U.S. patent application number 10/239134 was filed with the patent office on 2004-02-19 for ecdysone receptor-based inducible gene expression system.
Invention is credited to Cress, Dean Ervin, Kapitskaya, Marianna Zinovjevna, Palli, Subba Reddy.
Application Number | 20040033600 10/239134 |
Document ID | / |
Family ID | 31715347 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033600 |
Kind Code |
A1 |
Palli, Subba Reddy ; et
al. |
February 19, 2004 |
Ecdysone receptor-based inducible gene expression system
Abstract
This invention relates to the field of biotechnology or genetic
engineering. Specifically, this invention relates to the field of
gene expression More specifically, this invention relates to a
novel inducible gene expression system and methods of modulating
gene expression in a host cell for applications such as gene
therapy, large scale production of proteins and antibodies,
cell-based high throughput screening assays, functional genomics
and regulation of traits in transgenic plants and animals.
Inventors: |
Palli, Subba Reddy;
(Lansdale, PA) ; Kapitskaya, Marianna Zinovjevna;
(North Wales, PA) ; Cress, Dean Ervin;
(Sounderton, PA) |
Correspondence
Address: |
NEW RHEOGENE I LLC
2650 EISENHOWER AVENUE
NORRISTOWN
PA
19403
US
|
Family ID: |
31715347 |
Appl. No.: |
10/239134 |
Filed: |
September 19, 2002 |
PCT Filed: |
March 21, 2001 |
PCT NO: |
PCT/US01/09050 |
Current U.S.
Class: |
435/455 ;
435/320.1; 435/325; 435/69.1 |
Current CPC
Class: |
C12N 2510/00 20130101;
C07K 2319/71 20130101; C12N 15/1055 20130101; C12N 2830/002
20130101; C12N 2830/15 20130101; C07K 2319/80 20130101; C07K
2319/715 20130101; C12N 15/63 20130101; C12N 15/85 20130101 |
Class at
Publication: |
435/455 ;
435/69.1; 435/320.1; 435/325 |
International
Class: |
C12P 021/02; C12N
005/06; C12N 015/85 |
Claims
We claim:
1. A gene expression modulation system comprising: a) a first gene
expression cassette that is capable of being expressed in a host
cell comprising a polynucleotide sequence that encodes a first
polypeptide comprising: i) a DNA-binding domain that recognizes a
response element associated with a gene whose expression is to be
modulated; i) a ligand binding domain comprising a ligand binding
domain from a nuclear receptor; b) a second gene expression
cassette that is capable of being expressed in the host cell
comprising a polynucleotide sequence that encodes a second
polypeptide comprising: i) a transactivation domain; and ii) a
ligand binding domain comprising a ligand binding domain from a
nuclear receptor other than ultraspiracle (USP); wherein the
transactivation domain is from a nuclear receptor other than an
ecdysone receptor, a retinoid X receptor, or an ultraspiracle
receptor; and wherein the ligand binding domains from the first
polypeptide and the second polypeptide are different and
dimerize.
2. The gene expression modulation system according to claim 1,
further comprising a third gene expression cassette comprising: i)
a response element to which the DNA-binding domain of the first
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) the gene
whose expression is to be modulated.
3. The gene expression modulation system according to claim 1,
wherein the ligand binding domain of the first polypeptide is an
ecdysone receptor polypeptide.
4. The gene expression modulation system according to claim 1,
wherein the ligand binding domain of the second polypeptide is a
retinoid X receptor polypeptide.
5. A gene expression modulation system comprising: a) a first gene
expression cassette that is capable of being expressed in a host
cell comprising a polynucleotide sequence that encodes a first
polypeptide comprising: i) a DNA-binding domain that recognizes a
response element associated with a gene whose expression is to be
modulated; and ii) a ligand binding domain comprising a ligand
binding domain from an ecdysone receptor; and b) a second gene
expression cassette that is capable of being expressed in the host
cell comprising a polynucleotide sequence that encodes a second
polypeptide comprising: i) a transactivation domain; and ii) a
ligand binding domain comprising a ligand binding domain from a
retinoid X receptor; wherein the ligand binding domains from the
first polypeptide and the second polypeptide are different and
dimerize.
6. The gene expression modulation system according to claim 5,
further comprising a third gene expression cassette comprising: i)
a response element to which the DNA-binding domain of the fust
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) the gene
whose expression is to be modulated.
7. The gene expression modulation system according to claim 5,
wherein the ligand binding domain of the first polypeptide is
encoded by a polynucleotide comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ D NO: 10.
8. The gene expression modulation system according to claim 5,
wherein the ligand binding domain of the first polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19, and SEQ ID NO:20.
9. The gene expression modulation system according to claim 5,
wherein the ligand binding domain of the second polypeptide is
encoded by a polynucleotide comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO:29, and SEQ ID NO: 30.
10. The gene expression modulation system according to claim 5,
wherein the ligand binding domain of the second polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,, SEQ ID NO: 34, SEQ
ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:
39, and SEQ ID NO: 40.
11. A gene expression modulation system comprising: a) a first gene
expression cassette that is capable of being expressed in a host
cell comprising a polynucleotide sequence that encodes a first
polypeptide comprising: i) a DNA-binding domain that recognizes a
response element associated with a gene whose expression is to be
modulated; and ii) a ligand binding domain comprising a ligand
binding domain from a retinoid X receptor; and b) a second gene
expression cassette that is capable of being expressed in the host
cell comprising a polynucleotide sequence that encodes a second
polypeptide comprising: i) a transactivation domain; and ii) a
ligand binding domain comprising a ligand binding domain from an
ecdysone receptor; wherein the ligand binding domains from the
first polypeptide and the second polypeptide are different and
dimerize.
12. The gene expression modulation system according to claim 11,
further comprising a third gene expression cassette comprising: i)
a response element to which the DNA-binding domain of the first
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) the gene
whose expression is to be modulated.
13. The gene expression modulation system according to claim 11,
wherein the ligand binding domain of the first polypeptide is
encoded by a polynucleotide comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.
14. The gene expression modulation system according to claim 11,
wherein the ligand binding domain of the first polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:
39, and SEQ ID NO: 40.
15. The gene expression modulation system according to claim 11,
wherein the ligand binding domain of the second polypeptide is
encoded by a polynucleotide comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
16. The gene expression modulation system according to claim 11,
wherein the ligand binding domain of the second polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19, and SEQ ID NO: 20.
17. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a DNA-binding domain and an
ecdysone receptor ligand binding domain, wherein the DNA binding
domain is from a nuclear receptor other than an ecdysone
receptor.
18. The gene expression cassette according to claim 18, wherein the
DNA-binding domain is a GAL4 DNA-binding domain or a LexA
DNA-binding domain.
19. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a DNA-binding domain and a retinoid
X receptor ligand binding domain, wherein the DNA binding domain is
from a nuclear receptor other than a retinoid X receptor.
20. The gene expression cassette according to claim 19, wherein the
DNA-binding domain is a GALA DNA-binding domain or a LexA
DNA-binding domain.
21. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a transactivation domain and an
ecdysone receptor ligand binding domain, wherein the
transactivation domain is from a nuclear receptor other than an
ecdysone receptor.
22. The gene expression cassette according to claim 21, wherein the
transactivation domain is a VP16 transactivation domain.
23. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a transactivation domain and a
retinoid X receptor ligand binding domain, wherein the
transactivation domain is from a nuclear receptor other than a
retinoid X receptor.
24. The gene expression cassette according to claim 22, wherein the
transactivation domain is a VP16 transactivation domain.
25. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a DNA-binding domain encoded by a
polynucleotide comprising a nucleic acid sequence selected from the
group consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ
ID NO: 43) and an ecdysone receptor ligand binding domain encoded
by a polynucleotide comprising a nucleic acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
26. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a DNA-binding domain comprising an
amino acid sequence selected from the group consisting of a GAL4
DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and an ecdysone
receptor ligand binding domain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12,
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
27. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a DNA-binding domain encoded by a
polynucleotide comprising a nucleic acid sequence selected from the
group consisting of a GALA DBD (SEQ ID NO: 41) or a LexA DBD (SEQ
ID NO: 43) and a retinoid X receptor ligand binding domain encoded
by a polynucleotide comprising a nucleic acid sequence selected
from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID
NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.
28. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a DNA-binding domain comprising an
amino acid sequence selected from the group consisting of a GAL4
DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and a retinoid X
receptor ligand binding domain comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID
NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
29. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a transactivation domain encoded by
a polynucleotide comprising a nucleic acid sequence of SEQ ID NO:
45 and an ecdysone receptor ligand binding domain encoded by a
polynucleotide comprising a nucleic acid sequence selected from the
group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, and SEQ ID NO:10.
30. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a transactivation domain comprising
an amino acid sequence of SEQ ID NO: 46 and an ecdysone receptor
ligand binding domain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
31. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a transactivation domain encoded by
a polynucleotide comprising a nucleic acid sequence of SEQ ID NO:
45 and a retinoid X receptor ligand binding domain encoded by a
polynucleotide comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID
NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.
32. A gene expression cassette comprising a polynucleotide encoding
a hybrid polypeptide comprising a transactivation domain comprising
an amino acid sequence of SEQ ID NO: 46 and a retinoid X receptor
ligand binding domain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID
NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,
SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
33. An isolated polynucleotide encoding an ecdysone receptor
polypeptide or a retinoid X receptor polypeptide comprising a
truncation mutation, wherein the truncation mutation reduces ligand
binding activity of the ecdysone receptor polypeptide or the
retinoid X receptor polypeptide.
34. An isolated polynucleotide encoding an ecdysone receptor
polypeptide or a retinoid X receptor polypeptide comprising a
truncation mutation, wherein the truncation mutation reduces
steroid binding activity of the ecdysone receptor polypeptide or
the retinoid X receptor polypeptide.
35. An isolated polynucleotide encoding an ecdysone receptor
polypeptide or a retinoid X receptor polypeptide comprising a
truncation mutation, wherein the truncation mutation reduces
non-steroid binding activity of the ecdysone receptor polypeptide
or the retinoid X receptor polypeptide.
36. An isolated polynucleotide encoding an ecdysone receptor
polypeptide or a retinoid X receptor polypeptide comprising a
truncation mutation, wherein the truncation mutation enhances
ligand binding activity of the ecdysone receptor polypeptide or the
retinoid X receptor polypeptide.
37. An isolated polynucleotide encoding an ecdysone receptor
polypeptide or a retinoid X receptor polypeptide comprising a
truncation mutation, wherein the truncation mutation enhances
steroid binding activity of the ecdysone receptor polypeptide or
the retinoid X receptor polypeptide.
38. An isolated polynucleotide encoding an ecdysone receptor
polypeptide or a retinoid X receptor polypeptide comprising a
truncation mutation, wherein the truncation mutation enhances
non-steroid binding activity of the ecdysone receptor polypeptide
or the retinoid X receptor polypeptide.
39. An isolated polynucleotide encoding a retinoid X receptor
polypeptide comprising a truncation mutation, wherein the
truncation mutation increases ligand sensitivity of the retinoid X
receptor polypeptide.
40. An isolated polynucleotide encoding a retinoid X receptor
polypeptide comprising a truncation mutation, wherein the
truncation mutation increases ligand sensitivity of a heterodimer,
wherein the heterodimer comprises said retinoid X receptor
polypeptide and a dimerization partner.
41. The isolated polynucleotide according to claim 40, wherein the
dimerization partner is an ecdysone receptor polypeptide.
42. An isolated polynucleotide encoding a truncated ecdysone
receptor polypeptide, wherein the polynucleotide comprises a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:
10.
43. An isolated polypeptide encoded by the isolated polynucleotide
according to claim 42.
44. An isolated truncated ecdysone receptor polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ
ID NO: 20.
45. An isolated polynucleotide encoding a truncated retinoid X
receptor polypeptide, wherein the polynucleotide comprises a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ
ID NO: 30.
46. An isolated polypeptide encoded by the isolated polynucleotide
according to claim 45.
47. An isolated truncated retinoid X receptor polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID
NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,
SEQ ID NO: 39, and SEQ ID NO: 40.
48. A method of modulating the expression of a gene in a host cell
comprising the gene to be modulated comprising the steps of: a)
introducing into the host cell the gene expression modulation
system according to claim 1; and b) introducing into the host cell
a ligand that independently combines with the ligand binding
domains of the first polypeptide and the second polypeptide;
wherein the gene to be expressed is a component of a chimeric gene
comprising: i) a response element to which the DNA binding domain
from the first polypeptide binds; ii) a promoter that is activated
by the transactivation domain of the second polypeptide; and iii) a
gene whose expression is to be modulated, whereby a complex is
formed comprising the ligand, the first polypeptide, and the second
polypeptide, and whereby the complex modulates expression of the
gene in the host cell.
49. The method according to claim 48, wherein the ligand is a
compound of the formula: 3wherein: E is a (C.sub.4-C.sub.6)alkyl
containing a tertiary carbon or a cyano(C.sub.3-C.sub.5)alkyl
containing a tertiary carbon; R.sup.1 is H, Me, Et, i-Pr, F,
formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl,
CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH, 1-propynyl,
2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF.sub.2CF.sub.3,
CH.dbd.CHCN, allyl, azido, SCN, or SCHF.sub.2; R.sup.2 is H, Me,
Et, n-Pr, i-Pr, formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F,
CH.sub.2Cl, CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH,
1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr,
OAc, NMe.sub.2, NEt.sub.2, SMe, SEt, SOCF.sub.3,
OCF.sub.2CF.sub.2H, COEt, cyclopropyl, CF.sub.2CF.sub.3,
CH.dbd.CHCN, allyl, azido, OCF.sub.3, OCHF.sub.2, O-i-Pr, SCN,
SCHF.sub.2, SOMe, NH--CN, or joined with R.sup.3 and the phenyl
carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a
phenyl carbon; R.sup.3 is H, Et, or joined with R.sup.2 and the
phenyl carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a
phenyl carbon; R.sup.4, R.sup.5, and R.sup.6 are independently H,
Me, Et, F, Cl, Br, formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2,
CH.sub.2F, CH.sub.2Cl, CH.sub.2OH, CN, C.degree.CH, 1-propynyl,
2-propynyl, vinyl, OMe, OEt, SMe, or SEt.
50. A method of modulating the expression of a gene in a host cell
comprising the gene to be modulated comprising the steps of: a)
introducing into the host cell the gene expression modulation
system of claim 5; and b) introducing into the host cell a ligand
that independently combines with the ligand binding domains of the
first polypeptide and the second polypeptide; wherein the gene to
be expressed is a component of a chimeric gene comprising: i) a
response element to which the DNA binding domain from the first
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) a gene
whose expression is to be modulated, whereby a complex is formed
comprising the ligand, the first polypeptide, and the second
polypeptide, and whereby the complex modulates expression of the
gene in the host cell.
51. The method according to claim 50, wherein the ligand is a
compound of the formula: 4wherein: E is a (C.sub.4-C)alkyl
containing a tertiary carbon or a cyano(C.sub.3-C.sub.5)alkyl
containing a tertiary carbon; R.sup.1 is H, Me, Et, i-Pr, F,
formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl,
CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH, 1-propynyl,
2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF.sub.2CF.sub.3,
CH.dbd.CHCN, allyl, azido, SCN, or SCHF.sub.2; R.sup.2 is H, Me,
Et, n-Pr, i-Pr, formyl, CF.sub.3, CHF.sub.2, CHCl.sub.7, CH.sub.2F,
CH.sub.2Cl, CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH,
1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-nPr, OAc,
NMe2, NEt.sub.2, SMe, SEt, SOCF.sub.3, OCF.sub.2CF.sub.2H, COEt,
cyclopropyl, CF.sub.2CF.sub.3, CH.dbd.CHCN, allyl, azido,
OCF.sub.3, OCHF.sub.2, O-i-Pr, SCN, SCHF.sub.2, SOMe, NH--CN, or
joined with R.sup.3 and the phenyl carbons to which R.sup.2 and
R.sup.3 are attached to form an ethylenedioxy, a dihydrofuryl ring
with the oxygen adjacent to a phenyl carbon, or a dilydropyryl ring
with the oxygen adjacent to a phenyl carbon; R.sup.3 is H, Et, or
joined with R.sup.2 and the phenyl carbons to which R.sup.2 and
R.sup.3 are attached to form an ethylenedioxy, a dihydrofuryl ring
with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring
with the oxygen adjacent to a phenyl carbon; R.sup.4, R.sup.5, and
R.sup.6 are independently H, Me, Et, F, Cl, Br, formyl, CF.sub.3,
CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2OH, CN,
C.degree.CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or
SEt.
52. A method of modulating the expression of a gene in a host cell
comprising the gene to be modulated comprising the steps of: a)
introducing into the host cell the gene expression modulation
system of claim 11; and b) introducing into the host cell a ligand
that independently conibines with the ligand binding domains of the
first polypeptide and the second polypeptide; wherein the gene to
be expressed is a component of a chimeric gene comprising: i) a
response element to which the DNA binding domain from the first
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) a gene
whose expression is to be modulated, whereby a complex is formed
comprising the ligand, the first polypeptide, and the second
polypeptide, and whereby the complex modulates expression of the
gene in the host cell.
53. The method according to claim 52, wherein the ligand is a
compound of the formula: 5wherein: E is a (C.sub.4-C.sub.6)alkyl
containing a tertiary carbon or a cyano(C.sub.3-C.sub.5)alkyl
containing a tertiary carbon; R.sup.1 is H, Me, Et, i-Pr, F,
formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl,
CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH, 1-propynyl,
2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF.sub.2CF.sub.3,
CH.dbd.CHCN, allyl, azido, SCN, or SCHF.sub.2; R.sup.2 is H, Me,
Et, n-Pr, i-Pr, formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F,
CH.sub.2Cl, CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH,
1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-nPr, OAc,
NMe.sub.2, NEt.sub.2, SMe, SEt, SOCF.sub.3, OCF.sub.2CF.sub.2H,
COEt, cyclopropyl, CF2CF3, CH.dbd.CHCN, allyl, azido, OCF.sub.3,
OCHF.sub.2, O-i-Pr, SCN, SCHF.sub.2, SOMe, NH--CN, or joined with
R.sup.3 and the phenyl carbons to which R.sup.2 and R.sup.3 are
attached to form an ethylenedioxy, a dihydrofuryl ring with the
oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the
oxygen adjacent to a phenyl carbon; R.sup.3 is H, Et, or joined
with R.sup.2 and the phenyl carbons to which R.sup.2 and R.sup.3
are attached to form an ethylenedioxy, a dihydrofuryl ring with the
oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the
oxygen adjacent to a phenyl carbon; R.sup.4, R.sup.5, and R.sup.6
are independently H, Me, Et, F, Cl, Br, formyl, CF.sub.3,
CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2OH, CN,
C.degree.CH, 1-propynyl, 2-propynyL vinyl, OMe, OEt, SMe, or
SEt.
54. An isolated host cell into which the gene expression modulation
system according to claim 1 has been introduced.
55. The isolated host cell according to claim 54, wherein the host
cell is selected from the group consisting of abacterial cell, a
fungal cell, a yeast cell, a plant cell, an animal cell, and a
mammalian cell.
56. The isolated host cell according to claim 55, wherein the host
cell is a plant cell, a murine cell, or a human cell.
57. An isolated host cell into which the gene expression modulation
system according to claim 5 has been introduced.
58. The isolated host cell according to claim 57, wherein the host
cell is selected from the group consisting of a bacterial cell, a
fungal cell, a yeast cell, a plant cell, an animal cell, and a
mammalian cell.
59. The isolated host cell according to claim 58, wherein the host
cell is a plant cell, a murine cell, or a human cell.
60. An isolated host cell into which the gene expression modulation
system according to claim 11 has been introduced.
61. The isolated host cell according to claim 60, wherein the host
cell is selected from the group consisting of a bacterial cell, a
fungal cell, a yeast cell, a plant cell, an animal cell, and a
mammalian cell.
62. The isolated host cell according to claim 61, wherein the host
cell is a plant cell, a murine cell, or a human cell.
63. A non-human organism comprising a host cell into which the gene
expression modulation system according to claim 1 has been
introduced.
64. The non-human organism according to claim 63, wherein the
non-human organism is selected from the group consisting of a
bacterium, a fungus, a yeast, a plant, an animal, and a mammal.
65. The non-human organism according to claim 64, wherein the
non-human organism is selected from the group consisting of a
plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a
pig, a horse, a sheep, a monkey, and a chimpanzee.
66. A non-human organism comprising a host cell into which the gene
expression modulation system according to claim S has been
introduced.
67. The non-human organism according to claim 66, wherein the
non-human organism is selected from the group consisting of a
bacterium, a fungus, a yeast, a plant, an animal, and a mammal.
68. The non-human organism according to claim 67, wherein the
non-human organism is selected from the group consisting of a
plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a
pig, a horse, a sheep, a monkey, and a chimpanzee.
69. A non-human organism comprising a host cell into which the gene
expression modulation system according to claim 11 has been
introduced.
70. The non-human organism according to claim 69, wherein the
non-human organism is selected from the group consisting of a
bacterium, a fungus, a yeast, a plant, an animal, and a mammal.
71. The non-human organism according to claim 70, wherein the
non-human organism is selected from the group consisting of a
plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a
pig, a horse, a sheep, a monkey, and a chimpanzee.
Description
[0001] This application claims priority to co-pending U.S.
provisional application Serial No. 60/191,355, filed Mar. 22, 2000
and to co-pending U.S. provisional application Serial No.
60/269,799, filed Feb. 20, 2001.
FIELD OF THE INVENTION
[0002] This invention relates to the field of biotechnology or
genetic engineering. Specifically, this invention relates to the
field of gene expression. More specifically, this invention relates
to a novel ecdysone receptor-based inducible gene expression system
and methods of modulating the expression of a gene within a host
cell using this inducible gene expression system.
BACKGROUND OF THE INVENTION
[0003] In the field of genetic engineering, precise control of gene
expression is a valuable tool for studying, manipulating, and
controlling development and other physiological processes. Gene
expression is a complex biological process involving a number of
specific protein-protein interactions. In order for gene expression
to be triggered, such that it produces the RNA necessary as the
first step in protein synthesis, a transcriptional activator must
be brought into proximity of a promoter that controls gene
transcription. Typically, the transcriptional activator itself is
associated with a protein that has at least one DNA binding domain
that binds to DNA binding sites present in the promoter regions of
genes. Thus, for gene expression to occur, a protein comprising a
DNA binding domain and a transactivation domain located at an
appropriate distance from the DNA binding domain must be brought
into the correct position in the promoter region of the gene.
[0004] The traditional transgenic approach utilizes a cell-type
specific promoter to drive the expression of a designed transgene.
A DNA construct containing the transgene is first incorporated into
a host genome. When triggered by a transcriptional activator,
expression of the transgene occurs in a given cell type.
[0005] Another means to regulate expression of foreign genes in
cells is through inducible promoters. Examples of the use of such
inducible promoters include the PR1-a promoter, prokaryotic
repressor-operator systems, immunosuppressive-immunophilin systems,
and higher eukaryotic transcription activation systems such as
steroid hormone receptor systems and are described below.
[0006] The PR1-a promoter from tobacco is induced during the
systemic acquired resistance response following pathogen attack.
The use of PR1-a may be limited because it often responds to
endogenous materials and external factors such as pathogens, UV-B
radiation, and pollutants. Gene regulation systems based on
promoters induced by heat shock, interferon and heavy metals have
been described (Wurn et al., 1986, Proc. Natl. Acad. Sci. USA
83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61; Filmus et al.,
1992 Nucleic Acids Research 20:27550-27560). However, these systems
have limitations due to their effect on expression of non-target
genes. These systems are also leaky.
[0007] Prokaryotic repressor-operator systems utilize bacterial
repressor proteins and the unique operator DNA sequences to which
they bind. Both the tetracycline ("Tet") and lactose ("Lac")
repressor-operator systems from the bacterium Escherichia coli have
been used in plants and animals to control gene expression. In the
Tet system, tetracycline binds to the TetR repressor protein,
resulting in a conformational change which releases the repressor
protein from the operator which as a result allows transcription to
occur. In the Lac system, a lac operon is activated in response to
the presence of lactose, or synthetic analogs such as
isopropyl-b-D-thiogalactoside. Unfortunately, the use of such
systems is restricted by unstable chemistry of the ligands, i.e.
tetracycline and lactose, their toxicity, their natural presence,
or the relatively high levels required for induction or repression.
For similar reasons, utility of such systems in animals is
limited.
[0008] Immunosuppressive molecules such as FK506, rapamycin and
cyclosporine A can bind to immunophilins FKBP12, cyclophilin, etc.
Using this information, a general strategy has been devised to
bring together any two proteins simply by placing FK506 on each of
the two proteins or by placing FK506 on one and cyclosporine A on
another one. A synthetic homodimer of FK506 (FK1012) or a compound
resulted from fusion of FK506-cyclosporine (FKCsA) can then be used
to induce dimerization of these molecules (Spencer et al., 1993,
Science 262:1019-24; Belshaw et al., 1996 Proc Natl Acad Sci USA
93:4604-7). Gal4 DNA binding domain fused to FKBP12 and VP16
activator domain fused to cyclophilin, and FKCsA compound were used
to show heterodimerization and activation of a reporter gene under
the control of a promoter containing Gal4 binding sites.
Unfortunately, this system includes immunosuppressants that can
have unwanted side effects and therefore, limits its use for
various mammalian gene switch applications.
[0009] Higher eukaryotic transcription activation systems such as
steroid hormone receptor systems have also been employed. Steroid
hormone receptors are members of the nuclear receptor superfamily
and are found in vertebrate and invertebrate cells. Unfortunately,
use of steroidal compounds that activate the receptors for the
regulation of gene expression, particularly in plants and mammals,
is limited due to their involvement in many other natural
biological pathways in such organisms. In order to overcome such
difficulties, an alternative system has been developed using insect
ecdysone receptors (EcR).
[0010] Growth, molting, and development in insects are regulated by
the ecdysone steroid hormone (molting hormone) and the juvenile
hormones (Dhadialla, et al., 1998. Annu. Rev. Entomol. 43:
545-569). The molecular target for ecdysone in insects consists of
at least ecdysone receptor (EcR) and ultraspiracle protein (USP).
EcR is a member of the nuclear steroid receptor super family that
is characterized by signature DNA and ligand binding domains, and
an activation domain (Koelle et al. 1991, Cell, 67:59-77). EcR
receptors are responsive to a number of steroidal compounds such as
ponasterone A and muristerone A. Recently, non-steroidal compounds
with ecdysteroid agonist activity have been described, including
the commercially available insecticides tebufenozide and
methoxyfenozide that are marketed world wide by Rohm and Haas
Company (see International Patent Application No. PCT/EP96/00686
and U.S. Pat. No. 5,530,028). Both analogs have exceptional safety
profiles to other organisms.
[0011] International Patent Application No. PCT/US97/05330 (WO
97/38117) discloses methods for modulating the expression of an
exogenous gene in which a DNA construct comprising the exogenous
gene and an ecdysone response element is activated by a second DNA
construct comprising an ecdysone receptor that, in the presence of
a ligand therefor, and optionally in the presence of a receptor
capable of acting as a silent partner, binds to the ecdysone
response element to induce gene expression. The ecdysone receptor
of choice was isolated from Drosophila melanogaster. Typically,
such systems require the presence of the silent partner, preferably
retinoid X receptor (RXR), in order to provide optimum activation.
In mammalian cells, insect ecdysone receptor (EcR) heterodimerizes
with retinoid X receptor (RXR) and regulates expression of target
genes in a ligand dependent manner. International Patent
Application No. PCT/US98/14215 (WO 99/02683) discloses that the
ecdysone receptor isolated from the silk moth Bombyx mori is
functional in mammalian systems without the need for an exogenous
dimer partner.
[0012] U.S. Pat. No. 5,880,333 discloses a Drosophila melanogaster
EcR and ultraspiracle (USP) heterodimer system used in plants in
which the transactivation domain and the DNA binding domain are
positioned on two different hybrid proteins. Unfortunately, this
system is not effective for inducing reporter gene expression in
animal cells (for comparison, see Example 1.2, below).
[0013] In each of these cases, the transactivation domain and the
DNA binding domain (either as native EcR as in International Patent
Application No. PCT/US98/14215 or as modified EcR as in
International Patent Application No. PCT/US97/05330) were
incorporated into a single molecule and the other heterodimeric
partners, either USP or RXR, were used in their native state.
[0014] Drawbacks of the above described EcR-based gene regulation
systems include a considerable background activity in the absence
of ligands and that these systems are not applicable for use in
both plants and animals (see U.S. Pat. No. 5,880,333). For most
applications that rely on modulating gene expression, these
EcR-based systems are undesirable. Therefore, a need exists in the
art for improved systems to precisely modulate the expression of
exogenous genes in both plants and animals. Such improved systems
would be useful for applications such as gene therapy, large scale
production of proteins and antibodies, cell-based high throughput
screening assays, functional genomics and regulation of traits in
transgenic animals. Improved systems that are simple, compact, and
dependent on ligands that are relatively inexpensive, readily
available, and of low toxicity to the host would prove useful for
regulating biological systems.
[0015] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties. However,
the citation of any reference herein should not be construed as an
admission that such reference is available as "Prior Art" to the
instant application.
SUMMARY OF THE INVENTION
[0016] The present invention relates to a novel ecdysone
receptor-based inducible gene expression system, novel receptor
polynucleotides and polypeptides for use in the novel inducible
gene expression system, and methods of modulating the expression of
a gene within a host cell using this inducible gene expression
system. In particular, Applicants' invention relates to an improved
gene expression modulation system comprising a polynucleotide
encoding a receptor polypeptide comprising a truncation
mutation.
[0017] Specifically, the present invention relates to a gene
expression modulation system comprising: a) a first gene expression
cassette that is capable of being expressed in a host cell
comprising a polynucleotide that encodes a first polypeptide
comprising: i) a DNA-binding domain that recognizes a response
element associated with a gene whose expression is to be modulated;
and ii) a ligand binding domain comprising a ligand binding domain
from a nuclear receptor; and b) a second gene expression cassette
that is capable of being expressed in the host cell comprising a
polynucleotide sequence that encodes a second polypeptide
comprising: i) a transactivation domain; and ii) a ligand binding
domain comprising a ligand binding domain from a nuclear receptor
other than an ultraspiracle receptor; wherein the DNA binding
domain and the transactivation domain are from a polypeptide other
than an ecdysone receptor, a retinoid X receptor, or an
ultraspiracle receptor, wherein the ligand binding domains from the
first polypeptide and the second polypeptide are different and
dimerize.
[0018] In a specific embodiment, the ligand binding domain of the
first polypeptide comprises an ecdysone receptor (EcR) ligand
binding domain In another specific embodiment, the ligand binding
domain of the second polypeptide comprises a retinoid X receptor
(RXR) ligand binding domain.
[0019] In a preferred embodiment, the ligand binding domain of the
first polypeptide comprises an ecdysone receptor ligand binding
domain and the ligand binding domain of the second polypeptide
comprises a retinoid X receptor ligand binding domain.
[0020] The present invention also relates to a gene expression
modulation system according to the invention further comprising c)
a third gene expression cassette comprising: i) a response element
to which the DNA-binding domain of the first polypeptide binds; ii)
a promoter that is activated by the transactivation domain of the
second polypeptide; and iii) the gene whose expression is to be
modulated.
[0021] The present invention also relates to an isolated
polynucleotide encoding a truncated EcR or a truncated RXR
polypeptide, wherein the truncation mutation affects ligand binding
activity or ligand sensitivity.
[0022] In particular, the present invention relates to an isolated
polynucleotide encoding a truncated EcR or a truncated RXR
polypeptide comprising a truncation mutation that reduces ligand
binding activity or ligand sensitivity of said EcR or RXR
polypeptide. In a specific embodiment, the present invention
relates to an isolated polynucleotide encoding a truncated EcR or a
truncated RXR polypeptide comprising a truncation mutation that
reduces steroid binding activity or steroid sensitivity of said EcR
or RXR polypeptide. In another specific embodiment, the present
invention relates to an isolated polynucleotide encoding a
truncated EcR or a truncated RXR polypeptide comprising a
truncation mutation that reduces non-steroid binding activity or
non-steroid sensitivity of said EcR or RXR polypeptide.
[0023] The present invention also relates to an isolated
polynucleotide encoding a truncated EcR or a truncated RXR
polypeptide comprising a truncation mutation that enhances ligand
binding activity or ligand sensitivity of said EcR or RXR
polypeptide. In a specific embodiment, the present invention
relates to an isolated polynucleotide encoding a truncated EcR or a
truncated RXR polypeptide comprising a truncation mutation that
enhances steroid binding activity or steroid sensitivity of said
EcR or RXR polypeptide. In another specific embodiment, the present
invention relates to an isolated polynucleotide encoding a
truncated EcR or a truncated RXR polypeptide comprising a
truncation mutation that enhances non-steroid binding activity or
non-steroid sensitivity of said EcR or RXR polypeptide.
[0024] The present invention also relates to an isolated
polynucleotide encoding a truncated RXR polypeptide comprising a
truncation mutation that increases ligand sensitivity of a
heterodimer comprising the truncated retinoid X receptor
polypeptide and a dimerization partner. In a specific embodiment,
the dimerization partner is an ecdysone receptor polypeptide.
[0025] The present invention also relates to an isolated
polypeptide encoded by a polynucleotide according to Applicants'
invention. In particular, the present invention relates to an
isolated truncated EcR or truncated RXR polypeptide comprising a
truncation mutation, wherein the EcR or RXR polypeptide is encoded
by a polynucleotide according to the invention.
[0026] Thus, the present invention also relates to an isolated
truncated EcR or truncated RXR polypeptide comprising a truncation
mutation that affects ligand binding activity or ligand sensitivity
of said EcR or RXR polypeptide.
[0027] Applicants' invention also relates to methods of modulating
gene expression in a host cell using a gene expression modulation
system according to the invention. Specifically, Applicants'
invention provides a method of modulating the expression of a gene
in a host cell comprising the gene to be modulated comprising the
steps of: a) introducing into the host cell a gene expression
modulation system according to the invention; and b) introducing
into the host cell a ligand that independently combines with the
ligand binding domains of the first polypeptide and the second
polypeptide of the gene expression modulation system; wherein the
gene to be expressed is a component of a chimeric gene comprising:
i) a response element comprising a domain to which the DNA binding
domain from the first polypeptide binds; ii) a promoter that is
activated by the transactivation domain of the second polypeptide;
and iii) the gene whose expression is to be modulated, whereby a
complex is formed comprising the ligand, the first polypeptide, and
the second polypeptide, and whereby the complex modulates
expression of the gene in the host cell.
[0028] Applicants' invention also provides an isolated host cell
comprising an inducible gene expression system according to the
invention. The present invention also relates to an isolated host
cell comprising a polynucleotide or polypeptide according to the
invention Accordingly, Applicants' invention also relates to a
non-human organism comprising a host cell according to the
invention
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a Gal4
DBD-CfEcRDEF chimeric polypeptide and a second gene expression
cassette encoding a VP16AD-MmRXRDEF chimeric polypeptide; prepared
as described in Example 1 (switch 1.1).
[0030] FIG. 2: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a Gal4
DBD-CfEcRDEF chimeric polypeptide and a second gene expression
cassette encoding a VP16AD-CfUSPDEF chimeric polypeptide; prepared
as described in Example 1 (switch 1.2).
[0031] FIG. 3: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a Gal4
DBD-MmRXRDEF chimeric polypeptide and a second gene expression
cassette encoding a VP16AD-CfEcRCDEF chimeric polypeptide; prepared
as described in Example 1 (switch 1.3).
[0032] FIG. 4: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a Gal4
DBD-MmRXRDEF chimeric polypeptide and a second gene expression
cassette encoding a VP16AD-DmEcRCDEF chimeric polypeptide; prepared
as described in Example 1 (switch 1.4).
[0033] FIG. 5: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a Gal4
DBD-CfUSPDEF chimeric polypeptide and a second gene expression
cassette encoding a VP16AD-CfEcRCDEF chimeric polypeptide; prepared
as described in Example 1 (switch 1.5).
[0034] FIG. 6: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a Gal4
DBD-CfEcRDEF-VP16AD chimeric polypeptide; prepared as described in
Example 1 (switch 1.6).
