U.S. patent number RE39,247 [Application Number 10/622,201] was granted by the patent office on 2006-08-22 for glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases.
This patent grant is currently assigned to Monsanto Technology LLC. Invention is credited to Gerard F. Barry, Ganesh M. Kishore, Stephen R. Padgette, William C. Stallings.
United States Patent |
RE39,247 |
Barry , et al. |
August 22, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate
synthases
Abstract
Genes encoding Class II EPSPS enzymes are disclosed. The genes
are useful in producing transformed bacteria and plants which are
tolerant to glyphosate herbicide. Class II EPSPS genes share little
homology with known, Class I EPSPS genes, and do not hybridize to
probes from Class I EPSPS's. The Class II EPSPS enzymes are
characterized by being more kinetically efficient than Class I
EPSPS's in the presence of glyphosate. Plants transformed with
Class II EPSPS genes are also disclosed as well as a method for
selectively controlling weeds in a planted transgenic crop
field.
Inventors: |
Barry; Gerard F. (St. Louis,
MO), Kishore; Ganesh M. (Creve Coeur, MO), Padgette;
Stephen R. (Wildwood, MO), Stallings; William C.
(Wildwood, MO) |
Assignee: |
Monsanto Technology LLC (St.
Louis, MO)
|
Family
ID: |
27405130 |
Appl.
No.: |
10/622,201 |
Filed: |
July 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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07749611 |
Aug 28, 1991 |
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07576537 |
Aug 31, 1990 |
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Reissue of: |
08306063 |
Sep 13, 1994 |
05633435 |
May 27, 1997 |
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Current U.S.
Class: |
800/300;
435/320.1; 435/419; 536/23.2; 536/23.4; 536/23.7; 800/278;
800/288 |
Current CPC
Class: |
C12N
9/1092 (20130101); C12N 15/8275 (20130101); Y10S
435/816 (20130101); Y10S 435/874 (20130101); Y10S
435/839 (20130101); Y10S 435/822 (20130101); Y10S
435/883 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
A01H
5/00 (20060101); A01H 5/10 (20060101); C12N
15/82 (20060101) |
Field of
Search: |
;800/300,278,288,312,298
;536/23.2,23.7 ;435/419,320.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 193 259 |
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Sep 1986 |
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EP |
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0 218 571 |
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Apr 1987 |
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EP |
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0 293 358 |
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Nov 1988 |
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EP |
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0 426 641 |
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May 1991 |
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EP |
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0 546 090 |
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Jun 1996 |
|
EP |
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WO 91/04323 |
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Apr 1991 |
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WO |
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WO 92/04449 |
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Mar 1992 |
|
WO |
|
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Defendants' Motion for Establishment of Certain Facts, Invalidation
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Primary Examiner: Kruse; David H
Attorney, Agent or Firm: Howrey LLP
Parent Case Text
This is a continuation-in-part of a U.S. patent application Ser.
No. 07/749,611, filed Aug. 28, 1991 now abandoned, which is a
continuation-in-part of U.S. patent application Ser. No.
07/576,537, filed Aug. 31, 1990, now abandoned.
Claims
We claim:
1. An isolated DNA molecule which encodes an EPSPS enzyme having
the sequence of SEQ ID NO:3.
2. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 1 having the
sequence of SEQ ID NO:2.
3. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 1 having the
sequence of SEQ ID NO:9.
4. A recombinant, double-stranded DNA molecule comprising in
sequence: a) a promoter which functions in plant cells to cause the
production of an RNA sequence; b) a structural DNA sequence that
causes the production of an RNA sequence which encodes a EPSPS
enzyme having the sequence domains: -R-X.sub.1-H-X.sub.2-E-(SEQ ID
NO:37), in which X.sub.1 is G, S, T, C, Y, N, Q, D or E; X.sub.2 is
S or T; and -G-D-K-X.sub.3-(SEQ ID NO:38), in which X.sub.3 is S or
T; and -S-A-Q-X.sub.4-K-(SEQ ID NO:39), in which X.sub.4 is A, R,
N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V; and
-N-X.sub.5-T-R-(SEQ ID NO:40), in which X.sub.5 is A, R, N, D, C,
Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.Iadd., .Iaddend.
.Iadd.provided that when X.sub.1 is D, X.sub.2 is T, X.sub.3 is S,
and X.sub.4 is V, then X.sub.5 is A, R, N, D, C, Q, E, G, H, I, L,
K, M, F, S, T, W, Y, or V.Iaddend.; and c) a 3' non-translated
region which functions in plant cells to cause the addition of a
stretch of polyadenyl nucleotides to the 3' end of the RNA
sequence; where the promoter is heterologous with respect to the
structural DNA sequence and adapted to cause sufficient expression
of the encoded EPSPS enzyme to enhance the glyphosate tolerance of
a plant cell transformed with the DNA molecule.
5. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 4 in which the
structural DNA sequence encodes a fusion polypeptide comprising an
amino-terminal chloroplast transit peptide and the EPSPS
enzyme.
6. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 4 in which
X.sub.1 is D or N; X.sub.2 is S or T; X.sub.3 is S or T; X.sub.4 is
V, I or L; and X.sub.5 is P or Q.Iadd., provided that when X.sub.1
is D, X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V, then X.sub.5 is
Q.Iaddend..
.[.7. A DNA molecule of claim 6 in which the structural DNA
sequence encodes an EPSPS enzyme selected from the group consisting
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID
NO:43..].
8. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 5 in which
X.sub.1 is D or N; X.sub.2 is S or T; X.sub.3 is S or T; X.sub.4 is
V, I or L; and X.sub.5 is P or Q.Iadd., provided that when X.sub.1
is D, X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V, then X.sub.5 is
Q.Iaddend..
.[.9. A DNA molecule of claim 8 in which the structural DNA
sequence encodes an EPSPS enzyme selected from the group consisting
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID
NO:43..].
10. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim .[.8.].
.Iadd.137 .Iaddend.in which the EPSPS .[.sequence is.].
.Iadd.enzyme has the sequence set forth in .Iaddend.SEQ ID
NO:3.
11. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim .[.10.].
.Iadd.4 .Iaddend.in which the promoter is a plant DNA virus
promoter.
12. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 11 in which
the promoter is selected from the group consisting of CaMV35S and
FMV35S promoters.
13. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim .[.10.].
.Iadd.5 .Iaddend.in which the structural DNA sequence encodes a
chloroplast transit peptide selected from the group consisting of
SEQ ID NO:11 and SEQ ID NO:15.
14. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 13 in which
the 3' non-translated region is selected from the group consisting
of the NOS 3' and the E9 3' non-translated regions.
15. A method of producing genetically transformed plants which are
tolerant toward glyphosate herbicide, comprising the steps of: a)
inserting into the genome of a plant cell a recombinant,
double-stranded DNA molecule comprising: i) a promoter which
functions in plant cells to cause the production of an RNA
sequence, ii) a structural DNA sequence that causes the production
of an RNA sequence which encodes an EPSPS enzyme having the
sequence domains: -R-X.sub.1-H-X.sub.2-E-(SEQ ID NO:37), in which
X.sub.1 is G, S, T, C, Y, N, Q, D or E; X.sub.2 is S or T; and
-G-D-K-X.sub.3-(SEQ ID NO:38), in which X.sub.3 is S or T; and
-S-A-Q-X.sub.4-K-(SEQ ID NO:39), in which X.sub.4 is A, R, N, D, C,
Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V; and
-N-X.sub.5-T-R-(SEQ ID NO:40), in which X.sub.5 is A, R, N, D, C,
Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, .Iadd.provided that
when X.sub.1 is D, X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V,
then X.sub.5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W,
Y or V.Iaddend.; and iii) a 3' non-translated DNA sequence which
functions in plant cells to cause the addition of a stretch of
polyadenyl nucleotides to the 3' end of the RNA sequence; where the
promoter is heterologous with respect to the structural DNA
sequence and adapted to cause sufficient expression of the
polypeptide to enhance the glyphosate tolerance of a plant cell
transformed with the DNA molecule; b) obtaining a transformed plant
cell; and c) regenerating from the transformed plant cell a
genetically transformed plant which has increased tolerance to
glyphosate herbicide.
16. .[.A.]. .Iadd.The .Iaddend.method of claim 15 in which X.sub.1
is D or N; X.sub.2 is S or T; X.sub.3 is S or T; X.sub.4 is V, I or
L; and X.sub.5 is P or Q.Iadd., provided that when X.sub.1 is D,
X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V, then X.sub.5 is
Q.Iaddend..
.[.17. A method of claim 16 in which the structural DNA sequence
encodes an EPSPS enzyme selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID
NO:43..].
18. .[.A.]. .Iadd.The .Iaddend.method of claim 15 in which the
structural DNA sequence encodes a fusion polypeptide comprising an
amino-terminal chloroplast transit peptide and the EPSPS
enzyme.
19. .[.A.]. .Iadd.The .Iaddend.method of claim 18 in which X.sub.1
is D or N; X.sub.2 is S or T; X.sub.3 is S or T; X.sub.4 is V, I or
L; and X.sub.5 is P or Q.Iadd., provided that when X.sub.1 is D,
X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V, then X.sub.5 is
Q.Iaddend..
.[.20. A method of claim 19 in which the structural DNA sequence
encodes an EPSPS enzyme selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:42 and SEQ ID
NO:44..].
21. .[.A.]. .Iadd.The .Iaddend.method of claim .[.19.]. .Iadd.143
.Iaddend.in which the EPSPS enzyme is that set forth in SEQ ID
NO:3.
22. .[.A.]. .Iadd.The .Iaddend.method of claim .[.21.]. .Iadd.15
.Iaddend.in which the promoter is from a plant DNA virus.
23. .[.A.]. .Iadd.The .Iaddend.method of claim 22 in which the
promoter is selected from the group consisting of CaMV35S and
FMV35S promoters.
