can be introduced alone or in
conjunction with a second polynucleotide encoding a selectable
marker. In a preferred embodiment, the host comprises a mammalian
cell. Such host cells can then be used not only for production of
human RNase III, but also to identify agents which increase or
decrease levels of expression or activity of human RNase III in the
cell. In these assays, the host cell would be exposed to an agent
suspected of altering levels of expression or activity of human
RNase III in the cells. The level or activity of human RNase III in
the cell would then be determined in the presence and absence of
the agent. Assays to determine levels of protein in a cell are well
known to those of skill in the art and include, but are not limited
to, radioimmunoassays, competitive binding assays, Western blot
analysis and enzyme linked immunosorbent assays (ELISAs). Methods
of determining increased activity of the enzyme, and in particular
increased cleavage of dsRNA substrate can be performed in
accordance with the teachings of the examples of the present
application. Agents identified as modulators of the level or
activity of this enzyme may be useful.
[0068] Antisense modulators of human RNase III are provided herein
and may be used diagnostically, therapeutically and for research
purposes.
[0069] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
[0070] cDNA Cloning
[0071] An internet search of the XREF database in the National
Center of Biotechnology Information (NCBI) yielded a 393 base pair
(bp) human expressed sequenced tag (EST, GenBank accession
AA083888), homologous to the yeast RNase III (RNT1) gene (GenBank
accession #AAB04172; SEQ ID NO: 5) and the C. elegans RNase III
gene (GenBank accession 001326; SEQ ID NO: 3). Three sets of
oligonucleotide primers encoding the human RNase H EST sequence
were synthesized. Sequence-specific primer sets listed in Table 2
were designed based on the human expressed tag sequence or early
cloned cDNA fragments. These are shown in Table 2. These primers
were used in polymerase chain reaction for 3' and 5' RACE and/or
for detection on Southern blots.
2TABLE 2 RNase III Oligonucleotide Primers Position in full SEQ
Primer Sequence length Primer ID name source cDNA Sequence NO
NIII-2 EST AA083888 3516-3550 CCAAATACTGATCGACAACTTATTGAAACTTCTCC
21 NIII-4 EST AA083888 3569-3606
GAGTTTGAAGAAGCAATTGGAGTAATTTTTACTCATG 22 NIII-6 EST AA083888
3607-3634 TCGACTTCTGGCAAGGGCATTCACATT 23 3RACE3 Clone #3-4
2708-2683 CCTCTGTGCCAGCTTCTGTTTGTCAG 24 3RACE2 Clone #3-4 2688-2663
TGTCAGTTTGTTTGACTTTGGGACTA 25 3RACE1 Clone #3-4 2662-2637
TTTGCTAGGAGGTGGCGAAGTTTCAC 26 RACE4 Clone #L40 1923-1894
GCTTGATGGCCTCTTCTCCAGGATAAATGC 27 RACE5 Clone #L40 1898-1869
AATGCTGTGCCTAATTCCTGTGCGTCTTGC 28 RACE Det Clone #L40 1723-1676
CAGGTGCTGTCCTCATCAGACTCACAC- TCGGATTCACTGGAACTCTCT 29 33G Clone #25
831-306 CACTGGGCAGGAAAGAACTAGGGTTG 30 33H Clone #25 802-776
TGGAAACTATTAAAACTGGGAGGTGG 31 33 Det Clone #25 701-652
AGGCATGGAGGGAGGGGGCATCATGAAGGGGAAAGTGCCTTGTCCAGGAG 32
[0072] By 3' RACE (rapid amplification of 3' cDNA), the human RNase
III cDNA 3' from the expressed tag sequence was amplified by PCR
using human Marathon ready cDNA (Clontech, Palo Alto Calif.) as
templates, and NIII-2/AP1 (for the first amplification) and
NIII-4/AP2 (for the second amplification) as primers. AP1 and AP2
are primers provided with the Marathon ready cDNA by the
manufacturer. The standard DNA polymerase chain reaction (PCR)
procedure was performed using native pfu DNA polymerase
(Stratagene, San Diego Calif.) and its reaction buffer. The
annealing temperature was 55-60.degree. C. The elongation time was
approximately 6-8 min. The fragments were subjected to agarose gel
electrophoresis. The fragments were subjected to agarose gel
electrophoresis in the TAE buffer, denatured in 0.5 M NaOH and then
electronically transferred to a nitrocellulose membrane (Bio-Rad,
Hercules, Calif.) for confirmation by Southern blot. Southern blots
were performed using [.sup.32P]-end labeled NIII-6 oligonucleotide
as a probe in hybridization buffer (6.times. SSC, 5.times.
Denhardts solution) containing 100 .mu.g/ml sheared denatured
salmon sperm DNA, 0.5% SDS, 10 mM EDTA at 46.degree. C. for 4 hr,
then washed twice with 1.times. SSC and 0.1% SDS at 42-59.degree.
C. for 20 min. The confirmed fragments were excised from the
agarose gel and purified by gel extraction (Qiagen, Germany), then
subcloned into a zero-blunt vector (Invitrogen, Carlsbad, Calif.)
and subjected to DNA sequencing.
Example 2
[0073] Screening of the cDNA Libraries, DNA Sequencing and Sequence
Analysis
[0074] A human liver cDNA lambda phage Uni-ZAP library (Stratagene,
La Jolla, Calif.) was screened using the RACE products as specific
probes. Several positive clones were isolated. The two longest
clones, 3-1 and 3-4, correspond to the COOH-terminal region,
nucleotides 2636-3912 and 3350-4764, respectively, of the full
length cDNA. With primers (3RACE1, 3RACE2 and 3RACE3) based on the
NH.sub.2-terminal portion of the clone 3-4, 5' RACE was performed
to clone a cDNA (clone L40) of approximately 1 kb, which encodes
the middle part (nucleotides 1661-2688) of the full length cDNA. In
the same way, a cDNA (clone 25) of the NH.sub.2-terminal portion
(nucleotides 645-1898) was cloned. Using clone 25 to screen the
liver library again, several clones were isolated, but none
included additional NH.sub.2-terminal sequence. The most
NH.sub.2-terminal clone (328) corresponded to nucleotides 799-2191.
The last 5' RACE was performed with primers 33G, 33H and 33Dec,
based on clone 25, and the NH.sub.2-terminal portion of the cDNA
(clone 81, corresponding to nucleotides 1-802) was generated.
[0075] The positive cDNA clones were excised into pBluescript
phagemid from lambda phage and subjected to DNA sequencing.
Sequencing of the positive clones was performed with an automatic
DNA sequencer by Retrogen Inc. (San Diego, Calif.). The overlapping
sequences were aligned and combined by the assembling program of
MacDNASISv3.0 (Hitachi Software Engineering Co., America, Ltd.) to
give the full length (4764 nucleotides) polynucleotide sequence
(SEQ ID NO: 1). Protein structure and analysis were performed by
the program MacVector v6.0 (Oxford Molecular Group, UK). A homology
search was performed on the NCBI database.