[0035] FIG. 7: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a
VP16AD-CfEcRCDEF chimeric polypeptide; prepared as described in
Example 1 (switch 1.7).
[0036] FIG. 8: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a
VP16AD-DmEcRCDEF chimeric polypeptide and a second gene expression
cassette encoding a MmRXR polypeptide; prepared as described in
Example 1 (switch 1.8).
[0037] FIG. 9: An ecdysone receptor-based gene expression system
comprising a first gene expression cassette encoding a
VP16AD-CfEcRCDEF chimeric polypeptide and a second gene expression
cassette encoding a XR polypeptide; prepared as described in
Example 1 (switch 1.9).
[0038] FIG. 10: An ecdysone receptor-based gene expression system
comprising a gene expression cassette encoding a Gal4 DBD-CfEcRCDEF
chimeric polypeptide; prepared as described in Example 1 (switch
1.10).
[0039] FIG. 11: Expression data of GAIACfEcRA/BCDEF, GAL4CfEcRCDEF,
GAL4CfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAL4CfEcRDE truncation
mutants transfected into NIH3T3 cells along with VP16MmRXRDE,
pFRLUc and pTKRL plasmid DNAs.
[0040] FIG. 12: Expression data of GAIACfEcRA/BCDEF, GAL4CfEcRCDEF,
GALACfEcR1/2CDEF, GAL4CfEcRDEF, GAL4CfEcREF, GAIACfEcRDE truncation
mutants transfected into 3T3 cells along with VP16MmRXRE, pFRLUc
and pTKRL plasmid DNAs.
[0041] FIG. 13: Expression data of VP16MmRXRA/BCDEF, VP16mMRXRCDEF,
VP16mRXRDEF, VP16 MmRXREF, VP16MmRXRBam-EF, VP16MmRXRAF2del
constructs transfected into NIH3T3 cells along with GAL4CfEcRCDEF,
pFRLUc and pTKRL plasmid DNAs.
[0042] FIG. 14: Expression data of VP16MmRXRCDEF, VP16MmRXRCDEF,
VP16MmRXRDEF, VP16MmRXREF, VP16MmRXRBam-EF, VP16AF2del constructs
transfected into NIH3T3 cells along with GAL4CfEcRDEF, pFRLUc and
pTKRL plasmid DNAs.
[0043] FIG. 15: Expression data of various truncated CfEcR and
MmRXR receptor pairs transfected into NIH3T3 cells along with
GAL4CfEcRDEF, pFRLUc and pTKRL plasmid DNAs.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Applicants have now developed an improved ecdysone
receptor-based inducible gene expression system comprising a
truncation mutant of an ecdysone receptor or a retinoid X receptor
(RXR) polypeptide that affects ligand binding activity or ligand
sensitivity. This mutational effect may increase or reduce ligand
binding activity or ligand sensitivity and may be steroid or
non-steroid specific. Thus, Applicants' invention provides an
improved ecdysone receptor-based inducible gene expression system
useful for modulating expression of a gene of interest in a host
cell. In a particularly desirable embodiment, Applicants' invention
provides an inducible gene expression system that has a reduced
level of background gene expression and responds to submicromolar
concentrations of non-steroidal ligand. Thus, Applicants' novel
inducible gene expression system and its use in methods of
modulating gene expression in a host cell overcome the limitations
of currently available inducible expression systems and provide the
skilled artisan with an effective means to control gene
expression.
[0045] The present invention provides a novel inducible gene
expression system that can be used to modulate gene expression in
both prokaryotic and eukaryotic host cells. Applicants' invention
is useful for applications such as gene therapy, large scale
production of proteins and antibodies, cell-based high throughput
screening assays, functional genomics and regulation of traits in
transgenic organisms.
[0046] Definitions
[0047] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided and should be helpful
in understanding the scope and practice of the present
invention.
[0048] In a specific embodiment, the term "about" or
"approximately" means within 20%, preferably within 10%, more
preferably within 5%, and even more preferably within 1% of a given
value or range.
[0049] The term "substantially free" means that a composition
comprising "A" (where "A" is a single protein, DNA molecule,
vector, reconbinant host cell, etc.) is substantially free of "B"
(where "B" comprises one or more contaminating proteins, DNA
molecules, vectors, etc.) when at least about 75% by weight of the
proteins, DNA, vectors (depending on the category of species to
which A and B belong) in the composition is "A". Preferably, "A"
comprises at least about 90% by weight of the A+B species in the
composition, most preferably at least about 99% by weight. It is
also preferred that a composition, which is substantially free of
contamination, contain only a single molecular weight species
having the activity or characteristic of the species of
interest.
[0050] The term "isolated" for the purposes of the present
invention designates a biological material (nucleic acid or
protein) that has been removed from its original environment (the
environment in which it is naturally present).
[0051] For example, a polynucleotide present in the natural state
in a plant or an animal is not isolated. The same polynucleotide
separated from the adjacent nucleic acids in which it is naturally
present. The term "purified" does not require the material to be
present in a form exhibiting absolute purity, exclusive of the
presence of other compounds. It is rather a relative
definition.
[0052] A polynucleotide is in the "purified" state after
purification of the starting material or of the natural material by
at least one order of magnitude, preferably 2 or 3 and preferably 4
or 5 orders of magnitude.
[0053] A "nucleic acid" is a polymeric compound comprised of
covalently linked subunits called nucleotides. Nucleic acid
includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid
(DNA), both of which may be single-stranded or double-stranded. DNA
includes but is not limited to cDNA, genomic DNA, plasmids DNA,
synthetic DNA, and semi-synthetic DNA. DNA may be linear, circular,
or supercoiled.
[0054] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine; "RNA molecules") or deoxynbonucleosides (deoxyadenosine,
deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"),
or any phosphoester anologs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alia, in linear or circular DNA molecules (e.g., restriction
fragments), plasmids, and chromosomes. In discussing the structure
of particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA). A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
[0055] The term "fragment" will be understood to mean a nucleotide
sequence of reduced length relative to the reference nucleic acid
and comprising, over the common portion, a nucleotide sequence
identical to the reference nucleic acid. Such a nucleic acid
fragment according to the invention may be, where appropriate,
included in a larger polynucleotide of which it is a constituent.
Such fragments comprise, or alternatively consist of,
oligonucleotides ranging in length from at least 8, 10, 12, 15, 18,
20 to 25, 30, 40, 50, 70, 80, 100, 200, 500, 1000 or 1500
consecutive nucleotides of a nucleic acid according to the
invention.
[0056] As used herein, an "isolated nucleic acid fragment" is a
polymer of RNA or DNA that is single- or double-stranded,
optionally containng synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0057] A "gene" refers to an assembly of nucleotides that encode a
polypeptide, and includes cDNA and genomic DNA nucleic acids.
"Gene" also refers to a nucleic acid fragment that expresses a
specific protein or polypeptide, including regulatory sequences
preceding (5' noncoding sequences) and following (3' non-coding
sequences) the coding sequence. "Native gene" refers to a gene as
found in nature with its own regulatory sequences. "Chimeric gene"
refers to any gene that is not a native gene, comprising regulatory
and/or coding sequences that are not found together in nature.
Accordingly, a chimeric gene may comprise regulatory sequences and
coding sequences that are derived from different sources, or
regulatory sequences and coding sequences derived from the same
source, but arranged in a manner different than that found in
nature. A chimeric gene may comprise coding sequences derived from
different sources and/or regulatory sequences derived from
different sources. "Endogenous gene" refers to a native gene in its
natural location in the genome of an organism A "foreign" gene or
"heterologous" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure.
[0058] "Heterologous" DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the
heterologous DNA includes a gene foreign to the cell.
[0059] The term "genome" includes chromosomal as well as
mitochondrial, chloroplast and viral DNA or RNA.
[0060] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al., 1989
infra). Hybridization and washing conditions are well known and
exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T.
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor (1989), particularly
Chapter 11 and Table 11.1 therein (entirely incorporated herein by
reference). The conditions of temperature and ionic strength
determine the "stringency" of the hybridization Stringency
conditions can be adjusted to screen for moderately similar
fragments, such as homologous sequences from distantly related
organisms, to highly similar fragments, such as genes that
duplicate functional enzymes from closely related organisms. For
preliminary screening for homologous nucleic acids, low stringency
hybridization conditions, corresponding to a T.sub.m of 55.degree.,
can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25% milk, and no
formamide; or 30% formamide, 5.times.SSC, 0.5% SDS). Moderate
stringency hybridization conditions correspond to a higher T.sub.m,
e.g., 40% formamide, with 5.times. or 6.times.SCC. High stringency
hybridization conditions correspond to the highest T.sub.m, e.g.,
50% formamide, 5.times. or 6.times.SCC. Hybridization requires that
the two nucleic acids contain complementary sequences, although
depending on the stringency of the hybridization, mismatches
between bases are possible.
[0061] The term "complementary" is used to describe the
relationship between nucleotide bases that are capable of
hybridizing to one another. For example, with respect to DNA,
adenosine is complementary to thymine and cytosine is complementary
to guanine. Accordingly, the instant invention also includes
isolated nucleic acid fragments that are complementary to the
complete sequences as disclosed or used herein as well as those
substantially similar nucleic acid sequences.
[0062] In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 65.degree. C.
[0063] Post-hybridization washes also determine stringency
conditions. One set of preferred conditions uses a series of washes
starting with 6.times.SSC, 0.5% SDS at room temperature for 15
minutes (min), then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 minutes, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 minutes. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C. Hybridization requires that the two nucleic
acids comprise complementary sequences, although depending on the
stringency of the hybridization mismatches between bases are
possible.
[0064] The appropriate stringency for hybridizing nucleic acids
depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m of nucleic acid hybridizations decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating T.sub.m
have been derived (see Sambrook et al., supra, 9.50-0.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides,
the position of mismatches becomes more important, and the length
of the oligonucleotide determines its specificity (see Sambrook et
al., supra, 11.7-11.8).
[0065] In one embodiment the length for a hybridizable nucleic acid
is at least about 10 nucleotides. Preferable a minimum length for a
hybridizable nucleic acid is at least about 15 nucleotides; more
preferably at least about 20 nucleotides; and most preferably the
length is at least 30 nucleotides. Furthermore, the skilled artisan
will recognize that the temperature and wash solution salt
concentration may be adjusted as necessary according to factors
such as lengh of the probe.
[0066] The term "probe" refers to a single-stranded nucleic acid
molecule that can base pair with a complementary single stranded
target nucleic acid to form a double-stranded molecule.
[0067] As used herein, the tern "oligonucleotide" refers to a
nucleic acid, generally of at least 18 nucleotides, that is
hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid
DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g.,
with .sup.32P-nucleotides or nucleotides to which a label, such as
biotin, has been covalently conjugated. A labeled oligonucleotide
can be used as a probe to detect the presence of a nucleic acid.
Oligonucleotides (one or both of which may be labeled) can be used
as PCR primers, either for cloning full length or a fragment of a
nucleic acid, or to detect the presence of a nucleic acid. An
oligonucleotide can also be used to form a triple helix with a DNA
molecule. Generally, oligonucleotides are prepared synthetically,
preferably on a nucleic acid synthesizer. Accordingly,
oligonucleotides can be prepared with non-naturally occurring
phosphoester analog bonds, such as thioester bonds, etc.
[0068] A "primer" is an oligonucleotide that hybridizes to a target
nucleic acid sequence to create a double stranded nucleic acid
region that can serve as an initiation point for DNA synthesis
under suitable conditions. Such primers may be used in a polymerase
chain reaction.
[0069] "Polymerase chain reaction" is abbreviated PCR and means an
in vitro method for enzymatically amplifying specific nucleic acid
sequences. PCR involves a repetitive series of temperature cycles
with each cycle comprising three stages: denaturation of the
template nucleic acid to separate the strands of the target
molecule, annealing a single stranded PCR oligonucleotide primer to
the template nucleic acid, and extension of the annealed primer(s)
by DNA polymerase. PCR provides a means to detect the presence of
the target molecule and, under quantitative or semi-quantitative
conditions, to determine the relative amount of that target
molecule within the starting pool of nucleic acids.
[0070] "Reverse transcription-polymerase chain reaction" is
abbreviated RT-PCR and means an in vitro method for enzymatically
producing a target cDNA molecule or molecules from an RNA molecule
or molecules, followed by enzymatic amplification of a specific
nucleic acid sequence or sequences within the target cDNA molecule
or molecules as described above. RT-PCR also provides a means to
detect the presence of the target molecule and, under quantitative
or semi-quantitative conditions, to determine the relative amount
of that target molecule within the starting pool of nucleic
acids.
[0071] A DNA "coding sequence" is a double-stranded DNA sequence
that is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. "Suitable regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, polyadenylation recognition sequences, RNA
processing site, effector binding site and stem-loop structure. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA
sequences, and even synthetic DNA sequences. If the coding sequence
is intended for expression in a eukaryotic cell, a polyadenylation
signal and transcription termination sequence will usually be
located 3' to the coding sequence.
[0072] "Open reading frame" is abbreviated ORF and means a length
of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a
translation start signal or initiation codon such as an ATG or AUG,
and a termination codon and can be potentially translated into a
polypeptide sequence.
[0073] The term "head-to-head" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a head-to-head
orientation when the 5' end of the coding strand of one
polynucleotide is adjacent to the 5' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds away from the 5' end of the other
polynucleotide. The term "head-to-head" may be abbreviated
(5')-to-(5') and may also be indicated by the symbols (.rarw.
.fwdarw.) or (3'.rarw.5'5'.fwdarw.3').
[0074] The term "tail-to-tail" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a tail-to-tail
orientation when the 3' end of the coding strand of one
polynucleotide is adjacent to the 3' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds toward the other polynucleotide. The
term "tail-to-tail" may be abbreviated (3')-to-(3') and may also be
indicated by the symbols (.fwdarw. .rarw.) or
(5'.fwdarw.3'3'.rarw.5').
[0075] The term "head-to-tail" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a head-to-tail
orientation when the 5' end of the coding strand of one
polynucleotide is adjacent to the 3' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds in the same direction as that of the
other polynucleotide. The term "head-to-tail" may be abbreviated
(5')-to-(3') and may also be indicated by the symbols (.fwdarw.
.fwdarw.) or (5'.fwdarw.3'5'.fwdarw.3').
[0076] The term "downstream" refers to a nucleotide sequence that
is located 3' to reference nucleotide sequence. In particular,
downstream nucleotide sequences generally relate to sequences that
follow the starting point of transcription. For example, the
translation initiation codon of a gene is located downstream of the
start site of transcription.
[0077] The term "upstream" refers to a nucleotide sequence that is
located 5' to reference nucleotide sequence. In particular,
upstream nucleotide sequences generally relate to sequences that
are located on the 5' side of a coding sequence or starting point
of transcription. For example, most promoters are located upstream
of the start site of transcription.
[0078] The terms "restriction endonuclease" and "restriction
enzyme" refer to an enzyme that binds and cuts within a specific
nucleotide sequence within double stranded DNA.
[0079] "Homologous recombination" refers to the insertion of a
foreign DNA sequence into another DNA molecule, e.g., insertion of
a vector in a chromosome. Preferably, the vector targets a specific
chromosomal site for homologous recombination. For specific
homologous recombination, the vector will contain sufficiently long
regions of homology to sequences of the chromosome to allow
complementary binding and incorporation of the vector into the
chromosome. Longer regions of homology, and greater degrees of
sequence similarity, may increase the efficiency of homologous
recombination.
[0080] Several methods known in the art may be used to propagate a
polynucleotide according to the invention Once a suitable host
system and growth conditions are established, recombinant
expression vectors can be propagated and prepared in quantity. As
described herein, the expression vectors which can be used include,
but are not limited to, the following vectors or their derivatives:
human or animal viruses such as vaccinia virus or adenovirus;
insect viruses such as baculovirus; yeast vectors; bacteriophage
vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name
but a few.
[0081] A "vector" is any means for the cloning of and/or transfer
of a nucleic acid into a host cell. A vector may be a replicon to
which another DNA segment may be attached so as to bring about the
replication of the attached segment. A "replicon" is any genetic
element (e.g., plasmid, phage, cosmid, chromosome, virus) that
functions as an autonomous unit of DNA replication in vivo, i.e.,
capable of replication under its own control. The term "vector"
includes both viral and nonviral means for introducing the nucleic
acid into a cell in vitro, ex vivo or in vivo. A large nunber of
vectors known in the art may be used to manipulate nucleic acids,
incorporate response elements and promoters into genes, etc.
Possible vectors include, for example, plasmids or modified viruses
including, for example bacteriophages such as lambda derivatives,
or plasmids such as PBR322 or pUC plasmid derivatives, or the
Bluescript vector. For example, the insertion of the DNA fragments
corresponding to response elements and promoters into a suitable
vector can be accomplished by ligating the appropriate DNA
fragments into a chosen vector that has complementary cohesive
termini. Alternatively, the ends of the DNA molecules may be
enzymatically modified or any site may be produced by ligating
nucleotide sequences (linkers) into the DNA termini. Such vectors
may be engineered to contain selectable marker genes that provide
for the selection of cells that have incorporated the marker into
the cellular genome. Such markers allow identification and/or
selection of host cells that incorporate and express the proteins
encoded by the marker.
[0082] Viral vectors, and particularly retroviral vectors, have
been used in a wide variety of gene delivery applications in cells,
as well as living animal subjects. Viral vectors that can be used
include but are not limited to retrovirus, adeno-associated virus,
pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr,
adenovirus, geminivirus, and caulimovirus vectors. Non-viral
vectors include plasmids, liposomes, electrically charged lipids
(cytofectins), DNAprotein complexes, and biopolymers. In addition
to a nucleic acid, a vector may also comprise one or more
regulatory regions, and/or selectable markers useful in selecting,
measuring, and monitoring nucleic acid transfer results (transfer
to which tissues, duration of expression, etc.).
[0083] The term "plasmid" refers to an extra chromosomal element
often carrying a gene that is not part of the central metabolism of
the cell, and usually in the form of circular double-stranded DNA
molecules. Such elements may be autonomously replicating sequences,
genome integrating sequences, phage or nucleotide sequences,
linear, circular, or supercoiled, of a single- or double-stranded
DNA or RNA, derived from any source, in which a number of
nucleotide sequences have been joined or recombined into a unique
construction which is capable of introducing a promoter fragment
and DNA sequence for a selected gene product along with appropriate
3' untranslated sequence into a cell.
[0084] A "cloning vector" is a "replicon", which is a unit length
of a nucleic acid, preferably DNA, that replicates sequentially and
which comprises an origin of replication, such as a plasmid, phage
or cosmid, to which another nucleic acid segment may be attached so
as to bring about the replication of the attached segment. Cloning
vectors may be capable of replication in one cell type and
expression in another ("shuttle vector").
[0085] Vectors may be introduced into the desired host cells by
methods known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, lipofection (lysosome fusion), use of a
gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992,
J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem.
263:14621-14624; and Hartmut et al., Canadian Patent Application
No. 2,012,311, filed Mar. 15, 1990).
[0086] A polynucleotide according to the invention can also be
introduced in vivo by lipofection. For the past decade, there has
been increasing use of liposomes for encapsulation and transfection
of nucleic acids in vitro. Synthetic cationic lipids designed to
limit the difficulties and dangers encountered with liposome
mediated transfection can be used to prepare liposomes for in vivo
transfection of a gene encoding a marker (Felgner et al., 1987.
PNAS 84:7413; Mackey, et al., 1988. Proc. Natl. Acad. Sci. U.S.A.
85:8027-8031; and Ulmer et al., 1993. Science 259:1745-1748). The
use of cationic lipids may promote encapsulation of negatively
charged nucleic acids, and also promote fusion with negatively
charged cell membranes (Felgner and Ringold, 1989. Science
337:387-388). Particularly useful lipid compounds and compositions
for transfer of nucleic acids are described in International Patent
Publications WO95/18863 and WO96/17823, and in U.S. Pat. No.
5,459,127. The use of lipofection to introduce exogenous genes into
the specific organs in vivo has certain practical advantages.
Molecular targeting of liposomes to specific cells represents one
area of benefit. It is clear that directing transfection to
particular cell types would be particularly preferred in a tissue
with cellular heterogeneity, such as pancreas, liver, kidney, and
the brain. Lipids may be chemically coupled to other molecules for
the purpose of targeting (Mackey, et al., 1988, supra). Targeted
peptides, e.g., hormones or neurotransmitters, and proteins such as
antibodies, or non-peptide molecules could be coupled to liposomes
chemically.
[0087] Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as a cationic
oligopeptide (e.g., WO95/21931), peptides derived from DNA binding
proteins (e.g., WO96/25508), or a cationic polymer (e.g.,
WO95/21931).
[0088] It is also possible to introduce a vector in vivo as a naked
DNA plasmid (see U.S. Pat. Nos. 5,693,622,5,589,466 and 5,580,859).
Receptor-mediated DNA delivery approaches can also be used (Curiel
et al., 1992. Hum. Gene Ther. 3:147-154; and Wu and Wu, 1987. J.
Biol. Chem. 262:4429-4432).
[0089] The term "transfection" means the uptake of exogenous or
heterologous RNA or DNA by a cell. A cell has been "transfected" by
exogenous or heterologous RNA or DNA when such RNA or DNA has been
introduced inside the cell. A cell has been "transformed" by
exogenous or heterologous RNA or DNA when the transfected RNA or
DNA effects a phenotypic change. The transforming RNA or DNA can be
integrated (covalently linked) into chromosomal DNA making up the
genome of the cell.
[0090] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0091] The term "genetic region" will refer to a region of a
nucleic acid molecule or a nucleotide sequence that comprises a
gene encoding a polypeptide.
[0092] In addition, the recombinant vector comprising a
polynucleotide according to the invention may include one or more
origins for replication in the cellular hosts in which their
amplification or their expression is sought, markers or selectable
markers.
[0093] The term "selectable marker" means an identifying factor,
usually an antibiotic or chemical resistance gene, that is able to
be selected for based upon the marker gene's effect, i.e.,
resistance to an antibiotic, resistance to a herbicide,
colorimetric markers, enzymes, fluorescent markers, and the like,
wherein the effect is used to track the inheritance of a nucleic
acid of interest and/or to identify a cell or organism that has
inherited the nucleic acid of interest. Examples of selectable
marker genes known and used in the art include: genes providing
resistance to ampicillin, streptomycin, gentamycin, kanamycin,
hygromycin, bialaphos herbicide, sulfonamide, and the like; and
genes that are used as phenotypic markers, i.e., anthocyanin
regulatory genes, isopentanyl transferase gene, and the like.
[0094] The term "reporter gene" means a nucleic acid encoding an
identifying factor that is able to be identified based upon the
reporter gene's effect, wherein the effect is used to track the
inheritance of a nucleic acid of interest, to identify a cell or
organism that has inherited the nucleic acid of interest, and/or to
measure gene expression induction or transcription. Examples of
reporter genes known and used in the art include: luciferase (Luc),
green fluorescent protein (GFP), chloramphenicol acetyltransferase
(CAT), .beta.-galactosidase (LacZ), .beta.-glucuronidase (Gus), and
the like. Selectable marker genes may also be considered reporter
genes.
[0095] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. In general,
a coding sequence is located 3' to a promoter sequence. Promoters
may be derived in their entirety from a native gene, or be composed
of different elements derived from different promoters found in
nature, or even comprise synthetic DNA segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental or physiological conditions. Promoters that cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters". Promoters that cause a
gene to be expressed in a specific cell type are commonly referred
to as "cell-specific promoters" or "tissue-specific promoters".
Promoters that cause a gene to be expressed at a specific stage of
development or cell differentiation are commonly referred to as
"developmentally-specific promoters" or "cell
differentiation-specific promoters". Promoters that are induced and
cause a gene to be expressed following exposure or treatment of the
cell with an agent, biological molecule, chemical, ligand, light,
or the like that induces the promoter are commonly referred to as
"inducible promoters" or "regulatable promoters". It is further
recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, DNA
fragments of different lengths may have identical promoter
activity.
[0096] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined for example, by
mapping with nuclease S1), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0097] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-RNA
spliced (if the coding sequence contains introns) and translated
into the protein encoded by the coding sequence.
[0098] "Transcriptional and translational control sequences" are
DNA regulatory sequences, such as promoters, enhancers,
terminators, and the like, that provide for the expression of a
coding sequence in a host cell. In eukaryotic cells,
polyadenylation signals are control sequences.
[0099] The term "response element" means one or more cis-acting DNA
elements which confer responsiveness on a promoter mediated through
interaction with the DNA-binding domains of the first chimeric
gene. This DNA element may be either palindromic (perfect or
imperfect) in its sequence or composed of sequence motifs or half
sites separated by a variable number of nucleotides. The half sites
can be similar or identical and arranged as either direct or
inverted repeats or as a single half site or multimers of adjacent
half sites in tandem. The response element may comprise a minimal
promoter isolated from different organisms depending upon the
nature of the cell or organism into which the response element will
be incorporated. The DNA binding domain of the first hybrid protein
binds, in the presence or absence of a ligand, to the DNA sequence
of a response element to initiate or suppress transcription of
downstream gene(s) under the regulation of this response element
Examples of DNA sequences for response elements of the natural
ecdysone receptor include: RRGG/TTCANTGAC/ACYY (see Cherbas L., et.
al., (1991), Genes Dev. 5, 120-131); AGGTCAN.sub.(n)AGGTCA, where
N.sub.(n) can be one or more spacer nucleotides (see D'Avino P P.,
et. al., (1995), Mol. Cell. Endocrinol, 113, 1-9); and
GGGTTGAATGAATTT (see Antoniewski C., et. al., (1994). Mol. Cell
Biol. 14, 4465-4474).
[0100] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0101] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from a nucleic acid or polynucleotide. Expression may
also refer to translation of mRNA into a protein or
polypeptide.
[0102] The terms "cassette", "expression cassette" and "gene
expression cassette" refer to a segment of DNA that can be inserted
into a nucleic acid or polynucleotide at specific restriction sites
or by homologous recombination. The segment of DNA comprises a
polynucleotide that encodes a polypeptide of interest, and the
cassette and restriction sites are designed to ensure insertion of
the cassette in the proper reading frame for transcription and
translation. "Transformation cassette" refers to a specific vector
comprising a polynucleotide that encodes a polypeptide of interest
and having elements in addition to the polynucleotide that
facilitate transformation of a particular host cell. Cassettes,
expression cassettes, gene expression cassettes and transformation
cassettes of the invention may also comprise elements that allow
for enhanced expression of a polynucleotide encoding a polypeptide
of interest in a host cell. These elements may include, but are not
limited to: a promoter, a minimal promoter, an enhancer, a response
element, a terminator sequence, a polyadenylation sequence, and the
like.
[0103] For purposes of this invention, the term "gene switch"
refers to the combination of a, response element associated with a
promoter, and an EcR based system which, in the presence of one or
more ligands, modulates the expression of a gene into which the
response element and promoter are incorporated.
[0104] The terms "modulate" and "modulates" mean to induce, reduce
or inhibit nucleic acid or gene expression, resulting in the
respective induction, reduction or inhibition of protein or
polypeptide production.
[0105] The plasmids or vectors according to the invention may
further comprise at least one promoter suitable for driving
expression of a gene in a host cell. The term "expression vector"
means a vector, plasmid or vehicle designed to enable the
expression of an inserted nucleic acid sequence following
transformation into the host The cloned gene, i.e., the inserted
nucleic acid sequence, is usually placed under the control of
control elements such as a promoter, a minimal promoter, an
enhancer, or the like. Initiation control regions or promoters,
which are useful to drive expression of a nucleic acid in the
desired host cell are numerous and familiar to those skilled in the
art. Virtually any promoter capable of driving these genes is
suitable for the present invention including but not limited to:
viral promoters, plant promoters, bacterial promoters, animal
promoters, mammalian promoters, synthetic promoters, constitutive
promoters, tissue specific promoter, developmental specific
promoters, inducible promoters, light regulated promoters; CYC1,
HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3,
LEU2, ENO, TPI, alkaline phosphatase promoters (useful for
expression in Saccharomyces); AOX1 promoter (useful for expression
in Pichia); b-lactamase, lac, ara, tet, tryp, lP.sub.L, lP.sub.R,
T7, tac, and trc promoters (useful for expression in Escherichia
coli); and light regulated-, seed specific-, pollen specific-,
ovary specific-, pathogenesis or disease related-, cauliflower
mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus
(CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate
carboxylase, shoot-specific, root specific, chitinase, stress
inducible, rice tungro bacilliform virus, plant superpromoter,
potato leucine aminopeptidase, nitrate reductase, mannopine
synthase, nopaline synthase, ubiquitin, zein protein, and
anthocyanin promoters (useful for expression in plant cells);
animal and mammalian promoters known in the art include, but are
not limited to, the SV40 early (SV40e) promoter region, the
promoter contained in the 3' long termnal repeat (LTR) of Rous
sarcoma virus (RSV), the promoters of the E1A or major late
promoter (MLP) genes of adenoviruses, the cytomegalovirus early
promoter, the herpes simplex virus (HSV) thymidine kinase (TK)
promoter, an elongation factor 1 alpha (EF1) promoter, a
phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter,
an albumin promoter, the regulatory sequences of the mouse
metallothionein-L promoter, and transcriptional control regions,
the ubiquitous promoters (HPRT, vimentin, .alpha.-actin, tubulin
and the like), the promoters of the intermediate filaments (desmin,
neurofilaments, keratin, GFAP, and the like), the promoters of
therapeutic genes (of the MDR, CFTR or factor VIII type, and the
like), and promoters that exhibit tissue specificity and have been
utilized in transgenic animals, such as the elastase I gene control
region which is active in pancreatic acinar cells; insulin gene
control region active in pancreatic beta cells, immunoglobulin gene
control region active in lymphoid cells, mouse mammary tumor virus
control region active in testicular, breast, lymphoid and mast
cells; albumin gene, Apo AI and Apo AII control regions active in
liver, alpha-fetoprotein gene control region active in liver, alpha
1-antitrypsin gene control region active in the liver, beta-globin
gene control region active in myeloid cells, myelin basic protein
gene control region active in oligodendrocyte cells in the brain,
myosin light chain-2 gene control region active in skeletal muscle,
and gonadotropic releasing hormone gene control region active in
the hypothalamus, pyruvate kinase promoter, villin promoter,
promoter of the fatty acid binding intestinal protein, promoter of
the smooth muscle cell .alpha.-actin, and the like. In a preferred
embodiment of the invention, the promoter is selected from the
group consisting of a cauliflower mosaic virus .sup.35S promoter, a
cassaya vein mosaic virus promoter, and a cauliflower mosaic virus
.sup.35S minimal promoter, an elongation factor 1 alpha (EF1)
promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin
(Ubc) promoter, and an albumin promoter. In addition, these
expression sequences may be modified by addition of enhancer or
regulatory sequences and the like.
[0106] Enhancers that may be used in embodiments of the invention
include but are not limited to: tobacco mosaic virus enhancer,
cauliflower mosaic virus .sup.35S enhancer, tobacco etch virus
enhancer, ribulose 1,5-bisphosphate carboxylase enhancer, rice
tungro bacilliform virus enhancer, and other plant and viral gene
enhancers, and the like.
[0107] Termination control regions, i.e., terminator or
polyadenylation sequences, may also be derived from various genes
native to the preferred hosts. Optionally, a termination site may
be unnecessary, however, it is most preferred if included. In a
preferred embodiment of the invention, the termination control
region may be comprise or be derived from a synthetic sequence,
synthetic polyadenylation signal, an SV40 late polyadenylation
signal, an SV40 polyadenylation signal, a bovine growth hormone
(BGH) polyadenylation signal, nopaline synthase (nos), cauliflower
mosaic virus (CaMV), octopine synthase (ocs), Agrocateum, viral,
and plant terminator sequences, or the like.
[0108] The terms "3' non-coding sequences" or "3' untranslated
region (UTR)" refer to DNA sequences located downstream (3') of a
coding sequence and may comprise polyadenylation [poly(A)]
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor.
[0109] "Regulatory region" means a nucleic acid sequence which
regulates the expression of a second nucleic acid sequence. A
regulatory region may include sequences which are naturally
responsible for expressing a particular nucleic acid (a homologous
region) or may include sequences of a different origin that are
responsible for expressing different proteins or even synthetic
proteins (a heterologous region). In particular, the sequences can
be sequences of prokaryotic, eukaryotic, or viral genes or derived
sequences that stimulate or repress transcription of a gene in a
specific or non-specific manner and in an inducible or
non-inducible manner. Regulatory regions include origins of
replication, RNA splice sites, promoters, enhancers,
transcriptional termination sequences, and signal sequences which
direct the polypeptide into the secretory pathways of the target
cell.
[0110] A regulatory region from a "heterologous source" is a
regulatory region that is not naturally associated with the
expressed nucleic acid. Included among the heterologous regulatory
regions are regulatory regions from a different species, regulatory
regions from a different gene, hybrid regulatory sequences, and
regulatory sequences which do not occur m nature, but which are
designed by one having ordinary skill in the art.
[0111] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA (mRNA)" refers to the RNA that is without introns and that can
be translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene. The complementarity of an antisense RNA may be with
any part of the specific gene transcript, i.e., at the 5'
non-coding sequence, 3' non-coding sequence, or the coding
sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA,
or other RNA that is not translated yet has an effect on cellular
processes.
[0112] A "polypeptide" is a polymeric compound comprised of
covalently linked amino acid residues. Amino acids have the
following general structure: 1
[0113] Amino acids are classified into seven groups on the basis of
the side chain R: (1) aliphatic side chains, (2) side chains
containing a hydroxylic (OH) group, (3) side chains containing
sulfur atoms, (4) side chains containing an acidic or amide group,
(5) side chains containing a basic group, (6) side chains
containing an aromatic ring, and (7) proline, an imino acid in
which the side chain is fused to the amino group. A polypeptide of
the invention preferably comprises at least about 14 amino
acids.
[0114] A "protein" is a polypeptide that performs a structural or
functional role in a living cell.
[0115] An "isolated polypeptide" or "isolated protein" is a
polypeptide or protein that is substantially free of those
compounds that are normally associated therewith in its natural
state (e.g., other proteins or polypeptides, nucleic acids,
carbohydrates, lipids). "Isolated" is not meant to exclude
artificial or synthetic mixtures with other compounds, or the
presence of impurities which do not interfere with biological
activity, and which may be present, for example, due to incomplete
purification, addition of stabilizers, or compounding into a
pharmaceutically acceptable preparation.
[0116] "Fragment" of a polypeptide according to the invention will
be understood to mean a polypeptide whose amino acid sequence is
shorter than that of the reference polypeptide and which comprises,
over the entire portion with these reference polypeptides, an
identical amino acid sequence. Such fragments may, where
appropriate, be included in a larger polypeptide of which they are
a part. Such fragments of a polypeptide according to the invention
may have a length of 10, 15, 20, 30 to 40, 50, 100, 200 or 300
amino acids.
[0117] A "variant" of a polypeptide or protein is any analogue,
fragment, derivative, or mutant which is derived from a polypeptide
or protein and which retains at least one biological property of
the polypeptide or protein. Different variants of the polypeptide
or protein may exist in nature. These variants may be allelic
variations characterized by differences in the nucleotide sequences
of the structural gene coding for the protein, or may involve
differential splicing or post-translational modification. The
skilled artisan can produce variants having single or multiple
amino acid substitutions, deletions, additions, or replacements.
These variants may include, inter alia: (a) variants in which one
or more amino acid residues are substituted with conservative or
non-conservative amino acids, (b) variants in which one or more
amino acids are added to the polypeptide or protein, (c) variants
in which one or more of the amino acids includes a substituent
group, and (d) variants in which the polypeptide or protein is
fused with another polypeptide such as serum albumin. The
techniques for obtaining these variants, including genetic
(suppressions, deletions, mutations, etc.), chemical, and enzymatic
techniques, are known to persons having ordinary skill in the art.
A variant polypeptide preferably comprises at least about 14 amino
acids.
[0118] A "heterologous protein" refers to a protein not naturally
produced in the cell.
[0119] A "mature protein" refers to a post-translationally
processed polypeptide; i.e., one from which any pre- or propeptides
present in the primary translation product have been removed.