24. A glyphosate-tolerant plant cell comprising .[.a.]. .Iadd.the
.Iaddend.DNA molecule of .[.claims.]. .Iadd.claim 4, .Iaddend.5
.Iadd.or .Iaddend.8.[.or 10.]. .
25. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 24 in which the promoter is a plant DNA virus promoter.
26. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 25 in which the promoter is selected from the group
consisting of CaMV35S and FMV35S promoters.
27. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 24 selected from the group consisting of corn, wheat, rice,
barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax,
sunflower, potato, tobacco, tomato, alfalfa, poplar, pine,
.[.eukalyptus.]. .Iadd.eucalyptus.Iaddend., apple, lettuce, peas,
lentils, grape and turf grasses.
28. A glyphosate-tolerant plant comprising .Iadd.the .Iaddend.plant
.[.cells.]. .Iadd.cell .Iaddend.of claim 27.
29. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
28 in which the promoter is from a DNA plant virus promoter.
30. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
29 in which the promoter is selected from the group consisting of
CaMV35S and FMV35S promoters.
31. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
30 selected from the group consisting of corn, wheat, rice, barley,
soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower,
potato, tobacco, tomato, alfalfa, poplar, pine, .[.eukalyptus.].
.Iadd.eucalyptus.Iaddend., apple, lettuce, peas, lentils, grape and
turf grasses.
32. A method for selectively controlling weeds in a field
containing a crop having plant crop seeds or plants comprising the
steps of: a) planting the crop seeds or plants which are
glyphosate-tolerant as a result of a recombinant double-stranded
DNA molecule being inserted into the crop seed or plant, the DNA
molecule having: i) a promoter which functions in plant cells to
cause the production of an RNA sequence, ii) a structural DNA
sequence that causes the production of an RNA sequence which
encodes an EPSPS enzyme having the sequence domains:
-R-X.sub.1-H-X.sub.2-E-(SEQ ID NO:37), in which X.sub.1 is G, S, T,
C, Y, N, Q, D or E; X.sub.2 is S or T; and -G-D-K-X.sub.3-(SEQ ID
NO:38), in which X.sub.3 is S or T; and -S-A-Q-X.sub.4-K-(SEQ ID
NO:39), in which X.sub.4 is A, R, N, D, C, Q, E, G, H, I, L, K, M,
F, P, S, T, W, Y or V; and -N-X.sub.5-T-R-(SEQ ID NO:40), in which
X.sub.5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y
or V, .Iadd.provided that when X.sub.1 is D, X.sub.2 is T, X.sub.3
is S, and X.sub.4 is V, then X.sub.5 is A, R, N, D, C, Q, E, G, H,
I, L, K, M, F, S, T, W, Y or V.Iaddend.; and iii) a 3'
non-translated DNA sequence which functions in plant cells to cause
the addition of a stretch of polyadenyl nucleotides to the 3' end
of the RNA sequence where the promoter is heterologous with respect
to the structural DNA sequence and adapted to cause sufficient
expression of the EPSPS enzyme to enhance the glyphosate tolerance
of the crop plant transformed with the DNA molecule; and b)
applying to the crop and weeds in the field a sufficient amount of
glyphosate herbicide to control the weeds without significantly
affecting the crop.
33. .[.A.]. .Iadd.The .Iaddend.method of claim 32 in which X.sub.1
is D or N; X.sub.2 is S or T; X.sub.3 is S or T; X.sub.4 is V, I or
L; and X.sub.5 is P or Q.Iadd., provided that when X.sub.1 is D,
X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V, then X.sub.5 is
Q.Iaddend..
.[.34. A method of claim 33 in which the structural DNA sequence
encodes an EPSPS enzyme selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:42 and SEQ ID
NO:44..].
35. .[.A.]. .Iadd.The .Iaddend.method of claim 32 in which the
structural DNA sequence encodes a fusion polypeptide comprising an
amino-terminal chloroplast transit peptide and the EPSPS
enzyme.
36. .[.A.]. .Iadd.The .Iaddend.method of claim 35 in which X.sub.1
is D or N; X.sub.2 is S or T; X.sub.3 is S or T; X.sub.4 is V, I or
L; and X.sub.5 is P or Q.Iadd., provided that when X.sub.1 is D,
X.sub.2 is T, X.sub.3 is S, and X.sub.4 is V, then X.sub.5 is
Q.Iaddend..
.[.37. A method of claim 36 in which the structural DNA sequence
encodes an EPSPS enzyme selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID
NO:43..].
38. .[.A.]. .Iadd.The .Iaddend.method of claim .[.36.]. .Iadd.155
.Iaddend.in which the DNA molecule encodes an EPSPS enzyme as set
forth in SEQ ID NO:3.
39. .[.A.]. .Iadd.The .Iaddend.method of claim .[.38.]. .Iadd.32
.Iaddend.in which the DNA molecule further comprises a promoter
selected from the group consisting of the CAMV35S and FMV35S
promoters.
40. .[.A.]. .Iadd.The .Iaddend.method of claim 39 in which the crop
plant is selected from the group consisting of corn, wheat, rice,
barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax,
sunflower, potato, tobacco, tomato, alfalfa, poplar, pine,
.[.eukalyptus.]. .Iadd.eucalyptus.Iaddend., apple, lettuce, peas,
lentils, grape and turf grasses.
41. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 5 in which the
structural DNA sequence encodes a chloroplast transit peptide
selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15 and SEQ ID NO:17.
42. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 41 in which
the chloroplast transit peptide is encoded by a DNA sequence
selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14 and SEQ ID NO:16.
43. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 5 in which the
structural DNA sequence encodes a chloroplast transit peptide
selected from the group consisting of SEQ ID NO:11 and SEQ ID
NO:15.
44. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 43 in which
the chloroplast transit peptide is encoded by a DNA sequence
selected from the group consisting of SEQ ID NO:10 and SEQ ID
NO:14.
45. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 41 in which
the promoter is selected from the group consisting of CaMV 35S and
FMV 35S promoters.
46. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 42 in which
the promoter is selected from the group consisting of CaMV 35S and
FMV 35S promoters.
47. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 43 in which
the promoter is selected from the group consisting of CaMV 35S and
FMV 35S promoters.
48. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 44 in which
the promoter is selected from the group consisting of CaMV 35S and
FMV 35S promoters.
49. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 45 in which
the 3' non-translated region is selected from the group consisting
of the NOS 3' and the E9 3' non-translated regions.
50. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 46 in which
the 3' non-translated region is selected from the group consisting
of the NOS 3' and the E9 3' non-translated regions.
51. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 47 in which
the 3' non-translated region is selected from the group consisting
of the NOS 3' and the E9 3' non-translated regions.
52. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim 48 in which
the 3' non-translated region is selected from the group consisting
of the NOS 3' and the E9 3' non-translated regions.
.[.53. A DNA molecule of claim 49 in which the structural DNA
sequence encodes an EPSPS enzyme selected from the group consisting
of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID
NO:44..].
.[.54. A DNA molecule of claim 50 in which the structural DNA
sequence encodes an EPSPS enzyme selected from the group consisting
of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID
NO:44..].
.[.55. A DNA molecule of claim 51 in which the structural DNA
sequence encodes an EPSPS enzyme selected from the group consisting
of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID
NO:44..].
.[.56. A DNA molecule of claim 52 in which the structural DNA
sequence encodes an EPSPS enzyme selected from the group consisting
of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:42 and SEQ ID
NO:44..].
57. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim .[.53.].
.Iadd.137 .Iaddend.in which the structural DNA sequence contains an
EPSPS encoding sequence selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, .Iadd.and .Iaddend.SEQ ID NO:6.[., SEQ ID
NO:41 and SEQ ID NO:43.]. .
58. .[.A.]. .Iadd.The .Iaddend.DNA molecule of claim .[.54.].
.Iadd.137 .Iaddend.in which the structural DNA sequence contains an
EPSPS encoding sequence .[.selected from the group consisting of
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41 and SEQ ID
NO:43.]. .Iadd.as set forth in SEQ ID NO:9.Iaddend..
.[.59. A DNA molecule of claim 55 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:41 and SEQ ID NO:43..].
.[.60. A DNA molecule of claim 56 in which the structural DNA
sequence contains an EPSPS coding sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:41
and SEQ ID NO:43..].
.[.61. A DNA molecule of claim 49 in which the structural DNA
sequence encodes an EPSPS enzyme having the sequence of SEQ ID
NO:3..].
.[.62. A DNA molecule of claim 50 in which the structural DNA
sequence encodes an EPSPS enzyme having the sequence of SEQ ID
NO:3..].
.[.63. A DNA molecule of claim 51 in which the structural DNA
sequence encodes an EPSPS enzyme having the sequence of SEQ ID
NO:3..].
.[.64. A DNA molecule of claim 52 in which the structural DNA
sequence encodes an EPSPS enzyme having the sequence of SEQ ID
NO:3..].
.[.65. A DNA molecule of claim 61 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2 and SEQ ID NO:9..].
.[.66. A DNA molecule of claim 62 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2 and SEQ ID NO:9..].
.[.67. A DNA molecule of claim 63 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2 and SEQ ID NO:9..].
.[.68. A DNA molecule of claim 64 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2 and SEQ ID NO:9..].
69. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim .[.25.]. .Iadd.149 .Iaddend.in which: (a) the promoter is
selected from the group consisting of CaMV 35S and FMV 35S
promoters; (b) the structural DNA sequence encodes: (i) a
chloroplast transit peptide selected from the group consisting of
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and (ii)
an EPSPS enzyme selected from the group consisting of SEQ ID NO:3,
SEQ ID NO:5, .Iadd.and .Iaddend.SEQ ID NO:7.[., SEQ ID NO:42 and
SEQ ID NO:44.]. ; and (c) the 3' non-translated region is selected
from the group consisting of the NOS 3' and the E9 3'
non-translated regions.
70. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 69 in which the structural DNA sequence comprises: (a) a
chloroplast transit peptide encoding DNA sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and
SEQ ID NO:16; and (b) an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, .Iadd.and
.Iaddend.SEQ ID NO:6.[., SEQ ID NO:41 and SEQ ID NO:43.]. .
71. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 69 in which the structural DNA sequence comprises: (a) a
chloroplast transist peptide encoding DNA sequence selected from
the group consisting of SEQ ID NO:10 and SEQ ID NO:14; and (b) a
DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID
NO:3.
72. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 71 in which the structural DNA sequence comprises an EPSPS
encoding sequence .[.selected from the group consisting of SEQ ID
NO:2 and.]. .Iadd.as set forth in .Iaddend.SEQ ID NO:9.
73. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant cell of
claim 71 selected from the group consisting of corn, wheat, rice,
barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax,
sunflower, potato, tobacco, tomato, alfalfa, poplar, pine,
.[.eukalyptus.]. .Iadd.eucalyptus.Iaddend., apple, lettuce, peas,
lentils, grape and turf grasses.
74. A glyphosate-tolerant plant comprising .[.a.]. .Iadd.the
.Iaddend.DNA molecule of .[.claims 5, 8 or 10.]. .Iadd.claim 131
.Iaddend.in which: (a) the promoter is selected from the group
consisting of CaMV 35S and FMV 35S promoters; (b) the structural
DNA sequence encodes.[.:.]. .Iadd.;.Iaddend. (i) a chloroplast
transit peptide selected from the group consisting of SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and (ii) an EPSPS
enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID
NO:5, .Iadd.and .Iaddend.SEQ ID NO:7.[., SEQ ID NO:42 and SEQ ID
NO:44.]. ; and (c) the 3' non-translated region is selected from
the group consisting of the NOS 3' and the E9 3' non-translated
regions.
75. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
74 in which the structural DNA sequence comprises: (a) a
chloroplast transit peptide encoding DNA sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and
SEQ ID NO:16; and (b) an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, .Iadd.and
.Iaddend.SEQ ID NO:6.[., SEQ ID NO:41 and SEQ ID NO:43.]. .
76. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
75 in which the structural DNA sequence comprises: (a) a
chloroplast transit peptide encoding DNA sequence selected from the
group consisting of SEQ ID NO:10 and SEQ ID NO:14; and (b) a DNA
sequence encoding an EPSPS enzyme having the sequence of SEQ ID
NO:3.
77. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
.[.76.]. .Iadd.74 .Iaddend.in which the structural DNA sequence
comprises an EPSPS encoding sequence .[.selected from the group
consisting of SEQ ID NO:2 and.]. .Iadd.as set forth in .Iaddend.SEQ
ID NO:9.
78. .[.A.]. .Iadd.The .Iaddend.glyphosate-tolerant plant of claim
.[.77.]. .Iadd.74 .Iaddend.selected from the group consisting of
corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed
rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa,
poplar, pine, .[.eukalyptus.]. .Iadd.eucalyptus.Iaddend., apple,
lettuce, peas, lentils, grape and turf grasses.
79. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim 28.Iadd., wherein the seed comprises the recombinant DNA
molecule.Iaddend..
80. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim 31.Iadd., wherein the seed comprises the recombinant DNA
molecule.Iaddend..
81. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim 75.Iadd., wherein the seed comprises the recombinant DNA
molecule.Iaddend..
82. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim 76.Iadd., wherein the seed comprises the recombinant DNA
molecule.Iaddend..
83. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim 77.Iadd., wherein the seed comprises the recombinant DNA
molecule.Iaddend..
84. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim .[.78.]. .Iadd.129, wherein the seed comprises the
recombinant DNA molecule.Iaddend..
85. A seed of .[.a.]. .Iadd.the .Iaddend.glyphosate-tolerant plant
of claim .[.79.]. .Iadd.144, wherein the seed comprises the
recombinant DNA molecule.Iaddend..
.[.86. A transgenic soybean plant which contains a heterologous
gene which encodes an EPSPS enzyme having a K.sub.m for
phosphoenolpyruvate (PEP) between 1 and 150 .mu.M and a
K.sub.i(glyphosate)/K.sub.m(PEP) ratio between about 2 and 500,
said plant exhibiting tolerance to N-phosphonomethylglycine
herbicide at a rate of 1 lb/acre without significant yield
reduction due to herbicide application..].
.[.87. Seed of a soybean plant of claim 86..].
.Iadd.88. The DNA molecule of claim 6 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:41 and SEQ ID NO:43..Iaddend.
.Iadd.89. The DNA molecule of claim 8 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:41 and SEQ ID NO:43..Iaddend.
.Iadd.90. The method of claim 16 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:41 and SEQ ID NO: 43..Iaddend.
.Iadd.91. The method of claim 19 in which the structural DNA
sequence encodes an EPSPS enzyme having a sequence selected from
the group consisting of SEQ ID NO:42 and SEQ ID NO:44..Iaddend.
.Iadd.92. The method of claim 33 in which the structural DNA
sequence encodes an EPSPS enzyme having a sequence selected from
the group consisting of SEQ ID NO:42 and SEQ ID NO:44..Iaddend.
.Iadd.93. The method of claim 36 in which the structural DNA
sequence contains an EPSPS encoding sequence selected from the
group consisting of SEQ ID NO:41 and SEQ ID NO:43..Iaddend.
.Iadd.94. The DNA molecule of claim 49 in which the structural DNA
sequence encodes an EPSPS enzyme having a sequence selected from
the group consisting of SEQ ID NO: 42 and SEQ ID NO:
44..Iaddend.
.Iadd.95. The DNA molecule of claim 50 in which the structural DNA
sequence encodes an EPSPS enzyme having a sequence selected from
the group consisting of SEQ ID NO: 42 and SEQ ID NO:
44..Iaddend.
.Iadd.96. The DNA molecule of claim 51 in which the structural DNA
sequence encodes an EPSPS enzyme having a sequence selected from
the group consisting of SEQ ID NO: 42 and SEQ ID NO:
44..Iaddend.
.Iadd.97. The DNA molecule of claim 52 in which the structural DNA
sequence encodes an EPSPS enzyme having a sequence selected from
the group consisting of SEQ ID NO: 42 and SEQ ID NO:
44..Iaddend.
.Iadd.98. The glyphosate-tolerant plant cell of claim 25 in which:
a) the promoter is selected from the group consisting of CaMV 35S
and FMV 35S promoters; b) the structural DNA sequence encodes: i) a
chloroplast transit peptide selected from the group consisting of
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17; and ii)
an EPSPS enzyme selected from the group consisting of SEQ ID NO:42
and SEQ ID NO:44; and c) the 3' non-translated region is selected
from the group consisting of the NOS 3' and the E93' non-translated
regions..Iaddend.
.Iadd.99. The glyphosate-tolerant plant cell of claim 26 in which
the structural DNA sequence comprises: a) a chloroplast transit
peptide encoding DNA sequence selected from the group consisting of
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; and b)
an EPSPS encoding sequence selected from the group consisting of
SEQ ID NO:41 and SEQ ID NO:43..Iaddend.
.Iadd.100. The glyphosate-tolerant plant comprising the DNA
molecule of claim 4, 5 or 8 in which: a) the promoter is selected
from the group consisting of CaMV 35S and FMV 35S promoters; b) the
structural DNA sequence encodes: i) a chloroplast transit peptide
selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15 and SEQ ID NO:17; and ii) an EPSPS enzyme selected
from the group consisting of SEQ ID NO:42 and SEQ ID NO:44; and c)
the 3' non-translated region is selected from the group consisting
of the NOS 3' and the E93' non-translated regions..Iaddend.
.Iadd.101. The glyphosate-tolerant plant of claim 28 in which the
structural DNA sequence comprises: a) a chloroplast transit peptide
encoding DNA sequence selected from the group consisting of SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16; and b) an EPSPS
encoding sequence selected from the group consisting of SEQ ID
NO:41 and SEQ ID NO:43..Iaddend.
.Iadd.102. An isolated DNA molecule that encodes a
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme having
the sequence of SEQ ID NO:70..Iaddend.
.Iadd.103. A recombinant, double-stranded DNA molecule comprising
in sequence: a) a promoter which functions in plant cells to cause
the production of an RNA sequence; b) a structural DNA sequence
that causes the production of an RNA sequence which encodes an
EPSPS enzyme having the sequence of SEQ ID NO:70; and c) a 3'
non-translated region that functions in plant cells to cause the
addition of a stretch of polyadenyl nucleotides to the 3' end of
the RNA sequence; where the promoter is heterologous with respect
to the structural DNA sequence and adapted to cause sufficient
expression of the encoded EPSPS enzyme to enhance the glyphosate
tolerance of a plant cell transformed with the DNA
molecule..Iaddend.
.Iadd.104. The DNA molecule of claim 103, wherein the structural
DNA sequence further causes the production of an RNA sequence that
encodes an amino-terminal chloroplast transit peptide that is fused
to the EPSPS enzyme..Iaddend.
.Iadd.105. The DNA molecule of claim 104, wherein the chloroplast
transit peptide has the sequence of SEQ ID NO:11 or SEQ ID
NO:15..Iaddend.
.Iadd.106. The DNA molecule of claim 103, wherein the promoter is a
plant DNA virus promoter..Iaddend.
.Iadd.107. The DNA molecule of claim 106, wherein the promoter is a
CaMV35S promoter or an FMV35S promoter..Iaddend.
.Iadd.108. The DNA molecule of claim 103, wherein the 3'
non-translated region is a NOS 3' or an E93' non-translated
region..Iaddend.