Example 3
[0076] Antisense Treatment
[0077] HeLa cells were transfected with oligonucleotide mixed with
Lipofectin (GIBcO BRL, Gaithersburg, Md.) at a concentration of
37.5-300 nM for 5 hours in Opti-MEM (GIBco BRL). After removing the
medium containing oligonucleotide, cells were cultured in DMEM for
times indicated and harvested for analysis. Inhibition by antisense
oligonucleotides is expressed compared to control (without
oligonucleotide treatment).
Example 4
[0078] Northern Hybridization
[0079] Total RNA was isolated from HeLa cells using the guanidine
isothiocyanate method (R. E. Kingston, in Current protocols in
molecular biology, F. M. Ausubel, et al., Eds., John Wiley &
Sons Inc., New York, 1997, vol. 1, pp. 4.2.3-4.2.5.). Fifteen .mu.g
of total RNA was separated on a 1% agarose/formaldehyde gel and
transferred to Hybond-N+ (Amersham, Arlington Heights, Ill.)
followed by fixing using UV crosslinker (Stratagene, La Jolla,
Calif.). To detect RNase III mRNA, hybridization was performed by
using .sup.32P-labeled human RNase III cDNA in Quik-Hyb buffer
(Stratagene, La Jolla, Calif.) at 68.degree. C. for 2 hours. After
hybridization, membranes were washed in a final stringency of
0.1.times. SSC/0.1% SDS at 60.degree. C. for 30 minutes. Membranes
were analyzed using a PhosphorImager Storm 860 (Molecular Dynamics,
Sunnyvale, Calif.). The level of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA was used to normalize the amount of
total RNA loaded.
[0080] For Northern hybridization of pre-rRNAs, HeLa cells were
treated with ISIS 25691 and ISIS 27110 for 24 hours using
.sup.32P-end labeled oligo probes 5'ETS-1 (5'-CAA GGC ACG CCT CTC
AGA TCG CTA GAG AAG GCT TTT CTC A-3'; SEQ ID NO: 33), corresponding
to 5'ETS and 5.8S-1(5'-CAT TAA TTC TCG CAG CTA GCG CTG CGT TCT TCA
TCG ACG C-3'; SEQ ID NO: 34), corresponding to 5.8S rRNA.
Hybridizations were performed at 40.degree. C. for 2 hours and
washed in 2.times. SSC/0.1%SDS at 40.degree. C. for 1 hour. All
others were as described above. Data were mean .+-.SD of triplicate
determination of representative experiment.
Example 5
[0081] Western Blot Analysis of Human RNase III
[0082] Nuclear and non-nuclear fractions from HeLa cells were
prepared as described (Dignam et al., Nucleic Acids Res 1983, 11,
1475-89. Whole cell, non-nuclear and nuclear fractions were boiled
in SDS-sample buffer. Then the samples were separated by SDS-PAGE
using 4-20% Tris-glycine gels (NOVEX, San Diego, Calif.) under
reducing conditions. Molecular weight prestained markers were used
(NOVEX) to determine the protein sizes. The proteins were
electrophoretically transfered to a PVDF-membrane and processed for
immunoblotting using affinity purified anti-SR peptide antibody at
5.mu.g/ml. The immunoreactive bands were visualized using the
enhanced chemiluminescence method (Amersham, Arlington Heights,
Ill.) and analyzed using a PhosphorImager Storm 860 (Molecular
Dynamics, Sunnyvale, Calif.).
Example 6
[0083] Antibody Production
[0084] Antibodies were prepared to peptides synthesized having
amino acid sequences contained within the SR domain and the III
domain of human RNase III. The SR domain peptide
(H-CRSDYDRGRTPSRHRSYERS-OH, amino acids 226 to 284; SEQ ID NO: 35)
and the III region peptide (H-CRWEREHQEREPDETEDIKK-OH, amino acids
1356 to 1374; SEQ ID NO: 36) were synthesized, coupled to
diphtheria toxoid through maleimidocaproyl-N-hydr- oxysuccinamide
(MCS), mixed with Freund's adjuvant (complete for first
immunization, incomplete for remaining immunizations) and injected
intramuscularly into New Zealand White rabbits. Serum was collected
after the second immunization. Antibody titer was measured by
ELISA. Anti-SR and anti-III peptide IgGs were affinity purified
with SR and III peptides coupled to thiopropyl-Sepharose 6B,
respectively.
Example 7
[0085] Indirect Immunofluorescence Staining of Human RNase III
[0086] HeLa cells were cultured in chamber slides for
immunostaining. Cells were washed once with Dulbecco's Phosphate
Buffered Saline (D-PBS, pH 7.0), and then fixed in 10%
neutral-buffered formalin for 10 minutes followed by washing three
times with D-PBS. Fixed cells were then blocked for 30 minutes with
20% fetal bovine serum plus 0.5% Tween 20. Cells were first stained
with anti-III peptide antibody (10 .mu.g/ml) for 1 hour at
37.degree. C., washed three times with D-PBS plus 0.1% NP-40, and
incubated for 1 hour at 37.degree. C. with the FITC goat
anti-rabbit IgG (Jackson ImmunoResearch Laboratory, Inc. West
Grove, Pa.). The cells were washed with D-PBS three times and
mounted in mounting medium (Vector, Burlingame, Calif.) for
examination under a fluorescence microscope. NR IgG: normal rabbit
IgG was used as control.
Example 8
[0087] Indirect Immunofluorescence Staining of Human RNase III in
HeLa Cells in Different Phases of the Cell Cycle.
[0088] HeLa cells were synchronized at early-S phase using the
double thymidine method (Johnson et al., in The Cell Cycle: A
Practical Approach P. Fantes, R. Brooks, Eds., IRL Press, 1993, pp.
1-24). Briefly, cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM, 10% fetal calf serum) containing 2 mM of thymidine
for 17 hours. After washing twice with D-PBS, cells were cultured
in DMEM for 9 hours followed by second thymidine treatment for 15
hours. Synchronized cells were then washed twice with D-PBS,
cultured and harvested at 0, 2, 4, 6, 8 and 24 hours for
immunofluorescence staining and FACS analysis.
[0089] HeLa cells were detached from culture flasks with
trypsin-EDTA and washed once with D-PBS containing 5 mM of EDTA.
Cells were then fixed in 70% ethanol for 1 to 24 hours at 4.degree.
C. followed by propidium iodine (PI, 50 .mu.g/ml) staining for 1
hour at room temperature. Cell counts (Y axis) and PI content (X
axis) were determined by FACS analysis (Becton Dickinson and Co.,
San Jose, Calif.).