"Precursor" protein refers to the primary product of translation of
mRNA; i.e., with pre- and propeptides still present. Pre- and
propeptides may be but are not limited to intracellular
localization signals.
[0120] The term "signal peptide" refers to an amino terminal
polypeptide preceding the secreted mature protein. The signal
peptide is cleaved from and is therefore not present in the mature
protein. Signal peptides have the function of directing and
translocating secreted proteins across cell membranes. Signal
peptide is also referred to as signal protein.
[0121] A "signal sequence" is included at the beginning of the
coding sequence of a protein to be expressed on the surface of a
cell. This sequence encodes a signal peptide, N-terminal to the
mature polypeptide, that directs the host cell to translocate the
polypeptide. The term "translocation signal sequence" is used
herein to refer to this sort of signal sequence. Translocation
signal sequences can be found associated with a variety of proteins
native to eukaryotes and prokaryotes, and are often functional in
both types of organisms.
[0122] The term "hornology" refers to the percent of identity
between two polynucleotide or two polypeptide moieties. The
correspondence between the sequence from one moiety to another can
be determined by techniques known to the art. For example, homology
can be determined by a direct comparison of the sequence
information between two polypeptide molecules by aligning the
sequence information and using readily available computer programs.
Alternatively, homology can be determined by hybridization of
polynucleotides under conditions that form stable duplexes between
homologous regions, followed by digestion with
single-stranded-specific nuclease(s) and size determination of the
digested fragments.
[0123] As used herein, the term "homologous" in all its grammatical
forms and spelling variations refers to the relationship between
proteins that possess a "common evolutionary origin," including
proteins from superfamilies (e.g., the immunoglobulin superfamily)
and homologous proteins from different species (e.g., myosin light
chain, etc.) (Reeck et al., 1987, Cell 50:667.). Such proteins (and
their encoding genes) have sequence homology, as reflected by their
high degree of sequence similarity.
[0124] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of
proteins that may or may not share a common evolutionary origin
(see Reeck et al., 1987, Cell 50:667). As used herein, the term
"homologous" in all its grammatical forms and spelling variations
refers to the relationship between proteins that possess a "common
evolutionary origin," including proteins from superfamilies and
homologous proteins from different species (Reeck et al., supra).
Such proteins (and their encoding genes) have sequence homology, as
reflected by their high degree of sequence similarity. However, in
common usage and in the instant application, the term "homologous,"
when modified with an adverb such as "highly," may refer to
sequence similarity and not a common evolutionary origin.
[0125] In a specific embodiment, two DNA sequences are
"substantially homologous" or "substantially similar" when at least
about 50% (preferably at least about 75%, and most preferably at
least about 90 or 95To) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Sambrook et al., 1989, supra.
[0126] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the protein encoded by the DNA
sequence. "Substantially similar" also refers to nucleic acid
fragments wherein changes in one or more nucleotide bases does not
affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by antisense or co-suppression
technology. "Substantially similar" also refers to modifications of
the nucleic acid fragments of the instant invention such as
deletion or insertion of one or more nucleotide bases that do not
substantially affect the functional properties of the resulting
transcript. It is therefore understood that the invention
encompasses more than the specific exemplary sequences. Each of the
proposed modifications is well within the routine skill in the art,
as is determination of retention of biological activity of the
encoded products.
[0127] Moreover, the skilled artisan recognizes that substantially
similar sequences encompassed by this invention are also defined by
their ability to hybridize, under stringent conditions
(0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with
2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS), with
the sequences exemplified herein. Substantially similar nucleic
acid fragments of the instant invention are those nucleic acid
fragments whose DNA sequences are at least 70% identical to the DNA
sequence of the nucleic acid fragments reported herein. Preferred
substantially nucleic acid fragments of the instant invention are
thos nucleic acid fragments whose DNA sequences are at least 80%
identical to the DNA sequence of the nucleic acid fragments
reported herein. More preferred nucleic acid fragments are at least
90% identical to the DNA sequence of the nucleic acid fragments
reported herein. Even more preferred are nucleic acid fragments
that are at least 95% identical to the DNA sequence of the nucleic
acid fragments reported herein.
[0128] Two amino acid sequences are "substantially homologous" or
"substantially similar" when greater than about 40% of the amino
acids are identical, or greater than 60% are similar (functionally
identical). Preferably, the similar or homologous sequences are
identified by alignment using, for example, the GCG (Genetics
Computer Group, Program Manual for the GCG Package, Version 7,
Madison, Wis.) pileup program.
[0129] The term "corresponding to" is used herein to refer to
similar or homologous sequences, whether the exact position is
identical or different from the molecule to which the similarity or
homology is measured. A nucleic acid or amino acid sequence
alignment may include spaces. Thus, the term "corresponding to"
refers to the sequence similarity, and not the numbering of the
amino acid residues or nucleotide bases.
[0130] A "substantial portion" of an amino acid or nucleotide
sequence comprises enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to putatively
identify that polypeptide or gene, either by manual evaluation of
the sequence by one skilled in the art, or by computer-automated
sequence comparison and identification using algorithms such as
BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al.,
(1993) J. Mol. Biol. 215:403-410; see also www
ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more
contiguous amino acids or thirty or more nucleotides is necessary
in order to putatively identify a polypeptide or nucleic acid
sequence as homologous to a known protein or gene. Moreover, with
respect to nucleotide sequences, gene specific oligonucleotide
probes comprising 20-30 contiguous nucleotides may be used in
sequence-dependent methods of gene identification (e.g., Southern
hybridization) and isolation (e.g., in situ hybridization of
bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of 12-15 bases may be used as amplification
primers in PCR in order to obtain a particular nucleic acid
fragment comprising the primers. Accordingly, a "substantial
portion" of a nucleotide sequence comprises enough of the sequence
to specifically identify and/or isolate a nucleic acid fragment
comprising the sequence.
[0131] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects (Smith, D. W., ed.) Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,
and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press
(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,
J., eds.) Stockton Press, New York (1991). Preferred methods to
determine identity are designed to give the best match between the
sequences tested. Methods to determine identity and similarity are
codified in publicly available computer programs. Sequence
alignments and percent identity calculations may be performed using
the Megalign program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the
sequences may be performed using the Clustal method of alignment
(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default
parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default
parameters for pairwise alignments using the Clustal method may be
selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5.
[0132] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include but is not limited to the
GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol. 215:403-410 (1990), and DNASTAR
(DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA). Within
the context of this application it will be understood that where
sequence analysis software is used for analysis, that the results
of the analysis will be based on the "default values" of the
program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters which
originally load with the software when first initialized.
[0133] "Synthetic genes" can be assembled from oligonucleotide
building blocks that are chemically synthesized using procedures
known to those skilled in the art. These building blocks are
ligated and annealed to form gene segments that are then
enzymatically assembled to construct the entire gene. "Chemically
synthesized", as related to a sequence of DNA, means that the
component nucleotides were assembled in vitro. Manual chemical
synthesis of DNA may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the genes can be tailored for optimal gene expression based on
optimization of nucleotide sequence to reflect the codonbias of the
host cell. The skilled artisan appreciates the likelihood of
successful gene expression if codon usage is biased towards those
codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell where
sequence information is available.
[0134] Gene Expression Modulation System of the Invention
[0135] Applicants have now shown that separating the
transactivation and DNA binding domains by placing them on two
different proteins results in greatly reduced background activity
in the absence of a ligand and significantly increased activity
over background in the presence of a ligand. Applicants' improved
gene expression system comprises two chimeric gene expression; the
first encoding a DNA binding domain fused to a nuclear receptor
polypeptide and the second encoding a transactivation domain fused
to a nuclear receptor polypeptide. The interaction of the first
protein with the second protein effectively tethers the DNA binding
domain to the transactivation domain. Since the DNA binding and
transactivation domains reside on two different molecules, the
background activity in the absence of ligand is greatly
reduced.
[0136] In general, the inducible gene expression modulation system
of the invention comprises a) a first chimeric gene that is capable
of being expressed in a host cell comprising a polynucleotide
sequence that encodes a first hybrid polypeptide comprising i) a
DNA-binding domain that recognizes a response element associated
with a gene whose expression is to be modulated; and ii) a ligand
binding domain comprising the ligand binding domain from a nuclear
receptor; and b) a second chimeric gene that is capable of being
expressed in the host cell comprising a polynucleotide sequence
that encodes a second hybrid polypeptide comprising: i) a
transactivation domain; and ii) a ligand binding domain comprising
the ligand binding domain from a nuclear receptor other than
ultraspiracle (USP); wherein the transactivation domain are from
other than EcR, RXR, or USP; and wherein the ligand binding domains
from the first hybrid polypeptide and the second hybrid polypeptide
are different and dimerize.
[0137] This two-hybrid system exploits the ability of a pair of
interacting proteins to bring the transcription activation domain
into a more favorable position relative to the DNA binding domain
such that when the DNA binding domain binds to the DNA binding site
on the gene, the transactivation domain more effectively activates
the promoter (see, for example, U.S. Pat. No. 5,283,173). This
two-hybrid system is a significantly improved inducible gene
expression modulation system compared to the two systems disclosed
in International Patent Applications PCT/US97/05330 and
PCT/US98/14215. The ecdysone receptor-based gene expression
modulation system of the invention may be either heterodimeric and
homodimeric. A functional EcR complex generally refers to a
heterodimeric protein complex consisting of two members of the
steroid receptor family, an ecdysone receptor protein obtained from
various insects, and an ultraspiracle (USP) protein or the
vertebrate homolog of USP, retinoid X receptor protein (see Yao, et
al. (1993) Nature 366, 476-479; Yao, et al., (1992) Cell 71,
63-72). However, the complex may also be a homodimer as detailed
below. The functional ecdysteroid receptor complex may also include
additional protein(s) such as immunophilins. Additional members of
the steroid receptor family of proteins, known as transcriptional
factors (such as DHR38 or betaFTZ-1), may also be ligand dependent
or independent partners for EcR, USP, and/or RXR. Additionally,
other cofactors may be required such as proteins generally known as
coactivators (also termed adapters or mediators). These proteins do
not bind sequence-specifically to DNA and are not involved in basal
transcription. They may exert their effect on transcription
activation through various mechanisms, including stimulation of
DNA-binding of activators, by affecting chromatin structure, or by
mediating activator-initiation complex interactions. Examples of
such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,
SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the
promiscuous coactivator C response element B binding protein,
CBP/p300 (for review see Glass et al, Curr. Opin. Cell Biol.
9:222-232, 1997). Also, protein cofactors generally known as
corepressors (also known as repressors, silencers, or silencing
mediators) may be required to effectively inhibit transcriptional
activation in the absence of ligand. These corepressors may
interact with the ullliganded ecdysone receptor to silence the
activity at the response element Current evidence suggests that
binding of ligand changes the conformation of the receptor, which
results in release of the corepressor and recruitment of the above
descnbed coactivators, thereby abolishing their silencing activity.
Examples of corepressors include N-CoR and SMRT (for review, see
Horwitz et al. Mol Endocrinol. 10: 1167-1177, 1996). These
cofactors may either be endogenous within the cell or organism, or
may be added exogenously as transgenes to be expressed in either a
regulated or unregulated fashion. Homodimer complexes of the
ecdysone receptor protein, USP, or RXR may also be functional under
some circumstances.
[0138] The ecdysone receptor complex typically includes proteins
which are members of the nuclear receptor superfamily wherein all
members are characterized by the presence of an amino-terminal
transactivation domain, a DNA binding domain ("DBD"), and a ligand
binding domain ("LBD") separated from the DBD by a hinge region. As
used herein, the term "DNA binding domain" comprises a minimal
peptide sequence of a DNA binding protein, up to the entire length
of a DNA binding protein, so long as the DNA binding domain
functions to associate with a particular response element. Members
of the nuclear receptor superfamily are also characterized by the
presence of four or five domains: A/B, C, D, E, and in some members
F (see Evans, Science 240:889-895 (1988)). The "A/B" domain
corresponds to the transactivation domain, "C" corresponds to the
DNA-binding domain, "D" corresponds to the hinge region, and "E"
corresponds to the ligand binding domain. Some members of the
family may also have another transactivation domain on the
carboxy-terminal side of the LBD corresponding to "F".
[0139] The DBD is characterized by the presence of two cysteine
zinc fingers between which are two amino acid motifs, the P-box and
the D-box, which confer specificity for ecdysone response elements.
These domains may be either native, modified, or chimeras of
different domains of heterologous receptor proteins. This EcR
receptor, like a subset of the steroid receptor family, also
possesses less well defined regions responsible for
heterodimerization properties. Because the domains of EcR, USP, and
RXR are modular in nature, the LBD, DBD, and transactivation
domains may be interchanged.
[0140] Gene switch systems are known that incorporate components
from the ecdysone receptor complex. However, in these known
systems, whenever EcR is used it is associated with native or
modified DNA binding domains and transactivation domains on the
same molecule. USP or RXR are typically used as silent partners. We
have now shown that when DNA binding domains and transactivation
domains are on the same molecule the background activity in the
absence of ligand is high and that such activity is dramatically
reduced when DNA binding domains and transactivation domains are on
different molecules, that is, on each of two partners of a
heterodimeric or homodimeric complex. This two-hybrid system also
provides improved sensitivity to non-steroidal ligands for example,
diacylhydrazines, when compared to steroidal ligands for example,
ponasterone A ("PonA") or muristegone A ("MurA"). That is, when
compared to steroids, the non-steroidal ligands provide higher
activity at a lower concentration. In addition, since
transactivation based on EcR gene switches is often cell-line
dependent, it is easier to tailor switching system to obtain
maximum transactivation capability for each application.
Furthermore, this two-hybrid system avoids some side effects due to
overexpression of RXR that often occur when unmodified RXR is used
as a switching partner. In this two-hybrid system, native DNA
binding and transactivation domains of EcR or RXR are eliminated.
As a result, these chimeric molecules have less chance of
interacting with other steroid hormone receptors present in the
cell resulting in reduced side effects.
[0141] Specifically, Applicants' invention relates to a gene
expression modulation system comprising: a) a first gene expression
cassette that is capable of being expressed in a host cell, wherein
the first gene expression cassette comprises a polynucleotide that
encodes a first polypeptide comprising i) a DNA-binding domain that
recognizes a response element associated with a gene whose
expression is to be modulated; and ii) a ligand binding domain
comprising a ligand binding domain from a nuclear receptor; and b)
a second gene expression cassette that is capable of being
expressed in the host cell, wherein the second gene expression
cassette comprises a polynucleotide sequence that encodes a second
polypeptide comprising i) a transactivation domain; and ii) a
ligand binding domain comprising a ligand binding domain from a
nuclear receptor other than ultraspiracle (USP); wherein the DNA
binding domain and the transactivation domain are from other than
EcR, RXR, or USP; wherein the ligand binding domains from the first
polypeptide and the second polypeptide are different and
dimerize.
[0142] The present invention also relates to a gene expression
modulation system according to the present invention further
comprising c) a third gene expression cassette comprising: i) the
response element to which the DNA-binding domain of the first
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) the gene
whose expression is to be modulated.
[0143] In a specific embodiment, the gene whose expression is to be
modulated is a homologous gene with respect to the host cell. In
another specific embodiment, the gene whose expression is to be
modulated is a heterologous gene with respect to the host cell.
[0144] In a specific embodiment, the ligand binding domain of the
first polypeptide comprises an ecdysone receptor ligand binding
domain.
[0145] In another specific embodiment, the ligand binding domain of
the first polypeptide comprises a retinoid X receptor ligand
binding domain.
[0146] In a specific embodiment, the ligand binding domain of the
second polypeptide comprises an ecdysone receptor ligand binding
domain.
[0147] In another specific embodiment, the ligand binding domain of
the second polypeptide comprises a retinoid X receptor ligand
binding domain.
[0148] In a preferred embodiment, the ligand binding domain of the
first polypeptide comprises an ecdysone receptor ligand binding
domain, and the ligand binding domain of the second polypeptide
comprises a retinoid X receptor ligand binding domain.
[0149] In another preferred embodiment, the ligand binding domain
of the first polypeptide is from a retinoid X receptor polypeptide,
and the ligand binding domain of the second polypeptide is from an
ecdysone receptor polypeptide.
[0150] Preferably, the ligand binding domain is an EcR or RXR
related steroid/thyroid hormone nuclear receptor family member
ligand binding domain, or analogs, combinations, or modifications
thereof. More preferably, the LBD is from EcR or RXR. Even more
preferably, the LBD is from a truncated EcR or RXR. A truncation
mutation may be made by any method used in the art, including but
not limited to restriction endonuclease digestion/deletion,
PCR-mediated/oligonucleotide directed deletion, chemical
mutagenesis, UV strand breakage, and the like.
[0151] Preferably, the EcR is an insect EcR selected from the group
consisting of a Lepidopteran EcR, a Dipteran EcR, an Arthropod EcR,
a Homopteran EcR and a Hemipteran EcR. More preferably, the EcR for
use is a spruce budworm Choristoneura fumiferana EcR ("CfEcR"), a
Tenebrio molitor EcR ("TmEcR"), a Manduca sexta EcR ("MsEcR"), a
Heliothies virescens EcR ("HvEcR"), a silk moth Bombyx mori EcR
("BnEcR"), a fruit fly Drosophila melanogaster EcR ("DmEcR"), a
mosquito Aedes aegypti EcR ("AaEcR"), a blowfly Lucilia capitata
EcR ("LcEcR"), a Mediterranean fruit fly Ceratitis capitata EcR
("CcEcR"), a locust Locusta migratoria EcR ("LmEcR"), an aphid
Myzus persicae EcR ("MpEcR"), a fiddler crab Uca pugilator EcR
("UpEcR"), or an ixodid tick Amblyomma americanum EcR ("AmaEcR").
Even more preferably, the LBD is from spruce budworm (Choristoneura
fumiferana) EcR ("CfEcR") or fruit fly Drosophila melanogaster EcR
("DmEcR").
[0152] Preferably, the LBD is from a truncated insect EcR. The
insect EcR polypeptide truncation comprises a deletion of at least
1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,
210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino
acids. More preferably, the insect EcR polypeptide truncation
comprises a deletion of at least a partial polypeptide domain. Even
more preferably, the insect EcR polypeptide truncation comprises a
deletion of at least an entire polypeptide domain. In a specific
embodiment, the insect EcR polypeptide truncation comprises a
deletion of at least an A/B-domain deletion, a C-domain deletion, a
D-domain deletion, an E-domain deletion, an F-domain deletion, an
A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an
AIB/C/Ddomains deletion, an A/B/C/D/F-domains deletion, an
A/B/F-domains, and an A/B/C/F-domains deletion. A combination of
several complete and/or partial domain deletions may also be
performed.
[0153] In a preferred embodiment, the ecdysone receptor ligand
binding domain is encoded by a polynucleotide comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
[0154] In another preferred embodiment, the ecdysone receptor
ligand binding domain comprises a polypeptide sequence selected
from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. Preferably, the
RXR polypeptide is a mouse Mus musculus RXR ("MmRXR") or a human
Homo sapiens RXR ("HsRXR"). The RXR polypeptide may be an
RXR.sub..alpha., RXR.sub..beta., or RXR.sub..gamma. isoform.
[0155] Preferably, the LBD is from a truncated RXR. The RXR
polypeptide truncation comprises a deletion of at least 1, 2, 3, 4,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,
160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,
225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More
preferably, the RXR polypeptide truncation comprises a deletion of
at least a partial polypeptide domain. Even more preferably, the
RXR polypeptide truncation comprises a deletion of at least an
entire polypeptide domain. In a specific embodiment, the RXR
polypeptide truncation comprises a deletion of at least an
A/B-domain deletion, a C-domain deletion, a D-domain deletion, an
E-domain deletion, an F-domain deletion, an A/B/C-domains deletion,
an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an
A/B/C/D/F-domains deletion, an A/B/F-domains, and an
A/B/C/F-domains deletion. A combination of several complete and/or
partial domain deletions may also be performed.
[0156] In a preferred embodiment, the retinoid X receptor ligand
binding domain is encoded by a polynucleotide comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO:
30.
[0157] In another preferred embodiment, the retinoid X receptor
ligand binding domain comprises a polypeptide sequence selected
from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID
NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,
SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
[0158] For purposes of this invention EcR and RXR also include
synthetic and chimeric EcR and RXR and their homologs.
[0159] The DNA binding domain can be any DNA binding domain with a
known response element, including synthetic and chimeric DNA
binding domains, or analogs, combinations, or modifications
thereof. Preferably, the DBD is a GAL4 DBD, a LexA DBD, a
transcription factor DBD, a steroid/thyroid hormone nuclear
receptor superfamily member DBD, a bacterial LacZ DBD, or a yeast
put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO: 41
(polynucleotide) or SEQ ID NO: 42 (polypeptide)] or a LexA DBD
[(SEQ ID NO: 43 (polynucleotide) or SEQ ID NO: 44
(polypeptide)].
[0160] The transactivation domain (abbreviated "AD" or "TA") may be
any steroid/thyroid hormone nuclear receptor AD, synthetic or
chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a
VP16 AD, a GAL4 AD, an NF-.kappa.B AD, a BP64 AD, or an analog,
combination, or modification thereof. Preferably, the AD is a
synthetic or chimeric AD, or is obtained from a VP16, GAL4, or
NF-kB. Most preferably, the AD is a VP16 AD [SEQ ID NO: 45
(polynucleotide) or SEQ ID NO: 46 (polypeptide)].
[0161] The response element ("RE") may be any response element with
a known DNA binding domain, or an analog, combination, or
modification thereof. Preferably, the RE is an RE from GALA
("GAL4RE"), LexA, a steroid/thyroid hormone nuclear receptor RE, or
a synthetic RE that recognizes a synthetic DNA binding domain. More
preferably, the RE is a GAL4RE comprising a polynucleotide sequence
of SEQ ID NO: 47 or a LexA 8.times. operon comprising a
polynucleotide sequence of SEQ ID NO: 48. Preferably, the first
hybrid protein is substantially free of a transactivation domain
and the second hybrid protein is substantially free of a DNA
binding domain. For purposes of this invention, "substantially
free" means that the protein in question does not contain a
sufficient sequence of the domain in question to provide activation
or binding activity.
[0162] The ligands for use in the present invention as described
below, when combined with the ligand binding domain of an EcR, USP,
RXR, or another polypeptide which in turn are bound to the response
element linked to a gene, provide the means for external temporal
regulation of expression of the gene. The binding mechanism or the
order in which the various components of this invention bind to
each other, that is, ligand to receptor, first polypeptide to
response element, second polypeptide to promoter, etc., is not
critical. Binding of the ligand to the ligand binding domains of an
EcR, USP, RXR, or another protein, enables expression or
suppression of the gene. This mechanism does not exclude the
potential for ligand binding to EcR, USP, or RXR, and the resulting
formation of active homodimer complexes (e.g. EcR+EcR or USP+USP).
Preferably, one or more of the receptor domains can be varied
producing a chimeric gene switch. Typically, one or more of the
three domains, DBD, LBD, and transactivation domain, may be chosen
from a source different than the source of the other domains so
that the chimeric genes and the resulting hybrid proteins are
optimized in the chosen host cell or organism for transactivating
activity, complementary binding of the ligand, and recognition of a
specific response element In addition, the response element itself
can be modified or substituted with response elements for other DNA
binding protein domains such as the GAL4 protein from yeast (see
Sadowski, et al. (1988) Nature, 335:563-564) or LexA protein from
E. coli (see Brent and Ptashne (1985), Cell, 43:729-736), or
synthetic response elements specific for targeted interactions with
proteins desigped, modified, and selected for such specific
interactions (see, for example, Kim, et al. (1997), Proc. Natl.
Acad. Sci., USA, 94:3616-3620) to accommodate chimeric receptors.
Another advantage of chimeric systems is that they allow choice of
a promoter used to drive the gene expression according to a desired
end result Such double control can be particularly important in
areas of gene therapy, especially when cytotoxic proteins are
produced, because both the timing of expression as well as the
cells wherein expression occurs can be controlled. When genes,
operatively linked to a suitable promoter, are introduced into the
cells of the subject, expression of the exogenous genes is
controlled by the presence of the system of this invention.
Promoters may be constitutively or inducibly regulated or may be
tissue-specific (that is, expressed only in a particular type of
cells) or specific to certain developmental stages of the
organism.
[0163] Gene Expression Cassettes of the Invention
[0164] The novel ecdysone receptor-based inducible gene expression
system of the invention comprises a novel gene expression cassette
that is capable of being expressed in a host cell, wherein the gene
expression cassette comprises a polynucleotide encoding a hybrid
polypeptide. Thus, Applicants' invention also provides novel gene
expression cassettes for use in the gene expression system of the
invention.
[0165] Specifically, the present invention provides a gene
expression cassette comprising a polynucleotide encoding a hybrid
polypeptide. The hybrid polypeptide comprises either 1) a
DNA-binding domain that recognizes a response element and a ligand
binding domain of a nuclear receptor or 2) a transactivation domain
and a ligand binding domain of a nuclear receptor, wherein the
transactivation domain is from a nuclear receptor other than an
EcR, an RXR, or a USP.
[0166] In a specific embodiment, the gene expression cassette
encodes a hybrid polypeptide comprising a DNA-binding domain that
recognizes a response element and an ecdysone receptor ligand
binding domain, wherein the DNA binding domain is from a nuclear
receptor other than an ecdysone receptor.
[0167] In another specific embodiment, the gene expression cassette
encodes a hybrid polypeptide comprising a DNA-binding domain that
recognizes a response element and a retinoid X receptor ligand
binding domain, wherein the DNA binding domain is from a nuclear
receptor other than a retinoid X receptor.
[0168] The DNA binding domain can be any DNA binding domain with a
known response element, including synthetic and chimeric DNA
binding domains, or analogs, combinations, or modifications
thereof. Preferably, the DBD is a GALA DBD, a LexA DBD, a
transcription factor DBD, a steroid/thyroid hormone nuclear
receptor superfamily member DBD, a bacterial LacZ DBD, or a yeast
put DBD. More preferably, the DBD is a GAL4 DBD [SEQ ID NO: 41
(polynucleotide) or SEQ ID NO: 42 (polypeptide)] or a LexA DBD
[(SEQ ID NO: 43 (polynucleotide) or SEQ ID NO: 44
(polypeptide)].
[0169] In another specific embodiment, the gene expression cassette
encodes a hybrid polypeptide comprising a transactivation domain
and an ecdysone receptor ligand binding domain, wherein the
transactivation domain is from a nuclear receptor other than an
ecdysone receptor.
[0170] In another specific embodiment, the gene expression cassette
encodes a hybrid polypeptide comprising a transactivation domain
and a retinoid X receptor ligand binding domain, wherein the
transactivation domain is from a nuclear receptor other than a
retinoid X receptor.
[0171] The transactivation domain (abbreviated "AD" or "TA") may be
any steroid/thyroid hormone nuclear receptor AD, synthetic or
chimeric AD, polyglutamine AD, basic or acidic amino acid AD, a
VPl6 AD, a GALA AD, an NF-.kappa.B AD, a BP64 AD, or an analog,
combination, or modification thereof. Preferably, the AD is a
synthetic or chimeric AD, or is obtained from a VP16, GAL4, or
NF-kB. Most preferably, the AD is a VP16 AD [SEQ ID NO: 45
(polynucleotide) or SEQ ID NO: 46 (polypeptide)].
[0172] Preferably, the ligand binding domain is an EcR or RXR
related steroid/thyroid hormone nuclear receptor family member
ligand binding domain, or analogs, combinations, or modifications
thereof. More preferably, the LBD is from EcR or RXR. Even more
preferably, the LBD is from a truncated EcR or RXR.
[0173] Preferably, the EcR is an insect EcR selected from the group
consisting of a Lepidopteran EcR, a Dipteran EcR, an Arthropod EcR,
a Homopteran EcR and a Hemipteran EcR. More preferably, the EcR for
use is a spruce budworm Choristoneura fumiferana EcR ("CfEcR"), a
Tenebrio molitor EcR ("TmEcR"), a Manduca sexta EcR ("MsEcR"), a
Heliothies virescens EcR ("HvEcR"), a silk moth Bombyx mori EcR
("BmEcR"), a fruit fly Drosophila melanogaster EcR ("DmEcR"), a
mosquito Aedes aegypti EcR ("AaEcR"), a blowfly Lucilia capitata
EcR ("LcEcR"), a Mediterranean fruit fly Ceratitis capitata EcR
("CcEcR"), a locust Locusta migratoria EcR ("ImEcR"), an aphid
Myzus persicae EcR ("MpEcR"), a fiddler crab Uca pugilator EcR
("UpEcR"), or an ixodid tick Amblyomma americanum EcR ("AmaEcR").
Even more preferably, the LBD is from spruce budworm (Choristoneura
fumiferana) EcR ("CfEcR") or fruit fly Drosophila melanogaster EcR
("DmEcR").
[0174] Preferably, the LBD is from a truncated insect EcR. The
insect EcR polypeptide truncation comprises a deletion of at least
1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,
210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino
acids. More preferably, the insect EcR polypeptide truncation
comprises a deletion of at least a partial polypeptide domain. Even
more preferably, the insect EcR polypeptide truncation comprises a
deletion of at least an entire polypeptide domain. In a specific
embodiment, the insect EcR polypeptide truncation comprises a
deletion of at least an A/B-domain deletion, a C-domain deletion, a
D-domain deletion, an E-domain deletion, an F-domain deletion, an
A/B/C-domains deletion, an A/B1/2-C-domains deletion, an
A/B/C/D-domains deletion, an A/B/C/D/F-domains deletion, an
A/B/F-domains, and an A/B/C/F-domains deletion. A combination of
several complete and/or partial domain deletions may also be
performed.
[0175] In a preferred embodiment, the ecdysone receptor ligand
binding domain is encoded by a polynucleotide comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
[0176] In another preferred embodiment, the ecdysone receptor
ligand binding domain comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
[0177] Preferably, the RXR polypeptide is a mouse Mus musculus RXR
("MmRXR") or a human Homo sapiens RXR ("HsRXR"). The RXR
polypeptide may be an RXR.sub..alpha., RXR.sub..beta., or
RXR.sub..gamma. isoform.
[0178] Preferably, the LBD is from a truncated RXR. The RXR
polypeptide truncation comprises a deletion of at least 1, 2, 3, 4,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,
160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,
225, 230, 235, 240, 245, 250, 255, 260, or 265 amino acids. More
preferably, the RXR polypeptide truncation comprises a deletion of
at least a partial polypeptide domain. Even more preferably, the
RXR polypeptide truncation comprises a deletion of at least an
entire polypeptide domain. In a specific embodiment, the RXR
polypeptide truncation comprises a deletion of at least an
A/B-domain deletion, a C-domain deletion, a D-domain deletion, an
E-domain deletion, an F-domain deletion, an A/B/C-domains deletion,
an A/B/1/2-C-domains deletion, an A/B/C/D-domains deletion, an
A/B/C/D/F-domains deletion, an A/B/F-domains, and an
A/B/C/F-domains deletion A combination of several complete and/or
partial domain deletions may also be performed.
[0179] In a preferred embodiment, the retinoid X receptor ligand
binding domain is encoded by a polynucleotide comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO:
30.
[0180] In another preferred embodiment, the retinoid X receptor
ligand binding domain comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID
NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,
SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
[0181] In a preferred embodiment, the gene expression cassette
encodes a hybrid polypeptide comprising a DNA-binding domain
encoded by a polynucleotide comprising a nucleic acid sequence
selected from the group consisting of a GAL4 DBD (SEQ ID NO: 41) or
a LexA DBD (SEQ ID NO: 43) and an ecdysone receptor ligand binding
domain encoded by a polynucleotide comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
[0182] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a DNA-binding
domain comprising a polypeptide sequence selected from the group
consisting of a GAL4 DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO:
44) and an ecdysone receptor ligand binding domain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ
ID NO: 20.
[0183] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a DNA-binding
domain encoded by a polynucleotide comprising a nucleic acid
sequence selected from the group consisting of a GAL4 DBD (SEQ ID
NO: 41) or a LexA DBD (SEQ ID NO: 43) and a retinoid X receptor
ligand binding domain encoded by a polynucleotide comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ
ID NO: 30.
[0184] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a DNA-binding
domain comprising a polypeptide sequence selected from the group
consisting of a GALA DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO:
44) and a retinoid X receptor ligand binding domain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35,
SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ
ID NO:40.
[0185] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a transactivation
domain encoded by a polynucleotide comprising a nucleic acid
sequence of SEQ ID NO: 45 and an ecdysone receptor ligand binding
domain encoded by a polynucleotide comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ
ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
[0186] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a transactivation
domain comprising a polypeptide sequence of SEQ ID NO: 46 and an
ecdysone receptor ligand binding domain comprising a polypeptide
sequence selected from the group consisting of SEQ ID NO: 11, SEQ
ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:
20.
[0187] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a transactivation
domain encoded by a polynucleotide comprising a nucleic acid
sequence of SEQ ID NO: 45 and a retinoid X receptor ligand binding
domain encoded by a polynucleotide comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 21, SEQ
ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO:
30.
[0188] In another preferred embodiment, the gene expression
cassette encodes a hybrid polypeptide comprising a transactivation
domain comprising a polypeptide sequence of SEQ ID NO: 46 and a
retinoid X receptor ligand binding domain comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 31, SEQ
ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:
36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO:
40.
[0189] For purposes of this invention EcR and RXR also include
synthetic and chimeric EcR and RXR and their homologs.
[0190] Polynucleotides of the Invention
[0191] The novel ecdysone receptor-based inducible gene expression
system of the invention comprises a gene expression cassette
comprising a polynucleotide that encodes a truncated EcR or RXR
polypeptide comprising a truncation mutation and is useful in
methods of modulating the expression of a gene within a host
cell.
[0192] Thus, the present invention also relates to a polynucleotide
that encodes an EcR or RXR polypeptide comprising a truncation
mutation Specifically, the present invention relates to an isolated
polynucleotide encoding an EcR or RXR polypeptide comprising a
truncation mutation that affects ligand binding activity or ligand
sensitivity.
[0193] Preferably, the truncation mutation results in a
polynucleotide that encodes a truncated EcR polypeptide or a
truncated RXR polypeptide comprising a deletion of at least 1, 2,
3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,
150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,
215, 220, 225, 230, 235, 240, 245, 250, 255, 260, or 265 amino
acids. More preferably, the EcR or RXR polypeptide truncation
comprises a deletion of at least a partial polypeptide domain Even
more preferably, the EcR or RXR polypeptide truncation comprises a
deletion of at least an entire polypeptide domain. In a specific
embodiment, the EcR or RXR polypeptide truncation comprises a
deletion of at least an A/B-domain deletion, a C-domain deletion, a
D-domain deletion, an E-domain deletion, an F-domain deletion, an
A/B/C-domains deletion, an A/B/1/2-C-domains deletion, an
A/B/C/D-domains deletion, an A/B/C/D/Fdomains deletion, an
A/B/F-domains, and an A/B/C/F-domains deletion. A combination of
several complete and/or partial domain deletions may also be
performed.
[0194] In a specific embodiment, the EcR polynucleotide according
to the invention comprises a polynucleotide sequence selected from
the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, and SEQ ID NO: 10. In a specific embodiment, the
polynucleotide according to the invention encodes a ecdysone
receptor polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO:
12 (CfEcR-1/2CDEF, which comprises the last 33 carboxy-terminal
amino acids of C domain), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14
(CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DmEcR-CDEF),
SEQ ID NO: 17 (DnEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID
NO: 19 (DmEcR-EF), and SEQ ID NO: 20 (DmEc-DE).
[0195] In another specific embodiment, the RXR polynucleotide
according to the invention comprises a polynucleotide sequence
selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. In another
specific embodiment, the polynucleotide according to the invention
encodes a truncated RXR polypeptide comprising an amino acid
sequence consisting of SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32
(MmRXR-DEF), SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34
(MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36
(HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF),
SEQ ID NO: 39 (HSRXR-truncated EF), and SEQ ID NO: 40
(HsRXR-E).