.Iadd.109. A method of producing a genetically transformed plant
which is tolerant toward glyphosate herbicide, comprising the steps
of: a) inserting into the genome of a plant cell a recombinant
double-stranded DNA molecule comprising: i) a promoter that
functions in plant cells to cause the production of an RNS
sequence; ii) a structural DNA sequence that causes the production
of an RNS sequence which encodes an EPSPS enzyme having the
sequence of SEQ ID NO: 70; and iii) a 3' non-translated DNA
sequence that functions in plant cells to cause the addition of a
stretch of polyadenyl nucleotides to the 3' end of the RNS
sequence; wherein the promoter is heterologous with respect to the
structural DNA sequence and adapted to cause sufficient expression
of the polypeptide to enhance the glyphosate tolerance of a plant
cell transformed with the DNA molecule; b) obtaining a transformed
plant cell; and c) regenerating from the transformed plant cell a
genetically transformed plant which has increased tolerance to
glyphosate herbicide..Iaddend.
.Iadd.110. The method of claim 109, wherein the structural DNA
further causes the production of an RNA sequence that encodes an
amino-terminal chloroplast transit peptide that is fused to the
EPSPS enzyme..Iaddend.
.Iadd.111. The method of claim 110, wherein the chloroplast transit
peptide has the sequence of SEQ ID NO:11 or SEQ ID
NO:15..Iaddend.
.Iadd.112. The method of claim 109, in which the promoter is a
plant DNA virus promoter..Iaddend.
.Iadd.113. The method of claim 112, in which the promoter is a
CaMV35S promoter or an FMV35S promoter..Iaddend.
.Iadd.114. The method of claim 109, wherein the 3' non-translated
DNA sequence is a NOS 3' or an E93' non-translated
sequence..Iaddend.
.Iadd.115. A glyphosate-tolerant plant cell comprising a DNA
sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:
70..Iaddend.
.Iadd.116. A glyphosate-tolerant plant comprising a DNA sequence
encoding an EPSPS enzyme having the sequence of SEQ ID NO:
70..Iaddend.
.Iadd.117. The plant of claim 116, wherein the plant is corn,
wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape,
canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar,
pine, eucalyptus, apple, lettuce, peas, lentils, grape or turf
grasses..Iaddend.
.Iadd.118. The plant of claim 117, wherein the plant is
corn..Iaddend.
.Iadd.119. The plant of claim 117, wherein the plant is
soybean..Iaddend.
.Iadd.120. The plant of claim 117, wherein the plant is
canola..Iaddend.
.Iadd.121. The plant of claim 117, wherein the plant is
cotton..Iaddend.
.Iadd.122. A seed of the plant of claim 116, wherein the seed
comprises the DNA sequence encoding an EPSPS enzyme having the
sequence of SEQ ID NO: 70..Iaddend.
.Iadd.123. The seed of claim 122, wherein the seed is corn, wheat,
rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola,
flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine,
eucalyptus, apple, lettuce, peas, lentils, grape or turf grass
seed..Iaddend.
.Iadd.124. The seed of claim 123, wherein the seed is corn
seed..Iaddend.
.Iadd.125. The seed of claim 123, wherein the seed is soybean
seed..Iaddend.
.Iadd.126. The seed of claim 123, wherein the seed is canola
seed..Iaddend.
.Iadd.127. The seed of claim 123, wherein the seed is cotton
seed..Iaddend.
.Iadd.128. A glyphosate tolerant plant cell comprising the
recombinant DNA molecule of claim 103..Iaddend.
.Iadd.129. A plant comprising the glyphosate tolerant plant cell of
claim 128..Iaddend.
.Iadd.130. A method for selectively controlling weeds in a field
containing a crop having planted crop seeds or plants comprising
the steps of: a) planting the crop seeds or plants which are
glyphosate-tolerant as a result of a recombinant double-stranded
DNA molecule being inserted into the crop seed or plant, the DNA
molecule having: i) a promoter which functions in plant cells to
cause the production of an RNA sequence, ii) a structural DNA
sequence that causes the production of an RNA sequence which
encodes an EPSPS enzyme having the sequence of SEQ ID NO:70; and
iii) a 3' non-translated DNA sequence which functions in plant
cells to cause the addition of a stretch of polyadenyl nucleotides
to the 3' end of the RNA sequence, where the promoter is
heterologous with respect to the structural DNA sequence and
adapted to cause sufficient expression of the EPSPS enzyme to
enhance the glyphosate tolerance of the crop plant transformed with
the DNA molecule; and b) applying to the crop and weeds in the
field a sufficient amount of glyphosate herbicide to control the
weeds without significantly affecting the crop..Iaddend.
.Iadd.131. A recombinant, double-stranded DNA molecule comprising
in sequence: a) a promoter which functions in plant cells to cause
the production of an RNA sequence; b) a structural DNA sequence
that causes the production of an RNA sequence which encodes an
EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ
ID NO: 7; c) a 3' non-translated region which functions in plant
cells to cause the addition of a stretch of polyadenyl nucleotides
to the 3' end of the RNA sequence; where the promoter is
heterologous with respect to the structural DNA sequence and
adapted to cause sufficient expression of the encoded EPSPS enzyme
to enhance the glyphosate tolerance of a plant cell transformed
with the DNA molecule..Iaddend.
.Iadd.132. The DNA molecule of claim 131 in which the structural
DNA sequence encodes a fusion polypeptide comprising an
amino-terminal chloroplast transit peptide and the EPSPS
enzyme..Iaddend.
.Iadd.133. The DNA molecule of claim 131 in which the promoter is a
plant DNA virus promoter..Iaddend.
.Iadd.134. The DNA molecule of claim 133 in which the promoter is
selected from the group consisting of CaMV35S and FMV35S
promoters..Iaddend.
.Iadd.135. The DNA molecule of claim 132 in which the structural
DNA sequence encodes a chloroplast transit peptide selected from
the group consisting of SEQ ID NO: 11 and SEQ ID NO:
15..Iaddend.
.Iadd.136. The DNA molecule of claim 131 in which the 3'
non-translated region is selected from the group consisting of the
NOS 3' and the E93' non-translated regions..Iaddend.
.Iadd.137. A method of producing genetically transformed plants
which are tolerant toward glyphosate herbicide, comprising the
steps of: a) inserting into the genome of a plant cell a
recombinant, double-stranded DNA molecule comprising: i) a promoter
which functions in plant cells to cause the production of an RNA
sequence, ii) a structural DNA sequence that causes the production
of an RNA sequence which encodes an EPSPS enzyme having the
sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7; and iii) a 3'
non-translated DNA sequence which functions in plant cells to cause
the addition of a stretch of polyadenyl nucleotides to the 3' end
of the RNA sequence; where the promoter is heterologous with
respect to the structural DNA sequence and adapted to cause
sufficient expression of the polypeptide to enhance the glyphosate
tolerance of a plant cell transformed with the DNA molecule; b)
obtaining a transformed plant cell; and c) regenerating from the
transformed plant cell a genetically transformed plant which has
increased tolerance to glyphosate herbicide..Iaddend.
.Iadd.138. The method of claim 137 in which the structural DNA
sequence encodes a fusion polypeptide comprising an amino-terminal
chloroplast transit peptide and the EPSPS enzyme..Iaddend.
.Iadd.139. The method of claim 130, wherein the chloroplast transit
peptide has the sequence of SEQ ID NO: 11 or SEQ ID NO:
15..Iaddend.
.Iadd.140. The method of claim 137 in which the promoter is a plant
DNA virus..Iaddend.
.Iadd.141. The method of claim 140 in which the promoter is a
CaMV35S promoter or a FMV35S promoter..Iaddend.
.Iadd.142. The method of claim 137, wherein the 3' non-translated
DNA sequence is a NOS 3' or an e93' non-translated
sequence..Iaddend.
.Iadd.143. A glyphosate-tolerant plant cell comprising the DNA
molecule of claim 131..Iaddend.
.Iadd.144. A plant comprising the glyphosate-tolerant plant cell of
claim 143..Iaddend.
.Iadd.145. A glyphosate-tolerant plant cell comprising an EPSPS
enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID
NO:7..Iaddend.
.Iadd.146. A glyphosate-tolerant plant comprising an EPSPS enzyme
having the sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:
7..Iaddend.
.Iadd.147. The glyphosate-tolerant plant cell of claim 143 or 145
selected from the group consisting of corn, wheat, rice, barley,
soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower,
potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple,
lettuce, peas, lentils, grape, and turf grasses..Iaddend.
.Iadd.148. The glyphosate-tolerant plant of claim 144 or 146
selected from the group consisting of corn, wheat, rice, barley,
soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower,
potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple,
lettuce, peas, lentils, grapes, and turf grasses..Iaddend.
.Iadd.149. A method for selectively controlling weeds in a field
containing a crop having planted crop seeds or plants comprising
the steps of: a) planting the crop seeds or plants which are
glyphosate-tolerant as a result of a recombinant double-stranded
DNA molecule being inserted into the crop seed or plant, the DNA
molecule having: i) a promoter which functions in plant cells to
cause the production of an RNA sequence, ii) a structural DNA
sequence that causes the production of an RNA sequence which
encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID
NO:5, or SEQ ID NO:7; and iii) a 3' non-translated DNA sequence
which functions in plant cells to cause the addition of a stretch
of polyadenyl nucleotides to the 3' end of the RNA sequence,
wherein the promoter is heterologous with respect to the structural
DNA sequence and adapted to cause sufficient expression of the
EPSPS enzyme to enhance the glyphosate tolerance of the crop plant
transformed with the DNA molecule; and b) applying to the crop and
weeds in the field a sufficient amount of glyphosate herbicide to
control the weeds without significantly affecting the
crop..Iaddend.
Description
BACKGROUND OF THE INVENTION
This invention relates in general to plant molecular biology and,
more particularly, to a new class of glyphosate-tolerant
5-enolpyruvylshikimate-3-phosphate synthases.