Example 9
[0090] Expression of GST-RNase III Domain Fusion Protein
[0091] A cDNA fragment encoding the human RNase III-like domain
(C-terminal-most 466 amino acids) was amplified by PCR and
introduced into a BamH I site upstream and Not I site downstream.
This fragment was further subcloned into the sites of the
expression vector pGEX-4T-1 (Pharmacia Biotech, Piscataway, N.J.)
to produce the RNase III fusion protein with Glutathione
S-transferase (GST) at its N-terminus. The identity of the
construct was proven by DNA sequencing. The GST-RNase III fusion
protein was expressed in E. coli strain BL21 and purified using
glutathione agarose (Pharmacia Biotech, Piscataway, N.J.) under
native conditions with B-PER bacterial protein extraction reagent
(Pierce, Rockford, Ill.). Control GST protein was also prepared in
parallel from the pGEX-4T-1 plasmid. The purified products were
identified by Coomassie staining after 12% SDS-polyacrylamide gel
electrophoresis and Western blot analyses with anti-RNase III
peptide antibody (see examples above).
Example 10
[0092] In Vitro Cleavage of dsRNA
[0093] The dsRNA substrate was generated by hybridization of two
complementary strands of RNA produced with T7 and T3 polymerase
transcription of the polylinker region of the pBluscript II KS(-)
plasmid (Stratagen, San Diego, Calif.). The plasmid was digested
with either Sst I or Kpn I and further purified with
phenol/chloroform extraction and ethanol precipitation. The Sst I
or Kpn I-digested plasmids were then transcribed using T7 or T3 RNA
polymerase respectively (Stratagene, San Diego, Calif.) with or
without .sup.32P-AUTP. The resulting transcribed RNAs (about 100
nt) were purified by electrophoresis on 6% denaturing
polyacrylamide gel. The .sup.32P radiolabeled T7 transcript and
unlabeled T3 transcript fragments were mixed and heated for 5 min
at 90.degree. C. in a buffer containing 20 mM KCl, 50 mM Tris-HCl
(pH 7.5), 0.1 mM EDTA. MgCl, BSA and RNase inhibitor were added to
the mixture after heating (final concentrations were 10 mM. 100
ng/ml and 10 unit/ml respectively). The mixture was incubated at
37.degree. C. for 2 hr and the duplex RNA was purified on 6%
non-denaturing gels. The .sup.32P-labelled T7 transcript was also
used as the ssRNA control substrate. To evaluate cleavage, 0.4
.mu.g of GST protein or GST-RNase III (approximately 5-10 pmole of
purified GST-RNase III) fusion protein was incubated with labeled
dsRNA (250,000 cpm) (approximately 5-10 fmole) and ssRNA (250,000
cpm) at 37.degree. C. in a buffer containing 20 mM KCl, 50 mM
Tris-HCl (pH 7.5), 5 mM MgCl, 50 mM NaCl, 0.1 mM DTT, 0.1 mg/ml
yeast tRNA and 10 unit/ml RNase inhibitor in the total volume of 60
.mu.l. The digested samples were quenched at specific times and
analyzed using non-denaturing polyacrylamide gel electrophoresis
and PhosphorImager analysis.
Sequence CWU 1
1
36 1 4764 DNA Homo sapiens 1 ctgtcttggt acctgcggta gtagcctggc
tttgctctga cggcgatctc gcggcccgag 60 agccttttat aggttgcttt
tcccggggat gtgaaggata cagaaatgac tgtgaatcaa 120 cccatatcat
caaggagctg ataatctagt ggaagagtta gacgtgtgca tacttcacta 180
tgatatgagg cagtctctga gcttatattc tctgtggaag atgtgacata tccaggcgga
240 acatcatgat gcagggaaac acatgtcaca gaatgtcgtt ccacccggga
cgagggcgtc 300 cccgaggacg aggaggacat ggagccagac cctcagcacc
atcctttagg ccccaaaatc 360 tgaggctgct tcaccctcag cagcctcctg
tgcaatatca atatgaacct ccaagtgccc 420 cttccaccac tttctcaaac
tctccagccc ccaattttct ccctccacga ccagactttg 480 tacccttccc
cccacccatg cctccgtcag cgcaaggccc tcttcccccc tgcccaatca 540
ggccgccttt ccccaaccac cagatgaggc accccttccc agttcctcct tgttttcctc
600 ccatgccacc accaatgcct tgtcctaata accccccagt ccctggggca
cctcctggac 660 aaggcacttt ccccttcatg atgccccctc cctccatgcc
tcatcccccg ccccctccag 720 tcatgccgca gcaggttaat tatcagtacc
ctccgggcta ttctcaccac aacttcccac 780 ctcccagttt taatagtttc
cagaacaacc ctagttcttt cctgcccagt gctaataaca 840 gcagtagtcc
tcatttcaga catctccctc catacccact cccaaaggct cccagtgaga 900
gaaggtcccc agaaaggctg aaacactatg atgaccacag gcaccgagac cacagtcatg
960 ggcgaggtga gaggcatcgg tccctggatc ggcgggagcg aggccgcagt
cccgacagga 1020 gaagacaaga cagccggtac agatctgatt atgaccgagg