[0196] In particular, the present invention relates to an isolated
polynucleotide encoding an EcR or RXR polypeptide comprising a
truncation mutation, wherein the mutation reduces ligand binding
activity or ligand sensitivity of the EcR or RXR polypeptide. In a
specific embodiment, the present invention relates to an isolated
polynucleotide encoding an ECR or RXR polypeptide comprising a
truncation mutation that reduces steroid binding activity or
steroid sensitivity of the EcR or RXR polypeptide. In a preferred
embodiment, the present invention relates to an isolated
polynucleotide encoding an EcR polypeptide comprising a truncation
mutation that reduces steroid binding activity or steroid
sensitivity of the EcR polypeptide, wherein the polynucleotide
comprises a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF), SEQ
ID NO: 4 (CfEcR-EF), SEQ ID NO: 8 (DmEcR-DEF), or SEQ ID NO: 9
(DmEcR-EF). In another specific embodiment, the present invention
relates to an isolated polynucleotide encoding an EcR or RXR
polypeptide comprising a truncation mutation that reduces
non-steroid binding activity or non-steroid sensitivity of the EcR
or RXR polypeptide. In a preferred embodiment, the present
invention relates to an isolated polynucleotide encoding an EcR
polypeptide comprising a truncation mutation that reduces
non-steroid binding activity or non-steroid sensitivity of the EcR
polypeptide, wherein the polynucleotide comprises a nucleic acid
sequence of SEQ ID NO: 4 (CfEcR-EF) or SEQ ID NO: 9 (DmEcR-EF).
[0197] The present invention also relates to an isolated
polynucleotide encoding an EcR or RXR polypeptide comprising a
truncation mutation, wherein the mutation enhances ligand binding
activity or ligand sensitivity of the EcR or RXR polypeptide. In a
specific embodiment, the present invention relates to an isolated
polynucleotide encoding an EcR or RXR polypeptide comprising a
truncation mutation that enhances steroid binding activity or
steroid sensitivity of the EcR or RXR polypeptide. In another
specific embodiment, the present invention relates to an isolated
polynucleotide encoding an EcR or RXR polypeptide comprising a
truncation mutation that enhances non-steroid binding activity or
non-steroid sensitivity of the EcR or RXR polypeptide. In a
preferred embodiment, the present invention relates to an isolated
polynucleotide encoding an EcR polypeptide comprising a truncation
mutation that enhances non-steroid binding activity or non-steroid
sensitivity of the EcR polypeptide, wherein the polynucleotide
comprises a nucleic acid sequence of SEQ ID NO: 3 (CfEcR-DEF) or
SEQ ID NO: 8 (DmEcR-DEF).
[0198] The present invention also relates to an isolated
polynucleotide encoding a retinoid X receptor polypeptide
comprising a truncation mutation that increases ligand sensitivity
of a heterodimer comprising the mutated retinoid X receptor
polypeptide and a dimerization partner. Preferably, the isolated
polynucleotide encoding a retinoid X receptor polypeptide
comprising a truncation mutation that increases ligand sensitivity
of a heterodimer comprises a polynucleotide sequence selected from
the group consisting of SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24
(MmRXR-truncatedEF), SEQ ID NO: 28 (HsRXR-EF), or SEQ ID NO: 29
(HsRXR-truncated EF). In a specific embodiment, the dimerization
partner is an ecdysone receptor polypeptide. Preferably, the
dimerization partner is a truncated EcR polypeptide. More
preferably, the dimerization partner is an EcR polypeptide in which
domains A/B/C have been deleted. Even more preferably, the
dimerization partner is an EcR polypeptide comprising an amino acid
sequence of SEQ ID NO: 13 (CfEcR-DEF) or SEQ ID NO: 18
(DmEcR-DEF).
[0199] Polypeptides of the Invention
[0200] The novel ecdysone receptor-based inducible gene expression
system of the invention comprises a polynucleotide that encodes a
truncated EcR or RXR polypeptide and is useful in methods of
modulating the expression of a gene within a host cell. Thus, the
present invention also relates to an isolated truncated EcR or RXR
polypeptide encoded by a polynucleotide or a gene expression
cassette according to the invention. Specifically, the present
invention relates to an isolated truncated EcR or RXR polypeptide
comprising a truncation mutation that affects ligand binding
activity or ligand sensitivity encoded by a polynucleotide
according to the invention.
[0201] The present invention also relates to an isolated truncated
EcR or RXR polypeptide comprising a truncation mutation.
Specifically, the present invention relates to an isolated EcR or
RXR polypeptide comprising a truncation mutation that affects
ligand binding activity or ligand sensitivity.
[0202] Preferably, the truncation mutation results in a truncated
EcR polypeptide or a truncated RXR polypeptide comprising a
deletion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,
125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,
190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,
255, 260, or 265 amino acids. More preferably, the EcR or RXR
polypeptide truncation comprises a deletion of at least a partial
polypeptide domain. Even more preferably, the EcR or RXR
polypeptide truncation comprises a deletion of at least an entire
polypeptide domain. In a specific embodiment, the EcR or RXR
polypeptide truncation comprises a deletion of at least an
A/B-domain deletion, a C-domain deletion, a D-domain deletion, an
E-domain deletion, an F-domain deletion, an A/B/C-domains deletion,
an A/B/1/2C-domains deletion, an A/B/C/D-domains deletion, an
A/B/C/D/F-domains deletion, an A/B/F-domains, and an
A/B/C/F-domains deletion. A combination of several complete and/or
partial domain deletions may also be performed.
[0203] In a preferred embodiment, the isolated truncated ecdysone
receptor polypeptide is encoded by a polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO: 1 (CfEcR-CDEF), SEQ ID NO: 2 (CfEcR-1/2CDEF), SEQ ID NO: 3
(CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF), SEQ ID NO: 5 (CfEcR-DE), SEQ
ID NO: 6 (DnEcR-CDEF), SEQ ID NO: 7 (DmEcR-1/2CDEF), SEQ ID NO: 8
(DmEcR-DEF), SEQ ID NO: 9 (DmEcR-EF), and SEQ ID NO: 10 (DmEcR-DE).
In another preferred embodiment, the isolated truncated ecdysone
receptor polypeptide comprises an amino acid sequence selected from
the group consisting of SEQ ID NO: 11 (CfEcR-CDEF), SEQ ID NO: 12
(CfEcR-1/2CDEF), SEQ ID NO: 13 (CfEcR-DEF), SEQ ID NO: 14
(CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE), SEQ ID NO: 16 (DnEcR-CDEF),
SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID NO: 18 (DmEcR-DEF), SEQ ID
NO: 19 (DmEcR-EF), and SEQ ID NO: 20 (DmEcR-DE).
[0204] In a preferred embodiment, the isolated truncated RXR
polypeptide is encoded by a polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO: 21 (MmRXR-CDEF), SEQ ID NO: 22 (MmRXR-DEF), SEQ ID NO: 23
(MmRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 25
(MmRXR-E), SEQ ID NO: 26 (HsRXR-CDEF), SEQ ID NO: 27.(HsRXR-DEF),
SEQ ID NO: 28 (HsRXR-EF), SEQ ID NO: 29 (HsRXR-truncatedEF) and SEQ
ID NO: 30 (HsRXR-E). In another preferred embodiment, the isolated
truncated RXR polypeptide comprises an amino acid sequence selected
from the group consisting of SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO:
32 (MmRXR-DEF), SEQ ID NO: 33 (RxR-EF), SEQ ID NO: 34
(MmRXR-truncatedEF), SEQ ID NO: 35 (RXR-E), SEQ ID NO: 36
(HsRXR-CDEP), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF),
SEQ ID NO: 39 (HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).
[0205] The present invention relates to an isolated EcR or RXR
polypeptide comprising a truncation mutation that reduces ligand
binding activity or ligand sensitivity of the EcR or RXR
polypeptide, wherein the polypeptide is encoded by a polynucleotide
comprising a polynucleotide sequence selected from the group
consisting of SEQ ID NO: 1 (CfEcR-CDEF), SEQ ID NO: 2
(CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF),
SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6 (DmEcR-CDEF), SEQ ID NO: 7
(DmEcR1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF), SEQ ID NO: 9 (DmEcR-EF),
SEQ ID NO: 10 (DmEcR-DE), SEQ ID NO: 21 (MmRXR-CDEF), SEQ ID NO: 22
(MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24
(MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26
(HsRXR-CDEF), SEQ ID NO: 27 (HsRXR-DEF), SEQ ID NO: 28 (HsRXR-EF),
SEQ ID NO: 29 (HsRXR-truncatedEF), and SEQ ID NO: 30 (HsRXR-E).
[0206] Thus, the present invention relates to an isolated truncated
EcR or RXR polypeptide comprising a truncation mutation that
reduces ligand binding activity or ligand sensitivity of the EcR or
RXR polypeptide, wherein the polypeptide comprises an amino acid
sequence selected from the group consisting of SEQ ID NO: 11
(CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF), SEQ ID NO: 13
(CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE),
SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID
NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), SEQ ID NO: 20
(DmEcR10 DE), SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32
(MmRXR-DEF), SEQ ID NO: 33 (MmR-EF), SEQ ID NO: 34
(MmRXR-truncatedEF), SEQ ID NO: 35 (MmRXR-E), SEQ ID NO: 36
(HsRXR-CDEF), SEQ ID NO: 37 (HsRXR-DEF), SEQ ID NO: 38 (HsRXR-EF),
SEQ ID NO: 39 (HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).
[0207] In a specific embodiment, the present invention relates to
an isolated EcR or RXR polypeptide comprising a truncation mutation
that reduces steroid binding activity or steroid sensitivity of the
EcR or RXR polypeptide. In a preferred embodiment, the present
invention relates to an isolated EcR polypeptide comprising a
truncation mutation that reduces steroid binding activity or
steroid sensitivity of the EcR polypeptide, wherein the EcR
polypeptide is encoded by a polynucleotide comprising a nucleic
acid sequence of SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF),
SEQ ID NO: 8 (DmEcR-DEF), or SEQ ID NO: 9 (DmEcR-EF). Accordingly,
the present invention also relates to an isolated truncated EcR or
RXR polypeptide comprising a truncation nmtation that reduces
steroid binding activity or steroid sensitivity of the EcR or RXR
polypeptide. In a preferred embodiment, the present invention
relates to an isolated EcR polypeptide comprising a truncation
mutation that reduces steroid binding activity or steroid
sensitivity of the EcR polypeptide, wherein the EcR polypeptide
comprises an amino acid sequence of SEQ ID NO: 13 (CfEcR-DEF), SEQ
ID NO: 14 (CfEcR-EF), SEQ ID NO: 18 (DmEcR-DEF), or SEQ ID NO: 19
(DmEcR-EF).
[0208] In another specific embodiment, the present invention
relates to an isolated EcR or RXR polypeptide comprising a
truncation mutation that reduces non-steroid binding activity or
non-steroid sensitivity of the EcR or RXR polypeptide. In a
preferred embodiment, the present invention relates to an isolated
EcR polypeptide comprising a truncation mutation that reduces
non-steroid binding activity or non-steroid sensitivity of the EcR
polypeptide, wherein the EcR polypeptide is encoded by a
polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 4
(CfEcR-EF) or SEQ ID NO: 9 (DmEcR-EF). Accordingly, the present
invention also relates to an isolated truncated EcR or RXR
polypeptide comprising a truncation mutation that reduces
non-steroid binding activity or steroid sensitivity of the EcR or
RXR polypeptide. In a preferred embodiment, the present invention
relates to an isolated EcR polypeptide comprising a truncation
mutation that reduces non-steroid binding activity or non-steroid
sensitivity of the EcR polypeptide, wherein the EcR polypeptide
comprises an amino acid sequence of SEQ ID NO: 14 (CfEcR-EF) or SEQ
ID NO: 19 (DmEcR-EF).
[0209] In particular, the present invention relates to an isolated
EcR or RXR polypeptide comprising a truncation mutation that
enhances ligand binding activity or ligand sensitivity of the EcR
or RXR polypeptide, wherein the polypeptide is encoded by a
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO: 1 (CfEcR-CDEF), SEQ ID NO: 2
(CfEcR-1/2CDEF), SEQ ID NO: 3 (CfEcR-DEF), SEQ ID NO: 4 (CfEcR-EF),
SEQ ID NO: 5 (CfEcR-DE), SEQ ID NO: 6 (DmEcR-CDEF), SEQ ID NO: 7
(DmEcR-1/2CDEF), SEQ ID NO: 8 (DmEcR-DEF), SEQ ID NO: 9 (DmEcR-EF),
SEQ ID NO: 10 (DmEcR-DE), SEQ ID NO: 21 (MmRXR-CDEP), SEQ ID NO: 22
(MmRXR-DEF), SEQ ID NO: 23 (MmRXR-EF), SEQ ID NO: 24
(MmRXR-truncatedEF), SEQ ID NO: 25 (MmRXR-E), SEQ ID NO: 26
(HsRXR-CDEF), SEQ ID NO: 27 (HsRXRDEF), SEQ ID NO: 28 (HsRXR-EF),
SEQ ID NO: 29 (HsRXR-truncated EF), and SEQ ID NO: 30
(HsRXR-E).
[0210] The present invention relates to an isolated EcR or RXR
polypeptide comprising a truncation mutation that enhances ligand
binding activity or ligand sensitivity of the EcR or RXR
polypeptide, wherein the polypeptide comprises an amino acid
sequence selected from the group consisting of SEQ ID NO: 11
(CfEcR-CDEF), SEQ ID NO: 12 (CfEcR-1/2CDEF), SEQ ID NO: 13
(CfEcR-DEF), SEQ ID NO: 14 (CfEcR-EF), SEQ ID NO: 15 (CfEcR-DE),
SEQ ID NO: 16 (DmEcR-CDEF), SEQ ID NO: 17 (DmEcR-1/2CDEF), SEQ ID
NO: 18 (DmEcR-DEF), SEQ ID NO: 19 (DmEcR-EF), SEQ ID NO: 20
(DmEcR-DE), SEQ ID NO: 31 (MmRXR-CDEF), SEQ ID NO: 32 (MmRXR-DEF),
SEQ ID NO: 33 (MmRXR-EF), SEQ ID NO: 34 (MmRXR-runcatedEF), SEQ ID
NO: 35 (MmRXR-E), SEQ ID NO: 36 (HsRXR-CDEF), SEQ ID NO: 37
(HsRXR-DEF), SEQ ID NO: 39 (HsRXR-EF), SEQ ID NO: 39
(HsRXR-truncatedEF), and SEQ ID NO: 40 (HsRXR-E).
[0211] The present invention relates to an isolated EcR or RXR
polypeptide comprising a truncation mutation that enhances ligand
binding activity or ligand sensitivity of the EcR or RXR
polypeptide. In a specific embodiment, the present invention
relates to an isolated EcR or RXR polypeptide comprising a
truncation mutation that enhances steroid binding activity or
steroid sensitivity of the EcR or RXR polypeptide. Accordingly, the
present invention also relates to an isolated EcR or RXR
polypeptide comprising a truncation mutation that enhances steroid
binding activity or steroid sensitivity of the EcR or RXR
polypeptide.
[0212] In another specific embodiment, the present invention
relates to an isolated EcR or RXR polypeptide comprising a
truncation mutation that enhances non-steroid binding activity or
non-steroid sensitivity of the EcR or RXR polypeptide. In a
preferred embodiment, the present invention relates to an isolated
EcR polypeptide comprising a truncation mutation that enhances
non-steroid binding activity or non-steroid sensitivity of the EcR
polypeptide, wherein the EcR polypeptide is encoded by a
polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3
(CfEcR-DEF) or SEQ ID NO: 8 (DmEcR-DEF). Accordingly, the present
invention also relates to an isolated EcR or RXR polypeptide
comprising a truncation mutation that enhances non-steroid binding
activity or steroid sensitivity of the EcR or RXR polypeptide. In a
preferred embodiment, the present invention relates to an isolated
EcR polypeptide comprising a truncation mutation that enhances
non-steroid binding activity or non-steroid sensitivity of the EcR
polypeptide, wherein the EcR polynucleotide comprises an amino acid
sequence of SEQ ID NO: 13 (CfEcR-DEF) or SEQ ID NO: 18
(DmEcR-DEF).
[0213] The present invention also relates to an isolated retinoid X
receptor polypeptide comprising a truncation mutation that
increases ligand sensitivity of a heterodimer comprising the
mutated retinoid X receptor polypeptide and a dimerization partner.
Preferably, the isolated retinoid X receptor polypeptide comprising
a truncation mutation that increases ligand sensitivity of a
heterodimer is encoded by a polynucleotide comprising a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 23
(MmRXR-EF), SEQ ID NO: 24 (MmRXR-truncatedEF), SEQ ID NO: 28
(HsRXR-EF), or SEQ ID NO: 29 (HsRXR-truncatedEF). More preferably,
the isolated polynucleotide encoding a retinoid X receptor
polypeptide comprising a truncation mutation that increases ligand
sensitivity of a heterodimer comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 33 (MnRXR-EF), SEQ
ID NO: 34 (nRXR-trancatedEF), SEQ ID NO: 38 (HsRXR-EF), or SEQ ID
NO: 39 (HsRXR-truncatedEF).
[0214] In a specific embodiment, the dimerization partner is an
ecdysone receptor polypeptide. Preferably, the dimerization partner
is a truncated EcR polypeptide. More preferably, the dimerization
partner is an EcR polypeptide in which domains A/B/C have been
deleted. Even more preferably, the dimerization partner is an EcR
polypeptide comprising an amino acid sequence of SEQ ID NO: 13
(CfEcR-DEF) or SEQ ID NO: 18 (DmEcR-DEF).
[0215] Method of Modulating Gene Expression of the Invention
[0216] Applicants' invention also relates to methods of modulating
gene expression in a host cell using a gene expression modulation
system according to the invention. Specifically, Applicants'
invention provides a method of modulating the expression of a gene
in a host cell comprising the steps of: a) introducing into the
host cell a gene expression modulation system according to the
invention; and b) introducing into the host cell a ligand that
independently combines with the ligand binding domains of the first
polypeptide and the second polypeptide of the gene expression
modulation system, wherein the gene to be expressed is a component
of a gene expression cassette comprising: i) a response element
comprising a domain to which the DNA binding domain of the first
polypeptide binds; ii) a promoter that is activated by the
transactivation domain of the second polypeptide; and iii) a gene
whose expression is to be modulated, whereby a complex is formed
comprising the ligand, the first polypeptide of the gene expression
modulation system and the second polypeptide of the gene expression
modulation system, and whereby the complex modulates expression of
the gene in the host cell.
[0217] Genes of interest for expression in a host cell using
Applicants' methods may be endogenous genes or heterologous genes.
Nucleic acid or amino acid sequence information for a desired gene
or protein can be located in one of many public access databases,
for example, GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology
related journal publications. Thus, those skilled in the art have
access to nucleic acid sequence information for virtually all known
genes. Such information can then be used to construct the desired
constructs for the insertion of the gene of interest within the
gene expression cassettes used in Applicants' methods described
herein.
[0218] Examples of genes of interest for expression in a host cell
using Applicants' methods include, but are not limited to: antigens
produced in plants as vaccines, enzymes like alphaamylase, phytase,
glucanes, and xylanse, genes for resistance against insects,
nematodes, fungi, bacteria, viruses, and abiotic stresses,
nutraceuticals, pharmaceuticals, vitamins, genes for modifying
amino acid content, herbicide resistance, cold, drought, and heat
tolerance, industrial products, oils, protein, carbohydrates,
antioxidants, male sterile plants, flowers, fuels, other output
traits, genes encoding therapeutically desirable polypeptides or
products, such as genes that can provide, modulate, alleviate,
correct and/or restore polypeptides important in treating a
condition, a disease, a disorder, a dysfunction, a genetic defect,
and the like.
[0219] Acceptable ligands are any that modulate expression of the
gene when binding of the DNA binding domain of the two hybrid
system to the response element in the presence of the ligand
results in activation or suppression of expression of the genes.
Preferred ligands include ponasterone, muristerone A,
N,N'-diacylhydrazines such as those disclosed in U.S. Pat. Nos.
6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkyl
cyanohydrazines such as those disclosed in European Application No.
461,809; N-alkyl-N,N'-diaroylhydrazines such as those disclosed in
U.S. Pat. No. 5,225,443; N-acyl-N-alkylarbonylhydrazines such as
those disclosed in European Application No. 234,994;
N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S.
Pat. No. 4,985,461; each of which is incorporated herein by
reference and other similar materials including
3,5-di-tert-butyl-4-hydroxy-N-isobutylb- enzamide,
8-O-acetylharpagide, and the like.
[0220] Preferably, the ligand for use in Applicants' method of
modulating expression of gene is a compound of the formula: 2
[0221] wherein:
[0222] E is a (C.sub.4-C.sub.6)alkyl containing a tertiary carbon
or a cyano(C.sub.3-Cs)alkyl containing a tertiary carbon;
[0223] R.sup.1 is H, Me, Et, i-Pr, F, formyl, CF.sub.3, CHF.sub.2,
CHCl.sub.2, CH2F, CH.sub.2Cl, CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN,
CN, C.degree.CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt,
cyclopropyl, CF.sub.2CF.sub.3, CH.dbd.CHCN, allyl, azido, SCN, or
SCHF.sub.2;
[0224] R.sup.2 is H, Me, Et, n-Pr, i-Pr, formyl, CF.sub.3,
CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2OH,
CH.sub.2OMe, CH.sub.2CN, CN, C.degree.CH, 1-propynyl, 2-propynyl,
vinyl, Ac, F, Cl, OH, OMe, OEt, O-nPr, OAc, NMe.sub.2, NEt.sub.2,
SMe, SEt, SOCF.sub.3, OCF.sub.2CF.sub.2H, COEt, cyclopropyl,
CF.sub.2CF.sub.3, CH.dbd.CHCN, allyl, azido, OCF3, OCHF.sub.2,
O-i-Pr, SCN, SCHF.sub.2, SOMe, NH--CN, or joined with R.sup.3 and
the phenyl carbons to which R.sup.2 and R.sup.3 are attached to
form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent
to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent
to a phenyl carbon;
[0225] R.sup.3 is H, Et, or joined with R.sup.2 and the phenyl
carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a
phenyl carbon;
[0226] R.sup.4, R.sup.5, and R.sup.6 are independently H, Me, Et,
F, Cl, Br, formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH2F,
CH.sub.2Cl, CH.sub.2OH, CN, C.degree.CH, 1-propynyl, 2-propynyl,
vinyl, OMe, OEt, SMe, or SEt.
[0227] Applicants' invention provides for modulation of gene
expression in prokaryotic and eukaryotic host cells. Thus, the
present invention also relates to a method for modulating gene
expression in a host cell selected from the group consisting of a
bacterial cell, a fungal cell, a yeast cell, a plant cell, an
animal cell, and a mamalian cell. Preferably, the host cell is a
yeast cell, a plant cell, a murine cell, or a human cell.
[0228] Expression in transgenic host cells may be useful for the
expression of various polypeptides of interest including but not
limited to therapeutic polypeptides, pathway intermediates; for the
modulation of pathways already existing in the host for the
synthesis of new products heretofore not possible using the host;
cell based assays; and the like. Additionally the gene products may
be useful for conferring higher growth yields of the host or for
enabling alternative growth mode to is utilized.
[0229] Host Cells and Non-Human Organisms of the Inveniion
[0230] As described above, the gene expression modulation system of
the present invention may be used to modulate gene expression in a
host cell. Expression in transgenic host cells may be useful for
the expression of various genes of interest. Thus, Applicants'
invention also provides an isolated host cell comprising a gene
expression system according to the invention. The present invention
also provides an isolated host cell comprising a gene expression
cassette according to the invention. Applicants' invention also
provides an isolated host cell comprising a polynucleotide or
polypeptide according to the invention The isolated host cell may
be either a prokaryotic or a eukaryotic host cell.
[0231] Preferably, the host cell is selected from the group
consisting of a bacterial cell, a fungal cell, a yeast cell, a
plant cell, an animal cell, and a mammalian cell. Examples of
preferred host cells include, but are not limited to, fungal or
yeast species such as Aspergillus, Trichoderna, Saccharomyces,
Pichia, Candida, Hansenula, or bacterial species such as those in
the genera Synechocystis, Synechococcus, Salmonella, Bacillus,
Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas,
Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena,
Thiobacillus, Methanobacteriuin and Klebsiella, plant, animal, and
mammalian host cells. More preferably, the host cell is a yeast
cell, a plant cell, a murine cell, or a human cell.
[0232] In a specific embodiment, the host cell is a yeast cell
selected from the group consisting of a Saccharomyces, a Pichia,
and a Candida host cell.
[0233] In another specific embodiment, the host cell is a plant
cell selected from the group consisting of an apple, Arabidopsis,
bajra, banana, barley, bean, beet, blackgram, chickpea, chili,
cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat,
okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,
Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash,
sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato,
tobacco, watermelon, and wheat host cell.
[0234] In another specific embodiment, the host cell is a murine
cell.
[0235] In another specific embodiment, the host cell is a human
cell.
[0236] Host cell transformation is well known in the art and may be
achieved by a variety of methods including but not limited to
electroporation, viral infection, plasmid/vector transfection,
non-viral vector mediated transfection, Agrobacterium-mediated
transformation, particle bombardment, and the like. Expression of
desired gene products involves culturing the transformed host cells
under suitable conditions and inducing expression of the
transformed gene. Culture conditions and gene expression protocols
in prokaryotic and eukaryotic cells are well known in the art (see
General Methods section of Examples). Cells may be harvested and
the gene products isolated according to protocols specific for the
gene product.
[0237] In addition, a host cell may be chosen which modulates the
expression of the inserted polynucleotide, or modifies and
processes the polypeptide product in the specific fashion desired.
Different host cells have characteristic and specific mechanisms
for the translational and post-translational processing and
modification (e.g., glycosylation, cleavage [e.g., of signal
sequence]) of proteins. Appropriate cell lines or host systems can
be chosen to ensure the desired modification and processing of the
foreign protein expressed. For example, expression in a bacterial
system can be used to produce a non-glycosylated core protein
product. However, a polypeptide expressed in bacteria may not be
properly folded. Expression in yeast can produce a glycosylated
product. Expression in eukaryotic cells can increase the likelihood
of "native" glycosylation and folding of a heterologous protein.
Moreover, expression in mammalian cells can provide a tool for
reconstituting, or constituting, the polypeptide's activity.
Furthermore, different vector/host expression systems may affect
processing reactions, such as proteolytic cleavages, to a different
extent.
[0238] Applicants' invention also relates to a non-human organism
comprising an isolated host cell according to the invention.
Preferably, the non-human organism is selected from the group
consisting of a bacterium, a fungus, a yeast, a plant, an animal,
and a mammal. More preferably, the non-human organism is a yeast, a
plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a
pig, a horse, a sheep, a monkey, or a chimpanzee.
[0239] In a specific embodiment, the non-human organism is a yeast
selected from the group consisting of Saccharomyces, Pichia, and
Candida.
[0240] In another specific embodiment, the non-human organism is a
plant selected from the group consisting of an apple, Arabidopsis,
bajra, banana, barley, beans, beet, blackgram, chickpea, chili,
cucumber, eggplant, favabean, maize, melon, millet, mungbean, oat,
okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,
Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash,
sugarcane, sugarbeet, sunflower, sweet potato, tea, tomato,
tobacco, watermelon, and wheat.
[0241] In another specific embodiment, the non-human organism is a
Mus musculus mouse.
[0242] Measuring Gene Expression/Transcription
[0243] One useful measurement of Applicants' methods of the
invention is that of the transcriptional state of the cell
including the identities and abundances of RNA, preferably mRNA
species. Such measurements are conveniently conducted by measuring
cDNA abundances by any of several existing gene expression
technologies.
[0244] Nucleic acid array technology is a useful technique for
determining differential mRNA expression Such technology includes,
for example, oligonucleotide chips and DNA microarrays. These
techniques rely on DNA fragments or oligonucleotides which
correspond to different genes or cDNAs which are immobilized on a
solid support and hybridized to probes prepared from total mRNA
pools extracted from cells, tissues, or whole organisms and
converted to cDNA. Oligonucleotide chips are arrays of
oligonucleotides synthesized on a substrate using photolithographic
techniques. Chips have been produced which can analyze for up to
1700 genes. DNA microarrays are arrays of DNA samples, typically
PCR products, that are robotically printed onto a microscope slide.
Each gene is analyzed by a full or partial-length target DNA
sequence. Microarrays with up to 10,000 genes are now routinely
prepared commercially. The primary difference between these two
techniques is that oligonucleotide chips typically utilize 25-mer
oligonucleotides which allow fractionation of short DNA molecules
whereas the larger DNA targets of microarrays, approximately 1000
base pairs, may provide more sensitivity in fractionating complex
DNA mixtures.
[0245] Another useful measurement of Applicants' methods of the
invention is that of determining the translation state of the cell
by measuring the abundances of the constituent protein species
present in the cell using processes well known in the art.
[0246] Where identification of genes associated with various
physiological functions is desired, an assay may be employed in
which changes in such functions as cell growth, apoptosis,
senescence, differentiation, adhesion, binding to a specific
molecules, binding to another cell, cellular organization,
organogenesis, intracellular transport, transport facilitation,
energy conversion, metabolism, myogenesis, neurogenesis, and/or
hematopoiesis is measured.
[0247] In addition, selectable marker or reporter gene expression
may be used to measure gene expression modulation using Applicants'
invention.
[0248] Other methods to detect the products of gene expression are
well known in the art and include Southern blots (DNA detection),
dot or slot blots (DNA, RNA), Northem blots (RNA), and RT-PCR(RNA)
analyses. Although less preferred, labeled proteins can be used to
detect a particular nucleic acid sequence to which it
hybidizes.
[0249] In some cases it is necessary to amplify the amount of a
nucleic acid sequence. This may be carried out using one or more of
a number of suitable methods including, for example, polymerase
chain reaction ("PCR"), ligase chain reaction ("LCR"), strand
displacement amplification ("SDA"), transcription-based
amplification, and the like. PCR is carried out in accordance with
known techniques in which, for example, a nucleic acid sample is
treated in the presence of a heat stable DNA polymerase, under
hybridizing conditions, with one oligonucleotide primer for each
strand of the specific sequence to be detected. An extension
product of each primer that is synthesized is complementary to each
of the two nucleic acid strands, with the primers sufficiently
complementary to each strand of the specific sequence to hybridize
therewith. The extension product synthesized from each primer can
also serve as a template for further synthesis of extension
products using the same pmers. Following a sufficient number of
rounds of synthesis of extension products, the sample may be
analyzed as described above to assess whether the sequence or
sequences to be detected are present
[0250] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention.
EXAMPLES
[0251] General Methods
[0252] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and
L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience (1987).
[0253] Methods for plant tissue culture, transformation, plant
molecular biology, and plant, general molecular biology may be
found in Plant Tissue Culture Concepts and Laboratory Exercises
edited by RN Trigiano and DJ Gray, 2.sup.nd edition, 2000, CRC
press, New York; Agrobacterium Protocols edited by KMA Gartland and
MR Davey, 1995, Humana Press, Totowa, N.J.; Methods in Plant
Molecular Biology, P. Maliga et al., 1995, Cold Spring Harbor Lab
Press, New York; and Molecular Cloning, J. Sambrook et al., 1989,
Cold Spring Harbor Lab Press, New York.
[0254] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society
for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All
reagents, restriction enzymes and materials used for the growth and
maintenance of host cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St Louis, Mo.)
unless otherwise specified.
[0255] Manipulations of genetic sequences may be accomplished using
the suite of programs available from the Genetics Computer Group
Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),
Madison, Wis.). Where the GCG program "Pileup" is used the gap
creation default value of 12, and the gap extension default value
of 4 may be used. Where the CGC "Gap" or "Bestfit" programs is used
the default gap creation penalty of 50 and the default gap
extension penalty of 3 may be used. In any case where GCG program
parameters are not prompted for, in these or any other GCG program,
default values may be used.
[0256] The meaning of abbreviations is as follows: "1" means
hour(s), "min" means minute(s), "sec" means second(s), "d" means
day(s), ".mu.l" means microliter(s), "ml" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
".mu.g" means microgram(s), "mg" means milligram(s), "A" means
adenine or adenosine, "T" means thymine or thymidine, "G" means
guanine or guanosine, "C" means cytidine or cytosine, "x g" means
times gravity, "nt" means nucleotide(s), "aa" means amino acid(s),
"bp" means base pair(s), "kb" means kilobase(s), "k" means kilo,
".mu." means micro, and ".degree. C." means degrees Celsius.
Example 1
[0257] Applicants' improved EcR-based inducible gene modulation
system was developed for use in various applications including gene
therapy, expression of proteins of interest in host cells,
production of transgenic organisms, and cell-based assays. This
Example describes the construction and evaluation of several gene
expression cassettes for use in the EcR-based inducible gene
expression system of the invention.
[0258] In various cellular backgrounds, including mammalian cells,
insect ecdysone receptor (EcR) heterodimerizes with retinoid X
receptor (RXR) and, upon binding of ligand, transactivates genes
under the control of ecdysone response elements. Applicants
constructed several EcR-based gene expression cassettes based on
the spruce budworm Choristoneura fumiferana EcR ("CfEcR"; full
length polynucleotide and amino acid sequences are set forth in SEQ
ID NO: 49 and SEQ ID NO: 50, respectively), C. fumiferana
ultraspiracle ("CfUSP"; full length polynucleotide and amino acid
sequences are set forth in SEQ ID NO: 51 and SEQ ID NO: 52,
respectively), and mouse Mus musculus RXRA (MmRXR.alpha.; full
length polynucleotide and amino acid sequences are set forth in SEQ
ID NO: 53 and SEQ ID NO: 54, respectively). The prepared receptor
constructs comprise a ligand binding domain of EcR and of RXR or of
USP; a DNA binding domain of GAL4 or of EcR; and an activation
domain of VP16. The reporter constructs include a reporter gene,
luciferase or LacZ, operably linked to a synthetic promoter
construct that comprises either GAIA or EcR/USP binding sites
(response elements). Various combinations of these receptor and
reporter constructs were cotransfected into CHO, NIH3T3, CV1 and
293 cells. Gene induction potential (magnitude of induction) and
ligand specificity and sensitivity were examined using four
different ligands: two steroidal ligands (ponasterone A and
muristerone A) and two non-steroidal ligands
(N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzo-
yl)-N'-tert-butylhydrazine and
N-(3,4(1,2-ethylenedioxy)-2-methylbenzoyl)--
N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine) in a
dose-dependent induction of reporter gene expression in the
transfected cells. Reporter gene expression activities were assayed
at 24 hr or 48 hr after ligand addition.
[0259] Gene Expression Cassettes: Ecdysone receptor-based,
chemically inducible gene expression cassettes (switches) were
constructed as followed, using standard cloning methods available
in the art. The following is brief description of preparation and
compositionof each switch.
[0260] 11.1--GALAEcRNP16RXR: The D, E, and F domains from spruce
budworm Choristoneura fumiferana EcR ("CfEcRDEF"; SEQ ID NO: 3)
were fused to GAL4 DNA binding domain ("DNABD"; SEQ ID NO: 41) and
placed under the control of an SV40e promoter (SEQ ID NO: 55). The
DEF domains from mouse (Mus musculus) RXR ("MmRXRDEF"; SEQ ID NO:
22) were fused to the activation domain from VP16 ("VP16AD"; SEQ ID
NO: 45) and placed under the control of an SV40e promoter (SEQ ID
NO: 55). Five consensus GAL4 binding sites ("5XGALARE"; comprising
5, GAL4RE comprising SEQ ID NO: 47) were fused to a synthetic E1b
minimal promoter (SEQ ID NO: 56) and placed upstream of the
luciferase gene (SEQ ID NO: 57).
[0261] 1.2--GALAEcR/VP16USP: This construct was prepared in the
same way as in switch 1.1 above except MmRXRDEF was replaced with
the D, E and F domains from spruce budworm USP ("CfUSPDEF"; SEQ ID
NO: 58). The constructs used in this example are similar to those
disclosed in U.S. Pat. No. 5,880,333 except that Choristoneura
fumiferana USP rather than Drosophila melanogaster USP was
utilized.
[0262] 1.3--GAIARXR/VP16CfEcR: MmRXRDEF (SEQ ID NO: 22) was fused
to a GAL4DNABD (SEQ ID NO: 41) and CfEcRCDEF (SEQ ID NO: 1) was
fused to a VP16AD (SEQ ID NO: 45).