Recent advances in genetic engineering have provided the requisite
tools to transform plants to contain foreign genes. It is now
possible to produce plants which have unique characteristics of
agronomic importance. Certainly, one such advantageous trait is
more cost effective, environmentally compatible weed control via
herbicide tolerance. Herbicide-tolerant plants may reduce the need
for tillage to control weeds thereby effectively reducing soil
erosion.
One herbicide which is the subject of much investigation in this
regard is N-phosphonomethylglycine commonly referred to as
glyphosate. Glyphosate inhibits the shikimic acid pathway which
leads to the biosynthesis of aromatic compounds including amino
acids, plant hormones and vitamins. Specifically, glyphosate curbs
the conversion of phosphoenolpyruvic acid (PEP) and
3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by
inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase
(hereinafter referred to as EPSP synthase or EPSPS). For purposes
of the present invention, the term "glyphosate" should be
considered to include any herbicidally effective form of
N-phosphonomethylglycine (including any salt thereof) and other
forms which result in the production of the glyphosate anion in
plants.
It has been shown that glyphosate-tolerant plants can be produced
by inserting into the genome of the plant the capacity to produce a
higher level of EPSP synthase in the chloroplast of the cell (Shah
et al., 1986) which enzyme is preferably glyphosate-tolerant
(Kishore et al. 1988). Variants of the wild-type EPSPS enzyme have
been isolated which are glyphosate-tolerant as a result of
alterations in the EPSPS amino acid coding sequence (Kishore and
Shah, 1988; Schulz et al., 1984; Sost et al., 1984; Kishore et al.,
1986). These variants typically have a higher K.sub.i for
glyphosate than the wild-type EPSPS enzyme which confers the
glyphosate-tolerant phenotype, but these variants are also
characterized by a high K.sub.m for PEP which makes the enzyme
kinetically less efficient (Kishore and Shah, 1988; Sost et al.,
1984; Schulz et al., 1984; Kishore et al., 1986; Sost and Amrhein,
1990). For example, the apparent K.sub.m for PEP and the apparent
K.sub.i for glyphosate for the native EPSPS from E. coli are 10
.mu.M and 0.5 .mu.M while for a glyphosate-tolerant isolate having
a single amino acid substitution of an alanine for the glycine at
position 96 these values are 220 .mu.M and 4.0 mM, respectively. A
number of glyphosate-tolerant plant variant EPSPS genes have been
constructed by mutagenesis. Again, the glyphosate-tolerant EPSPS
was impaired due to an increase in the K.sub.m for PEP and a slight
reduction of the V.sub.max of the native plant enzyme (Kishore and
Shah, 1988) thereby lowering the catalytic efficiency
(V.sub.max/K.sub.m) of the enzyme. Since the kinetic constants of
the variant enzymes are impaired with respect to PEP, it has been
proposed that high levels of overproduction of the variant enzyme,
40-80 fold, would be required to maintain normal catalytic activity
in plants in the presence of glyphosate (Kishore et al., 1988).
While such variant EPSP synthases have proved useful in obtaining
transgenic plants tolerant to glyphospate, it would be increasingly
beneficial to obtain an EPSP synthase that is highly
glyphosate-tolerant while still kinetically efficient such that the
amount of the glyphosate-tolerant EPSPS needed to be produced to
maintain normal catalytic activity in the plant is reduced or that
improved tolerance be obtained with the same expression level.
Previous studies have shown that EPSPS enzymes from different
sources vary widely with respect to their degree of sensitivity to
inhibition by glyphosate. A study of plant and bacterial EPSPS
enzyme activity as a function of glyphosate concentration showed
that there was a very wide range in the degree of sensitivity to
glyphosate. The degree of sensitivity showed no correlation with
any genus or species tested (Schulz et al., 1985). Insensitivity to
glyphosate inhibition of the activity of the EPSPS from the
Pseudomonas sp. PG2982 has also been reported but with no details
of the studies (Fitzgibbon, 1988). In general, while such natural
tolerance has been reported, there is no report suggesting the
kinetic superiority of the naturally occurring bacterial
phosphosate-tolerant EPSPS enzymes over those of mutated EPSPS
enzymes nor have any of the genes been characterized. Similarly,
there are no reports on the expression of naturally
glyphosate-tolerant EPSPS enzymes in plants to confer glyphosate
tolerance.
For purposes of the present invention the term "mature EPSP
synthase" relates to the EPSPS polypeptide without the N-terminal
chloroplast transit peptide. It is now known that the precursor
form of the EPSP synthase in plants (with the transit peptide) is
expressed and upon delivery to the chloroplast, the transit peptide
is cleaved yielding the mature EPSP synthase. All numbering of
amino acid positions are given with respect to the mature EPSP
synthase (without chloroplast transit peptide leader) to facilitate
comparison of EPSPS sequences from sources which have chloroplast
transit peptides (i.e., plants and fungi) to sources which do not
utilize a chloroplast targeting signal (i.e., bacteria).
In the amino acid sequences which follow, the standard single
letter or three letter nomenclature are used. All peptide
structures represented in the following description are shown in
conventional format in which the amino group at the N-terminus
appears to the left and the carboxyl group at the C-terminus at the
right. Likewise, amino acid nomenclature for the naturally
occurring amino acids found in protein is as follows: alanine
(Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine
(Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine
(Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I),
leucine (Leu;L), lysine (Lys;k), methionine (Met;M), phenylalanine
(Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T),
tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V). An "X" is
used when the amino acid residue is unknown and parentheses
designate that an unambiguous assignment is not possible and the
amino acid designation within the parentheses is the most probable
estimate based on known information.
The term "nonpolar" amino acids include alanine, valine, leucine,
isoleucine, proline, phenylalanine, tryptophan, and methionine. The
term "uncharged polar" amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine and glutamine. The term
"charged polar" amino acids includes the "acidic" and "basic" amino
acids. The term "acidic" amino acids includes aspartic acid and
glutamic acid. The term "basic" amino acid includes lysine,
arginine and histidine. The term "polar" amino acids includes both
"charged polar" and "uncharged polar" amino acids.
Deoxyribonucleic acid (DNA) is a polymer comprising four
mononucleotide units, dAMP (2'-Deoxyadenosine-5-monophosphate),
dGMP (2'-Deoxyguanosine-5-monophosphate), dCMP
(2'-Deoxycytosine-5-monophosphate) and dTMP
(2'-Deoxythymosine-5-monophosphate) linked in various sequences by
3',5'-phosphodiester bridges. The structural DNA consists of
multiple nucleotide triplets called "codons" which code for the
amino acids. The codons correspond to the various amino acids as
follows: Arg (CGA, CGC, CGG, CGT, AGA, AGG); Leu (CTA, CTC, CTG,
CTT, TTA, TTG); Ser (TCA, TCC, TCG, TCT, AGC, AGT); Thr (ACA, ACC,
ACG, ACT); Pro (CCA, CCC, CCG, CCT); Ala (GCA, GCC, GCG, GCT); Gly
(GGA, GGC, GGG, GGT); Ile (ATA, ATC, ATT); Val (GTA, GTC, GTG,
GTT); Lys (AAA, AAG); Asn (AAC, AAT); Gla (CAA, CAG); His (CAC,
CAT); Glu (GAA, GAG); Asp (GAC, GAT); Tyr (TAC, TAT); Cys (TGC,
TGT); Phe (TTC, TTT); Met (ATG); and Trp (UGG). Moreover, due to
the redundancy of the genetic code (i.e., more than one codon for
all but two amino acids), there are many possible DNA sequences
which may code for a particular amino acid sequence.
SUMMARY OF THE INVENTION
DNA molecules comprising DNA encoding kinetically efficient,
glyphosate-tolerant EPSP synthases are disclosed. The EPSP
synthases of the present invention reduce the amount of
overproduction of the EPSPS enzyme in a transgenic plant necessary
for the enzyme to maintain catalytic activity while still
conferring glyphosate tolerance. The EPSP synthases described
herein represent a new class of EPSPS enzymes, referred to
hereinafter as Class II EPSPS enzymes. Class II EPSPS enzymes of
the present invention usually share only between about 47% and 55%
amino acid similarity or between about 22% and 30% amino acid
identity to other known bacterial or plant EPSPS enzymes and
exhibit tolerance to glyphosate while maintaining suitable K.sub.m
(PEP) ranges. Suitable ranges of K.sub.m (PEP) for EPSPS for
enzymes of the present invention are between 1-150 .mu.M, with a
more preferred range of between 1-35 .mu.M, and a most preferred
range between 2-25 .mu.M. These kinetic constants are determined
under the assay conditions specified hereinafter. An EPSPS of the
present invention preferably has a K.sub.i for glyphosate range of
between 15-10000 .mu.M. The K.sub.i/K.sub.m ratio should be between
about 2-500, and more preferably between 25-500. The V.sub.max of
the purified enzyme should preferably be in the range of 2-100
units/mg (.mu.moles/minute.mg at 25.degree. C.) and the K.sub.m for
shikimate-3-phosphate should preferably be in the range of 0.1 to
50 .mu.M.
Genes coding for Class II EPSPS enzymes have been isolated from
five (5) different bacteria:Agrobacterium tumefaciens sp. strain
CP4, Achromobacter sp. strain LBAA, Pseudomonas sp. strain PG2982,
Bacillus subtilis, and Staphylococcus aureus. The LBAA and PG2982
Class II EPSPS genes have been determined to be identical and the
proteins encoded by these two genes are very similar to the CP4
protein and share approximately 84% amino acid identity with it.
Class II EPSPS enzymes often may be distinguished from Class I
EPSPS's by their inability to react with polyclonal antibodies
prepared from Class I EPSPS enzymes under conditions where other
Class I EPSPS enzymes would readily react with the Class I
antibodies as well as the presence of certain unique regions of
amino acid homology which are conserved in Class II EPSP synthases
as discussed hereinafter.