gagaacacca tctcgccacc 1080 gcagctacga acggagcaga gagcgagaac
gggagagaca caggcatcga gacaaccgaa 1140 gatcaccatc tctggaaagg
tcctacaaaa aagagtataa gagatctgga aggagttacg 1200 gtttatcggt
tgttcctgaa cctgctggat gcacaccaga attacctggg gagattatta 1260
aaaatacaga ttcttgggcc ccacccctgg agattgtgaa tcatcgctcc ccaagtaggg
1320 agaagaagag agctcgttgg gaggaagaaa aagaccgttg gagtgacaac
cagagttctg 1380 gcaaagacaa gaactatacc tcaatcaagg aaaaagagcc
cgaggagacc atgcctgaca 1440 agaatgagga ggaagaagaa gaacttctta
agcctgtgtg gattcgatgc actcattcag 1500 aaaactacta ctccagtgac
cccatggatc aggtgggaga ttctacagtg gttggaacga 1560 gtaggcttcg
tgacttatat gacaaatttg aggaggagtt ggggagcagg caagaaaagg 1620
ccaaagctgc tcggcctccg tgggaacctc caaagacgaa gctcgatgaa gatttagaga
1680 gttccagtga atccgagtgt gagtctgatg aggacagcac ctgttctagc
agctcagact 1740 ctgaagtttt tgacgttatt gcagaaatca aacgcaaaaa
ggcccaccct gaccgacttc 1800 atgatgaact ttggtacaac gatccaggcc
agatgaatga tggaccactc tgcaaatgca 1860 gcgcaaaggc aagacgcaca
ggaattaggc acagcattta tcctggagaa gaggccatca 1920 agccctgtcg
tcctatgacc aacaatgctg gcagactttt ccactaccgg atcacagtct 1980
ccccgcctac gaacttttta actgacaggc caactgttat agaatacgat gatcacgagt
2040 atatctttga aggattttct atgtttgcac atgcccccct gaccaatatt
ccactgtgta 2100 aagtaattag attcaacata gactacacga ttcatttcat
tgaagagatg atgccggaga 2160 atttttgtgt gaaagggctt gaactctttt
cactgttcct attcagagat attttggaat 2220 tatatgactg gaatcttaaa
ggtcctttgt ttgaagacag ccctccctgc tgcccaagat 2280 ttcatttcat
gccacgtttt gtaagatttc ttccagatgg aggaaaggaa gtgctgtcca 2340
tgcaccagat tctcctgtac ttgttaaggt gcagcaaagc cctggtgcct gaggaggaga
2400 ttgccaatat gcttcagtgg gaggagctgg agtggcagaa atatgcagaa
gaatgcaaag 2460 gcatgattgt taccaaccct gggacgaaac caagctctgt
ccgtatcgat caactggatc 2520 gtgaacagtt caaccccgat gtgattactt
ttccgattat cgtccacttt gggatacgcc 2580 ctgcacagtt gagttatgca
ggagacccac agtaccaaaa actgtggaag agttatgtga 2640 aacttcgcca
cctcctagca aatagtccca aagtcaaaca aactgacaaa cagaagctgg 2700
cacagaggga ggaagccctc caaaaaatac ggcagaagaa tacaatgaga cgagaagtaa
2760 cggtggagct aagtagccaa ggattctgga aaactggcat ccgttctgat
gtctgtcagc 2820 atgcaatgat gctacctgtt ctgacccatc atatccgcta
ccaccaatgc ctaatgcatt 2880 tggacaagtt gataggatat actttccaag
atcgttgtct gttgcagctg gccatgactc 2940 atccaagtca tcatttaaat
tttggaatga atcctgatca tgccaggaat tcattatcta 3000 actgtggaat
tcggcagccc aaatacggag acagaaaagt tcatcacatg cacatgcgga 3060
agaaagggat taacaccttg ataaatatca tgtcacgcct tggccaagat gacccaactc
3120 cctcgaggat taaccacaat gaacggttgg aattcctggg tgatgctgtt
gttgaatttc 3180 tgaccagcgt ccatttgtac tatttgtttc ctagtctgga
agaaggagga ttagcaacct 3240 atcggactgc cattgttcag aatcagcacc
ttgccatgct agcaaagaaa cttgaactgg 3300 atccatttat gctgtatgct
cacgggcctg acctttgtag agaatcggac cttcgacatg 3360 caatggccaa
ttgttttgaa gcgttaatag gagctgttta cttggaggga agcctggagg 3420
aagccaagca gttatttgga cgcttgctct ttaatgatcc ggacctgcgc gaagtctggc
3480 tcaattatcc tctccaccca ctccaactac aagagccaaa tactgatcga
caacttattg 3540 aaacttctcc agttctacaa aaacttactg agtttgaaga
agcaattgga gtaattttta 3600 ctcatgttcg acttctggca agggcattca
cattgagaac tgtgggattt aaccatctga 3660 ccctaggcca caatcagaga
atggaattcc taggtgactc cataatgcaa ctggtagcca 3720 cagagtactt
attcattcat ttcccagatc atcatgaagg acacttaact ttgttgcgaa 3780
gctctttggt gaataataga actcaggcca aggtagcgga ggagctgggc atgcaggagt
3840 acgccataac caacgacaag accaagaggc ctgtggcgct tcgcaccaag
accttggcgg 3900 accttttgga atcatttatt gcagcgctgt acactgataa
ggatttggaa tatgttcata 3960 ctttcatgaa tgtctgcttc tttccacgat
tgaaagaatt cattttgaat caggattgga 4020 atgaccccaa atcccagctt
cagcagtgtt gcttgacact taggacagaa ggaaaagagc 4080 cagacattcc
tctgtacaag actctgcaga cagtgggccc atcccatgcc cgaacctaca 4140
ctgtggctgt ttatttcaag ggagaaagaa taggctgtgg gaaaggacca agtattcagc
4200 aagcggaaat gggagcagca atggatgcgc ttgaaaaata taattttccc
cagatggccc 4260 atcagaagcg gttcatcgaa cggaagtaca gacaagagtt
aaaagaaatg aggtgggaaa 4320 gagagcatca agagagagag ccagatgaga
ctgaagacat caagaaataa aggagggcat 4380 gcaagtgtgg agtatttact
tgctcagtaa ctgtgactgt tgtctattga gacctagcct 4440 agttttcctg
cagacaatga acgaagtgtg ctcattgaaa taaaatacag agtcaaatcg 4500
ctattgttgt tttaatgatc tgtttttagc tggatggtct ttattacaaa gtattagatt