[0263] 1.4--GAL4RXR/VP16DmEcR: This construct was prepared in the
same way as switch 1.3 except CfEcRCDEF was replaced with DmEcRCDEF
(SEQ ID NO: 6).
[0264] 1.5--GAl4USP/VP16CfEcR: This construct was prepared in the
same way as switch 1.3 except MmRXRDEF was replaced with CFUSPDEF
(SEQ ID NO: 58).
[0265] 1.6--GALARXRCfEcRVP16: This construct was prepared so that
both the GAL4 DNABD and the VP16AD were placed on the same
molecule. GAL4DNABD (SEQ ID NO: 41) and VP16AD (SEQ ID NO: 45) were
fused to CfEcRDEF (SEQ ID NO: 3) at N-and C-termini respectively.
The fusion was placed under the control of an SV40e promoter (SEQ
ID NO: 55).
[0266] 1.7--VP16CfEcR: This construct was prepared such that
CfEcRCDEF (SEQ ID NO: 1) was fused to VP16AD (SEQ ID NO: 45) and
placed under the control of an SV40e promoter (SEQ ID NO: 55). Six
ecdysone response elements ("EcRE"; SEQ ID NO: 59) from the hsp27
gene were placed upstream of the promoter and a luciferase gene
(SEQ ID NO: 57). This switch most probably uses endogenous RXR.
[0267] 1.8--DmVgRXR: This system was purchased from Invitrogen
Corp., Carlsbad, Calif. It comprises a Drosophila melanogaster EcR
("DnEcR") with a modified DNABD fused to VP16AD and placed under
the control of a CMV promoter (SEQ D) NO: 60). Full length MmRXR
(SEQ ID NO: 53) was placed under the control of the RSV promoter
(SEQ ID NO: 61). The reporter, pIND(SP1)LacZ, contains five copies
of a modified ecdysone response element ("EcRE", E/GRE), three
copies of an SP1 enhancer, and a minimal heat shock promoter, all
of which were placed upstream to the LacZ reporter gene.
[0268] 1.9--CfVgRXR: This example was prepared in the same way as
switch 1.8 except DmEcR was replaced with a truncated CfER
comprising a partial A/B domain and the complete CDEF domains [SEQ
ID NO: 62 (polynucleotide) and SEQ ID NO: 63 (polypeptide)].
[0269] 1.10--CfVgRXRdel: This example was prepared in the same way
as switch 1.9 except MmRXR (SEQ ID NO: 53) was deleted.
[0270] Cell lines: Four cell lines: CHO, Chinese hamster Cricetulus
griseus ovarian cell line; NIH3T3 (3T3) mouse Mus musculus cell
line; 293 human Homo sapiens kidney cell line, and CV1 Afican green
monkey kidney cell line were used in these experiments. Cells were
maintained in their respective media and were subcultured when they
reached 60% confluency. Standard methods for culture and
maintenance of the cells were followed.
[0271] Transfections: Several commercially available lipofactors as
well as electroporation methods were evaluated and the best
conditions for transfection of each cell line were developed. CHO,
NIH3T3, 293 and CV1 cells were grown to 60% confluency. DNAs
corresponding to the various switch constructs outlined in Examples
1.1 through 1.10 were transfected into CHO cells, NIH3T3 cells, 293
cells, or CV1 cells as follows.
[0272] CHO cells: Cells were harvested when they reach 60-80%
confluency and plated in 6- or 12 or 24-well plates at 250,000,
100,000, or 50,000 cells in 2.5, 1.0, or 0.5 ml of growth medium
containing 10% Fetal bovine serum respectively. The next day, the
cells were rinsed with growth medium and transfected for four
hours. LipofectAMINE.TM. 2000 (Life Technologies Inc,) was found to
be the best transfection reagent for these cells. For 12-well
plates, 4 .mu.l of LipofectAMINEm 2000 was mixed with 100 .mu.l of
growth medium 1.0 .mu.g of reporter construct and 0.25 .mu.g of
receptor construct(s) were added to the transfection mix. A second
reporter construct was added [pTKRL (Promega), 0.1
.mu.g/transfection mix] and comprised a Renilla luciferase gene
(SEQ ID NO: 64) operably linked and placed under the control of a
thyrnidine kinase (IK) constitutive promoter and was used for
normalization. The contents of the transfection mix were mixed in a
vortex mixer and let stand at room temperature for 30 min. At the
end of incubation, the transfection mix was added to the cells
maintained in 400 .mu.l growth medium. The cells were maintained at
37.degree. C. and 5% CO.sub.2 for four hours. At the end of
incubation, 500 .mu.l of growth medium containing 20% FBS and
either DMSO (control) or a DMSO solution of appropriate ligands
were added and the cells were maintained at 37.degree. C. and 5%
CO.sub.2 for 24-48 hr. The cells were harvested and reporter
activity was assayed. The same procedure was followed for 6 and 24
well plates as well except all the reagents were doubled for 6 well
plates and reduced to half for 24-well plates.
[0273] NIH3T3 Cells: Superfect.TM. (Qiagen Inc.) was found to be
the best trasfection reagent for 3T3 cells. The same procedures
described for CHO cells were followed for 3T3 cells as well with
two modifications. The cells were plated when they reached 50%
confluency. 125,000 or 50,000 or 25,000 cells were plated per well
of 6 or 12- or 24-well plates respectively. The GA14EcR/VP16RXR and
reporter vector DNAs were transfected into NIH3T3 cells, the
transfected cells were grown in medium containing PonA, MurA,
N-(2-ethyl-3methoxybenzoyl)-N'-(3,5-dimethylbenzoy-
l)-N'-t-butylhydrazine, or
N-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)-N'--
(3,5-dimethybenzoyl)-N'-tert-butylhydrazine for 48 hr. The ligand
treatments were performed as described in the CHO cell section
above.
[0274] 293 Cells: LipofectAMINE.TM. 2000 (Life Technologies) was
found to be the best lipofactor for 293 cells. The same procedures
described for CHO were followed for 293 cells except that the cells
were plated in biocoated plates to avoid clumping. The ligand
treatments were performed as described in the CHO cell section
above.
[0275] CV1 Cells: LipofectAMiNE.TM. plus (Life Technologies) was
found to be the best lipofactor for CV1 cells. The same procedures
described for NIH3T3 cells were followed for CV1 cells
[0276] Ligands: Ponasterone A and Muristerone A were purchased from
Sigma Chemical Company. The two non-steroids
N-(2-ethyl-3-methoxybenzoyl)-N'-(3-
,5-dimethylbenzoyl)-N'-t-butylhydrazine, or
N-(3,4-(1,2-ethylenedioxy)-2-m-
ethylbenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine are
synthetic stable ecdysteroids synthesized at Rohm and Haas Company.
All ligands were dissolved in DMSO and the final concentration of
DMSO was maintained at 0.1% in both controls and treatments.
[0277] Reporter Assays: Cells were harvested 24-48 hr after adding
ligands. 0.125, 250, or 500 .mu.l of passive lysis buffer (part of
Dual-luciferase.TM. reporter assay system from Promega Corporation)
were added to each well of 24- or 12- or 24well plates
respectively. The plates were placed on a rotary shaker for 15 min.
Twenty .mu.l of lysate was assayed. Luciferase activity was
measured using Dual-luciferase.TM. reporter assay system from
Promega Corporation following the manufacturer's instructions.
.beta.-Galactosidase was measured using Galacto-Star.TM. assay kit
from TROPIX following the manufacturer's instructions. All
luciferase and .beta.-galactosidase activities were normalized
using Renilla luciferase as a standard. Fold activities were
calculated by dividing normalized relative light units ("RLU") in
ligand treated cells with normlized RLU in DMSO treated cells
(untreated control).
[0278] The results of these experiments are provided in the
following tables.
1TABLE 1 Transactivation of reporter genes through various switches
in CHO cells Mean Fold Activation with 50 .mu.M
N-(2-ethyl-3-methoxybenzoyl)-N'- (3,5-dimethylbenzoyl)-N'-t-
Composition of Switch butylhydrazine 1.1 GAL4EcR + VP16RXR 267
pGAL4RELuc 1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR +
VP16CfEcR 85 pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 312 pGAL4RELuc 1.5
GAL4USP + VP16CfEcR 2 pGAL4RELuc 1.6 GAL4CfEcRVP16 9 pGAL4RELuc 1.7
VP16CfEcR 36 pEcRELuc 1.8 DmVgRXR + MmRXR 14 pIND(SP1)LacZ 1.9
CfVgRXR + MmRXR 27 pIND(SP1)LacZ 1.10 CfVgRXR 29 pIND(SP1)LacZ
[0279]
2TABLE 2 Transactivation of reporter genes through various switches
in 3T3 cells Mean Fold Activation Through
N-(2-ethyl-3-methoxybenzoyl)-N'- (3,5-dimethylbenzoyl)- Composition
of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 1118 pGAL4RELuc
1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 47
pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 269 pGAL4RELuc 1.5 GAL4USP +
VP16CfEcR 3 pGAL4RELuc 1.6 GAL4CfEcRVP16 7 pGAL4RELuc 1.7 VP16CfEcR
1 pEcRELuc 1.8 DmVgRXR + MmRXR 21 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR
19 pIND(SP1)LacZ 1.10 CfVgRXR 2 pIND(SP1)LacZ
[0280]
3TABLE 3 Transactivation of reporter genes through various switches
in 293 cells Mean Fold Activation Through
N-(2-ethyl-3-methoxybenzoyl)-N'- (3,5-dimethylbenzoyl)- Composition
of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 125 pGAL4RELuc
1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 17
pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 3 pGAL4RELuc 1.5 GAL4USP +
VP16CfEcR 2 pGAL4RELuc 1.6 GAL4CfEcRVP16 3 pGAL4RELuc 1.7 VP16CfEcR
2 pEcRELuc 1.8 DmVgRXR + MmRXR 21 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR
12 pIND(SP1)LacZ 1.10 CfVgRXR 3 pIND(SP1)LacZ
[0281]
4TABLE 4 Transactivation of reporter genes through various switches
in CV1 cells Mean Fold Activation Through
N-(2-ethyl-3-methoxybenzoyl)-N'- (3,5-dimethylbenzoyl)- Composition
of Switch N'-t-butylhydrazine 1.1 GAL4EcR + VP16RXR 279 pGAL4RELuc
1.2 GAL4EcR + VP16USP 2 pGAL4RELuc 1.3 GAL4RXR + VP16CfEcR 25
pGAL4RELuc 1.4 GAL4RXR + VP16DmEcR 80 pGAL4RELuc 1.5 GAL4USP +
VP16CfEcR 3 pGAL4RELuc 1.6 GAL4CfEcRVP16 6 pGAL4RELuc 1.7 VP16CfEcR
1 pEcRELuc 1.8 DmVgRXR + MmRXR 12 pIND(SP1)LacZ 1.9 CfVgRXR + MmRXR
7 pIND(SP1)LacZ 1.10 CfVgRXR 1 pIND(SP1)LacZ
[0282]
5TABLE 5 Transactivation of reporter gene GAL4CfEcRDEF/VP16MmRXRDEF
(switch 1.1) through steroids and non-steroids in 3T3 cells. Mean
Fold Induction at 1.0 .mu.M Ligand Concentration 1. Ponasterone A
1.0 2. Muristerone A 1.0 3. N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-
116 dimethylbenzoyl)-N'-tert-butylhydrazine 4.
N'-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)- 601
N'-(3,5-dimethylbenzoyl)-N'-tert- butylhydrazine
[0283]
6TABLE 6 Transactivation of reporter gene
GAL4MmRXRDEF/VP16CfEcRCDEF (switch 1.3) through steroids and
non-steroids in 3T3 cells. Mean Fold Induction at 1.0 .mu.M Ligand
Concentration 1. Ponasterone A 1.0 2. Muristerone A 1.0 3.
N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5- 71
dimethylbenzoyl)-N'-tert-butylhydrazine 4.
N'-(3,4-(1,2-ethylenedioxy)-2-methylbenzoyl)- 54
N'-(3,5-dimethylbenzoyl)-N'-tert- butylhydrazine
[0284] Applicants' results demonstrate that the non-steroidal
ecdysone agonists,
N-(2-ethyl-3methoxybenzoyl)N'-(3,5-dimethylbenzoyl)-N'-tert-but-
ylhydrazine and
N'-(3,4(1,2-ethyenedioxy)-2-methylbenzoyl)-N'-(3,5-dimethy-
lbenzoyl)-N'-tert-butylhydrazine, were more potent activators of
CfEcR as compared to Drosophila melanogaster EcR (DmEcR). (see
Tables 14). Also, in the mammalian cell lines tested, MmRXR
performed better than CfUSP as a heterodimeric partner for CfEcR.
(see Tables 14). Additionally, Applicants' inducible gene
expression modulation system performed better when exogenous MmRXR
was used than when the system relied only on endogenous RXR levels
(see Tables 14).
[0285] Applicants' results also show that in a CfEcR-based
inducible gene expression system, the non-steroidal ecdysone
agonist induced reporter gene expression at a lower concentration
(i.e., increased ligand sensitivity) as compared to the steroid
ligands, ponasterone A and muristerone A (see Tables 5 and 6).
[0286] Out of 10 EcR based gene switches tested, the
GAL4EcR/VP16RXR switch (Switch 1.1) performed better than any other
switch in all four cell lines examined and was more sensitive to
non-steroids than steroids. The results also demonstrate that
placing the activation domain (AD) and DNA binding domain (DNABD)
on each of the two partners reduced background when compared to
placing both AD and DNABD together on one of the two partners.
Therefore, a switch format where the AD and DNABD are separated
between two partners, works well for EcR-based gene switch
applications.
[0287] In addition, the MmRXR/EcR-based switches performed better
than CfUSP/EcR-based switches, which have a higher background
activity than the MmRXR/EoR switches in the absence of ligand.
[0288] Finally, the GAL4EcR/VP16RXR switch (Switch 1.1) was more
sensitive to nonsteroid ligands than to the steroid ligands (see
Tables 5 and 6). In particular, steroid ligands initiated
transactivation at concentrations of 50 .mu.M, whereas the
non-steroid ligands initiated transactivation at less than 1 .mu.M
(submicromolar) concentration.
Example 2
[0289] This Example describes Applicants' further analysis of
truncated EcR and RXR polypeptides in the improved EcR-based
inducible gene expression system of the invention. To identify the
best combination and length of two receptors that give a switch
with a) maximum induction in the presence of ligand; b) minimum
background in the absence of ligand; c) highly sensitive to ligand
concentration; and d) minimum cross-talk among ligands and
receptors, Applicants made and analyzed several truncation
mutations of the CfEcR and MMRXR receptor polypeptides in NIH3T3
cells.
[0290] Briefly, polynucleotides encoding EcR or RXR receptors were
truncated at the junctions of A/B, C, D, E and F domains and fused
to either a GAL4 DNA binding domain encoding polynucleotide (SEQ ID
NO: 41) for CfEcR, or a VP16 activation domain encoding
polynucleotide (SEQ ID NO: 45) for MmRXR as described in Example 1.
The resulting receptor truncation/fusion polypeptides were assayed
in NIH3T3 cells. Plasmid pFRLUC (Stratagene) encoding a luciferase
polypeptide was used as a reporter gene construct and pTKRL
(Promega) encoding a Renilla luciferase polypeptide under the
control of the constitutive TK promoter was used to normalize the
transfections as described above. The analysis was performed in
triplicates and mean luciferase counts were determined as described
above.
[0291] Gene Expression Cassettes Encoding Truncated Ecdysone
Receptor Polypeptides
[0292] Gene expression cassettes comprising polynucleotides
encoding either fill length or truncated CfEcR polypeptides fused
to a GAL4 DNA binding domain (SEQ ID NO: 41): GAL4CfEcRA/BCDEF
(full length CfEcRA/BCDEF; SEQ ID NO: 49), GAL4CfEcRCDEF
(CfEcRCDEF; SEQ ID NO: 1), GAIACfEcR1/2CDEF (CfEcR1/2CDEF; SEQ ID
NO: 2), GAL4CfEcRDEF (CfEcRDEF; SEQ ID NO: 3), GALACfEcREF
(CfEcREF; SEQ ID NO: 4), and GAL4CfEcRDE (CfEcRDE; SEQ ID NO: 5)
were wansfected into NIH3T3 cells along with VP16MmRXRDEF
(constructed as in Example 1.1; FIG. 11) or VP16MmR EF [constructed
as in Example 1.1 except that MmRXRDEF was replaced with MmRXREF
(SEQ ID NO: 23); FIG. 12], and pFRLUc and pTKRL plasmid DNAs. The
transfected cells were grown in the presence 0, 1, 5 or 25 uM of
N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydraz-
ine or PonA for 48 hr. The cells were harvested, lysed and
luciferase reporter activity was measured in the cell lysates.
Total fly luciferase relative light units are presented. The number
on the top of each bar is the maximun fold induction for that
treatment.
[0293] Applicants' results show that the EF domain of MmRXR is
sufficient and performs better than DEF domains of this receptor
(see FIGS. 11 and 12). Applicants have also shown that, in general,
EcR/RXR receptor combinations are insensitive to PonA (see FIGS. 11
and 12). As shown in the FIGS. 11 and 12, the GAL4CfEcRCDEF hybrid
polypeptide (SEQ ID NO: 7) performed better than any other CfEcR
hybrid polypeptide.
[0294] Gene Expression Cassettes Encoding Truncated Retinoid X
Receptor Pol peptides
[0295] Gene expression cassettes comprising polynucleotides
encoding either full length or truncated MmRXR polypeptides fused
to a VP16 transactivation domain (SEQ ID NO: 45): VP16MmR A/BCDEF
(full length MmRXRA/BCDEF; SEQ ID NO: 53), VP16MmRXRCDEF
(MmRXRCDEF; SEQ ID NO: 21), VP16MmRDEF (MmRXRDEF; SEQ ID NO: 22),
VP16MmRXREF RXREF; SEQ ID NO: 23), VP16MmRXRBam-EF ("MmRXRBam-EF"
or "MmRXR-truncatedEF"; SEQ ID NO: 24), and VP16MmRXRAdel
("MmRXRAF2del" or "MmRxR-E"; SEQ ID NO: 25) constructs were
transfected into NIH3T3 cells along with GAL4CfEcRCDEF (constructed
as in Example 1.1; FIG. 13) or GAL4CfEcRDEF [constructed as in
Example 1.1 except CfEcRCDEF was replaced with CfEcRDEF (SEQ ID NO:
3); FIG. 14], pFRLUc and pTKRL plasmid DNAs as described above. The
transfected cells were grown in the presence 0, 1, 5 and 25 uM of
N-(2-ethyl-3-methoxybenzo-
yl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine or PonA for 48
hr. The cells were harvested and lysed and reporter activity was
measured in the cell lysate. Total fly luciferase relative light
units are presented. The nuiber on top of each bar is the maximum
fold induction in that treatment Of all the truncations of MmRXR
tested, Applicants' results show that the MmRXREF receptor was the
best partner for CfEcR (FIGS. 13 and 14). CfEcRCDEF showed better
induction than CfEcRDEF using MmRXREF. Deleting AF2 (abbreviated
"EF-AF2del") or helices 1-3 of the E domain (abbreviated
"EF-Bamdel") resulted in an RXR receptor that reduced gene
induction and ligand sensitivity when partnered with either
CfEcRCDEF (FIG. 13) or CfEcRDEF (FIG. 14) in NIH3T3 cells. In
general, the CfEcR/RXR-based switch was much more sensitive to the
non-steroid N-(2-ethyl-3-methoxyben-
zoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydrazine than to the
steroid PonA.
Example 3
[0296] This Example describes Applicants' further analysis of gene
expression cassettes encoding truncated EcR or RXR receptor
polypeptides that affect either ligand binding activity or ligand
sensitivity, or both. Briefly, six different combinations of
chimeric receptor pairs, constructed as described in Examples 1 and
2, were further analyzed in a single experiment in NIH3T3 cells.
These six receptor pair combinations and their corresponding sample
numbers are depicted in Table 7.
7TABLE 7 CfEcR + MmRXR Truncation Receptor Combinations in NIH3T3
Cells EcR Polypeptide RXR Polypeptide X-Axis Sample No. Construct
Construct Samples 1 and 2 GAL4CfEcRCDEF VP16RXRA/BCDEF (Full
length) Samples 3 and 4 GAL4CfEcRCDEF VP16RXRDEF Samples 5 and 6
GAL4CfEcRCDEF VP16RXREF Samples 7 and 8 GAL4CfEcRDEF VP16RXRA/BCDEF
(Full length) Samples 9 and 10 GAL4CfEcRDEF VP16RXRDEF Samples 11
and 12 GAL4CfEcRDEF VP16RXREF
[0297] The above receptor construct pairs, along with the reporter
plasmid pFRLuc were transfected into NIH3T3 cells as described
above. The six CfEcR truncation receptor combinations were
duplicated into two groups and treated with either steroid (odd
numbers on x-axis of FIG. 15) or non-steroid (even numbers on
x-axis of FIG. 15). In particular, the cells were grown in media
containing 0, 1, 5 or 25 uM PonA (steroid) or
N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydraz-
ine (non-steroid) ligand. The reporter gene activity was measured
and total RLU are shown. Ihe number on top of each bar is the
maximum fold induction for that treatment and is the mean of three
replicates.
[0298] As shown in FIG. 15, the CfEcRCDEF/MmRXREF receptor
combinations were the best switch pairs both in terms of total RLU
and fold induction (compare columns 1-6 to columns 7-12). This
confirms Applicants' earlier findings as described in Example 2
(FIGS. 11-14). The same gene expression cassettes encoding the
truncated EcR and RXR polypeptides were also assayed in a human
lung carcinoma cell line A549 (ATCC) and similar results were
observed (data not shown).
Sequence CWU 1
1
64 1 1288 DNA Artificial Sequence misc_feature Novel Sequence 1
aagggccctg cgccccgtca gcaagaggaa ctgtgtctgg tatgcgggga cagagcctcc
60 ggataccact acaatgcgct cacgtgtgaa gggtgtaaag ggttcttcag
acggagtgtt 120 accaaaaatg cggtttatat ttgtaaattc ggtcacgctt
gcgaaatgga catgtacatg 180 cgacggaaat gccaggagtg ccgcctgaag
aagtgcttag ctgtaggcat gaggcctgag 240 tgcgtagtac ccgagactca
gtgcgccatg aagcggaaag agaagaaagc acagaaggag 300 aaggacaaac
tgcctgtcag cacgacgacg gtggacgacc acatgccgcc cattatgcag 360
tgtgaacctc cacctcctga agcagcaagg attcacgaag tggtcccaag gtttctctcc
420 gacaagctgt tggagacaaa ccggcagaaa aacatccccc agttgacagc
caaccagcag 480 ttccttatcg ccaggctcat ctggtaccag gacgggtacg
agcagccttc tgatgaagat 540 ttgaagagga ttacgcagac gtggcagcaa
gcggacgatg aaaacgaaga gtctgacact 600 cccttccgcc agatcacaga
gatgactatc ctcacggtcc aacttatcgt ggagttcgcg 660 aagggattgc
cagggttcgc caagatctcg cagcctgatc aaattacgct gcttaaggct 720
tgctcaagtg aggtaatgat gctccgagtc gcgcgacgat acgatgcggc ctcagacagt
780 gttctgttcg cgaacaacca agcgtacact cgcgacaact accgcaaggc
tggcatggcc 840 tacgtcatcg aggatctact gcacttctgc cggtgcatgt
actctatggc gttggacaac 900 atccattacg cgctgctcac ggctgtcgtc
atcttttctg accggccagg gttggagcag 960 ccgcaactgg tggaagaaat
ccagcggtac tacctgaata cgctccgcat ctatatcctg 1020 aaccagctga
gcgggtcggc gcgttcgtcc gtcatatacg gcaagatcct ctcaatcctc 1080
tctgagctac gcacgctcgg catgcaaaac tccaacatgt gcatctccct caagctcaag
1140 aacagaaagc tgccgccttt cctcgaggag atctgggatg tggcggacat
gtcgcacacc 1200 caaccgccgc ctatcctcga gtcccccacg aatctctagc
ccctgcgcgc acgcatcgcc 1260 gatgccgcgt ccggccgcgc tgctctga 1288 2
1110 DNA Artificial Sequence misc_feature Novel Sequence 2
gcggtttata tttgtaaatt cggtcacgct tgcgaaatgg acatgtacat gcgacggaaa
60 tgccaggagt gccgcctgaa gaagtgctta gctgtaggca tgaggcctga
gtgcgtagta 120 cccgagactc agtgcgccat gaagcggaaa gagaagaaag
cacagaagga gaaggacaaa 180 ctgcctgtca gcacgacgac ggtggacgac
cacatgccgc ccattatgca gtgtgaacct 240 ccacctcctg aagcagcaag
gattcacgaa gtggtcccaa ggtttctctc cgacaagctg 300 ttggagacaa
accggcagaa aaacatcccc cagttgacag ccaaccagca gttccttatc 360
gccaggctca tctggtacca ggacgggtac gagcagcctt ctgatgaaga tttgaagagg
420 attacgcaga cgtggcagca agcggacgat gaaaacgaag agtctgacac
tcccttccgc 480 cagatcacag agatgactat cctcacggtc caacttatcg
tggagttcgc gaagggattg 540 ccagggttcg ccaagatctc gcagcctgat
caaattacgc tgcttaaggc ttgctcaagt 600 gaggtaatga tgctccgagt
cgcgcgacga tacgatgcgg cctcagacag tgttctgttc 660 gcgaacaacc
aagcgtacac tcgcgacaac taccgcaagg ctggcatggc ctacgtcatc 720
gaggatctac tgcacttctg ccggtgcatg tactctatgg cgttggacaa catccattac
780 gcgctgctca cggctgtcgt catcttttct gaccggccag ggttggagca
gccgcaactg 840 gtggaagaaa tccagcggta ctacctgaat acgctccgca
tctatatcct gaaccagctg 900 agcgggtcgg cgcgttcgtc cgtcatatac
ggcaagatcc tctcaatcct ctctgagcta 960 cgcacgctcg gcatgcaaaa
ctccaacatg tgcatctccc tcaagctcaa gaacagaaag 1020 ctgccgcctt
tcctcgagga gatctgggat gtggcggaca tgtcgcacac ccaaccgccg 1080
cctatcctcg agtcccccac gaatctctag 1110 3 1054 DNA Artificial
Sequence misc_feature Novel Sequence 3 cctgagtgcg tagtacccga
gactcagtgc gccatgaagc ggaaagagaa gaaagcacag 60 aaggagaagg
acaaactgcc tgtcagcacg acgacggtgg acgaccacat gccgcccatt 120
atgcagtgtg aacctccacc tcctgaagca gcaaggattc acgaagtggt cccaaggttt
180 ctctccgaca agctgttgga gacaaaccgg cagaaaaaca tcccccagtt
gacagccaac 240 cagcagttcc ttatcgccag gctcatctgg taccaggacg
ggtacgagca gccttctgat 300 gaagatttga agaggattac gcagacgtgg
cagcaagcgg acgatgaaaa cgaagagtct 360 gacactccct tccgccagat
cacagagatg actatcctca cggtccaact tatcgtggag 420 ttcgcgaagg
gattgccagg gttcgccaag atctcgcagc ctgatcaaat tacgctgctt 480
aaggcttgct caagtgaggt aatgatgctc cgagtcgcgc gacgatacga tgcggcctca
540 gacagtgttc tgttcgcgaa caaccaagcg tacactcgcg acaactaccg
caaggctggc 600 atggcctacg tcatcgagga tctactgcac ttctgccggt
gcatgtactc tatggcgttg 660 gacaacatcc attacgcgct gctcacggct
gtcgtcatct tttctgaccg gccagggttg 720 gagcagccgc aactggtgga
agaaatccag cggtactacc tgaatacgct ccgcatctat 780 atcctgaacc
agctgagcgg gtcggcgcgt tcgtccgtca tatacggcaa gatcctctca 840
atcctctctg agctacgcac gctcggcatg caaaactcca acatgtgcat ctccctcaag
900 ctcaagaaca gaaagctgcc gcctttcctc gaggagatct gggatgtggc
ggacatgtcg 960 cacacccaac cgccgcctat cctcgagtcc cccacgaatc
tctagcccct gcgcgcacgc 1020 atcgccgatg ccgcgtccgg ccgcgctgct ctga
1054 4 735 DNA Artificial Sequence misc_feature Novel Sequence 4
taccaggacg ggtacgagca gccttctgat gaagatttga agaggattac gcagacgtgg
60 cagcaagcgg acgatgaaaa cgaagagtct gacactccct tccgccagat
cacagagatg 120 actatcctca cggtccaact tatcgtggag ttcgcgaagg
gattgccagg gttcgccaag 180 atctcgcagc ctgatcaaat tacgctgctt
aaggcttgct caagtgaggt aatgatgctc 240 cgagtcgcgc gacgatacga
tgcggcctca gacagtgttc tgttcgcgaa caaccaagcg 300 tacactcgcg
acaactaccg caaggctggc atggcctacg tcatcgagga tctactgcac 360
ttctgccggt gcatgtactc tatggcgttg gacaacatcc attacgcgct gctcacggct
420 gtcgtcatct tttctgaccg gccagggttg gagcagccgc aactggtgga
agaaatccag 480 cggtactacc tgaatacgct ccgcatctat atcctgaacc
agctgagcgg gtcggcgcgt 540 tcgtccgtca tatacggcaa gatcctctca
atcctctctg agctacgcac gctcggcatg 600 caaaactcca acatgtgcat
ctccctcaag ctcaagaaca gaaagctgcc gcctttcctc 660 gaggagatct
gggatgtggc ggacatgtcg cacacccaac cgccgcctat cctcgagtcc 720
cccacgaatc tctag 735 5 960 DNA Artificial Sequence misc_feature
Novel Sequence 5 cctgagtgcg tagtacccga gactcagtgc gccatgaagc
ggaaagagaa gaaagcacag 60 aaggagaagg acaaactgcc tgtcagcacg
acgacggtgg acgaccacat gccgcccatt 120 atgcagtgtg aacctccacc
tcctgaagca gcaaggattc acgaagtggt cccaaggttt 180 ctctccgaca
agctgttgga gacaaaccgg cagaaaaaca tcccccagtt gacagccaac 240
cagcagttcc ttatcgccag gctcatctgg taccaggacg ggtacgagca gccttctgat
300 gaagatttga agaggattac gcagacgtgg cagcaagcgg acgatgaaaa
cgaagagtct 360 gacactccct tccgccagat cacagagatg actatcctca
cggtccaact tatcgtggag 420 ttcgcgaagg gattgccagg gttcgccaag
atctcgcagc ctgatcaaat tacgctgctt 480 aaggcttgct caagtgaggt
aatgatgctc cgagtcgcgc gacgatacga tgcggcctca 540 gacagtgttc
tgttcgcgaa caaccaagcg tacactcgcg acaactaccg caaggctggc 600
atggcctacg tcatcgagga tctactgcac ttctgccggt gcatgtactc tatggcgttg
660 gacaacatcc attacgcgct gctcacggct gtcgtcatct tttctgaccg
gccagggttg 720 gagcagccgc aactggtgga agaaatccag cggtactacc
tgaatacgct ccgcatctat 780 atcctgaacc agctgagcgg gtcggcgcgt
tcgtccgtca tatacggcaa gatcctctca 840 atcctctctg agctacgcac
gctcggcatg caaaactcca acatgtgcat ctccctcaag 900 ctcaagaaca
gaaagctgcc gcctttcctc gaggagatct gggatgtggc ggacatgtcg 960 6 1878
DNA Artificial Sequence misc_feature Novel Sequence 6 ggacctgcgc
cacgggtgca agaggagctg tgcctggttt gcggcgacag ggcctccggc 60
taccactaca acgccctcac ctgtgagggc tgcaaggggt tctttcgacg cagcgttacg
120 aagagcgccg tctactgctg caagttcggg cgcgcctgcg aaatggacat
gtacatgagg 180 cgaaagtgtc aggagtgccg cctgaaaaag tgcctggccg
tgggtatgcg gccggaatgc 240 gtcgtcccgg agaaccaatg tgcgatgaag
cggcgcgaaa agaaggccca gaaggagaag 300 gacaaaatga ccacttcgcc
gagctctcag catggcggca atggcagctt ggcctctggt 360 ggcggccaag
actttgttaa gaaggagatt cttgacctta tgacatgcga gccgccccag 420
catgccacta ttccgctact acctgatgaa atattggcca agtgtcaagc gcgcaatata
480 ccttccttaa cgtacaatca gttggccgtt atatacaagt taatttggta
ccaggatggc 540 tatgagcagc catctgaaga ggatctcagg cgtataatga
gtcaacccga tgagaacgag 600 agccaaacgg acgtcagctt tcggcatata
accgagataa ccatactcac ggtccagttg 660 attgttgagt ttgctaaagg
tctaccagcg tttacaaaga taccccagga ggaccagatc 720 acgttactaa
aggcctgctc gtcggaggtg atgatgctgc gtatggcacg acgctatgac 780
cacagctcgg actcaatatt cttcgcgaat aatagatcat atacgcggga ttcttacaaa
840 atggccggaa tggctgataa cattgaagac ctgctgcatt tctgccgcca
aatgttctcg 900 atgaaggtgg acaacgtcga atacgcgctt ctcactgcca
ttgtgatctt ctcggaccgg 960 ccgggcctgg agaaggccca actagtcgaa
gcgatccaga gctactacat cgacacgcta 1020 cgcatttata tactcaaccg
ccactgcggc gactcaatga gcctcgtctt ctacgcaaag 1080 ctgctctcga
tcctcaccga gctgcgtacg ctgggcaacc agaacgccga gatgtgtttc 1140
tcactaaagc tcaaaaaccg caaactgccc aagttcctcg aggagatctg ggacgttcat