Other Class II EPSPS enzymes can be readily isolated and identified
by utilizing a nucleic acid probe from one of the Class II EPSPS
genes disclosed herein using standard hybridization techniques.
Such a probe from the CP4 strain has been prepared and utilized to
isolate the Class II EPSPS genes from strains LBAA and PG2982.
These genes may also optionally be adapted for enhanced expression
in plants by known methodology. Such a probe has also been used to
identify homologous genes is bacteria isolated de novo from
soil.
The Class II EPSPS enzymes are preferably fused to a chloroplast
transit peptide (CTP) to target the protein to the chloroplasts of
the plant into which it may be introduced. Chimeric genes encoding
this CTP-Class II EPSPS fusion protein may be prepared with an
appropriate promoter and 3' polyadenylation site for introduction
into a desired plant by standard methods.
To obtain the maximal tolerance to glyphosate herbicide it is
preferable to transform the desired plant with a plant-expressible
Class II EPSPS gene in conjunction with another plant-expressible
gene which expresses a protein capable of degrading glyphosate such
as a plant-expressible gene encoding a glyphosate oxidoreductase
enzyme as described in PCT Application No. WO 92/00377, the
disclosure of which is hereby incorporated by reference.
Therefore, in one aspect, the present invention provides a new
class of EPSP synthases that exhibit a low K.sub.m for
phosphoenolpyruvate (PEP), a high V.sub.max/K.sub.m ratio, and a
high K.sub.i for glyphosate such that when introduced into a plant,
the plant is made glyphosate-tolerant such that the catalytic
activity of the enzyme and plant metabolism are maintained in a
substantially normal state. For purposes of this discussion, a
highly efficient EPSPS refers to its efficiency in the presence of
glyphosate.
More particularly, the present invention provides EPSPS enzymes
having a K.sub.m for phosphoenolpyruvate (PEP) between 1-150 .mu.M
and a K.sub.i(glyphosate)/K.sub.m (PEP) ratio between 3-500, said
enzymes having the sequence domains: -R-X.sub.1-H-X.sub.2-E-(SEQ ID
NO:37), in which X.sub.1 is an uncharged polar or acidic amino
acid, X.sub.2 is serine or threonine; and -G-D-K-X.sub.3-(SEQ ID
NO:38), in which X.sub.3 is serine or threonine; and
-S-A-Q-X.sub.4-K-(SEQ ID NO:39), in which X.sub.4 is any amino
acid; and -N-X.sub.5-T-R-(SEQ ID NO:40), in which X.sub.5 is any
amino acid.
Exemplary Class II EPSPS enzyme sequences are disclosed from seven
sources: Agrobacterium sp. strain designated CP4, Achromobacter sp.
strain LBAA, Pseudomonas sp. strain PG2982, Bacillus subtilis 1A2,
Staphylococcus aureus (ATCC 35556), Synechocystis sp. PCC6803 and
Dichelobacter nodosus.
In another aspect of the present invention, a double-stranded DNA
molecule comprising DNA encoding a Class II EPSPS enzyme is
disclosed. Exemplary Class II EPSPS enzyme DNA sequences are
disclosed from seven sources: Agrobacterium sp. strain designated
CP4, Achromobacter sp. strain LBAA, Pseudomonas sp. strain PG2982,
Bacillus subtilis 1A2, Staphylococcus aureus (ATCC 35556),
Synechocystis sp. PCC6803 and Dichelobacter nodosus.
In a further aspect of the present invention, nucleic acid probes
from EPSPS Class II genes are presented that are suitable for use
in screening for Class II EPSPS genes in other sources by assaying
for the ability of a DNA sequence from the other source to
hybridize to the probe.
In yet another aspect of the present invention, a recombinant,
double-stranded DNA molecule comprising in sequence: a) a promoter
which functions in plant cells to cause the production of an RNA
sequence; b) a structural DNA sequence that causes the production
of an RNA sequence which encodes a Class II EPSPS enzyme having the
sequence domains; -R-X.sub.1-H-X.sub.2-E-(SEQ ID NO:37), in which
X.sub.1 is an uncharged polar or acidic amino acid, X.sub.2 is
serine or threonine; and -G-D-K-X.sub.3-(SEQ ID NO:38), in which
X.sub.3 is serine or threonine; and -S-A-Q-X.sub.4-K-(SEQ ID
NO:39), in which X.sub.4 is any amino acid; and -N-X.sub.5-T-R-(SEQ
ID NO:40), in which X.sub.5 is any amino acid; and c) a 3'
nontranslated region which functions in plant cells to cause the
addition of a stretch of polyadenyl nucleotides to the 3' end of
the RNA sequence where the promoter is heterologous with respect to
the structural DNA sequence and adapted to cause sufficient
expression of the EPSP synthase polypeptide to enhance the
glyphosate tolerance of a plant cell transformed with said DNA
molecule.
In still yet another aspect of the present invention, transgenic
plants and transformed plant cells are disclosed that are made
glyphosate-tolerant by the introduction of the above-described
plant-expressible Class II EPSPS DNA molecule into the plant's
genome.
In still another aspect of the present invention, a method for
selectively controlling weeds in a crop field is presented by
planting crop seeds or crop plants transformed with a
plant-expressible Class II EPSPS DNA molecule to confer glyphosate
tolerance to the plants which allows for glyphosate containing
herbicides to be applied to the crop to selectively kill the
glyphosate sensitive weeds, but not the crops.
Other and further objects, advantages and aspects of the invention
will become apparent from the accompanying drawing figures and the
description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B, show the DNA sequence (SEQ ID NO:1) for the
full-length promoter of figwort mosaic virus (FMV35S).
FIG. 2 shows the cosmid cloning vector pMON17020.
FIG. 3A, 3B, 3C, 3D and 3E show the structural DNA sequence (SEQ ID
NO:2) for the Class II EPSPS gene from bacterial isolate
Agrobacterium sp. strain CP4 and the deduced amino acid sequence
(SEQ ID NO:3).
FIG. 4A, 4B, 4C, 4D and 4E show the structural DNA sequence (SEQ ID
NO:4) for the Class II EPSPS gene from the bacterial isolate
Achromobacter sp. strain LBAA and the deduced amino acid sequence
(SEQ ID NO:5).
FIG. 5A, 5B, 5C, 5D and 5E show the structural DNA sequence (SEQ ID
NO:6) for the Class II EPSPS gene from the bacterial isolate
Pseudomonas sp. strain PG2982 and the deduced amino acid sequence
(SEQ ID NO:7).
FIG. 6A and 6B show the Bestfit comparison of the CP4 EPSPS amino
acid sequence (SEQ ID NO:3) with that for the E. coli EPSPS (SEQ ID
NO:8).
FIG. 7A and 7B show the Bestfit comparison of the CP4 EPSPS amino
acid sequence (SEQ ID NO:3) with that for the LBAA EPSPS (SEQ ID
NO:5).
FIG. 8A and 8B show the structural DNA sequence (SEQ ID NO:9) for
the synthetic CP4 Class II EPSPS gene.
FIG. 9 shows the DNA sequence (SEQ ID NO:10) of the chloroplast
transit peptide (CTP) and encoded amino acid sequence (SEQ ID
NO:11) derived from the Arabidopsis thaliana EPSPS CTP and
containing a SphI restriction site at the chloroplast processing
site, hereinafter referred to as CTP2.
FIG. 10A and 10B show the DNA sequence (SEQ ID NO:12) of the
chloroplast transit peptide and encoded amino acid sequence (SEQ ID
NO:13) derived from the Arabidopsis thaliana EPSPS gene and
containing an EcoRI restriction site within the mature region of
the EPSPS, hereinafter referred to as CTP3.
FIG. 11 shows the DNA sequence (SEQ ID NO:14) of the chloroplast
transit peptide and encoded amino acid sequence (SEQ ID NO:15)
derived from the Petunia hybrida EPSPS CTP and containing a SphI
restriction site at the chloroplast processing site and in which
the amino acids at the processing site are changed to -Cys-Met-,
hereinafter referred to as CTP4.
FIG. 12A and 12B show the DNA sequence (SEQ ID NO:16) of the
chloroplast transit peptide and encoded amino acid sequence (SEQ ID
NO:17) derived from the Petunia hybrida EPSPS gene with the
naturally occurring EcoRI site in the mature region of the EPSPS
gene, hereinafter referred to as CTP5.
FIG. 13 shows a plasmid map of CP4 plant transformation/expression
vector pMON17110.
FIG. 14 shows a plasmid map of CP4 synthetic EPSPS gene plant
transformation/expression vector pMON17131.
FIG. 15 shows a plasmid map of CP4 EPSPS free DNA plant
transformation expression vector pMON13640.
FIG. 16 shows a plasmid map of CP4 plant transformation/direct
selection vector pMON17227.
FIG. 17 shows a plasmid map of CP4 plant transformation/expression
vector pMON19653.
FIG. 18A, 18B, 18C and 18D show the structural DNA sequence (SEQ ID
NO:41) for the Class II EPSPS gene from the bacterial isolate
Bacillus subtilis and the deduced amino acid sequence (SEQ ID
NO:42).
FIG. 19A, 19B, 19C and 19D show the structural DNA sequence (SEQ ID
NO:43) for the Class II EPSPS gene from the bacterial isolate
Staphylococcus aureus and the deduced amino acid sequence (SEQ ID
NO:44).