4560 tttcttctat ttaacggaaa acttgacttt ggtgaatgtg cattacttcc
ttttattttg 4620 ctctttaaat aataaaattc aagaagcata ttctatgtgg
aatagatcct gtttttccat 4680 ctgtgtccca gattgtgacc ctagactttc
aattgacaag taaaaaattg actttactag 4740 taaaaaaaaa aaaaaaaaaa aaaa
4764 2 1374 PRT Homo sapiens 2 Met Met Gln Gly Asn Thr Cys His Arg
Met Ser Phe His Pro Gly Arg 1 5 10 15 Gly Cys Pro Arg Gly Arg Gly
Gly His Gly Ala Arg Pro Ser Ala Pro 20 25 30 Ser Phe Arg Pro Gln
Asn Leu Arg Leu Leu His Pro Gln Gln Pro Pro 35 40 45 Val Gln Tyr
Gln Tyr Glu Pro Pro Ser Ala Pro Ser Thr Thr Phe Ser 50 55 60 Asn
Ser Pro Ala Pro Asn Phe Leu Pro Pro Arg Pro Asp Phe Val Pro 65 70
75 80 Phe Pro Pro Pro Met Pro Pro Ser Ala Gln Gly Pro Leu Pro Pro
Cys 85 90 95 Pro Ile Arg Pro Pro Phe Pro Asn His Gln Met Arg His
Pro Phe Pro 100 105 110 Val Pro Pro Cys Phe Pro Pro Met Pro Pro Pro
Met Pro Cys Pro Asn 115 120 125 Asn Pro Pro Val Pro Gly Ala Pro Pro
Gly Gln Gly Thr Phe Pro Phe 130 135 140 Met Met Pro Pro Pro Ser Met
Pro His Pro Pro Pro Pro Pro Val Met 145 150 155 160 Pro Gln Gln Val
Asn Tyr Gln Tyr Pro Pro Gly Tyr Ser His His Asn 165 170 175 Phe Pro
Pro Pro Ser Phe Asn Ser Phe Gln Asn Asn Pro Ser Ser Phe 180 185 190
Leu Pro Ser Ala Asn Asn Ser Ser Ser Pro His Phe Arg His Leu Pro 195
200 205 Pro Tyr Pro Leu Pro Lys Ala Pro Ser Glu Arg Arg Ser Pro Glu
Arg 210 215 220 Leu Lys His Tyr Asp Asp His Arg His Arg Asp His Ser
His Gly Arg 225 230 235 240 Gly Glu Arg His Arg Ser Leu Asp Arg Arg
Glu Arg Gly Arg Ser Pro 245 250 255 Asp Arg Arg Arg Gln Asp Ser Arg
Tyr Arg Ser Asp Tyr Asp Arg Gly 260 265 270 Arg Thr Pro Ser Arg His
Arg Ser Tyr Glu Arg Ser Arg Glu Arg Glu 275 280 285 Arg Glu Arg His
Arg His Arg Asp Asn Arg Arg Ser Pro Ser Leu Glu 290 295 300 Arg Ser
Tyr Lys Lys Glu Tyr Lys Arg Ser Gly Arg Ser Tyr Gly Leu 305 310 315
320 Ser Val Val Pro Glu Pro Ala Gly Cys Thr Pro Glu Leu Pro Gly Glu
325 330 335 Ile Ile Lys Asn Thr Asp Ser Trp Ala Pro Pro Leu Glu Ile
Val Asn 340 345 350 His Arg Ser Pro Ser Arg Glu Lys Lys Arg Ala Arg
Trp Glu Glu Glu 355 360 365 Lys Asp Arg Trp Ser Asp Asn Gln Ser Ser
Gly Lys Asp Lys Asn Tyr 370 375 380 Thr Ser Ile Lys Glu Lys Glu Pro
Glu Glu Thr Met Pro Asp Lys Asn 385 390 395 400 Glu Glu Glu Glu Glu
Glu Leu Leu Lys Pro Val Trp Ile Arg Cys Thr 405 410 415 His Ser Glu
Asn Tyr Tyr Ser Ser Asp Pro Met Asp Gln Val Gly Asp 420 425 430 Ser
Thr Val Val Gly Thr Ser Arg Leu Arg Asp Leu Tyr Asp Lys Phe 435 440
445 Glu Glu Glu Leu Gly Ser Arg Gln Glu Lys Ala Lys Ala Ala Arg Pro
450 455 460 Pro Trp Glu Pro Pro Lys Thr Lys Leu Asp Glu Asp Leu Glu
Ser Ser 465 470 475 480 Ser Glu Ser Glu Cys Glu Ser Asp Glu Asp Ser
Thr Cys Ser Ser Ser 485 490 495 Ser Asp Ser Glu Val Phe Asp Val Ile
Ala Glu Ile Lys Arg Lys Lys 500 505 510 Ala His Pro Asp Arg Leu His
Asp Glu Leu Trp Tyr Asn Asp Pro Gly 515 520 525 Gln Met Asn Asp Gly
Pro Leu Cys Lys Cys Ser Ala Lys Ala Arg Arg 530 535 540 Thr Gly Ile
Arg His Ser Ile Tyr Pro Gly Glu Glu Ala Ile Lys Pro 545 550 555 560
Cys Arg Pro Met Thr Asn Asn Ala Gly Arg Leu Phe His Tyr Arg Ile 565
570 575 Thr Val Ser Pro Pro Thr Asn Phe Leu Thr Asp Arg Pro Thr Val
Ile 580 585 590 Glu Tyr Asp Asp His Glu Tyr Ile Phe Glu Gly Phe Ser
Met Phe Ala 595 600 605 His Ala Pro Leu Thr Asn Ile Pro Leu Cys Lys
Val Ile Arg Phe Asn 610 615 620 Ile Asp Tyr Thr Ile His Phe Ile Glu
Glu Met Met Pro Glu Asn Phe 625 630 635 640 Cys Val Lys Gly Leu Glu
Leu Phe Ser Leu Phe Leu Phe Arg Asp Ile 645 650 655 Leu Glu Leu Tyr
Asp Trp Asn Leu Lys Gly Pro Leu Phe Glu Asp Ser 660 665 670 Pro Pro
Cys Cys Pro Arg Phe His Phe Met Pro Arg Phe Val Arg Phe 675 680 685
Leu Pro Asp Gly Gly Lys Glu Val Leu Ser Met His Gln Ile Leu Leu 690
695 700 Tyr Leu Leu Arg Cys Ser Lys Ala Leu Val Pro Glu Glu Glu Ile
Ala 705 710 715 720 Asn Met Leu Gln Trp Glu Glu Leu Glu Trp Gln Lys
Tyr Ala Glu Glu 725 730 735 Cys Lys Gly Met Ile Val Thr Asn Pro Gly
Thr Lys Pro Ser Ser Val 740 745 750 Arg Ile Asp Gln Leu Asp Arg Glu
Gln Phe Asn Pro Asp Val Ile Thr 755 760 765 Phe Pro Ile Ile Val His
Phe Gly Ile Arg Pro Ala Gln Leu Ser Tyr 770 775 780 Ala Gly Asp Pro
Gln Tyr Gln Lys Leu Trp Lys Ser Tyr Val Lys Leu 785 790 795 800 Arg