1200 gccatcccgc catcggtcca gtcgcacctt cagattaccc aggaggagaa
cgagcgtctc 1260 gagcgggctg agcgtatgcg ggcatcggtt gggggcgcca
ttaccgccgg cattgattgc 1320 gactctgcct ccacttcggc ggcggcagcc
gcggcccagc atcagcctca gcctcagccc 1380 cagccccaac cctcctccct
gacccagaac gattcccagc accagacaca gccgcagcta 1440 caacctcagc
taccacctca gctgcaaggt caactgcaac cccagctcca accacagctt 1500
cagacgcaac tccagccaca gattcaacca cagccacagc tccttcccgt ctccgctccc
1560 gtgcccgcct ccgtaaccgc acctggttcc ttgtccgcgg tcagtacgag
cagcgaatac 1620 atgggcggaa gtgcggccat aggacccatc acgccggcaa
ccaccagcag tatcacggct 1680 gccgttaccg ctagctccac cacatcagcg
gtaccgatgg gcaacggagt tggagtcggt 1740 gttggggtgg gcggcaacgt
cagcatgtat gcgaacgccc agacggcgat ggccttgatg 1800 ggtgtagccc
tgcattcgca ccaagagcag cttatcgggg gagtggcggt taagtcggag 1860
cactcgacga ctgcatag 1878 7 1752 DNA Artificial Sequence
misc_feature Novel Sequence 7 gccgtctact gctgcaagtt cgggcgcgcc
tgcgaaatgg acatgtacat gaggcgaaag 60 tgtcaggagt gccgcctgaa
aaagtgcctg gccgtgggta tgcggccgga atgcgtcgtc 120 ccggagaacc
aatgtgcgat gaagcggcgc gaaaagaagg cccagaagga gaaggacaaa 180
atgaccactt cgccgagctc tcagcatggc ggcaatggca gcttggcctc tggtggcggc
240 caagactttg ttaagaagga gattcttgac cttatgacat gcgagccgcc
ccagcatgcc 300 actattccgc tactacctga tgaaatattg gccaagtgtc
aagcgcgcaa tataccttcc 360 ttaacgtaca atcagttggc cgttatatac
aagttaattt ggtaccagga tggctatgag 420 cagccatctg aagaggatct
caggcgtata atgagtcaac ccgatgagaa cgagagccaa 480 acggacgtca
gctttcggca tataaccgag ataaccatac tcacggtcca gttgattgtt 540
gagtttgcta aaggtctacc agcgtttaca aagatacccc aggaggacca gatcacgtta
600 ctaaaggcct gctcgtcgga ggtgatgatg ctgcgtatgg cacgacgcta
tgaccacagc 660 tcggactcaa tattcttcgc gaataataga tcatatacgc
gggattctta caaaatggcc 720 ggaatggctg ataacattga agacctgctg
catttctgcc gccaaatgtt ctcgatgaag 780 gtggacaacg tcgaatacgc
gcttctcact gccattgtga tcttctcgga ccggccgggc 840 ctggagaagg
cccaactagt cgaagcgatc cagagctact acatcgacac gctacgcatt 900
tatatactca accgccactg cggcgactca atgagcctcg tcttctacgc aaagctgctc
960 tcgatcctca ccgagctgcg tacgctgggc aaccagaacg ccgagatgtg
tttctcacta 1020 aagctcaaaa accgcaaact gcccaagttc ctcgaggaga
tctgggacgt tcatgccatc 1080 ccgccatcgg tccagtcgca ccttcagatt
acccaggagg agaacgagcg tctcgagcgg 1140 gctgagcgta tgcgggcatc
ggttgggggc gccattaccg ccggcattga ttgcgactct 1200 gcctccactt
cggcggcggc agccgcggcc cagcatcagc ctcagcctca gccccagccc 1260
caaccctcct ccctgaccca gaacgattcc cagcaccaga cacagccgca gctacaacct
1320 cagctaccac ctcagctgca aggtcaactg caaccccagc tccaaccaca
gcttcagacg 1380 caactccagc cacagattca accacagcca cagctccttc
ccgtctccgc tcccgtgccc 1440 gcctccgtaa ccgcacctgg ttccttgtcc
gcggtcagta cgagcagcga atacatgggc 1500 ggaagtgcgg ccataggacc
catcacgccg gcaaccacca gcagtatcac ggctgccgtt 1560 accgctagct
ccaccacatc agcggtaccg atgggcaacg gagttggagt cggtgttggg 1620
gtgggcggca acgtcagcat gtatgcgaac gcccagacgg cgatggcctt gatgggtgta
1680 gccctgcatt cgcaccaaga gcagcttatc gggggagtgg cggttaagtc
ggagcactcg 1740 acgactgcat ag 1752 8 1650 DNA Artificial Sequence
misc_feature Novel Sequence 8 cggccggaat gcgtcgtccc ggagaaccaa
tgtgcgatga agcggcgcga aaagaaggcc 60 cagaaggaga aggacaaaat
gaccacttcg ccgagctctc agcatggcgg caatggcagc 120 ttggcctctg
gtggcggcca agactttgtt aagaaggaga ttcttgacct tatgacatgc 180
gagccgcccc agcatgccac tattccgcta ctacctgatg aaatattggc caagtgtcaa
240 gcgcgcaata taccttcctt aacgtacaat cagttggccg ttatatacaa
gttaatttgg 300 taccaggatg gctatgagca gccatctgaa gaggatctca
ggcgtataat gagtcaaccc 360 gatgagaacg agagccaaac ggacgtcagc
tttcggcata taaccgagat aaccatactc 420 acggtccagt tgattgttga
gtttgctaaa ggtctaccag cgtttacaaa gataccccag 480 gaggaccaga
tcacgttact aaaggcctgc tcgtcggagg tgatgatgct gcgtatggca 540
cgacgctatg accacagctc ggactcaata ttcttcgcga ataatagatc atatacgcgg
600 gattcttaca aaatggccgg aatggctgat aacattgaag acctgctgca
tttctgccgc 660 caaatgttct cgatgaaggt ggacaacgtc gaatacgcgc
ttctcactgc cattgtgatc 720 ttctcggacc ggccgggcct ggagaaggcc
caactagtcg aagcgatcca gagctactac 780 atcgacacgc tacgcattta
tatactcaac cgccactgcg gcgactcaat gagcctcgtc 840 ttctacgcaa
agctgctctc gatcctcacc gagctgcgta cgctgggcaa ccagaacgcc 900
gagatgtgtt tctcactaaa gctcaaaaac cgcaaactgc ccaagttcct cgaggagatc
960 tgggacgttc atgccatccc gccatcggtc cagtcgcacc ttcagattac
ccaggaggag 1020 aacgagcgtc tcgagcgggc tgagcgtatg cgggcatcgg
ttgggggcgc cattaccgcc 1080 ggcattgatt gcgactctgc ctccacttcg
gcggcggcag ccgcggccca gcatcagcct 1140 cagcctcagc cccagcccca
accctcctcc ctgacccaga acgattccca gcaccagaca 1200 cagccgcagc
tacaacctca gctaccacct cagctgcaag gtcaactgca accccagctc 1260
caaccacagc ttcagacgca actccagcca cagattcaac cacagccaca gctccttccc
1320 gtctccgctc ccgtgcccgc ctccgtaacc gcacctggtt ccttgtccgc
ggtcagtacg 1380 agcagcgaat acatgggcgg aagtgcggcc ataggaccca
tcacgccggc aaccaccagc 1440 agtatcacgg ctgccgttac cgctagctcc
accacatcag cggtaccgat gggcaacgga 1500 gttggagtcg gtgttggggt
gggcggcaac gtcagcatgt atgcgaacgc ccagacggcg 1560 atggccttga
tgggtgtagc cctgcattcg caccaagagc agcttatcgg gggagtggcg 1620
gttaagtcgg agcactcgac gactgcatag 1650 9 1338 DNA Artificial
Sequence misc_feature Novel Sequence 9 tatgagcagc catctgaaga
ggatctcagg cgtataatga gtcaacccga tgagaacgag 60 agccaaacgg
acgtcagctt tcggcatata accgagataa ccatactcac ggtccagttg 120
attgttgagt ttgctaaagg tctaccagcg tttacaaaga taccccagga ggaccagatc
180 acgttactaa aggcctgctc gtcggaggtg atgatgctgc gtatggcacg
acgctatgac 240 cacagctcgg actcaatatt cttcgcgaat aatagatcat
atacgcggga ttcttacaaa 300 atggccggaa tggctgataa cattgaagac
ctgctgcatt tctgccgcca aatgttctcg 360 atgaaggtgg acaacgtcga
atacgcgctt ctcactgcca ttgtgatctt ctcggaccgg 420 ccgggcctgg
agaaggccca actagtcgaa gcgatccaga gctactacat cgacacgcta 480
cgcatttata tactcaaccg ccactgcggc gactcaatga gcctcgtctt ctacgcaaag
540 ctgctctcga tcctcaccga gctgcgtacg ctgggcaacc agaacgccga
gatgtgtttc 600 tcactaaagc tcaaaaaccg caaactgccc aagttcctcg
aggagatctg ggacgttcat 660 gccatcccgc catcggtcca gtcgcacctt
cagattaccc aggaggagaa cgagcgtctc 720 gagcgggctg agcgtatgcg
ggcatcggtt gggggcgcca ttaccgccgg cattgattgc 780 gactctgcct
ccacttcggc ggcggcagcc gcggcccagc atcagcctca gcctcagccc 840
cagccccaac cctcctccct gacccagaac gattcccagc accagacaca gccgcagcta
900 caacctcagc taccacctca gctgcaaggt caactgcaac cccagctcca
accacagctt 960 cagacgcaac tccagccaca gattcaacca cagccacagc
tccttcccgt ctccgctccc 1020 gtgcccgcct ccgtaaccgc acctggttcc
ttgtccgcgg tcagtacgag cagcgaatac 1080 atgggcggaa gtgcggccat
aggacccatc acgccggcaa ccaccagcag tatcacggct 1140 gccgttaccg
ctagctccac cacatcagcg gtaccgatgg gcaacggagt tggagtcggt 1200
gttggggtgg gcggcaacgt cagcatgtat gcgaacgccc agacggcgat ggccttgatg
1260 ggtgtagccc tgcattcgca ccaagagcag cttatcgggg gagtggcggt
taagtcggag 1320 cactcgacga ctgcatag 1338 10 969 DNA Artificial
Sequence misc_feature Novel Sequence 10 cggccggaat gcgtcgtccc
ggagaaccaa tgtgcgatga agcggcgcga aaagaaggcc 60 cagaaggaga
aggacaaaat gaccacttcg ccgagctctc agcatggcgg caatggcagc 120
ttggcctctg gtggcggcca agactttgtt aagaaggaga ttcttgacct tatgacatgc
180 gagccgcccc agcatgccac tattccgcta ctacctgatg aaatattggc
caagtgtcaa 240 gcgcgcaata taccttcctt aacgtacaat cagttggccg
ttatatacaa gttaatttgg 300 taccaggatg gctatgagca gccatctgaa
gaggatctca ggcgtataat gagtcaaccc 360 gatgagaacg agagccaaac
ggacgtcagc tttcggcata taaccgagat aaccatactc 420 acggtccagt
tgattgttga gtttgctaaa ggtctaccag cgtttacaaa gataccccag 480
gaggaccaga tcacgttact aaaggcctgc tcgtcggagg tgatgatgct gcgtatggca
540 cgacgctatg accacagctc ggactcaata ttcttcgcga ataatagatc
atatacgcgg 600 gattcttaca aaatggccgg aatggctgat aacattgaag
acctgctgca tttctgccgc 660 caaatgttct cgatgaaggt ggacaacgtc
gaatacgcgc ttctcactgc cattgtgatc 720 ttctcggacc ggccgggcct
ggagaaggcc caactagtcg aagcgatcca gagctactac 780 atcgacacgc
tacgcattta tatactcaac cgccactgcg gcgactcaat gagcctcgtc 840
ttctacgcaa agctgctctc gatcctcacc gagctgcgta cgctgggcaa ccagaacgcc
900 gagatgtgtt tctcactaaa gctcaaaaac cgcaaactgc ccaagttcct
cgaggagatc 960 tgggacgtt 969 11 412 PRT Artificial Sequence
misc_feature Novel Sequence 11 Lys Gly Pro Ala Pro Arg Gln Gln Glu
Glu Leu Cys Leu Val Cys Gly 1 5 10 15 Asp Arg Ala Ser Gly Tyr His
Tyr Asn Ala Leu Thr Cys Glu Gly Cys 20 25 30 Lys Gly Phe Phe Arg
Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys 35 40 45 Lys Phe Gly
His Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys 50 55 60 Gln
Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu 65 70
75 80 Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys
Lys 85 90 95 Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr
Thr Val Asp 100 105 110 Asp His Met Pro Pro Ile Met Gln Cys Glu Pro
Pro Pro Pro Glu Ala 115 120 125 Ala Arg Ile His Glu Val Val Pro Arg
Phe Leu Ser Asp Lys Leu Leu 130 135 140 Glu Thr Asn Arg Gln Lys Asn
Ile Pro Gln Leu Thr Ala Asn Gln Gln 145 150 155 160 Phe Leu Ile Ala
Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro 165 170 175 Ser Asp
Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp 180 185 190
Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met 195
200 205 Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu
Pro 210
215 220 Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu Lys
Ala 225 230 235 240 Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg
Arg Tyr Asp Ala 245 250 255 Ala Ser Asp Ser Val Leu Phe Ala Asn Asn
Gln Ala Tyr Thr Arg Asp 260 265 270 Asn Tyr Arg Lys Ala Gly Met Ala
Tyr Val Ile Glu Asp Leu Leu His 275 280 285 Phe Cys Arg Cys Met Tyr
Ser Met Ala Leu Asp Asn Ile His Tyr Ala 290 295 300 Leu Leu Thr Ala
Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln 305 310 315 320 Pro
Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg 325 330
335 Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser Val Ile
340 345 350 Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu
Gly Met 355 360 365 Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys
Asn Arg Lys Leu 370 375 380 Pro Pro Phe Leu Glu Glu Ile Trp Asp Val
Ala Asp Met Ser His Thr 385 390 395 400 Gln Pro Pro Pro Ile Leu Glu
Ser Pro Thr Asn Leu 405 410 12 412 PRT Artificial Sequence
misc_feature Novel Sequence 12 Lys Gly Pro Ala Pro Arg Gln Gln Glu
Glu Leu Cys Leu Val Cys Gly 1 5 10 15 Asp Arg Ala Ser Gly Tyr His
Tyr Asn Ala Leu Thr Cys Glu Gly Cys 20 25 30 Lys Gly Phe Phe Arg
Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys 35 40 45 Lys Phe Gly
His Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys 50 55 60 Gln
Glu Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu 65 70
75 80 Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys
Lys 85 90 95 Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr
Thr Val Asp 100 105 110 Asp His Met Pro Pro Ile Met Gln Cys Glu Pro
Pro Pro Pro Glu Ala 115 120 125 Ala Arg Ile His Glu Val Val Pro Arg
Phe Leu Ser Asp Lys Leu Leu 130 135 140 Glu Thr Asn Arg Gln Lys Asn
Ile Pro Gln Leu Thr Ala Asn Gln Gln 145 150 155 160 Phe Leu Ile Ala
Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro 165 170 175 Ser Asp
Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp 180 185 190
Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met 195
200 205 Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly Leu
Pro 210 215 220 Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu
Leu Lys Ala 225 230 235 240 Cys Ser Ser Glu Val Met Met Leu Arg Val
Ala Arg Arg Tyr Asp Ala 245 250 255 Ala Ser Asp Ser Val Leu Phe Ala
Asn Asn Gln Ala Tyr Thr Arg Asp 260 265 270 Asn Tyr Arg Lys Ala Gly
Met Ala Tyr Val Ile Glu Asp Leu Leu His 275 280 285 Phe Cys Arg Cys
Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala 290 295 300 Leu Leu
Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln 305 310 315
320 Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg
325 330 335 Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser
Val Ile 340 345 350 Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg
Thr Leu Gly Met 355 360 365 Gln Asn Ser Asn Met Cys Ile Ser Leu Lys
Leu Lys Asn Arg Lys Leu 370 375 380 Pro Pro Phe Leu Glu Glu Ile Trp
Asp Val Ala Asp Met Ser His Thr 385 390 395 400 Gln Pro Pro Pro Ile
Leu Glu Ser Pro Thr Asn Leu 405 410 13 334 PRT Artificial Sequence
misc_feature Novel Sequence 13 Pro Glu Cys Val Val Pro Glu Thr Gln
Cys Ala Met Lys Arg Lys Glu 1 5 10 15 Lys Lys Ala Gln Lys Glu Lys
Asp Lys Leu Pro Val Ser Thr Thr Thr 20 25 30 Val Asp Asp His Met
Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro 35 40 45 Glu Ala Ala
Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp Lys 50 55 60 Leu
Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn 65 70
75 80 Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr
Glu 85 90 95 Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr
Trp Gln Gln 100 105 110 Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro
Phe Arg Gln Ile Thr 115 120 125 Glu Met Thr Ile Leu Thr Val Gln Leu
Ile Val Glu Phe Ala Lys Gly 130 135 140 Leu Pro Gly Phe Ala Lys Ile
Ser Gln Pro Asp Gln Ile Thr Leu Leu 145 150 155 160 Lys Ala Cys Ser
Ser Glu Val Met Met Leu Arg Val Ala Arg Arg Tyr 165 170 175 Asp Ala
Ala Ser Asp Ser Val Leu Phe Ala Asn Asn Gln Ala Tyr Thr 180 185 190
Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile Glu Asp Leu 195
200 205 Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile
His 210 215 220 Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg
Pro Gly Leu 225 230 235 240 Glu Gln Pro Gln Leu Val Glu Glu Ile Gln
Arg Tyr Tyr Leu Asn Thr 245 250 255 Leu Arg Ile Tyr Ile Leu Asn Gln
Leu Ser Gly Ser Ala Arg Ser Ser 260 265 270 Val Ile Tyr Gly Lys Ile
Leu Ser Ile Leu Ser Glu Leu Arg Thr Leu 275 280 285 Gly Met Gln Asn
Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn Arg 290 295 300 Lys Leu
Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser 305 310 315
320 His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 325 330
14 244 PRT Artificial Sequence misc_feature Novel Sequence 14 Tyr
Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile 1 5 10
15 Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr
20 25 30 Pro Phe Arg Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln
Leu Ile 35 40 45 Val Glu Phe Ala Lys Gly Leu Pro Gly Phe Ala Lys
Ile Ser Gln Pro 50 55 60 Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser
Ser Glu Val Met Met Leu 65 70 75 80 Arg Val Ala Arg Arg Tyr Asp Ala
Ala Ser Asp Ser Val Leu Phe Ala 85 90 95 Asn Asn Gln Ala Tyr Thr
Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala 100 105 110 Tyr Val Ile Glu
Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met 115 120 125 Ala Leu
Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe 130 135 140
Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln 145
150 155 160 Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln
Leu Ser 165 170 175 Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile
Leu Ser Ile Leu 180 185 190 Ser Glu Leu Arg Thr Leu Gly Met Gln Asn
Ser Asn Met Cys Ile Ser 195 200 205 Leu Lys Leu Lys Asn Arg Lys Leu
Pro Pro Phe Leu Glu Glu Ile Trp 210 215 220 Asp Val Ala Asp Met Ser
His Thr Gln Pro Pro Pro Ile Leu Glu Ser 225 230 235 240 Pro Thr Asn
Leu 15 320 PRT Artificial Sequence misc_feature Novel Sequence 15
Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu 1 5
10 15 Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr
Thr 20 25 30 Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro
Pro Pro Pro 35 40 45 Glu Ala Ala Arg Ile His Glu Val Val Pro Arg
Phe Leu Ser Asp Lys 50 55 60 Leu Leu Glu Thr Asn Arg Gln Lys Asn
Ile Pro Gln Leu Thr Ala Asn 65 70 75 80 Gln Gln Phe Leu Ile Ala Arg
Leu Ile Trp Tyr Gln Asp Gly Tyr Glu 85 90 95 Gln Pro Ser Asp Glu
Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln 100 105 110 Ala Asp Asp
Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr 115 120 125 Glu
Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly 130 135
140 Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu Leu
145 150 155 160 Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala
Arg Arg Tyr 165 170 175 Asp Ala Ala Ser Asp Ser Val Leu Phe Ala Asn
Asn Gln Ala Tyr Thr 180 185 190 Arg Asp Asn Tyr Arg Lys Ala Gly Met
Ala Tyr Val Ile Glu Asp Leu 195 200 205 Leu His Phe Cys Arg Cys Met
Tyr Ser Met Ala Leu Asp Asn Ile His 210 215 220 Tyr Ala Leu Leu Thr
Ala Val Val Ile Phe Ser Asp Arg Pro Gly Leu 225 230 235 240 Glu Gln
Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr 245 250 255
Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser 260
265 270 Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr
Leu 275 280 285 Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu
Lys Asn Arg 290 295 300 Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp
Val Ala Asp Met Ser 305 310 315 320 16 625 PRT Artificial Sequence
misc_feature Novel Sequence 16 Gly Pro Ala Pro Arg Val Gln Glu Glu
Leu Cys Leu Val Cys Gly Asp 1 5 10 15 Arg Ala Ser Gly Tyr His Tyr
Asn Ala Leu Thr Cys Glu Gly Cys Lys 20 25 30 Gly Phe Phe Arg Arg
Ser Val Thr Lys Ser Ala Val Tyr Cys Cys Lys 35 40 45 Phe Gly Arg
Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln 50 55 60 Glu
Cys Arg Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu Cys 65 70
75 80 Val Val Pro Glu Asn Gln Cys Ala Met Lys Arg Arg Glu Lys Lys
Ala 85 90 95 Gln Lys Glu Lys Asp Lys Met Thr Thr Ser Pro Ser Ser
Gln His Gly 100 105 110 Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly Gln
Asp Phe Val Lys Lys 115 120 125 Glu Ile Leu Asp Leu Met Thr Cys Glu
Pro Pro Gln His Ala Thr Ile 130 135 140 Pro Leu Leu Pro Asp Glu Ile
Leu Ala Lys Cys Gln Ala Arg Asn Ile 145 150 155 160 Pro Ser Leu Thr
Tyr Asn Gln Leu Ala Val Ile Tyr Lys Leu Ile Trp 165 170 175 Tyr Gln
Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp Leu Arg Arg Ile 180 185 190
Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr Asp Val Ser Phe Arg 195
200 205 His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln Leu Ile Val Glu
Phe 210 215 220 Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile Pro Gln Glu
Asp Gln Ile 225 230 235 240 Thr Leu Leu Lys Ala Cys Ser Ser Glu Val
Met Met Leu Arg Met Ala 245 250 255 Arg Arg Tyr Asp His Ser Ser Asp
Ser Ile Phe Phe Ala Asn Asn Arg 260 265 270 Ser Tyr Thr Arg Asp Ser
Tyr Lys Met Ala Gly Met Ala Asp Asn Ile 275 280 285 Glu Asp Leu Leu
His Phe Cys Arg Gln Met Phe Ser Met Lys Val Asp 290 295 300 Asn Val
Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile Phe Ser Asp Arg 305 310 315
320 Pro Gly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr
325 330 335 Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn Arg His Cys Gly
Asp Ser 340 345 350 Met Ser Leu Val Phe Tyr Ala Lys Leu Leu Ser Ile
Leu Thr Glu Leu 355 360 365 Arg Thr Leu Gly Asn Gln Asn Ala Glu Met
Cys Phe Ser Leu Lys Leu 370 375 380 Lys Asn Arg Lys Leu Pro Lys Phe
Leu Glu Glu Ile Trp Asp Val His 385 390 395 400 Ala Ile Pro Pro Ser
Val Gln Ser His Leu Gln Ile Thr Gln Glu Glu 405 410 415 Asn Glu Arg
Leu Glu Arg Ala Glu Arg Met Arg Ala Ser Val Gly Gly 420 425 430 Ala
Ile Thr Ala Gly Ile Asp Cys Asp Ser Ala Ser Thr Ser Ala Ala 435 440
445 Ala Ala Ala Ala Gln His Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro
450 455 460 Ser Ser Leu Thr Gln Asn Asp Ser Gln His Gln Thr Gln Pro
Gln Leu 465 470 475 480 Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly Gln
Leu Gln Pro Gln Leu 485 490 495 Gln Pro Gln Leu Gln Thr Gln Leu Gln
Pro Gln Ile Gln Pro Gln Pro 500 505 510 Gln Leu Leu Pro Val Ser Ala
Pro Val Pro Ala Ser Val Thr Ala Pro 515 520 525 Gly Ser Leu Ser Ala
Val Ser Thr Ser Ser Glu Tyr Met Gly Gly Ser 530 535 540 Ala Ala Ile
Gly Pro Ile Thr Pro Ala Thr Thr Ser Ser Ile Thr Ala 545 550 555 560
Ala Val Thr Ala Ser Ser Thr Thr Ser Ala Val Pro Met Gly Asn Gly 565
570 575 Val Gly Val Gly Val Gly Val Gly Gly Asn Val Ser Met Tyr Ala
Asn 580 585 590 Ala Gln Thr Ala Met Ala Leu Met Gly Val Ala Leu His
Ser His Gln 595 600 605 Glu Gln Leu Ile Gly Gly Val Ala Val Lys Ser
Glu His Ser Thr Thr 610 615 620 Ala 625 17 583 PRT Artificial
Sequence misc_feature Novel Sequence 17 Ala Val Tyr Cys Cys Lys Phe
Gly Arg Ala Cys Glu Met Asp Met Tyr 1 5 10 15 Met Arg Arg Lys Cys
Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val 20 25 30 Gly Met Arg
Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys 35 40 45 Arg
Arg Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr Thr Ser 50 55
60 Pro Ser Ser Gln His Gly Gly Asn Gly Ser Leu Ala Ser Gly Gly Gly
65 70 75 80 Gln Asp Phe Val Lys Lys Glu Ile Leu Asp Leu Met Thr Cys
Glu Pro 85 90 95 Pro Gln His Ala Thr Ile Pro Leu Leu Pro Asp Glu
Ile Leu Ala Lys 100 105 110 Cys Gln Ala Arg Asn Ile Pro Ser Leu Thr
Tyr Asn Gln Leu Ala Val 115 120 125 Ile Tyr Lys Leu Ile Trp Tyr Gln
Asp Gly Tyr Glu Gln Pro Ser Glu 130 135 140 Glu Asp Leu Arg Arg Ile
Met Ser Gln Pro Asp Glu Asn Glu Ser Gln 145 150 155 160 Thr Asp Val
Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val 165 170 175 Gln
Leu Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr Lys Ile 180 185
190 Pro Gln Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys Ser Ser Glu Val
195 200 205 Met Met Leu Arg Met Ala Arg Arg Tyr Asp His Ser Ser Asp
Ser Ile 210 215 220 Phe Phe Ala Asn Asn Arg Ser Tyr Thr Arg Asp Ser
Tyr Lys Met Ala 225 230 235 240 Gly Met Ala Asp Asn Ile Glu Asp Leu
Leu His Phe Cys Arg Gln Met 245 250 255 Phe Ser Met Lys Val Asp Asn
Val Glu Tyr Ala Leu Leu Thr Ala Ile 260 265 270 Val Ile Phe Ser Asp
Arg Pro Gly Leu Glu Lys
Ala Gln Leu Val Glu 275 280 285 Ala Ile Gln Ser Tyr Tyr Ile Asp Thr
Leu Arg Ile Tyr Ile Leu Asn 290 295 300 Arg His Cys Gly Asp Ser Met
Ser Leu Val Phe Tyr Ala Lys Leu Leu 305 310 315 320 Ser Ile Leu Thr
Glu Leu Arg Thr Leu Gly Asn Gln Asn Ala Glu Met 325 330 335 Cys Phe
Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Lys Phe Leu Glu 340 345 350
Glu Ile Trp Asp Val His Ala Ile Pro Pro Ser Val Gln Ser His Leu 355
360 365 Gln Ile Thr Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala Glu Arg
Met 370 375 380 Arg Ala Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp
Cys Asp Ser 385 390 395 400 Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala
Gln His Gln Pro Gln Pro 405 410 415 Gln Pro Gln Pro Gln Pro Ser Ser
Leu Thr Gln Asn Asp Ser Gln His 420 425 430 Gln Thr Gln Pro Gln Leu
Gln Pro Gln Leu Pro Pro Gln Leu Gln Gly 435 440 445 Gln Leu Gln Pro
Gln Leu Gln Pro Gln Leu Gln Thr Gln Leu Gln Pro 450 455 460 Gln Ile
Gln Pro Gln Pro Gln Leu Leu Pro Val Ser Ala Pro Val Pro 465 470 475
480 Ala Ser Val Thr Ala Pro Gly Ser Leu Ser Ala Val Ser Thr Ser Ser
485 490 495 Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr Pro
Ala Thr 500 505 510 Thr Ser Ser Ile Thr Ala Ala Val Thr Ala Ser Ser
Thr Thr Ser Ala 515 520 525 Val Pro Met Gly Asn Gly Val Gly Val Gly
Val Gly Val Gly Gly Asn 530 535 540 Val Ser Met Tyr Ala Asn Ala Gln
Thr Ala Met Ala Leu Met Gly Val 545 550 555 560 Ala Leu His Ser His
Gln Glu Gln Leu Ile Gly Gly Val Ala Val Lys 565 570 575 Ser Glu His
Ser Thr Thr Ala 580 18 549 PRT Artificial Sequence misc_feature
Novel Sequence 18 Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala
Met Lys Arg Arg 1 5 10 15 Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys
Met Thr Thr Ser Pro Ser 20 25 30 Ser Gln His Gly Gly Asn Gly Ser
Leu Ala Ser Gly Gly Gly Gln Asp 35 40 45 Phe Val Lys Lys Glu Ile
Leu Asp Leu Met Thr Cys Glu Pro Pro Gln 50 55 60 His Ala Thr Ile
Pro Leu Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln 65 70 75 80 Ala Arg
Asn Ile Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr 85 90 95
Lys Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp 100
105 110 Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr
Asp 115 120 125 Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr
Val Gln Leu 130 135 140 Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe
Thr Lys Ile Pro Gln 145 150 155 160 Glu Asp Gln Ile Thr Leu Leu Lys
Ala Cys Ser Ser Glu Val Met Met 165 170 175 Leu Arg Met Ala Arg Arg
Tyr Asp His Ser Ser Asp Ser Ile Phe Phe 180 185 190 Ala Asn Asn Arg
Ser Tyr Thr Arg Asp Ser Tyr Lys Met Ala Gly Met 195 200 205 Ala Asp
Asn Ile Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser 210 215 220
Met Lys Val Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile 225
230 235 240 Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu
Ala Ile 245 250 255 Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile
Leu Asn Arg His 260 265 270 Cys Gly Asp Ser Met Ser Leu Val Phe Tyr
Ala Lys Leu Leu Ser Ile 275 280 285 Leu Thr Glu Leu Arg Thr Leu Gly
Asn Gln Asn Ala Glu Met Cys Phe 290 295 300 Ser Leu Lys Leu Lys Asn
Arg Lys Leu Pro Lys Phe Leu Glu Glu Ile 305 310 315 320 Trp Asp Val
His Ala Ile Pro Pro Ser Val Gln Ser His Leu Gln Ile 325 330 335 Thr
Gln Glu Glu Asn Glu Arg Leu Glu Arg Ala Glu Arg Met Arg Ala 340 345
350 Ser Val Gly Gly Ala Ile Thr Ala Gly Ile Asp Cys Asp Ser Ala Ser
355 360 365 Thr Ser Ala Ala Ala Ala Ala Ala Gln His Gln Pro Gln Pro
Gln Pro 370 375 380 Gln Pro Gln Pro Ser Ser Leu Thr Gln Asn Asp Ser
Gln His Gln Thr 385 390 395 400 Gln Pro Gln Leu Gln Pro Gln Leu Pro
Pro Gln Leu Gln Gly Gln Leu 405 410 415 Gln Pro Gln Leu Gln Pro Gln
Leu Gln Thr Gln Leu Gln Pro Gln Ile 420 425 430 Gln Pro Gln Pro Gln
Leu Leu Pro Val Ser Ala Pro Val Pro Ala Ser 435 440 445 Val Thr Ala
Pro Gly Ser Leu Ser Ala Val Ser Thr Ser Ser Glu Tyr 450 455 460 Met
Gly Gly Ser Ala Ala Ile Gly Pro Ile Thr Pro Ala Thr Thr Ser 465 470
475 480 Ser Ile Thr Ala Ala Val Thr Ala Ser Ser Thr Thr Ser Ala Val
Pro 485 490 495 Met Gly Asn Gly Val Gly Val Gly Val Gly Val Gly Gly
Asn Val Ser 500 505 510 Met Tyr Ala Asn Ala Gln Thr Ala Met Ala Leu
Met Gly Val Ala Leu 515 520 525 His Ser His Gln Glu Gln Leu Ile Gly
Gly Val Ala Val Lys Ser Glu 530 535 540 His Ser Thr Thr Ala 545 19
445 PRT Artificial Sequence misc_feature Novel Sequence 19 Tyr Glu
Gln Pro Ser Glu Glu Asp Leu Arg Arg Ile Met Ser Gln Pro 1 5 10 15
Asp Glu Asn Glu Ser Gln Thr Asp Val Ser Phe Arg His Ile Thr Glu 20
25 30 Ile Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly
Leu 35 40 45 Pro Ala Phe Thr Lys Ile Pro Gln Glu Asp Gln Ile Thr
Leu Leu Lys 50 55 60 Ala Cys Ser Ser Glu Val Met Met Leu Arg Met
Ala Arg Arg Tyr Asp 65 70 75 80 His Ser Ser Asp Ser Ile Phe Phe Ala
Asn Asn Arg Ser Tyr Thr Arg 85 90 95 Asp Ser Tyr Lys Met Ala Gly
Met Ala Asp Asn Ile Glu Asp Leu Leu 100 105 110 His Phe Cys Arg Gln
Met Phe Ser Met Lys Val Asp Asn Val Glu Tyr 115 120 125 Ala Leu Leu
Thr Ala Ile Val Ile Phe Ser Asp Arg Pro Gly Leu Glu 130 135 140 Lys
Ala Gln Leu Val Glu Ala Ile Gln Ser Tyr Tyr Ile Asp Thr Leu 145 150
155 160 Arg Ile Tyr Ile Leu Asn Arg His Cys Gly Asp Ser Met Ser Leu
Val 165 170 175 Phe Tyr Ala Lys Leu Leu Ser Ile Leu Thr Glu Leu Arg
Thr Leu Gly 180 185 190 Asn Gln Asn Ala Glu Met Cys Phe Ser Leu Lys
Leu Lys Asn Arg Lys 195 200 205 Leu Pro Lys Phe Leu Glu Glu Ile Trp
Asp Val His Ala Ile Pro Pro 210 215 220 Ser Val Gln Ser His Leu Gln
Ile Thr Gln Glu Glu Asn Glu Arg Leu 225 230 235 240 Glu Arg Ala Glu
Arg Met Arg Ala Ser Val Gly Gly Ala Ile Thr Ala 245 250 255 Gly Ile
Asp Cys Asp Ser Ala Ser Thr Ser Ala Ala Ala Ala Ala Ala 260 265 270
Gln His Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro Ser Ser Leu Thr 275
280 285 Gln Asn Asp Ser Gln His Gln Thr Gln Pro Gln Leu Gln Pro Gln
Leu 290 295 300 Pro Pro Gln Leu Gln Gly Gln Leu Gln Pro Gln Leu Gln
Pro Gln Leu 305 310 315 320 Gln Thr Gln Leu Gln Pro Gln Ile Gln Pro
Gln Pro Gln Leu Leu Pro 325 330 335 Val Ser Ala Pro Val Pro Ala Ser
Val Thr Ala Pro Gly Ser Leu Ser 340 345 350 Ala Val Ser Thr Ser Ser
Glu Tyr Met Gly Gly Ser Ala Ala Ile Gly 355 360 365 Pro Ile Thr Pro
Ala Thr Thr Ser Ser Ile Thr Ala Ala Val Thr Ala 370 375 380 Ser Ser
Thr Thr Ser Ala Val Pro Met Gly Asn Gly Val Gly Val Gly 385 390 395
400 Val Gly Val Gly Gly Asn Val Ser Met Tyr Ala Asn Ala Gln Thr Ala
405 410 415 Met Ala Leu Met Gly Val Ala Leu His Ser His Gln Glu Gln