FIG. 20A, 20B, 20C, 20D, 20E, 20F, 20G, 20H, 20I, 20J and 20K show
the Bestfit comparison of the representative Class II EPSPS amino
acid sequences Pseudomonas sp. stain PG2982 (SEQ ID NO:7),
Achromobacter sp. strain LBAA (SEQ ID NO:5), Agrobacterium sp.
strain designated CP4 (SEQ ID NO:3), Bacillus subtilis (SEQ ID
NO:42), and Staphylococcus aureus (SEQ ID NO:44) with that for
representative Class I EPSPS amino acid sequences [Sacchromyces
cerevisiae (SEQ ID NO:49), Aspergillus nidulans (SEQ ID NO:50),
Brassica napus (SEQ ID NO:51), Arabidopsis thaliana (SEQ ID NO:52),
Nicotina tobacum (SEQ ID NO:53), L. esculentum (SEQ ID NO:54),
Petunia hybrida (SEQ ID NO:55), Zea mays (SEQ ID NO:56), Solmenella
gallinarum (SEQ ID NO:57), Solmenella typhimurium (SEQ ID NO:58),
Solmenella typhi (SEQ ID NO:65), E. coli (SEQ ID NO:8), K.
pneumoniae (SEQ ID NO:59), Y. enterocolitica (SEQ ID NO:60), H.
influenzae (SEQ ID NO:61), P. multocida (SEQ ID NO:62), Aeromonas
salmonicida (SEQ ID NO:63), Bacillus pertussis (SEQ ID NO:64)] and
illustrates the conserved regions among Class II EPSPS sequences
which are unique to Class II EPSPS sequences. To aid in a
comparison of the EPSPS sequences, only mature EPSPS sequences were
compared. That is, the sequence corresponding to the chloroplast
transit peptide, if present in a subject EPSPS, was removed prior
to making the sequence alignment.
FIG. 21A, 21B, 21C, 21D and 21E show the structural DNA sequence
(SEQ ID NO:66) for the Class II EPSPS gene from the bacterial
isolate Synechocystis sp. PCC6803 and the deduced amino acid
sequence (SEQ ID NO:67).
FIG. 22A, 22B, 22C, 22D and 22E show the structural DNA sequence
(SEQ ID NO:68) for the Class II EPSPS gene from the bacterial
isolate Dichelobacter nodosus and the deduced amino acid sequence
(SEQ ID NO:69).
FIG. 23A, 23B, 23C and 23D show the Bestfit comparison of the
representative Class II EPSPS amino acid sequences Pseudomonas sp.
strain PG2982 (SEQ ID NO:7), Achromobacter sp. strain LBAA (SEQ ID
NO:5), Agrobacterium sp. strain designated CP4 (SEQ ID NO:3),
Synechocystis sp. PCC6803 (SEQ ID NO:67), Bacillus subtilis (SEQ ID
NO:42), Dichelobacter nodosus (SEQ ID NO:69) and Staphylococcus
aureus (SEQ ID NO:44).
FIG. 24 a plasmid map of canola plant transformation/expression
vector pMON17209.
FIG. 25 a plasmid map of canola plant transformation/expression
vector pMON17237.
STATEMENT OF THE INVENTION
The expression of a plant gene which exists in double-stranded DNA
form involves synthesis of messenger RNA (mRNA) from one strand of
the DNA by RNA polymerase enzyme, and the subsequent processing of
the mRNA primary transcript inside the nucleus. This processing
involves a 3' non-translated region which adds polyadenylate
nucleotides to the 3' end of the RNA.
Transcription of DNA into mRNA is regulated by a region of DNA
usually referred to as the "promoter." The promoter region contains
a sequence of bases that signals RNA polymerase to associate with
the DNA, and to initiate the transcription into mRNA using one of
the DNA strands as a template to make a corresponding complementary
strand of RNA. A number of promoters which are active in plant
cells have been described in the literature. These include the
nopaline synthase (NOS) and octopine synthase (OCS) promoters
(which are carried on tumor-inducing plasmids of Agrobacterium
tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S
promoters, the light-inducible promoter from the small subunit of
ribulose bis-phosphate carboxylase (ssRUBISCO, a very abundant
plant polypeptide) and the full-length transcript promoter from the
figwort mosaic virus (FMV35S), promoters from the maize ubiquitin
and rice actin genes. All of these promoters have been used to
create various types of DNA constructs which have been expressed in
plants; see, e.g., PCT publication WO 84/02913 (Rogers et al.,
Mosanto).
Promoters which are known or found to cause transcription of DNA in
plant cells can be used in the present invention. Such promoters
may be obtained from a variety of sources such as plants and plant
DNA virsues and include, but are not limited to, the CaMV35A and
FMV35S promoters and promoters isolated from plant genes such as
ssRUBISCO genes and the maize ubiquitin and rice actin genes. As
described below, it is preferred that the particular promoter
selected should be capable of causing sufficient expression to
result in the production of an effective amount of a Class II EPSPS
to render the plant substantially tolerant to glyphosate
herbicides. The amount of Class II EPSPS needed to induce the
desired tolerance may vary with the plant species. It is preferred
that the promoters utilized have relatively high expression in all
meristematic tissues in addition to other tissues inasmuch as it is
now known that glyphosate is translocated and accumulated in this
type of plant tissue. Alternatively, a combination of chimeric
genes can be used to cumulatively result in the necessary overall
expression level of the selected Class II EPSPS enzyme to result in
the glyphosate-tolerant phenotype.
The mRNA produced by a DNA construct of the present invention also
contains a 5' non-translated leader sequence. This sequence can be
derived from the promoter selected to express the gene, and can be
specifically modified so as to increase translation of the mRNA.
The 5' non-translated regions can also be obtained from viral RNAs,
from suitable eukaryotic genes, or from a synthetic gene sequence.
The present invention is not limited to constructs, as presented in
the following examples, wherein the non-translated region is
derived from both the 5' non-translated sequence that accompanies
the promoter sequence and part of the 5' non-translated region of
the virus coat protein gene. Rather, the non-translated leader
sequence can be derived from an unrelated promoter or coding
sequence as discussed above.
Preferred promoters for use in the present invention the
full-length transcript (SEQ ID NO:1) promoter from the figwort
mosaic virus (FMV35S) and the full-length transcript (35S) promoter
from cauliflower mosaic virus (CaMV), including the enhanced
CaMV35S promoter (Kay et al. 1987). The FMV35S promoter functions
as strong and uniform promoter with particularly good expression in
meristematic tissue for chimeric genes inserted into plants,
particularly dicotyledons. The resulting transgenic plant in
general expresses the protein encoded by the inserted gene at a
higher and more uniform level throughout the tissues and cells of
the transformed plant than the same gene driven by an enhanced
CaMV35S promoter. Referring to FIG. 1, the DNA sequence (SEQ ID
NO:1) of the FMV35S promoter is located between nucleotides 6368
and 6930 of the FMV genome. A 5' non-translated leader sequence is
preferably coupled with the promoter. The leader sequence can be
from the FMV35S genome itself or can be from a source other than
FMV35S.
For expression of heterologous genes in moncotyledonous plants the
use of an intron has been found to enhance expression of the
heterologous gene. While one may use any of a number of introns
which have been isolated from plant genes, the use of the first
intron from the maize heat shock 70 gene is preferred.
The 3' non-translated region of the chimeric plant gene contains a
polyadenylation signal which functions in plants to cause the
addition of polyadenylate nucleotides to the 3' end of the viral
RNA. Examples of suitable 3' regions are (1) the 3' transcribed,
non-translated regions containing the polyadenylated signal of
Agrobacterium tumor-inducing (Ti) plasmid genes, such as the
nopaline synthase (NOS) gene, and (2) plant genes like the soybean
storage protein genes and the small subunit of the
ribulose-1,5-biphosphate carboxylase (ssRUBISCO) gene. An example
of a preferred 3' region is that from the ssRUBISCO gene from pea
(E9), described in greater detail below.
The DNA constructs of the present invention also contain a
structural coding sequence in double-stranded DNA form which
encodes a glyphosate-tolerant, highly efficient Class II EPSPS
enzyme.
Identification of glyphosate-tolerant, highly efficient EPSPS
enzymes
In an attempt to identify and isolate glyphosate-tolerant, highly
efficient EPSPS enzymes, kinetic analysis of the EPSPS enzymes from
a number of bacteria exhibiting tolerance to glyphosate or that had
been isolated from suitable sources was undertaken. It was
discovered that in some cases the EPSPS enzymes showed no tolerance
to inhibition by glyphosate and it was concluded that the tolerance
phenotype of the bacterium was due to an impermeability to
glyphosate or other factors. In a number of cases, however,
microorganisms were identified whose EPSPS enzyme showed a greater
degree of tolerance to inhibition by glyphosate and that displayed
a low K.sub.m for PEP when compared to that previously reported for
other microbial and plant sources. The EPSPS enzymes from these
microorganisms were then subjected to further study and
analysis.
Table I displays the data obtained for the EPSPS enzymes identified
and isolated as a result of the above described analysis. Table I
includes data for three identified Class II EPSPS enzymes that were
observed to have a high tolerance to inhibition to glyphosate and a
low K.sub.m for PEP as well as data for the native Petunia EPSPS
and a glyphosate-tolerant variant of the Petunia EPSPS referred to
as GA101. The GA101 variant is so named because it exhibits the
substitution of an alanine residue for a glycine residue at
position 101 (with respect to Petunia). When the change introduced
into the Petunia EPSPS (GA101) was introduced into a number of
other EPSPS enzymes, similar changes in a kinetics were observed,
an elevation of the K.sub.i for glyphosate and of the K.sub.m for
PEP.