His Leu Leu Ala Asn Ser Pro Lys Val Lys Gln Thr Asp Lys Gln 805 810
815 Lys Leu Ala Gln Arg Glu Glu Ala Leu Gln Lys Ile Arg Gln Lys Asn
820 825 830 Thr Met Arg Arg Glu Val Thr Val Glu Leu Ser Ser Gln Gly
Phe Trp 835 840 845 Lys Thr Gly Ile Arg Ser Asp Val Cys Gln His Ala
Met Met Leu Pro 850 855 860 Val Leu Thr His His Ile Arg Tyr His Gln
Cys Leu Met His Leu Asp 865 870 875 880 Lys Leu Ile Gly Tyr Thr Phe
Gln Asp Arg Cys Leu Leu Gln Leu Ala 885 890 895 Met Thr His Pro Ser
His His Leu Asn Phe Gly Met Asn Pro Asp His 900 905 910 Ala Arg Asn
Ser Leu Ser Asn Cys Gly Ile Arg Gln Pro Lys Tyr Gly 915 920 925 Asp
Arg Lys Val His His Met His Met Arg Lys Lys Gly Ile Asn Thr 930 935
940 Leu Ile Asn Ile Met Ser Arg Leu Gly Gln Asp Asp Pro Thr Pro Ser
945 950 955 960 Arg Ile Asn His Asn Glu Arg Leu Glu Phe Leu Gly Asp
Ala Val Val 965 970 975 Glu Phe Leu Thr Ser Val His Leu Tyr Tyr Leu
Phe Pro Ser Leu Glu 980 985 990 Glu Gly Gly Leu Ala Thr Tyr Arg Thr
Ala Ile Val Gln Asn Gln His 995 1000 1005 Leu Ala Met Leu Ala Lys
Lys Leu Glu Leu Asp Pro Phe Met Leu 1010 1015 1020 Tyr Ala His Gly
Pro Asp Leu Cys Arg Glu Ser Asp Leu Arg His 1025 1030 1035 Ala Met
Ala Asn Cys Phe Glu Ala Leu Ile Gly Ala Val Tyr Leu 1040 1045 1050
Glu Gly Ser Leu Glu Glu Ala Lys Gln Leu Phe Gly Arg Leu Leu 1055
1060 1065 Phe Asn Asp Pro Asp Leu Arg Glu Val Trp Leu Asn Tyr Pro
Leu 1070 1075 1080 His Pro Leu Gln Leu Gln Glu Pro Asn Thr Asp Arg
Gln Leu Ile 1085 1090 1095 Glu Thr Ser Pro Val Leu Gln Lys Leu Thr
Glu Phe Glu Glu Ala 1100 1105 1110 Ile Gly Val Ile Phe Thr His Val
Arg Leu Leu Ala Arg Ala Phe 1115 1120 1125 Thr Leu Arg Thr Val Gly
Phe Asn His Leu Thr Leu Gly His Asn 1130 1135 1140 Gln Arg Met Glu
Phe Leu Gly Asp Ser Ile Met Gln Leu Val Ala 1145 1150 1155 Thr Glu
Tyr Leu Phe Ile His Phe Pro Asp His His Glu Gly His 1160 1165 1170
Leu Thr Leu Leu Arg Ser Ser Leu Val Asn Asn Arg Thr Gln Ala 1175
1180 1185 Lys Val Ala Glu Glu Leu Gly Met Gln Glu Tyr Ala Ile Thr
Asn 1190 1195 1200 Asp Lys Thr Lys Arg Pro Val Gly Leu Arg Thr Lys
Thr Leu Ala 1205 1210 1215 Asp Leu Leu Glu Ser Phe Ile Ala Ala Leu
Tyr Thr Asp Lys Asp 1220 1225 1230 Leu Glu Tyr Val His Thr Phe Met
Asn Val Cys Phe Phe Pro Arg 1235 1240 1245 Leu Lys Glu Phe Ile Leu
Asn Gln Asp Trp Asn Asp Pro Lys Ser 1250 1255 1260 Gln Leu Gln Gln
Cys Cys Leu Thr Leu Arg Thr Glu Gly Lys Glu 1265 1270 1275 Pro Asp
Ile Pro Leu Tyr Lys Thr Leu Gln Thr Val Gly Pro Ser 1280 1285 1290
His Ala Arg Thr Tyr Thr Val Ala Val Tyr Phe Lys Gly Glu Arg 1295
1300 1305 Ile Gly Cys Gly Lys Gly Pro Ser Ile Gln Gln Ala Glu Met
Gly 1310 1315 1320 Ala Ala Met Asp Ala Leu Glu Lys Tyr Asn Phe Pro
Gln Met Ala 1325 1330 1335 His Gln Lys Arg Phe Ile Gly Arg Lys Tyr
Arg Gln Glu Leu Lys 1340 1345 1350 Glu Met Arg Trp Glu Arg Glu His
Gln Glu Arg Glu Pro Asp Glu 1355 1360 1365 Thr Glu Asp Ile Lys Lys
1370 3 412 PRT Caenorhabditis elegans 3 Met Ser Leu Phe Asn Ile Met
Lys Gly Thr Ser Gly Gly Glu Pro Ile 1 5 10 15 Leu His Asn Glu Arg
Leu Glu Tyr Leu Gly Asp Ala Val Val Glu Leu 20 25 30 Ile Val Ser
His His Leu Tyr Phe Met Leu Thr His His Phe Glu Gly 35 40 45 Gly
Leu Ala Thr Tyr Arg Thr Ala Leu Val Gln Asn Arg Asn Leu Ala 50 55
60 Thr Leu Ala Lys Asn Cys Arg Ile Asp Glu Met Leu Gln Tyr Ser His
65 70 75 80 Gly Ala Asp Leu Ile Asn Val Ala Glu Phe Lys His Ala Leu
Ala Asn 85 90 95 Ala Phe Glu Ala Val Met Ala Ala Ile Tyr Leu Asp
Gly Gly Leu Ala 100 105 110 Pro Cys Asp Val Ile Phe Ser Lys Ala Met
Tyr Gly His Gln Pro Val 115 120 125 Leu Lys Glu Lys Trp Asp His Ile
Asn Glu His Glu Leu Lys Arg Glu 130 135 140 Asp Pro Gln Gly Asp Arg
Asp Leu Ser Phe Ile Thr Pro Thr Leu Ser 145 150 155 160 Thr Phe His
Ala Leu Glu Glu Arg Leu Gly Ile Gln Phe Asn Asn Ile 165 170 175 Arg
Leu Leu Ala Lys Ala Phe Thr Arg Arg Asn Ile Pro Asn Asn Asp 180 185
190 Leu Thr Lys Gly His Asn Gln Arg Leu Glu Trp Leu Gly Asp Ser Val
195 200 205 Leu Gln Leu Ile Val Ser Asp Phe Leu Tyr Arg Arg Phe Pro
Tyr His 210 215 220 His Glu Gly His Met Ser Leu Leu Arg Thr Ser Leu
Val Ser Asn Gln 225 230 235 240 Thr Gln Ala Val Val Cys Asp Asp Leu
Gly Phe Thr Glu Phe Val Ile 245 250 255 Lys Ala Pro Tyr Lys Thr Pro