Leu Ile 420 425 430 Gly Gly Val Ala Val Lys Ser Glu His Ser Thr Thr
Ala 435 440 445 20 323 PRT Artificial Sequence misc_feature Novel
Sequence 20 Arg Pro Glu Cys Val Val Pro Glu Asn Gln Cys Ala Met Lys
Arg Arg 1 5 10 15 Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Met Thr
Thr Ser Pro Ser 20 25 30 Ser Gln His Gly Gly Asn Gly Ser Leu Ala
Ser Gly Gly Gly Gln Asp 35 40 45 Phe Val Lys Lys Glu Ile Leu Asp
Leu Met Thr Cys Glu Pro Pro Gln 50 55 60 His Ala Thr Ile Pro Leu
Leu Pro Asp Glu Ile Leu Ala Lys Cys Gln 65 70 75 80 Ala Arg Asn Ile
Pro Ser Leu Thr Tyr Asn Gln Leu Ala Val Ile Tyr 85 90 95 Lys Leu
Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Glu Glu Asp 100 105 110
Leu Arg Arg Ile Met Ser Gln Pro Asp Glu Asn Glu Ser Gln Thr Asp 115
120 125 Val Ser Phe Arg His Ile Thr Glu Ile Thr Ile Leu Thr Val Gln
Leu 130 135 140 Ile Val Glu Phe Ala Lys Gly Leu Pro Ala Phe Thr Lys
Ile Pro Gln 145 150 155 160 Glu Asp Gln Ile Thr Leu Leu Lys Ala Cys
Ser Ser Glu Val Met Met 165 170 175 Leu Arg Met Ala Arg Arg Tyr Asp
His Ser Ser Asp Ser Ile Phe Phe 180 185 190 Ala Asn Asn Arg Ser Tyr
Thr Arg Asp Ser Tyr Lys Met Ala Gly Met 195 200 205 Ala Asp Asn Ile
Glu Asp Leu Leu His Phe Cys Arg Gln Met Phe Ser 210 215 220 Met Lys
Val Asp Asn Val Glu Tyr Ala Leu Leu Thr Ala Ile Val Ile 225 230 235
240 Phe Ser Asp Arg Pro Gly Leu Glu Lys Ala Gln Leu Val Glu Ala Ile
245 250 255 Gln Ser Tyr Tyr Ile Asp Thr Leu Arg Ile Tyr Ile Leu Asn
Arg His 260 265 270 Cys Gly Asp Ser Met Ser Leu Val Phe Tyr Ala Lys
Leu Leu Ser Ile 275 280 285 Leu Thr Glu Leu Arg Thr Leu Gly Asn Gln
Asn Ala Glu Met Cys Phe 290 295 300 Ser Leu Lys Leu Lys Asn Arg Lys
Leu Pro Lys Phe Leu Glu Glu Ile 305 310 315 320 Trp Asp Val 21 987
DNA Artificial Sequence misc_feature Novel Sequence 21 tgtgctatct
gtggggaccg ctcctcaggc aaacactatg gggtatacag ttgtgagggc 60
tgcaagggct tcttcaagag gacagtacgc aaagacctga cctacacctg ccgagacaac
120 aaggactgcc tgatcgacaa gagacagcgg aaccggtgtc agtactgccg
ctaccagaag 180 tgcctggcca tgggcatgaa gcgggaagct gtgcaggagg
agcggcagcg gggcaaggac 240 cggaatgaga acgaggtgga gtccaccagc
agtgccaacg aggacatgcc tgtagagaag 300 attctggaag ccgagcttgc
tgtcgagccc aagactgaga catacgtgga ggcaaacatg 360 gggctgaacc
ccagctcacc aaatgaccct gttaccaaca tctgtcaagc agcagacaag 420
cagctcttca ctcttgtgga gtgggccaag aggatcccac acttttctga gctgccccta
480 gacgaccagg tcatcctgct acgggcaggc tggaacgagc tgctgatcgc
ctccttctcc 540 caccgctcca tagctgtgaa agatgggatt ctcctggcca
ccggcctgca cgtacaccgg 600 aacagcgctc acagtgctgg ggtgggcgcc
atctttgaca gggtgctaac agagctggtg 660 tctaagatgc gtgacatgca
gatggacaag acggagctgg gctgcctgcg agccattgtc 720 ctgttcaacc
ctgactctaa ggggctctca aaccctgctg aggtggaggc gttgagggag 780
aaggtgtatg cgtcactaga agcgtactgc aaacacaagt accctgagca gccgggcagg
840 tttgccaagc tgctgctccg cctgcctgca ctgcgttcca tcgggctcaa
gtgcctggag 900 cacctgttct tcttcaagct catcggggac acgcccatcg
acaccttcct catggagatg 960 ctggaggcac cacatcaagc cacctag 987 22 789
DNA Artificial Sequence misc_feature Novel Sequence 22 aagcgggaag
ctgtgcagga ggagcggcag cggggcaagg accggaatga gaacgaggtg 60
gagtccacca gcagtgccaa cgaggacatg cctgtagaga agattctgga agccgagctt
120 gctgtcgagc ccaagactga gacatacgtg gaggcaaaca tggggctgaa
ccccagctca 180 ccaaatgacc ctgttaccaa catctgtcaa gcagcagaca
agcagctctt cactcttgtg 240 gagtgggcca agaggatccc acacttttct
gagctgcccc tagacgacca ggtcatcctg 300 ctacgggcag gctggaacga
gctgctgatc gcctccttct cccaccgctc catagctgtg 360 aaagatggga
ttctcctggc caccggcctg cacgtacacc ggaacagcgc tcacagtgct 420
ggggtgggcg ccatctttga cagggtgcta acagagctgg tgtctaagat gcgtgacatg
480 cagatggaca agacggagct gggctgcctg cgagccattg tcctgttcaa
ccctgactct 540 aaggggctct caaaccctgc tgaggtggag gcgttgaggg
agaaggtgta tgcgtcacta 600 gaagcgtact gcaaacacaa gtaccctgag
cagccgggca ggtttgccaa gctgctgctc 660 cgcctgcctg cactgcgttc
catcgggctc aagtgcctgg agcacctgtt cttcttcaag 720 ctcatcgggg
acacgcccat cgacaccttc ctcatggaga tgctggaggc accacatcaa 780
gccacctag 789 23 714 DNA Artificial Sequence misc_feature Novel
Sequence 23 gccaacgagg acatgcctgt agagaagatt ctggaagccg agcttgctgt
cgagcccaag 60 actgagacat acgtggaggc aaacatgggg ctgaacccca
gctcaccaaa tgaccctgtt 120 accaacatct gtcaagcagc agacaagcag
ctcttcactc ttgtggagtg ggccaagagg 180 atcccacact tttctgagct
gcccctagac gaccaggtca tcctgctacg ggcaggctgg 240 aacgagctgc
tgatcgcctc cttctcccac cgctccatag ctgtgaaaga tgggattctc 300
ctggccaccg gcctgcacgt acaccggaac agcgctcaca gtgctggggt gggcgccatc
360 tttgacaggg tgctaacaga gctggtgtct aagatgcgtg acatgcagat
ggacaagacg 420 gagctgggct gcctgcgagc cattgtcctg ttcaaccctg
actctaaggg gctctcaaac 480 cctgctgagg tggaggcgtt gagggagaag
gtgtatgcgt cactagaagc gtactgcaaa 540 cacaagtacc ctgagcagcc
gggcaggttt gccaagctgc tgctccgcct gcctgcactg 600 cgttccatcg
ggctcaagtg cctggagcac ctgttcttct tcaagctcat cggggacacg 660
cccatcgaca ccttcctcat ggagatgctg gaggcaccac atcaagccac ctag 714 24
536 DNA Artificial Sequence misc_feature Novel Sequence 24
ggatcccaca cttttctgag ctgcccctag acgaccaggt catcctgcta cgggcaggct
60 ggaacgagct gctgatcgcc tccttctccc accgctccat agctgtgaaa
gatgggattc 120 tcctggccac cggcctgcac gtacaccgga acagcgctca
cagtgctggg gtgggcgcca 180 tctttgacag ggtgctaaca gagctggtgt
ctaagatgcg tgacatgcag atggacaaga 240 cggagctggg ctgcctgcga
gccattgtcc tgttcaaccc tgactctaag gggctctcaa 300 accctgctga
ggtggaggcg ttgagggaga aggtgtatgc gtcactagaa gcgtactgca 360
aacacaagta ccctgagcag ccgggcaggt ttgccaagct gctgctccgc ctgcctgcac
420 tgcgttccat cgggctcaag tgcctggagc acctgttctt cttcaagctc
atcggggaca 480 cgcccatcga caccttcctc atggagatgc tggaggcacc
acatcaagcc acctag 536 25 672 DNA Artificial Sequence misc_feature
Novel Sequence 25 gccaacgagg acatgcctgt agagaagatt ctggaagccg
agcttgctgt cgagcccaag 60 actgagacat acgtggaggc aaacatgggg
ctgaacccca gctcaccaaa tgaccctgtt 120 accaacatct gtcaagcagc
agacaagcag ctcttcactc ttgtggagtg ggccaagagg 180 atcccacact
tttctgagct gcccctagac gaccaggtca tcctgctacg ggcaggctgg 240
aacgagctgc tgatcgcctc cttctcccac cgctccatag ctgtgaaaga tgggattctc
300 ctggccaccg gcctgcacgt acaccggaac agcgctcaca gtgctggggt
gggcgccatc 360 tttgacaggg tgctaacaga gctggtgtct aagatgcgtg
acatgcagat ggacaagacg 420 gagctgggct gcctgcgagc cattgtcctg
ttcaaccctg actctaaggg gctctcaaac 480 cctgctgagg tggaggcgtt
gagggagaag gtgtatgcgt cactagaagc gtactgcaaa 540 cacaagtacc
ctgagcagcc gggcaggttt gccaagctgc tgctccgcct gcctgcactg 600
cgttccatcg ggctcaagtg cctggagcac ctgttcttct tcaagctcat cggggacacg
660 cccatcgaca cc 672 26 1123 DNA Artificial Sequence misc_feature
Novel Sequence 26 tgcgccatct gcggggaccg ctcctcaggc aagcactatg
gagtgtacag ctgcgagggg 60 tgcaagggct tcttcaagcg gacggtgcgc
aaggacctga cctacacctg ccgcgacaac 120 aaggactgcc tgattgacaa
gcggcagcgg aaccggtgcc agtactgccg ctaccagaag 180 tgcctggcca
tgggcatgaa gcgggaagcc gtgcaggagg agcggcagcg tggcaaggac 240
cggaacgaga atgaggtgga gtcgaccagc agcgccaacg aggacatgcc ggtggagagg
300 atcctggagg ctgagctggc cgtggagccc aagaccgaga cctacgtgga
ggcaaacatg 360 gggctgaacc ccagctcgcc gaacgaccct gtcaccaaca
tttgccaagc agccgacaaa 420 cagcttttca ccctggtgga gtgggccaag
cggatcccac acttctcaga gctgcccctg 480 gacgaccagg tcatcctgct
gcgggcaggc tggaatgagc tgctcatcgc ctccttctcc 540 caccgctcca
tcgccgtgaa ggacgggatc ctcctggcca ccgggctgca cgtccaccgg 600
aacagcgccc acagcgcagg ggtgggcgcc atctttgaca gggtgctgac ggagcttgtg
660 tccaagatgc gggacatgca gatggacaag acggagctgg gctgcctgcg
cgccatcgtc 720 ctctttaacc ctgactccaa ggggctctcg aacccggccg
aggtggaggc
gctgagggag 780 aaggtctatg cgtccttgga ggcctactgc aagcacaagt
acccagagca gccgggaagg 840 ttcgctaagc tcttgctccg cctgccggct
ctgcgctcca tcgggctcaa atgcctggaa 900 catctcttct tcttcaagct
catcggggac acacccattg acaccttcct tatggagatg 960 ctggaggcgc
cgcaccaaat gacttaggcc tgcgggccca tcctttgtgc ccacccgttc 1020
tggccaccct gcctggacgc cagctgttct tctcagcctg agccctgtcc ctgcccttct
1080 ctgcctggcc tgtttggact ttggggcaca gcctgtcact gct 1123 27 925
DNA Artificial Sequence misc_feature Novel Sequence 27 aagcgggaag
ccgtgcagga ggagcggcag cgtggcaagg accggaacga gaatgaggtg 60
gagtcgacca gcagcgccaa cgaggacatg ccggtggaga ggatcctgga ggctgagctg
120 gccgtggagc ccaagaccga gacctacgtg gaggcaaaca tggggctgaa
ccccagctcg 180 ccgaacgacc ctgtcaccaa catttgccaa gcagccgaca
aacagctttt caccctggtg 240 gagtgggcca agcggatccc acacttctca
gagctgcccc tggacgacca ggtcatcctg 300 ctgcgggcag gctggaatga
gctgctcatc gcctccttct cccaccgctc catcgccgtg 360 aaggacggga
tcctcctggc caccgggctg cacgtccacc ggaacagcgc ccacagcgca 420
ggggtgggcg ccatctttga cagggtgctg acggagcttg tgtccaagat gcgggacatg
480 cagatggaca agacggagct gggctgcctg cgcgccatcg tcctctttaa
ccctgactcc 540 aaggggctct cgaacccggc cgaggtggag gcgctgaggg
agaaggtcta tgcgtccttg 600 gaggcctact gcaagcacaa gtacccagag
cagccgggaa ggttcgctaa gctcttgctc 660 cgcctgccgg ctctgcgctc
catcgggctc aaatgcctgg aacatctctt cttcttcaag 720 ctcatcgggg
acacacccat tgacaccttc cttatggaga tgctggaggc gccgcaccaa 780
atgacttagg cctgcgggcc catcctttgt gcccacccgt tctggccacc ctgcctggac
840 gccagctgtt cttctcagcc tgagccctgt ccctgccctt ctctgcctgg
cctgtttgga 900 ctttggggca cagcctgtca ctgct 925 28 850 DNA
Artificial Sequence misc_feature Novel Sequence 28 gccaacgagg
acatgccggt ggagaggatc ctggaggctg agctggccgt ggagcccaag 60
accgagacct acgtggaggc aaacatgggg ctgaacccca gctcgccgaa cgaccctgtc
120 accaacattt gccaagcagc cgacaaacag cttttcaccc tggtggagtg
ggccaagcgg 180 atcccacact tctcagagct gcccctggac gaccaggtca
tcctgctgcg ggcaggctgg 240 aatgagctgc tcatcgcctc cttctcccac
cgctccatcg ccgtgaagga cgggatcctc 300 ctggccaccg ggctgcacgt
ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 360 tttgacaggg
tgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 420
gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac
480 ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggc
ctactgcaag 540 cacaagtacc cagagcagcc gggaaggttc gctaagctct
tgctccgcct gccggctctg 600 cgctccatcg ggctcaaatg cctggaacat
ctcttcttct tcaagctcat cggggacaca 660 cccattgaca ccttccttat
ggagatgctg gaggcgccgc accaaatgac ttaggcctgc 720 gggcccatcc
tttgtgccca cccgttctgg ccaccctgcc tggacgccag ctgttcttct 780
cagcctgagc cctgtccctg cccttctctg cctggcctgt ttggactttg gggcacagcc
840 tgtcactgct 850 29 670 DNA Artificial Sequence misc_feature
Novel Sequence 29 atcccacact tctcagagct gcccctggac gaccaggtca
tcctgctgcg ggcaggctgg 60 aatgagctgc tcatcgcctc cttctcccac
cgctccatcg ccgtgaagga cgggatcctc 120 ctggccaccg ggctgcacgt
ccaccggaac agcgcccaca gcgcaggggt gggcgccatc 180 tttgacaggg
tgctgacgga gcttgtgtcc aagatgcggg acatgcagat ggacaagacg 240
gagctgggct gcctgcgcgc catcgtcctc tttaaccctg actccaaggg gctctcgaac
300 ccggccgagg tggaggcgct gagggagaag gtctatgcgt ccttggaggc
ctactgcaag 360 cacaagtacc cagagcagcc gggaaggttc gctaagctct
tgctccgcct gccggctctg 420 cgctccatcg ggctcaaatg cctggaacat
ctcttcttct tcaagctcat cggggacaca 480 cccattgaca ccttccttat
ggagatgctg gaggcgccgc accaaatgac ttaggcctgc 540 gggcccatcc
tttgtgccca cccgttctgg ccaccctgcc tggacgccag ctgttcttct 600
cagcctgagc cctgtccctg cccttctctg cctggcctgt ttggactttg gggcacagcc
660 tgtcactgct 670 30 672 DNA Artificial Sequence misc_feature
Novel Sequence 30 gccaacgagg acatgccggt ggagaggatc ctggaggctg
agctggccgt ggagcccaag 60 accgagacct acgtggaggc aaacatgggg
ctgaacccca gctcgccgaa cgaccctgtc 120 accaacattt gccaagcagc
cgacaaacag cttttcaccc tggtggagtg ggccaagcgg 180 atcccacact
tctcagagct gcccctggac gaccaggtca tcctgctgcg ggcaggctgg 240
aatgagctgc tcatcgcctc cttctcccac cgctccatcg ccgtgaagga cgggatcctc
300 ctggccaccg ggctgcacgt ccaccggaac agcgcccaca gcgcaggggt
gggcgccatc 360 tttgacaggg tgctgacgga gcttgtgtcc aagatgcggg
acatgcagat ggacaagacg 420 gagctgggct gcctgcgcgc catcgtcctc
tttaaccctg actccaaggg gctctcgaac 480 ccggccgagg tggaggcgct
gagggagaag gtctatgcgt ccttggaggc ctactgcaag 540 cacaagtacc
cagagcagcc gggaaggttc gctaagctct tgctccgcct gccggctctg 600
cgctccatcg ggctcaaatg cctggaacat ctcttcttct tcaagctcat cggggacaca
660 cccattgaca cc 672 31 328 PRT Artificial Sequence misc_feature
Novel Sequence 31 Cys Ala Ile Cys Gly Asp Arg Ser Ser Gly Lys His
Tyr Gly Val Tyr 1 5 10 15 Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys
Arg Thr Val Arg Lys Asp 20 25 30 Leu Thr Tyr Thr Cys Arg Asp Asn
Lys Asp Cys Leu Ile Asp Lys Arg 35 40 45 Gln Arg Asn Arg Cys Gln
Tyr Cys Arg Tyr Gln Lys Cys Leu Ala Met 50 55 60 Gly Met Lys Arg
Glu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp 65 70 75 80 Arg Asn
Glu Asn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met 85 90 95
Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr 100
105 110 Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro
Asn 115 120 125 Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln
Leu Phe Thr 130 135 140 Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe
Ser Glu Leu Pro Leu 145 150 155 160 Asp Asp Gln Val Ile Leu Leu Arg
Ala Gly Trp Asn Glu Leu Leu Ile 165 170 175 Ala Ser Phe Ser His Arg
Ser Ile Ala Val Lys Asp Gly Ile Leu Leu 180 185 190 Ala Thr Gly Leu
His Val His Arg Asn Ser Ala His Ser Ala Gly Val 195 200 205 Gly Ala
Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg 210 215 220
Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val 225
230 235 240 Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu
Val Glu 245 250 255 Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala
Tyr Cys Lys His 260 265 270 Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala
Lys Leu Leu Leu Arg Leu 275 280 285 Pro Ala Leu Arg Ser Ile Gly Leu
Lys Cys Leu Glu His Leu Phe Phe 290 295 300 Phe Lys Leu Ile Gly Asp
Thr Pro Ile Asp Thr Phe Leu Met Glu Met 305 310 315 320 Leu Glu Ala
Pro His Gln Ala Thr 325 32 262 PRT Artificial Sequence misc_feature
Novel Sequence 32 Lys Arg Glu Ala Val Gln Glu Glu Arg Gln Arg Gly
Lys Asp Arg Asn 1 5 10 15 Glu Asn Glu Val Glu Ser Thr Ser Ser Ala
Asn Glu Asp Met Pro Val 20 25 30 Glu Lys Ile Leu Glu Ala Glu Leu
Ala Val Glu Pro Lys Thr Glu Thr 35 40 45 Tyr Val Glu Ala Asn Met
Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro 50 55 60 Val Thr Asn Ile
Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val 65 70 75 80 Glu Trp
Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu Asp Asp 85 90 95
Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile Ala Ser 100
105 110 Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu Ala
Thr 115 120 125 Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly
Val Gly Ala 130 135 140 Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser
Lys Met Arg Asp Met 145 150 155 160 Gln Met Asp Lys Thr Glu Leu Gly
Cys Leu Arg Ala Ile Val Leu Phe 165 170 175 Asn Pro Asp Ser Lys Gly
Leu Ser Asn Pro Ala Glu Val Glu Ala Leu 180 185 190 Arg Glu Lys Val
Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr 195 200 205 Pro Glu
Gln Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala 210 215 220
Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe Phe Lys 225
230 235 240 Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met
Leu Glu 245 250 255 Ala Pro His Gln Ala Thr 260 33 237 PRT
Artificial Sequence misc_feature Novel Sequence 33 Ala Asn Glu Asp
Met Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala 1 5 10 15 Val Glu
Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25 30
Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp 35
40 45 Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His
Phe 50 55 60 Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg
Ala Gly Trp 65 70 75 80 Asn Glu Leu Leu Ile Ala Ser Phe Ser His Arg
Ser Ile Ala Val Lys 85 90 95 Asp Gly Ile Leu Leu Ala Thr Gly Leu
His Val His Arg Asn Ser Ala 100 105 110 His Ser Ala Gly Val Gly Ala
Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125 Val Ser Lys Met Arg
Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140 Leu Arg Ala
Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn 145 150 155 160
Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu 165
170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe Ala
Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly Leu
Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu Ile Gly Asp
Thr Pro Ile Asp Thr 210 215 220 Phe Leu Met Glu Met Leu Glu Ala Pro
His Gln Ala Thr 225 230 235 34 177 PRT Artificial Sequence
misc_feature Novel Sequence 34 Ile Pro His Phe Ser Glu Leu Pro Leu
Asp Asp Gln Val Ile Leu Leu 1 5 10 15 Arg Ala Gly Trp Asn Glu Leu
Leu Ile Ala Ser Phe Ser His Arg Ser 20 25 30 Ile Ala Val Lys Asp
Gly Ile Leu Leu Ala Thr Gly Leu His Val His 35 40 45 Arg Asn Ser
Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val 50 55 60 Leu
Thr Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr 65 70
75 80 Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser
Lys 85 90 95 Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu Arg Glu
Lys Val Tyr 100 105 110 Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr
Pro Glu Gln Pro Gly 115 120 125 Arg Phe Ala Lys Leu Leu Leu Arg Leu
Pro Ala Leu Arg Ser Ile Gly 130 135 140 Leu Lys Cys Leu Glu His Leu
Phe Phe Phe Lys Leu Ile Gly Asp Thr 145 150 155 160 Pro Ile Asp Thr
Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Ala 165 170 175 Thr 35
224 PRT Artificial Sequence misc_feature Novel Sequence 35 Ala Asn
Glu Asp Met Pro Val Glu Lys Ile Leu Glu Ala Glu Leu Ala 1 5 10 15
Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20
25 30 Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala
Asp 35 40 45 Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile
Pro His Phe 50 55 60 Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu
Leu Arg Ala Gly Trp 65 70 75 80 Asn Glu Leu Leu Ile Ala Ser Phe Ser
His Arg Ser Ile Ala Val Lys 85 90 95 Asp Gly Ile Leu Leu Ala Thr
Gly Leu His Val His Arg Asn Ser Ala 100 105 110 His Ser Ala Gly Val
Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125 Val Ser Lys
Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140 Leu
Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn 145 150
155 160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu
Glu 165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg
Phe Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile
Gly Leu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu Ile
Gly Asp Thr Pro Ile Asp Thr 210 215 220 36 328 PRT Artificial
Sequence misc_feature Novel Sequence 36 Cys Ala Ile Cys Gly Asp Arg
Ser Ser Gly Lys His Tyr Gly Val Tyr 1 5 10 15 Ser Cys Glu Gly Cys
Lys Gly Phe Phe Lys Arg Thr Val Arg Lys Asp 20 25 30 Leu Thr Tyr
Thr Cys Arg Asp Asn Lys Asp Cys Leu Ile Asp Lys Arg 35 40 45 Gln
Arg Asn Arg Cys Gln Tyr Cys Arg Tyr Gln Lys Cys Leu Ala Met 50 55
60 Gly Met Lys Arg Glu Ala Val Gln Glu Glu Arg Gln Arg Gly Lys Asp
65 70 75 80 Arg Asn Glu Asn Glu Val Glu Ser Thr Ser Ser Ala Asn Glu
Asp Met 85 90 95 Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala Val
Glu Pro Lys Thr 100 105 110 Glu Thr Tyr Val Glu Ala Asn Met Gly Leu
Asn Pro Ser Ser Pro Asn 115 120 125 Asp Pro Val Thr Asn Ile Cys Gln
Ala Ala Asp Lys Gln Leu Phe Thr 130 135 140 Leu Val Glu Trp Ala Lys
Arg Ile Pro His Phe Ser Glu Leu Pro Leu 145 150 155 160 Asp Asp Gln
Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu Leu Ile 165 170 175 Ala
Ser Phe Ser His Arg Ser Ile Ala Val Lys Asp Gly Ile Leu Leu 180 185
190 Ala Thr Gly Leu His Val His Arg Asn Ser Ala His Ser Ala Gly Val
195 200 205 Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser Lys
Met Arg 210 215 220 Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys Leu
Arg Ala Ile Val 225 230 235 240 Leu Phe Asn Pro Asp Ser Lys Gly Leu
Ser Asn Pro Ala Glu Val Glu 245 250 255 Ala Leu Arg Glu Lys Val Tyr
Ala Ser Leu Glu Ala Tyr Cys Lys His 260 265 270 Lys Tyr Pro Glu Gln
Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu 275 280 285 Pro Ala Leu
Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe 290 295 300 Phe
Lys Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu Met Glu Met 305 310
315 320 Leu Glu Ala Pro His Gln Met Thr 325 37 262 PRT Artificial
Sequence misc_feature Novel Sequence 37 Lys Arg Glu Ala Val Gln Glu
Glu Arg Gln Arg Gly Lys Asp Arg Asn 1 5 10 15 Glu Asn Glu Val Glu
Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val 20 25 30 Glu Arg Ile
Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr 35 40 45 Tyr
Val Glu Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro 50 55
60 Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu Val
65 70 75 80 Glu Trp Ala Lys Arg Ile Pro His Phe Ser Glu Leu Pro Leu
Asp Asp 85 90 95 Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu Leu
Leu Ile Ala Ser 100 105 110 Phe Ser His Arg Ser Ile Ala Val Lys Asp
Gly Ile Leu Leu Ala Thr 115 120 125 Gly Leu His Val His Arg Asn Ser
Ala His Ser Ala Gly Val Gly Ala 130 135 140 Ile Phe Asp Arg Val Leu
Thr Glu Leu Val Ser Lys Met Arg Asp Met 145 150 155 160 Gln Met Asp
Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe 165 170 175 Asn
Pro Asp Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu 180 185
190 Arg Glu Lys Val Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr
195 200
205 Pro Glu Gln Pro Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro Ala
210 215 220 Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His Leu Phe Phe
Phe Lys 225 230 235 240 Leu Ile Gly Asp Thr Pro Ile Asp Thr Phe Leu
Met Glu Met Leu Glu 245 250 255 Ala Pro His Gln Met Thr 260 38 237
PRT Artificial Sequence misc_feature Novel Sequence 38 Ala Asn Glu
Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala 1 5 10 15 Val
Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20 25
30 Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp
35 40 45 Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro
His Phe 50 55 60 Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu
Arg Ala Gly Trp 65 70 75 80 Asn Glu Leu Leu Ile Ala Ser Phe Ser His
Arg Ser Ile Ala Val Lys 85 90 95 Asp Gly Ile Leu Leu Ala Thr Gly
Leu His Val His Arg Asn Ser Ala 100 105 110 His Ser Ala Gly Val Gly
Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125 Val Ser Lys Met
Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140 Leu Arg
Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn 145 150 155
160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu Glu
165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg Phe
Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile Gly
Leu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu Ile Gly
Asp Thr Pro Ile Asp Thr 210 215 220 Phe Leu Met Glu Met Leu Glu Ala
Pro His Gln Met Thr 225 230 235 39 177 PRT Artificial Sequence
misc_feature Novel Sequence 39 Ile Pro His Phe Ser Glu Leu Pro Leu
Asp Asp Gln Val Ile Leu Leu 1 5 10 15 Arg Ala Gly Trp Asn Glu Leu
Leu Ile Ala Ser Phe Ser His Arg Ser 20 25 30 Ile Ala Val Lys Asp
Gly Ile Leu Leu Ala Thr Gly Leu His Val His 35 40 45 Arg Asn Ser
Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg Val 50 55 60 Leu
Thr Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys Thr 65 70
75 80 Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser
Lys 85 90 95 Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu Arg Glu
Lys Val Tyr 100 105 110 Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr
Pro Glu Gln Pro Gly 115 120 125 Arg Phe Ala Lys Leu Leu Leu Arg Leu
Pro Ala Leu Arg Ser Ile Gly 130 135 140 Leu Lys Cys Leu Glu His Leu
Phe Phe Phe Lys Leu Ile Gly Asp Thr 145 150 155 160 Pro Ile Asp Thr
Phe Leu Met Glu Met Leu Glu Ala Pro His Gln Met 165 170 175 Thr 40
224 PRT Artificial Sequence misc_feature Novel Sequence 40 Ala Asn
Glu Asp Met Pro Val Glu Arg Ile Leu Glu Ala Glu Leu Ala 1 5 10 15
Val Glu Pro Lys Thr Glu Thr Tyr Val Glu Ala Asn Met Gly Leu Asn 20
25 30 Pro Ser Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala
Asp 35 40 45 Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile
Pro His Phe 50 55 60 Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu
Leu Arg Ala Gly Trp 65 70 75 80 Asn Glu Leu Leu Ile Ala Ser Phe Ser
His Arg Ser Ile Ala Val Lys 85 90 95 Asp Gly Ile Leu Leu Ala Thr
Gly Leu His Val His Arg Asn Ser Ala 100 105 110 His Ser Ala Gly Val
Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu 115 120 125 Val Ser Lys
Met Arg Asp Met Gln Met Asp Lys Thr Glu Leu Gly Cys 130 135 140 Leu
Arg Ala Ile Val Leu Phe Asn Pro Asp Ser Lys Gly Leu Ser Asn 145 150
155 160 Pro Ala Glu Val Glu Ala Leu Arg Glu Lys Val Tyr Ala Ser Leu
Glu 165 170 175 Ala Tyr Cys Lys His Lys Tyr Pro Glu Gln Pro Gly Arg
Phe Ala Lys 180 185 190 Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile
Gly Leu Lys Cys Leu 195 200 205 Glu His Leu Phe Phe Phe Lys Leu Ile
Gly Asp Thr Pro Ile Asp Thr 210 215 220 41 441 DNA Artificial
Sequence misc_feature Novel Sequence 41 atgaagctac tgtcttctat
cgaacaagca tgcgatattt gccgacttaa aaagctcaag 60 tgctccaaag
aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac 120
tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg
180 ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct
tgacatgatt 240 ttgaaaatgg attctttaca ggatataaaa gcattgttaa
caggattatt tgtacaagat 300 aatgtgaata aagatgccgt cacagataga
ttggcttcag tggagactga tatgcctcta 360 acattgagac agcatagaat
aagtgcgaca tcatcatcgg aagagagtag taacaaaggt 420 caaagacagt
tgactgtatc g 441 42 147 PRT Artificial Sequence misc_feature Novel
Sequence 42 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys
Arg Leu 1 5 10 15 Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys
Ala Lys Cys Leu 20 25 30 Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro
Lys Thr Lys Arg Ser Pro 35 40 45 Leu Thr Arg Ala His Leu Thr Glu
Val Glu Ser Arg Leu Glu Arg Leu 50 55 60 Glu Gln Leu Phe Leu Leu
Ile Phe Pro Arg Glu Asp Leu Asp Met Ile 65 70 75 80 Leu Lys Met Asp
Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95 Phe Val
Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110
Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115
120 125 Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln
Leu 130 135 140 Thr Val Ser 145 43 606 DNA Artificial Sequence
misc_feature Novel Sequence 43 atgaaagcgt taacggccag gcaacaagag
gtgtttgatc tcatccgtga tcacatcagc 60 cagacaggta tgccgccgac
gcgtgcggaa atcgcgcagc gtttggggtt ccgttcccca 120 aacgcggctg
aagaacatct gaaggcgctg gcacgcaaag gcgttattga aattgtttcc 180
ggcgcatcac gcgggattcg tctgttgcag gaagaggaag aagggttgcc gctggtaggt
240 cgtgtggctg ccggtgaacc acttctggcg caacagcata ttgaaggtca
ttatcaggtc 300 gatccttcct tattcaagcc gaatgctgat ttcctgctgc
gcgtcagcgg gatgtcgatg 360 aaagatatcg gcattatgga tggtgacttg
ctggcagtgc ataaaactca ggatgtacgt 420 aacggtcagg tcgttgtcgc
acgtattgat gacgaagtta ccgttaagcg cctgaaaaaa 480 cagggcaata
aagtcgaact gttgccagaa aatagcgagt ttaaaccaat tgtcgtagat 540
cttcgtcagc agagcttcac cattgaaggg ctggcggttg gggttattcg caacggcgac
600 tggctg 606 44 202 PRT Artificial Sequence misc_feature Novel
Sequence 44 Met Lys Ala Leu Thr Ala Arg Gln Gln Glu Val Phe Asp Leu
Ile Arg 1 5 10 15 Asp His Ile Ser Gln Thr Gly Met Pro Pro Thr Arg
Ala Glu Ile Ala 20 25 30 Gln Arg Leu Gly Phe Arg Ser Pro Asn Ala
Ala Glu Glu His Leu Lys 35 40 45 Ala Leu Ala Arg Lys Gly Val Ile
Glu Ile Val Ser Gly Ala Ser Arg 50 55 60 Gly Ile Arg Leu Leu Gln
Glu Glu Glu Glu Gly Leu Pro Leu Val Gly 65 70 75 80 Arg Val Ala Ala
Gly Glu Pro Leu Leu Ala Gln Gln His Ile Glu Gly 85 90 95 His Tyr
Gln Val Asp Pro Ser Leu Phe Lys Pro Asn Ala Asp Phe Leu 100 105 110
Leu Arg Val Ser Gly Met Ser Met Lys Asp Ile Gly Ile Met Asp Gly 115
120 125 Asp Leu Leu Ala Val His Lys Thr Gln Asp Val Arg Asn Gly Gln
Val 130 135 140 Val Val Ala Arg Ile Asp Asp Glu Val Thr Val Lys Arg
Leu Lys Lys 145 150 155 160 Gln Gly Asn Lys Val Glu Leu Leu Pro Glu
Asn Ser Glu Phe Lys Pro 165 170 175 Ile Val Val Asp Leu Arg Gln Gln
Ser Phe Thr Ile Glu Gly Leu Ala 180 185 190 Val Gly Val Ile Arg Asn
Gly Asp Trp Leu 195 200 45 271 DNA Artificial Sequence misc_feature
Novel Sequence 45 atgggcccta aaaagaagcg taaagtcgcc cccccgaccg
atgtcagcct gggggacgag 60 ctccacttag acggcgagga cgtggcgatg