Glu Leu Lys Leu Lys Asp Lys Ala Asp 260 265 270 Leu Val Glu Ala Phe
Ile Gly
Ala Leu Tyr Val Asp Arg Gly Ile Glu 275 280 285 His Cys Arg Ala Phe
Ile Arg Ile Val Phe Cys Pro Arg Leu Lys His 290 295 300 Phe Ile Glu
Ser Glu Lys Trp Asn Asp Ala Lys Ser His Leu Gln Gln 305 310 315 320
Trp Cys Leu Ala Met Arg Asp Pro Ser Ser Ser Glu Pro Asp Met Pro 325
330 335 Glu Tyr Arg Val Leu Gly Ile Glu Gly Pro Thr Asn Asn Arg Ile
Phe 340 345 350 Lys Ile Ala Val Tyr Tyr Lys Gly Lys Arg Leu Ala Ser
Ala Ala Glu 355 360 365 Ser Asn Val His Lys Ala Glu Leu Arg Val Ala
Glu Leu Ala Leu Ala 370 375 380 Asn Leu Glu Ser Met Ser Phe Ser Lys
Met Lys Ala Lys Asn Asn Ser 385 390 395 400 Asn Met Arg Arg Arg Leu
Glu Gln Asp Thr Ser Asp 405 410 4 366 PRT Saccharomyces pombe 4 Met
Gly Arg Phe Lys Arg His His Glu Gly Asp Ser Asp Ser Ser Ser 1 5 10
15 Ser Ala Ser Asp Ser Leu Ser Arg Gly Arg Arg Ser Leu Gly His Lys
20 25 30 Arg Ser Ser His Ile Lys Asn Arg Gln Tyr Tyr Ile Leu Glu
Lys Lys 35 40 45 Ile Arg Lys Leu Met Phe Ala Met Lys Ala Leu Leu
Glu Glu Thr Lys 50 55 60 His Ser Thr Lys Asp Asp Val Asn Leu Val
Ile Pro Gly Ser Thr Trp 65 70 75 80 Ser His Ile Glu Gly Val Tyr Glu
Met Leu Lys Ser Arg His Asp Arg 85 90 95 Gln Asn Glu Pro Val Ile
Glu Glu Pro Ser Ser His Pro Lys Asn Gln 100 105 110 Lys Asn Gln Glu
Asn Asn Glu Pro Thr Ser Glu Glu Phe Glu Glu Gly 115 120 125 Glu Tyr
Pro Pro Pro Leu Pro Pro Leu Arg Ser Glu Lys Leu Lys Glu 130 135 140
Gln Val Phe Met His Ile Ser Arg Ala Tyr Glu Ile Tyr Pro Asn Gln 145
150 155 160 Ser Asn Pro Asn Glu Leu Leu Asp Ile His Asn Glu Arg Leu
Glu Phe 165 170 175 Leu Gly Asp Ser Phe Phe Asn Leu Phe Thr Thr Arg
Ile Ile Phe Ser 180 185 190 Lys Phe Pro Gln Met Asp Glu Gly Ser Leu
Ser Lys Leu Arg Ala Lys 195 200 205 Phe Val Gly Asn Glu Ser Ala Asp
Lys Phe Ala Arg Leu Tyr Gly Phe 210 215 220 Asp Lys Thr Leu Val Leu
Ser Tyr Ser Ala Glu Lys Asp Gln Leu Arg 225 230 235 240 Lys Ser Gln
Lys Val Ile Ala Asp Thr Phe Glu Ala Tyr Leu Gly Ala 245 250 255 Leu
Ile Leu Asp Gly Gln Glu Glu Thr Ala Phe Gln Trp Val Ser Arg 260 265
270 Leu Leu Gln Pro Lys Ile Ala Asn Ile Thr Val Gln Arg Pro Ile Asp
275 280 285 Lys Leu Ala Lys Ser Lys Leu Phe His Lys Tyr Ser Thr Leu
Gly His 290 295 300 Ile Glu Tyr Arg Trp Pro Ala Cys Val Asp Gly Ala
Gly Gly Ser Ala 305 310 315 320 Glu Gly Tyr Val Ile Ala Cys Ile Phe
Asn Gly Lys Glu Val Ala Arg 325 330 335 Ala Trp Gly Ala Asn Gln Lys
Asp Ala Gly Ser Arg Ala Ala Met Gln 340 345 350 Ala Leu Glu Val Leu
Ala Lys Asp Tyr Ser Lys Phe Ala Arg 355 360 365 5 471 PRT
Saccharomyces cerevisiae 5 Met Gly Ser Lys Val Ala Gly Lys Lys Lys
Thr Gln Asn Asp Asn Lys 1 5 10 15 Leu Asp Asn Glu Asn Gly Ser Gln
Gln Arg Glu Asn Ile Asn Thr Lys 20 25 30 Thr Leu Leu Lys Gly Asn
Leu Lys Ile Ser Asn Tyr Lys Tyr Leu Glu 35 40 45 Val Ile Gln Leu
Glu His Ala Val Thr Lys Leu Val Glu Ser Tyr Asn 50 55 60 Lys Ile
Ile Glu Leu Ser Pro Asn Leu Val Ala Tyr Asn Glu Ala Val 65 70 75 80
Asn Asn Gln Asp Arg Val Pro Val Gln Ile Leu Pro Ser Leu Ser Arg 85
90 95 Tyr Gln Leu Lys Leu Ala Ala Glu Leu Lys Thr Leu His Asp Leu
Lys 100 105 110 Lys Asp Ala Ile Leu Thr Glu Ile Thr Asp Tyr Glu Asn
Glu Phe Asp 115 120 125 Thr Glu Gln Lys Gln Pro Ile Leu Gln Glu Ile
Ser Lys Ala Asp Met 130 135 140 Glu Lys Leu Glu Lys Leu Glu Gln Val
Lys Arg Glu Lys Arg Glu Lys 145 150 155 160 Ile Asp Val Asn Val Tyr
Glu Asn Leu Asn Glu Lys Glu Asp Glu Glu 165 170 175 Glu Asp Glu Gly
Glu Asp Ser Tyr Asp Pro Thr Lys Ala Gly Asp Ile 180 185 190 Val Lys
Ala Thr Lys Trp Pro Pro Lys Leu Pro Glu Ile Gln Asp Leu 195 200 205
Ala Ile Arg Ala Arg Val Phe Ile His Lys Ser Thr Ile Lys Asp Lys 210
215 220 Val Tyr Leu Ser Gly Ser Glu Met Ile Asn Ala His Asn Glu Arg
Leu 225 230 235 240 Glu Phe Leu Gly Asp Ser Ile Leu Asn Ser Val Met
Thr Leu Ile Ile 245 250 255 Tyr Asn Lys Phe Pro Asp Tyr Ser Glu Gly
Gln Leu Ser Thr Leu Arg 260 265 270 Met Asn Leu Val Ser Asn Glu Gln
Ile Lys Gln Trp Ser Ile Met Tyr 275 280 285 Asn Phe His Glu Lys Leu
Lys Thr Asn Phe Asp Leu Lys Asp Glu Asn 290 295 300 Ser Asn Phe Gln
Asn Gly Lys Leu Lys Leu Tyr Ala Asp Val Phe Glu 305 310 315 320 Ala
Tyr Ile Gly Gly Leu Met Glu Asp Asp Pro Arg Asn Asn Leu Pro 325 330
335 Lys Ile Arg Lys Trp Leu Arg Lys Leu Ala Lys Pro Val Ile Glu Glu
340 345 350 Ala Thr Arg Asn Gln Val Ala Leu Glu Lys Thr Asp Lys Leu
Asp Met 355 360 365 Asn Ala Lys Arg Gln Leu Tyr Ser Leu Ile Gly Tyr
Ala Ser Leu Arg 370 375 380 Leu His Tyr Val Thr Val Lys Lys Pro Thr
Ala Val Asp Pro Asn Ser 385 390 395 400 Ile Val Glu Cys Arg Val Gly
Asp Gly Thr Val Leu Gly Thr Gly Val 405 410 415 Gly Arg Asn Ile Lys
Ile Ala Gly Ile Arg Ala Ala Glu Asn Ala Leu 420 425 430 Arg Asp Lys
Lys Met Leu Asp Phe Tyr Ala Lys Gln Arg Ala Ala Ile 435 440 445 Pro
Arg Ser Glu Ser Val Leu Lys Asp Pro Ser Gln Lys Asn Lys Lys 450 455
460 Arg Lys Phe Ser Asp Thr Ser 465 470 6 226 PRT Escherichia coli
6 Met Asn Pro Ile Val Ile Asn Arg Leu Gln Arg Lys Leu Gly Tyr Thr 1
5 10 15 Phe Asn His Gln Glu Leu Leu Gln Gln Ala Leu Thr His Arg Ser
Ala 20 25 30 Ser Ser Lys His Asn Glu Arg Leu Glu Phe Leu Gly Asp
Ser Ile Leu 35 40 45 Ser Tyr Val Ile Ala Asn Ala Leu Tyr His Arg
Phe Pro Arg Val Asp 50 55 60 Glu Gly Asp Met Ser Arg Met Arg Ala
Thr Leu Val Arg Gly Asn Thr 65 70 75 80 Leu Ala Glu Leu Ala Arg Glu
Phe Glu Leu Gly Glu Cys Leu Arg Leu 85 90 95 Gly Pro Gly Glu Leu
Lys Ser Gly Gly Phe Arg Arg Glu Ser Ile Leu 100 105 110 Ala Asp Thr
Val Glu Ala Leu Ile Gly Gly Val Phe Leu Asp Ser Asp 115 120 125 Ile
Gln Thr Val Glu Lys Leu Ile Leu Asn Trp Tyr Gln Thr Arg Leu 130 135
140 Asp Glu Ile Ser Pro Gly Asp Lys Gln Lys Asp Pro Lys Thr Arg Leu
145 150 155 160 Gln Glu Tyr Leu Gln Gly Arg His Leu Pro Leu Pro Thr
Tyr Leu Val 165 170 175 Val Gln Val Arg Gly Glu Ala His Asp Gln Glu
Phe Thr Ile His Cys 180 185 190 Gln Val Ser Gly Leu Ser Glu Pro Val
Val Gly Thr Gly Ser Ser Arg 195 200 205 Arg Lys Ala Glu Gln Ala Ala
Ala Glu Gln Ala Leu Lys Lys Leu Glu 210 215 220 Leu Glu 225 7 11
PRT Homo sapiens 7 His Asn Glu Arg Leu Glu Phe Leu Gly Asp Ser 1 5
10 8 20 DNA Artificial Sequence Synthetic 8 atccctttct tccgcatgtg
20 9 20 DNA Artificial Sequence Synthetic 9 gccaaggcgt gacatgatat
20 10 20 DNA Artificial Sequence Synthetic 10 cggatcatta aagagcaagc
20 11 20 DNA Artificial Sequence Synthetic 11 tattcaccaa agagcttcgc
20 12 20 DNA Artificial Sequence Synthetic 12 caatcgtgga aagaagcaga
20 13 20 DNA Artificial Sequence Synthetic 13 gctcccattt ccgcttgctg
20 14 20 DNA Artificial Sequence Synthetic 14 atgctctctt tcccacctca
20 15 20 DNA Artificial Sequence Synthetic 15 aaatactcca cacttgcatg
20 16 20 DNA Artificial Sequence Synthetic 16 tgcacattca ccaaagtcaa
20 17 20 DNA Artificial Sequence Synthetic 17 agtctagggt cacaatctgg
20 18 20 DNA Artificial Sequence Synthetic 18 ttcagttgta gtggtccgac
20 19 40 DNA Artificial Sequence Synthetic 19 caaggcacgc ctctcagatc
gctagagaag gcttttctca 40 20 40 DNA Artificial Sequence Synthetic 20
cattaattct cgcagctagc gctgcgttct tcatcgacgc 40 21 35 DNA Artificial
Sequence Synthetic 21 ccaaatactg atcgacaact tattgaaact tctcc 35 22
37 DNA Artificial Sequence Synthetic 22 gagtttgaag aagcaattgg
agtaattttt actcatg 37 23 27 DNA Artificial Sequence Synthetic 23
tcgacttctg gcaagggcat tcacatt 27 24 26 DNA Artificial Sequence
Synthetic 24 cctctgtgcc agcttctgtt tgtcag 26 25 26 DNA Artificial
Sequence Synthetic 25 tgtcagtttg tttgactttg ggacta 26 26 26 DNA
Artificial Sequence Synthetic 26 tttgctagga ggtggcgaag tttcac 26 27
30 DNA Artificial Sequence Synthetic 27 gcttgatggc ctcttctcca
ggataaatgc 30 28 30 DNA Artificial Sequence Synthetic 28 aatgctgtgc
ctaattcctg tgcgtcttgc 30 29 48 DNA Artificial Sequence Synthetic 29
caggtgctgt cctcatcaga ctcacactcg gattcactgg aactctct 48 30 26 DNA
Artificial Sequence Synthetic 30 cactgggcag gaaagaacta gggttg 26 31
26 DNA Artificial Sequence Synthetic 31 tggaaactat taaaactggg
aggtgg 26 32 50 DNA Artificial Sequence Synthetic 32 aggcatggag
ggagggggca tcatgaaggg gaaagtgcct tgtccaggag 50 33 40 DNA Artificial
Sequence Synthetic 33 caaggcacgc ctctcagatc gctagagaag gcttttctca
40 34 40 DNA Artificial Sequence Synthetic 34 cattaattct cgcagctagc
gctgcgttct tcatcgacgc 40 35 20 PRT Homo sapiens 35 Cys Arg Ser Asp
Tyr Asp Arg Gly Arg Thr Pro Ser Arg His Arg Ser 1 5 10 15 Tyr Glu
Arg Ser 20 36 20 PRT Homo sapiens 36 Cys Arg Trp Glu Arg Glu His
Gln Glu Arg Glu Pro Asp Glu Thr Glu 1 5 10 15 Asp Ile Lys Lys
20
* * * * *