gcgcatgccg acgcgctaga cgatttcgat 120 ctggacatgt tgggggacgg
ggattccccg gggccgggat ttacccccca cgactccgcc 180 ccctacggcg
ctctggatat ggccgacttc gagtttgagc agatgtttac cgatgccctt 240
ggaattgacg agtacggtgg ggaattcccg g 271 46 90 PRT Artificial
Sequence misc_feature Novel Sequence 46 Met Gly Pro Lys Lys Lys Arg
Lys Val Ala Pro Pro Thr Asp Val Ser 1 5 10 15 Leu Gly Asp Glu Leu
His Leu Asp Gly Glu Asp Val Ala Met Ala His 20 25 30 Ala Asp Ala
Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly Asp 35 40 45 Ser
Pro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly Ala 50 55
60 Leu Asp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala Leu
65 70 75 80 Gly Ile Asp Glu Tyr Gly Gly Glu Phe Pro 85 90 47 19 DNA
Artificial Sequence misc_feature Novel Sequence 47 ggagtactgt
cctccgagc 19 48 666 DNA Artificial Sequence misc_feature Novel
Sequence 48 ggatccccag cttggaattc gacaggttat cagcaacaac acagtcatat
ccattctcaa 60 ttagctctac cacagtgtgt gaaccaatgt atccagcacc
acctgtaacc aaaacaattt 120 tagaagtact ttcactttgt aactgagctg
tcatttatat tgaattttca aaaattctta 180 cttttttttt ggatggacgc
aaagaagttt aataatcata ttacatggca ttaccaccat 240 atacatatcc
atatacatat ccatatctaa tcttacctcg actgctgtat ataaaaccag 300
tggttatatg tacagtactg ctgtatataa aaccagtggt tatatgtaca gtacgtcgac
360 tgctgtatat aaaaccagtg gttatatgta cagtactgct gtatataaaa
ccagtggtta 420 tatgtacagt acgtcgaggg atgataatgc gattagtttt
ttagccttat ttctggggta 480 attaatcagc gaagcgatga tttttgatct
attaacagat atataaatgc aaaaactgca 540 taaccacttt aactaatact
ttcaacattt tcggtttgta ttacttctta ttcaaatgta 600 ataaaagtat
caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa 660 actata
666 49 1542 DNA Artificial Sequence misc_feature Novel Sequence 49
ctggacctga aacacgaagt ggcttaccga ggggtgctcc caggccaggt gaaggccgaa
60 ccgggggtcc acaacggcca ggtcaacggc cacgtgaggg actggatggc
aggcggcgct 120 ggtgccaatt cgccgtctcc gggagcggtg gctcaacccc
agcctaacaa tgggtattcg 180 tcgccactct cctcgggaag ctacgggccc
tacagtccaa atgggaaaat aggccgtgag 240 gaactgtcgc cagcttcaag
tataaatggg tgcagtacag atggcgaggc acgacgtcag 300 aagaagggcc
ctgcgccccg tcagcaagag gaactgtgtc tggtatgcgg ggacagagcc 360
tccggatacc actacaatgc gctcacgtgt gaagggtgta aagggttctt cagacggagt
420 gttaccaaaa atgcggttta tatttgtaaa ttcggtcacg cttgcgaaat
ggacatgtac 480 atgcgacgga aatgccagga gtgccgcctg aagaagtgct
tagctgtagg catgaggcct 540 gagtgcgtag tacccgagac tcagtgcgcc
atgaagcgga aagagaagaa agcacagaag 600 gagaaggaca aactgcctgt
cagcacgacg acggtggacg accacatgcc gcccattatg 660 cagtgtgaac
ctccacctcc tgaagcagca aggattcacg aagtggtccc aaggtttctc 720
tccgacaagc tgttggagac aaaccggcag aaaaacatcc cccagttgac agccaaccag
780 cagttcctta tcgccaggct catctggtac caggacgggt acgagcagcc
ttctgatgaa 840 gatttgaaga ggattacgca gacgtggcag caagcggacg
atgaaaacga agagtctgac 900 actcccttcc gccagatcac agagatgact
atcctcacgg tccaacttat cgtggagttc 960 gcgaagggat tgccagggtt
cgccaagatc tcgcagcctg atcaaattac gctgcttaag 1020 gcttgctcaa
gtgaggtaat gatgctccga gtcgcgcgac gatacgatgc ggcctcagac 1080
agtgttctgt tcgcgaacaa ccaagcgtac actcgcgaca actaccgcaa ggctggcatg
1140 gcctacgtca tcgaggatct actgcacttc tgccggtgca tgtactctat
ggcgttggac 1200 aacatccatt acgcgctgct cacggctgtc gtcatctttt
ctgaccggcc agggttggag 1260 cagccgcaac tggtggaaga aatccagcgg
tactacctga atacgctccg catctatatc 1320 ctgaaccagc tgagcgggtc
ggcgcgttcg tccgtcatat acggcaagat cctctcaatc 1380 ctctctgagc
tacgcacgct cggcatgcaa aactccaaca tgtgcatctc cctcaagctc 1440
aagaacagaa agctgccgcc tttcctcgag gagatctggg atgtggcgga catgtcgcac
1500 acccaaccgc cgcctatcct cgagtccccc acgaatctct ag 1542 50 513 PRT
Artificial Sequence misc_feature Novel Sequence 50 Leu Asp Leu Lys
His Glu Val Ala Tyr Arg Gly Val Leu Pro Gly Gln 1 5 10 15 Val Lys
Ala Glu Pro Gly Val His Asn Gly Gln Val Asn Gly His Val 20 25 30
Arg Asp Trp Met Ala Gly Gly Ala Gly Ala Asn Ser Pro Ser Pro Gly 35
40 45 Ala Val Ala Gln Pro Gln Pro Asn Asn Gly Tyr Ser Ser Pro Leu
Ser 50 55 60 Ser Gly Ser Tyr Gly Pro Tyr Ser Pro Asn Gly Lys Ile
Gly Arg Glu 65 70 75 80 Glu Leu Ser Pro Ala Ser Ser Ile Asn Gly Cys
Ser Thr Asp Gly Glu 85 90 95 Ala Arg Arg Gln Lys Lys Gly Pro Ala
Pro Arg Gln Gln Glu Glu Leu 100 105 110 Cys Leu Val Cys Gly Asp Arg
Ala Ser Gly Tyr His Tyr Asn Ala Leu 115 120 125 Thr Cys Glu Gly Cys
Lys Gly Phe Phe Arg Arg Ser Val Thr Lys Asn 130 135 140 Ala Val Tyr
Ile Cys Lys Phe Gly His Ala Cys Glu Met Asp Met Tyr 145 150 155 160
Met Arg Arg Lys Cys Gln Glu Cys Arg Leu Lys Lys Cys Leu Ala Val 165
170 175 Gly Met Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met
Lys 180 185 190 Arg Lys Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu
Pro Val Ser 195 200 205 Thr Thr Thr Val Asp Asp His Met Pro Pro Ile
Met Gln Cys Glu Pro 210 215 220 Pro Pro Pro Glu Ala Ala Arg Ile His
Glu Val Val Pro Arg Phe Leu 225 230 235 240 Ser Asp Lys Leu Leu Glu
Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu 245 250 255 Thr Ala Asn Gln
Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp 260 265 270 Gly Tyr
Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr 275 280 285
Trp Gln Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg 290
295 300 Gln Ile Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu
Phe 305 310 315 320 Ala Lys Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln
Pro Asp Gln Ile 325 330 335 Thr Leu Leu Lys Ala Cys Ser Ser Glu Val
Met Met Leu Arg Val Ala 340 345 350 Arg Arg Tyr Asp Ala Ala Ser Asp
Ser Val Leu Phe Ala Asn Asn Gln 355 360 365 Ala Tyr Thr Arg Asp Asn
Tyr Arg Lys Ala Gly Met Ala Tyr Val Ile 370 375 380 Glu Asp Leu Leu
His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp 385 390 395 400 Asn
Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg 405 410
415 Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr
420 425 430 Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly
Ser Ala 435 440 445 Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile
Leu Ser Glu Leu 450 455 460 Arg Thr Leu Gly Met Gln Asn Ser Asn Met
Cys Ile Ser Leu Lys Leu 465 470 475 480 Lys Asn Arg Lys Leu Pro Pro
Phe Leu Glu Glu Ile Trp Asp Val Ala 485 490 495 Asp Met Ser His Thr
Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn 500 505 510 Leu 51 4375
DNA Artificial Sequence misc_feature Novel Sequence 51 tgtaattttg
atgggcgccg tgatgcaccg tgtgccatat tgccatccag tcgaatagaa 60
aaaaaaaaaa aaaaaaaaat atcagttgtt ttgtccctcg ctcgctttcg agtgtattcg
120 gaatattaga cgtcataatt cacgagtgtc ttttaaattt atatagcgat
tagcggggcc 180 gtttgttgga cgtgcgcttg cgtttagtgg agtgcaggga
tagtgaggcg agtatggtag 240 ttcgtggtca tgtcaagtgt ggcgaagaaa
gacaagccga cgatgtcggt gacggcgctg 300 atcaactggg cgcggccggc
gccgccaggc ccgccgcagc cgcagtcagc gtcgcctgcg 360 ccggcagcca
tgctgcagca gctcccgacg cagtcaatgc agtcgttaaa ccacatccca 420
actgtcgatt gctcgctcga tatgcagtgg cttaatttag aacctggatt catgtcgcct
480 atgtcacctc ctgagatgaa accagacacc gccatgcttg atgggctacg
agacgacgcc 540 acttcgccgc ctaacttcaa gaactacccg cctaatcacc
ccctgagtgg ctccaaacac 600 ctatgctcta tatgcggcga cagggcgtct
gggaagcact atggggtgta cagttgcgaa 660 ggatgcaagg gtttcttcaa
gcggaccgtc cggaaggacc tgtcgtacgc ttgccgggag 720 gagcggaact
gcatcataga caagcgacaa aggaaccgat gccagtactg ccgctatcaa 780
aagtgtttgg cttgcggtat gaagcgagag gcggtgcaag aggagcgcca gaggaatgct
840 cgcggcgcgg aggatgcgca cccgagtagc tcggtgcagg taagcgatga
gctgtcaatc 900 gagcgcctaa cggagatgga gtctttggtg gcagatccca
gcgaggagtt ccagttcctc 960 cgcgtggggc ctgacagcaa cgtgcctcca
cgttaccgcg cgcccgtctc ctccctctgc 1020 caaataggca acaagcaaat
agcggcgttg gtggtatggg cgcgcgacat ccctcatttc 1080 gggcagctgg
agctggacga tcaagtggta ctcatcaagg cctcctggaa tgagctgcta 1140
ctcttcgcca tcgcctggcg ctctatggag tatttggaag atgagaggga gaacggggac
1200 ggaacgcgga gcaccactca gccacaactg atgtgtctca tgcctggcat
gacgttgcac 1260 cgcaactcgg cgcagcaggc gggcgtgggc gccatcttcg
accgcgtgct gtccgagctc 1320 agtctgaaga tgcgcacctt gcgcatggac
caggccgagt acgtcgcgct caaagccatc 1380 gtgctgctca accctgatgt
gaaaggactg aagaatcggc aagaagttga cgttttgcga 1440 gaaaaaatgt
tctcttgcct ggacgactac tgccggcggt cgcgaagcaa cgaggaaggc 1500
cggtttgcgt ccttgctgct gcggctgcca gctctccgct ccatctcgct caagagcttc
1560 gaacacctct acttcttcca cctcgtggcc gaaggctcca tcagcggata
catacgagag 1620 gcgctccgaa accacgcgcc tccgatcgac gtcaatgcca
tgatgtaaag tgcgatacac 1680 gccctgccga tgtgagaaga actatggcta
atagaagcga aactgaatac atctagggtg 1740 ggacttaact tgggactatc
attaaagtat cacgcaaatt atgcgtagtc agaaagtcgc 1800 gtcgatcaaa
cttttttata aacgaattga gtttctaacg actgcaacac agcggagttt 1860
tgcttctgat agtttttatt ctaatggtta agatgcttta cacgggcatt attgacattc
1920 aagtgtaagt ggaagttgac aaccttgaca tttatatcac gtttgtaatt
ggttaaataa 1980 attaattaat cacaagtaag actaacatca acgtcacgat
actaacgcca tttagtgata 2040 tttttcatgt caagaaactc attgttttga
taaaatattt ttctaattac tccagtgaac 2100 tcatccaaat gtgacccagt
ttcccgcaga gttgcccgtg taaaatcatc tttagggaca 2160 tatcccccgc
tatctcatga aattccaagg atcagtaggg gccaattccc ccgatgtgtt 2220
gggaggcaga attttcgata atctacgact attgttagcc tacgaattag ttgaattttt
2280 tgaaattatt tttattaagt cgccactttc caaacacatc agcagggtat
atgtgcaatt 2340 ttgtaacgat aactctattc atttctgata tttatcgaaa
ttttatctta cataacatgc 2400 tggctggtcc aggtgtttgg tagttacata
tgtatctacg gtttgtttta aattatagct 2460 tttttattgt aatctgtata
aaattgagtt atcttacttc acactacgat cgagtaaacc 2520 catcgtcagc
tacgaaaaac taatcgtata aggcgtaaga gtaaataact aattgacaac 2580
cagcaacgag gaccacctca gtcctcgtgc ttacattgtg ccgtagctta atatgatgga
2640 agctgtcgtc gttacgacat tagataaagt gcatgaatac caaaaatgta
ccatcccgta 2700 ctgatctctc atgctctcgc tgcgtgggac ccgtgtcgag
tgtcgtaagg actgactaat 2760 attttagact aggcgtctat gcttcagtaa
ttccttatac atattataag tcatccaaat 2820 aacgagtaag gcggcatgtt
gagatcagca ttccgagagt caaagagccc ctaacgtgac 2880 tgagaagtag
agacaataca ctgattttct gagatgaacg caaccgagat tgacactaaa 2940
aatctattta tggatttcaa aatggcgatg cttgattgtc tgcggcgtgg atagactgaa
3000 atgggtttgc ttaacactgg atattgtttt tattagttaa tagtcttaca
ttgcaagttg 3060 gtaattcggt gctaatatcg accggtttgt taactatcta
acggttccca gtgtcaggca 3120 cacatctttc ccaagcagac aacgcaagag
tgtacaaaat gtacatgtta caaaataagg 3180 aacattcgtc ggataagtgt
aacagttgat aggtaaagaa aatggggccg cctctttatt 3240 attacgtagc
cgtaaaatta ttaacgtatt tagtttagat gttcagctaa ttaggataat 3300
tctatttgtc gagtacctag atgtccatag tgaattaata taataattag actgttacgc
3360 gtaggtaatt ataaagttta ccaaatctct cttcaaagca aaaactttgt
acacttccgt 3420 actgagacgt cgtagcttat tctgattcac gaaatatttg
gatcacattg ttacaaggcg 3480 accgtcacgt agtatatgat tatttacaaa
tgacacgtat gtatcaatgc tataagtgtt 3540 ttcgttacat atgtcggtgc
tttaacgtgc atttcgatgt gcagattaaa aatagcaaga 3600 aatcttgaaa
ttgttttaga aaatatttga tttccttatt gaaagttatt tttaaatgta 3660
aatatttcgt aatcataata attatgtatt gtgtagttat ttcaccttta cggttgggat
3720 attatttaat ggtggcctac gaaagtgatt ataaccatcc gcgtcctcaa
aaaggccagt 3780 ttatttttgt acctcataca tactaattac gtaagtaata
tcaggcgaat ggttgactaa 3840 caactaacca gtattaaaaa ttaaaagact
tcgtcctaat aaaatgtaat atctatgtat 3900 aaaaatgaaa aatctggcgt
ataataggta aaattaaact agattgttaa tgaatgtgat 3960 gtctcataaa
cgtttagttt ttaatgagaa acatgtttag tcgcctacta taagacgaga 4020
cggcaagctc accgagttaa ctcgtaaaca ggaatgttga aaaagatgac acaatttata
4080 tttggtattg aaattatgac taaccatgcg ctctatcgtt tgttatggat
gcatagtatt 4140 gctgttgaaa ataatggaat taggtaatta ctgcattaat
gttgaaaact tgatattatt 4200 ctatggttgg gtatgaattc tatgttggaa
gtgttgcagc ggttgtaaag atgatttata 4260 atgatgttca ctaaatatct
gactaaatgt aagttatttt tttttgtata gacatagctt 4320 taagatgaag
gtgattaaac tttatcctta tcacaataaa aaaaaaaaaa aaaaa 4375 52 472 PRT
Artificial Sequence misc_feature Novel Sequence 52 Met Ser Ser Val
Ala Lys Lys Asp Lys Pro Thr Met Ser Val Thr Ala 1 5 10 15 Leu Ile
Asn Trp Ala Arg Pro Ala Pro Pro Gly Pro Pro Gln Pro Gln 20 25 30
Ser Ala Ser Pro Ala Pro Ala Ala Met Leu Gln Gln Leu Pro Thr Gln 35
40 45 Ser Met Gln Ser Leu Asn His Ile Pro Thr Val Asp Cys Ser Leu
Asp 50 55 60 Met Gln Trp Leu Asn Leu Glu Pro Gly Phe Met Ser Pro
Met Ser Pro 65 70 75 80 Pro Glu Met Lys Pro Asp Thr Ala Met Leu Asp
Gly Leu Arg Asp Asp 85 90 95 Ala Thr Ser Pro Pro Asn Phe Lys Asn
Tyr Pro Pro Asn His Pro Leu 100 105 110 Ser Gly Ser Lys His Leu Cys
Ser Ile Cys Gly Asp Arg Ala Ser Gly 115 120 125 Lys His Tyr Gly Val
Tyr Ser Cys Glu Gly Cys Lys Gly Phe Phe Lys 130 135 140 Arg Thr Val
Arg Lys Asp Leu Ser Tyr Ala Cys Arg Glu Glu Arg Asn 145 150 155 160
Cys Ile Ile Asp Lys Arg Gln Arg Asn Arg Cys Gln Tyr Cys Arg Tyr 165
170 175 Gln Lys Cys Leu Ala Cys Gly Met Lys Arg Glu Ala Val Gln Glu
Glu 180 185 190 Arg Gln Arg Asn Ala Arg Gly Ala Glu Asp Ala His Pro
Ser Ser Ser 195 200 205 Val Gln Val Ser Asp Glu Leu Ser Ile Glu Arg
Leu Thr Glu Met Glu 210 215 220 Ser Leu Val Ala Asp Pro Ser Glu Glu
Phe Gln Phe Leu Arg Val Gly 225 230 235 240 Pro Asp Ser Asn Val Pro
Pro Arg Tyr Arg Ala Pro Val Ser Ser Leu 245 250 255 Cys Gln Ile Gly
Asn Lys Gln Ile Ala Ala Leu Val Val Trp Ala Arg 260 265 270 Asp Ile
Pro His Phe Gly Gln Leu Glu Leu Asp Asp Gln Val Val Leu 275 280 285
Ile Lys Ala Ser Trp Asn Glu Leu Leu Leu Phe Ala Ile Ala Trp Arg 290
295 300 Ser Met Glu Tyr Leu Glu Asp Glu Arg Glu Asn Gly Asp Gly Thr
Arg 305 310 315 320 Ser Thr Thr Gln Pro Gln Leu Met Cys Leu Met Pro
Gly Met Thr Leu 325 330 335 His Arg Asn Ser Ala Gln Gln Ala Gly Val
Gly Ala Ile Phe Asp Arg 340 345 350 Val Leu Ser Glu Leu Ser Leu Lys
Met Arg Thr Leu Arg Met Asp Gln 355 360 365 Ala Glu Tyr Val Ala Leu
Lys Ala Ile Val Leu Leu Asn Pro Asp Val 370 375 380 Lys Gly Leu Lys
Asn Arg Gln Glu Val Asp Val Leu Arg Glu Lys Met 385 390 395 400 Phe
Ser Cys Leu Asp Asp Tyr Cys Arg Arg Ser Arg Ser Asn Glu Glu 405 410
415 Gly Arg Phe Ala Ser Leu Leu Leu Arg Leu Pro Ala Leu Arg Ser Ile
420 425 430 Ser Leu Lys Ser Phe Glu His Leu Tyr Phe Phe His Leu Val
Ala Glu 435 440 445 Gly Ser Ile Ser Gly Tyr Ile Arg Glu Ala Leu Arg
Asn His Ala Pro 450 455 460 Pro Ile Asp Val Asn Ala Met Met 465 470
53 1404 DNA Artificial Sequence misc_feature Novel Sequence 53
atggacacca aacatttcct gccgctcgac ttctctaccc aggtgaactc ttcgtccctc
60 aactctccaa cgggtcgagg ctccatggct gtcccctcgc tgcacccctc
cttgggtccg 120 ggaatcggct ctccactggg ctcgcctggg cagctgcact
ctcctatcag caccctgagc 180 tcccccatca atggcatggg tccgcccttc
tctgtcatca gctcccccat gggcccgcac 240 tccatgtcgg tacccaccac
acccacattg ggcttcggga ctggtagccc ccagctcaat 300 tcacccatga
accctgtgag cagcactgag gatatcaagc cgccactagg cctcaatggc 360
gtcctcaagg ttcctgccca tccctcagga aatatggcct ccttcaccaa gcacatctgt
420 gctatctgtg gggaccgctc ctcaggcaaa cactatgggg tatacagttg
tgagggctgc 480 aagggcttct tcaagaggac agtacgcaaa gacctgacct
acacctgccg agacaacaag 540 gactgcctga tcgacaagag acagcggaac
cggtgtcagt actgccgcta ccagaagtgc 600 ctggccatgg gcatgaagcg
ggaagctgtg caggaggagc ggcagcgggg caaggaccgg 660 aatgagaacg
aggtggagtc caccagcagt gccaacgagg acatgcctgt agagaagatt 720
ctggaagccg agcttgctgt cgagcccaag actgagacat acgtggaggc aaacatgggg
780 ctgaacccca gctcaccaaa tgaccctgtt accaacatct gtcaagcagc
agacaagcag 840 ctcttcactc ttgtggagtg ggccaagagg atcccacact
tttctgagct gcccctagac 900 gaccaggtca tcctgctacg ggcaggctgg
aacgagctgc tgatcgcctc cttctcccac 960 cgctccatag ctgtgaaaga
tgggattctc ctggccaccg gcctgcacgt acaccggaac 1020 agcgctcaca
gtgctggggt gggcgccatc tttgacaggg tgctaacaga gctggtgtct 1080
aagatgcgtg acatgcagat ggacaagacg gagctgggct gcctgcgagc cattgtcctg
1140 ttcaaccctg actctaaggg gctctcaaac cctgctgagg tggaggcgtt
gagggagaag 1200 gtgtatgcgt cactagaagc gtactgcaaa cacaagtacc
ctgagcagcc gggcaggttt 1260 gccaagctgc tgctccgcct gcctgcactg
cgttccatcg ggctcaagtg cctggagcac 1320 ctgttcttct tcaagctcat
cggggacacg cccatcgaca ccttcctcat ggagatgctg 1380 gaggcaccac
atcaagccac ctag 1404 54 467 PRT Artificial Sequence misc_feature
Novel Sequence 54 Met Asp Thr Lys His Phe Leu Pro Leu Asp Phe Ser
Thr Gln Val Asn 1 5 10 15 Ser Ser Ser Leu Asn Ser Pro Thr Gly Arg
Gly Ser Met Ala Val Pro 20 25 30 Ser Leu His Pro Ser Leu Gly Pro
Gly Ile Gly Ser Pro Leu Gly Ser 35 40 45 Pro Gly Gln Leu His Ser
Pro Ile Ser Thr Leu Ser Ser Pro Ile Asn 50 55 60 Gly Met Gly Pro
Pro Phe Ser Val Ile Ser Ser Pro Met Gly Pro His 65 70 75 80 Ser Met
Ser Val Pro Thr Thr Pro Thr Leu Gly Phe Gly Thr Gly Ser 85 90 95
Pro Gln Leu Asn Ser Pro Met Asn Pro Val Ser Ser Thr Glu Asp Ile 100
105 110 Lys Pro Pro Leu Gly Leu Asn Gly Val Leu Lys Val Pro Ala His
Pro 115 120 125 Ser Gly Asn Met Ala Ser Phe Thr Lys His Ile Cys Ala
Ile Cys Gly 130 135 140 Asp Arg Ser Ser Gly Lys His Tyr Gly Val Tyr
Ser Cys Glu Gly Cys 145 150 155 160 Lys Gly Phe Phe Lys Arg Thr Val
Arg Lys Asp Leu Thr Tyr Thr Cys 165 170 175 Arg Asp Asn Lys Asp Cys
Leu Ile Asp Lys Arg Gln Arg Asn Arg Cys 180 185 190 Gln Tyr Cys Arg
Tyr Gln Lys Cys Leu Ala Met Gly Met Lys Arg Glu 195 200 205 Ala Val
Gln Glu Glu Arg Gln Arg Gly Lys Asp Arg Asn Glu Asn Glu 210 215 220
Val Glu Ser Thr Ser Ser Ala Asn Glu Asp Met Pro Val Glu Lys Ile 225
230 235 240 Leu Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr Tyr
Val Glu 245 250 255 Ala Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp
Pro Val Thr Asn 260 265 270 Ile Cys Gln Ala Ala Asp Lys Gln Leu Phe
Thr Leu Val Glu Trp Ala 275 280 285 Lys Arg Ile Pro His Phe Ser Glu
Leu Pro Leu Asp Asp Gln Val Ile 290 295 300 Leu Leu Arg Ala Gly Trp
Asn Glu Leu Leu Ile Ala Ser Phe Ser His 305 310 315 320 Arg Ser Ile
Ala Val Lys Asp Gly Ile Leu Leu Ala Thr Gly Leu His 325 330 335 Val
His Arg Asn Ser Ala His Ser Ala Gly Val Gly Ala Ile Phe Asp 340 345
350 Arg Val Leu Thr Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp
355 360 365 Lys Thr Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn
Pro Asp 370 375 380 Ser Lys Gly Leu Ser Asn Pro Ala Glu Val Glu Ala
Leu Arg Glu Lys 385 390 395 400 Val Tyr Ala Ser Leu Glu Ala Tyr Cys
Lys His Lys Tyr Pro Glu Gln 405 410 415 Pro Gly Arg Phe Ala Lys Leu
Leu Leu Arg Leu Pro Ala Leu Arg Ser 420 425 430 Ile Gly Leu Lys Cys
Leu Glu His Leu Phe Phe Phe Lys Leu Ile Gly 435 440 445 Asp Thr Pro
Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ala Pro His 450 455 460 Gln
Ala Thr 465 55 309 DNA Artificial Sequence misc_feature Novel
Sequence 55 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
catctcaatt 60 agtcagcaac caggtgtgga aagtccccag gctccccagc
aggcagaagt atgcaaagca 120 tgcatctcaa ttagtcagca accatagtcc
cgcccctaac tccgcccatc ccgcccctaa 180 ctccgcccag ttccgcccat
tctccgcccc atggctgact aatttttttt atttatgcag 240 aggccgaggc
cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 300
gcctaggct 309 56 24 DNA Artificial Sequence misc_feature Novel
Sequence 56 tatataatgg atccccgggt accg 24 57 1653 DNA Artificial
Sequence misc_feature Novel Sequence 57 atggaagacg ccaaaaacat
aaagaaaggc ccggcgccat tctatcctct agaggatgga 60 accgctggag
agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120
gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc
180 gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag
aatcgtcgta 240 tgcagtgaaa actctcttca attctttatg ccggtgttgg
gcgcgttatt tatcggagtt 300 gcagttgcgc ccgcgaacga catttataat
gaacgtgaat tgctcaacag tatgaacatt 360 tcgcagccta ccgtagtgtt
tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420 aaaaaattac
caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat
540 tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa
ttcctctgga 600 tctactgggt tacctaaggg tgtggccctt ccgcatagaa
ctgcctgcgt cagattctcg 660 catgccagag atcctatttt tggcaatcaa
atcattccgg atactgcgat tttaagtgtt 720 gttccattcc atcacggttt
tggaatgttt actacactcg gatatttgat atgtggattt 780 cgagtcgtct
taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840
aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg
900 attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc
acctctttcg 960 aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc
cagggatacg acaaggatat 1020 gggctcactg agactacatc agctattctg
attacacccg agggggatga taaaccgggc 1080 gcggtcggta aagttgttcc
attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140 acgctgggcg
ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct
1260 ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg
cttgaagtct 1320 ttaattaaat acaaaggata tcaggtggcc cccgctgaat
tggaatcgat attgttacaa 1380 caccccaaca tcttcgacgc gggcgtggca
ggtcttcccg acgatgacgc cggtgaactt 1440 cccgccgccg ttgttgtttt
ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500 tacgtcgcca
gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560
gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata
1620 aaggccaaga agggcggaaa gtccaaattg taa 1653 58 867 DNA
Artificial Sequence misc_feature Novel Sequence 58 aagcgagagg
cggtgcaaga ggagcgccag aggaatgctc gcggcgcgga ggatgcgcac 60
ccgagtagct cggtgcaggt aagcgatgag ctgtcaatcg agcgcctaac ggagatggag
120 tctttggtgg cagatcccag cgaggagttc cagttcctcc gcgtggggcc
tgacagcaac 180 gtgcctccac gttaccgcgc gcccgtctcc tccctctgcc
aaataggcaa caagcaaata 240 gcggcgttgg tggtatgggc gcgcgacatc
cctcatttcg ggcagctgga gctggacgat 300 caagtggtac tcatcaaggc
ctcctggaat gagctgctac tcttcgccat cgcctggcgc 360 tctatggagt
atttggaaga tgagagggag aacggggacg gaacgcggag caccactcag 420
ccacaactga tgtgtctcat gcctggcatg acgttgcacc gcaactcggc gcagcaggcg
480 ggcgtgggcg ccatcttcga ccgcgtgctg tccgagctca gtctgaagat
gcgcaccttg 540 cgcatggacc aggccgagta cgtcgcgctc aaagccatcg
tgctgctcaa ccctgatgtg 600 aaaggactga agaatcggca agaagttgac
gttttgcgag aaaaaatgtt ctcttgcctg 660 gacgactact gccggcggtc
gcgaagcaac gaggaaggcc ggtttgcgtc cttgctgctg 720 cggctgccag
ctctccgctc catctcgctc aagagcttcg aacacctcta cttcttccac 780
ctcgtggccg aaggctccat cagcggatac atacgagagg cgctccgaaa ccacgcgcct
840 ccgatcgacg tcaatgccat gatgtaa 867 59 225 DNA Artificial
Sequence misc_feature Novel Sequence 59 tcgacattgg acaagtgcat
tgaacccttg tctctcgaga gacaaggggg ttcaatgcac 60 ttgtccaatg
tcgagagaca agggggttca atgcacttgt ccaatgtcga gagacaaggg 120
ggttcaatgc acttgtccaa tgtcgagaga caagggggtt caatgcactt gtccaatgtc
180 gagagacaag ggggttcaat gcacttgtcc aatgtcgact ctaga 225 60 619
DNA Artificial Sequence misc_feature Novel Sequence 60 cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
atcatatgcc 180 aagtacgccc cctattgacg
tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta
tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
360 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
aaaatcaacg 420 ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg gtaggcgtgt 480 acggtgggag gtctatataa gcagagctcg
tttagtgaac cgtcagatcg cctggagacg 540 ccatccacgc tgttttgacc
tccatagaag acaccgggac cgatccagcc tccgcggccg 600 ggaacggtgc
attggaacg 619 61 262 DNA Artificial Sequence misc_feature Novel
Sequence 61 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
ttagcaacat 60 gccttacaag gagagaaaaa gcaccgtgca tgccgatagg
tggaagtaag gtggtacgat 120 cgtgccttat taggaaggca acagacgggt
ctgacatgga ttggacgaac cactgaattc 180 cgcattgcag agatattgta
tttaagtgcc tagctcgata caataaacgc catttgacca 240 ttcaccacat
tggagtgcac ct 262 62 1247 DNA Artificial Sequence misc_feature
Novel Sequence 62 tctatttcct caggccgtga ggaactgtcg ccagcttcaa
gtataaatgg gtgcagtaca 60 gatggcgagg cacgacgtca gaagaagggc
cctgcgcccc gtcagcaaga ggaactgtgt 120 ctggtatgcg gggacagagc
ctccggatac cactacaatg cgctcacgtg tgaagggtgt 180 aaagggttct
tcagacggag tgttaccaaa aatgcggttt atatttgtaa attcggtcac 240
gcttgcgaaa tggacatgta catgcgacgg aaatgccagg agtgccgcct gaagaagtgc
300 ttagctgtag gcatgaggcc tgagtgcgta gtacccgaga ctcagtgcgc
catgaagcgg 360 aaagagaaga aagcacagaa ggagaaggac aaactgcctg
tcagcacgac gacggtggac 420 gaccacatgc cgcccattat gcagtgtgaa
cctccacctc ctgaagcagc aaggattcac 480 gaagtggtcc caaggtttct
ctccgacaag ctgttggaga caaaccggca gaaaaacatc 540 ccccagttga
cagccaacca gcagttcctt atcgccaggc tcatctggta ccaggacggg 600
tacgagcagc cttctgatga agatttgaag aggattacgc agacgtggca gcaagcggac
660 gatgaaaacg aagagtctga cactcccttc cgccagatca cagagatgac
tatcctcacg 720 gtccaactta tcgtggagtt cgcgaaggga ttgccagggt
tcgccaagat ctcgcagcct 780 gatcaaatta cgctgcttaa ggcttgctca
agtgaggtaa tgatgctccg agtcgcgcga 840 cgatacgatg cggcctcaga
cagtgttctg ttcgcgaaca accaagcgta cactcgcgac 900 aactaccgca
aggctggcat ggcctacgtc atcgaggatc tactgcactt ctgccggtgc 960
atgtactcta tggcgttgga caacatccat tacgcgctgc tcacggctgt cgtcatcttt
1020 tctgaccggc cagggttgga gcagccgcaa ctggtggaag aaatccagcg
gtactacctg 1080 aatacgctcc gcatctatat cctgaaccag ctgagcgggt
cggcgcgttc gtccgtcata 1140 tacggcaaga tcctctcaat cctctctgag
ctacgcacgc tcggcatgca aaactccaac 1200 atgtgcatct ccctcaagct
caagaacaga aagctgccgc ctttcct 1247 63 440 PRT Artificial Sequence
misc_feature Novel Sequence 63 Ser Ile Ser Ser Gly Arg Glu Glu Leu
Ser Pro Ala Ser Ser Ile Asn 1 5 10 15 Gly Cys Ser Thr Asp Gly Glu
Ala Arg Arg Gln Lys Lys Gly Pro Ala 20 25 30 Pro Arg Gln Gln Glu
Glu Leu Cys Leu Val Cys Gly Asp Arg Ala Ser 35 40 45 Gly Tyr His
Tyr Asn Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe 50 55 60 Arg
Arg Ser Val Thr Lys Asn Ala Val Tyr Ile Cys Lys Phe Gly His 65 70
75 80 Ala Cys Glu Met Asp Met Tyr Met Arg Arg Lys Cys Gln Glu Cys
Arg 85 90 95 Leu Lys Lys Cys Leu Ala Val Gly Met Arg Pro Glu Cys
Val Val Pro 100 105 110 Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys
Lys Ala Gln Lys Glu 115 120 125 Lys Asp Lys Leu Pro Val Ser Thr Thr
Thr Val Asp Asp His Met Pro 130 135 140 Pro Ile Met Gln Cys Glu Pro
Pro Pro Pro Glu Ala Ala Arg Ile His 145 150 155 160 Glu Val Val Pro
Arg Phe Leu Ser Asp Lys Leu Leu Glu Thr Asn Arg 165 170 175 Gln Lys
Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu Ile Ala 180 185 190
Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser Asp Glu Asp 195
200 205 Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp Asp Glu Asn
Glu 210 215 220 Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu Met Thr
Ile Leu Thr 225 230 235 240 Val Gln Leu Ile Val Glu Phe Ala Lys Gly
Leu Pro Gly Phe Ala Lys 245 250 255 Ile Ser Gln Pro Asp Gln Ile Thr
Leu Leu Lys Ala Cys Ser Ser Glu 260 265 270 Val Met Met Leu Arg Val
Ala Arg Arg Tyr Asp Ala Ala Ser Asp Ser 275 280 285 Val Leu Phe Ala
Asn Asn Gln Ala Tyr Thr Arg Asp Asn Tyr Arg Lys 290 295 300 Ala Gly
Met Ala Tyr Val Ile Glu Asp Leu Leu His Phe Cys Arg Cys 305 310 315
320 Met Tyr Ser Met Ala Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala
325 330 335 Val Val Ile Phe Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln
Leu Val 340 345 350 Glu Glu Ile Gln Arg Tyr Tyr Leu Asn Thr Leu Arg
Ile Tyr Ile Leu 355 360 365 Asn Gln Leu Ser Gly Ser Ala Arg Ser Ser
Val Ile Tyr Gly Lys Ile 370 375 380 Leu Ser Ile Leu Ser Glu Leu Arg
Thr Leu Gly Met Gln Asn Ser Asn 385 390 395 400 Met Cys Ile Ser Leu
Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe Leu 405 410 415 Glu Glu Ile
Trp Asp Val Ala Asp Met Ser His Thr Gln Pro Pro Pro 420 425 430 Ile
Leu Glu Ser Pro Thr Asn Leu 435 440 64 943 DNA Artificial Sequence
misc_feature Novel Sequence 64 atgacttcga aagtttatga tccagaacaa
aggaaacgga tgataactgg tccgcagtgg 60 tgggccagat gtaaacaaat
gaatgttctt gattcattta ttaattatta tgattcagaa 120 aaacatgcag
aaaatgctgt tattttttta catggtaacg cggcctcttc ttatttatgg 180
cgacatgttg tgccacatat tgagccagta gcgcggtgta ttataccaga ccttattggt
240 atgggcaaat caggcaaatc tggtaatggt tcttataggt tacttgatca
ttacaaatat 300 cttactgcat ggtttgaact tcttaattta ccaaagaaga
tcatttttgt cggccatgat 360 tggggtgctt gtttggcatt tcattatagc
tatgagcatc aagataagat caaagcaata 420 gttcacgctg aaagtgtagt
agatgtgatt gaatcatggg atgaatggcc tgatattgaa 480 gaagatattg
cgttgatcaa atctgaagaa ggagaaaaaa tggttttgga gaataacttc 540
ttcgtggaaa ccatgttgcc atcaaaaatc atgagaaagt tagaaccaga agaatttgca
600 gcatatcttg aaccattcaa agagaaaggt gaagttcgtc gtccaacatt
atcatggcct 660 cgtgaaatcc cgttagtaaa aggtggtaaa cctgacgttg
tacaaattgt taggaattat 720 aatgcttatc tacgtgcaag tgatgattta
ccaaaaatgt ttattgaatc ggacccagga 780 ttcttttcca atgctattgt
tgaaggtgcc aagaagtttc ctaatactga atttgtcaaa 840 gtaaaaggtc
ttcatttttc gcaagaagat gcacctgatg aaatgggaaa atatatcaaa 900
tcgttcgttg agcgagttct caaaaatgaa caataattct aga 943
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