TABLE-US-00001 TABLE I Kinetic characterization of EPSPS enzymes
ENZYME K.sub.m PEP K.sub.l Glyphosate SOURCE (.mu.M) (.mu.M)
K.sub.l/K.sub.m Petunia 5 0.4 0.08 Petunia GA101 200 2000 10 PG2982
2.1-3.1.sup.1 25-82 .about.8-40 LBAA .about.7.3-8.sup.2 60
(est).sup.7 .about.7.9 CP4 12.sup.3 2720 227 B. subtilis 1A2
13.sup.4 440 33.8 S. aureus 5.sup.5 200 40 .sup.1Range of PEP
tested = 1-40 .mu.M .sup.2Range of PEP tested = 5-80 .mu.M
.sup.3Range of PEP tested = 1.5-40 .mu.M .sup.4Range of PEP tested
= 1-60 .mu.M .sup.5Range of PEP tested = 1-50 .mu.M .sup.7(est) =
estimated
The Agrobacterium sp. strain CP4 was initially identified by its
ability to grow on glyphosate as a carbon source (10 mM) in the
presence of 1 mM phosphate. The strain CP4 was identified from a
collection obtained from a fixed-bed immobilized cell column that
employed Mannville R-635 diatomaceous earth beads. The column had
been run for three months on a waste-water feed from a glyphosate
production plant. The column contained 50 mg/ml glyphosate and
NH.sub.3 as NH.sub.4Cl. Total organic carbon was 300 mg/ml and
BOD's (Biological Oxygen Demand--a measure of "soft" carbon
availability) were less than 30 mg/ml. This treatment column has
been described (Heitkamp et al., 1990). Dworkin-Foster minimal
salts medium containing glyphosate at 10 mM and with phosphate at 1
mM was used to select for microbes from a wash of this column that
were capable of growing on glyphosate as sole carbon source.
Dworkin-Foster minimal medium was made up by combining in I liter
(with autoclaved H.sub.2O), 1 ml each of A, B and C and 10 ml of D
(as per below) and thiamine HCl (5 mg).
TABLE-US-00002 A. D-F Salts (1000X stock; per 100 ml; autoclaved):
H.sub.2BO.sub.3 1 mg MnSO.sub.4.7 H.sub.2O 1 mg ZnSO.sub.4.7
H.sub.2O 12.5 mg CuSO.sub.4.5 H.sub.2O 8 mg NaMoO.sub.3.3 H.sub.2O
1.7 mg B. FeSO.sub.4.7 H.sub.2O (1000X Stock; per 100 0.1 g ml;
autoclaved) C. MgSO.sub.4.7 H.sub.2O (1000X Stock; per 100 20 g ml;
autoclaved) D. (NH.sub.4).sub.2SO.sub.4 (100X stock; per 100 ml; 20
g autoclaved)
Yeast Extract (YE; Difco) was added to a final concentration of
0.01 or 0.001%. The strain CP4 was also grown on media composed of
D-F salts, amended as described above, containing glucose,
gluconate and citrate (each at 0.1%) as carbon sources and with
inorganic phosphate (0.2-1.0 mM) as the phosphorous source.
Other Class II EPSPS containing microorganisms were identified as
Achromobacter sp. strain LBAA (Hallas et al., 1988), Pseudomonas
sp. strain PG2982 (Moore et al., 1983; Fitzgibbon 1988), Bacillus
subtilis 1A2 (Henner et al., 1984) and Staphylococcus aureus
(O'Connell et al., 1993). It had been reported previously, from
measurements in crude lysates, that the EPSPS enzyme from strain
PG2982 was less sensitive to inhibition to glyphosate than that of
E. coli, but there has been no report of the details of this lack
of sensitivity and there has been no report on the K.sub.m for PEP
for this enzyme or of the DNA sequence for the gene for this enzyme
(Fitzgibbon, 1988; Fitzgibbon and Braymer, 1990). Relationship of
the Class II EPSPS to those previously studied.
All EPSPS proteins studied to date have shown a remarkable degree
of homology. For example, bacterial and plant EPSPS's are about 54%
identical and with similarity as high as 80%. Within bacterial
EPSPS's and plant EPSPS's themselves the degree of identity and
similarity is much greater (see Table II).
TABLE-US-00003 TABLE II Comparison between exemplary Class I EPSPS
protein sequences.sup.1 similarity identity E. coli vs. S.
typhaurium 93 88 P. hybrids vs. E. coli 72 55 P. hybrids vs. L.
excalentum 93 88 .sup.1The EPSPS sequences compared here were
obtained from the following reference: E. coli, Rogers et al.,
1983; S. typhourium, Smetzer et al, 1985; Petanoic hybrids; Shah et
al, 1986; and tomato (L. escalautum), Gasper et al, 1988.
When crude extracts of CP4 and LBAA bacteria (50 .mu.g protein)
were probed using rabbit anti-EPSPS antibody (Padgette et al.,
1987) to the Petunia EPSPS protein in a Western analysis, no
positive signal could be detected, even with extended exposure
times (Protein A--.sup.125I development system) and under
conditions where the control EPSPS (Petunia EPSPS, 20 ng; a Class I
EPSPS) was readily detected. The presence of EPSPS activity in
these extracts was confirmed by enzyme assay. This surprising
result, indicating a lack of similarity between the EPSPS's from
these bacterial isolates and those previously studied, coupled with
the combination of a low K.sub.m for PEP and a high K.sub.i for
glyphosate, illustrates that these new EPSPS enzymes are different
from known EPSPS enzymes (now referred to as Class I EPSPS).
Glyphosate-tolerant Enzymes is Microbial Isolates
For clarity and brevity of disclosure, the following description of
the isolation of genes encoding Class II EPSPS enzymes is directed
to the isolation of such a gene from a bacterial isolate. Those
skilled in the art will recognize that the same or similar strategy
can be utilized to isolate such genes from other microbial
isolates, plant or fungal sources.
Cloning of the Agrobacterium sp. strain CP4 EPSPS Gene(s) in E.
coli
Having established the existence of a suitable EPSPS in
Agrobacterium sp. strain CP4, two parallel approaches were
undertaken to clone the gene: cloning based on the expected
phenotype for a glyphosate-tolerant EPSPS; and purification of the
enzyme to provide material to raise antibodies and to obtain amino
acid sequences from the protein to facilitate the verification of
clones. Cloning and genetic techniques, unless otherwise indicated,
are generally those described in Maniatis et al., 1982 or Sambrook
et al., 1987. The cloning strategy was as follows: introduction of
a cosmid bank of strain Agrobacterium sp. strain CP4 into E. coli
and selection for the EPSPS gene by selection for growth on
inhibitory concentrations of glyphosate.
Chromosomal DNA was prepared from strain Agrobacterium sp. strain
CP4 as follows: The cell pellet from a 200 ml L-Broth (Miller,
1972), late log phase culture of Agrobacterium sp. strain CP4 was
resuspended in 10 ml of Solution I; 50 mM Glucose, 10 mM EDTA, 25
mM Tris-CL pH 8.0 (Birnboim and Doly, 1979). SDS was added to a
final concentration of 1% and the suspension was subjected to three
freeze-thaw cycles, each consisting of immersion in dry ice for 15
minutes and in water at 70.degree. C. for 10 minutes. The lysate
was then extracted four times with equal volumes of
phenol:chloroform (1:1; phenol saturated with TE; TE=10 mM Tris
pH8.0; 1.0 mM EDTA) and the phases separated by centrifugation
(15000 g; 10 minutes). The ethanol-precipitable material was
pelleted from the supernatant by brief centrifugation (8000 g; 5
minutes) following addition of two volumes of ethanol. The pellet
was resuspended in 5 ml TE and dialyzed for 16 hours at 4.degree.
C. against 2 liters TE. This preparation yielded a 5 ml DNA
solution of 552 .mu.g/ml.
Partially-restricted DNA was prepared as follows. Three 100 .mu.g
aliquot samples of CP4 DNA were treated for 1 hour at 37.degree. C.
with restriction endonuclease HindIII at rates of 4, 2 and 1 enzyme
unit/.mu.g DNA, respectively. The DNA samples were pooled, made
0.25 mM with EDTA and extracted with an equal volume of
phenol:chloroform. Following the addition of sodium acetate and
ethanol, the DNA was precipitated with two volumes of ethanol and
pelleted by centrifugation (12000 g; 10 minutes). The dried DNA
pellet was resuspended in 500 .mu.l TE and layered on a 10-40%
Sucrose gradient (in 5% increments of 5.5 ml each) in 0.5M NaCl, 50
mM Tris pH8.0, 5 mM EDTA. Following centrifugation for 20 hours at
26,000 rpm in a SW28 rotor, the tubes were punctured and .about.1.5
ml fractions collected. Samples (20 .mu.l) of each second fraction
were run on 0.7% agarose gel and the size of the DNA determined by
comparison with linearized lambda DNA and HindIII-digested lambda
DNA standards. Fractions containing DNA of 25-35 kb fragments were
pooled, desalted on AMICON10 columns (7000 rpm; 20.degree. C.; 45
minutes) and concentrated by precipitation. This procedure yielded
15 .mu.g of CP4 DNA of the required size. A cosmid bank was
constructed using the vector pMON17020. This vector, a map of which
is presented in FIG. 2, is based on the pBR327 replicon and
contains the spectinomycin/streptomycin (Sp.sup.r;spc) resistance
gene from Tn7 (Fling et al., 1985), the chloramphenicol resistance
gene (Cm.sup.r;cat) from Tn9 (Alton et al., 1979), the gene10
promoter region from phage T7 (Dunn et al., 1983), and the 1.6 kb
BglII phage lambda cos fragment from pHC79 (Hohn and Collins,
1980). A number of cloning sites are located downstream of the cat
gene. Since the predominant block to the expression of genes from
other microbial sources in E. coli appears to be at the level of
transcription, the use of the T7 promoter and supplying the T7
polymerase in trans from the pGP1-2 plasmid (Tabor and Richardson,
1985), enables the expression of large DNA segments of foreign DNA,
even those containing RNA polymerase transcription termination
sequences. The expression of the spc gene is impaired by
transcription from the T7 promoter such that only Cmr can be
selected in strains containing pGP1-2. The use of antibiotic
resistances such as Cm resistance which do not employ a membrane
component is preferred due to the observation that high level
expression of resistance genes that involve a membrane component,
i.e. .beta.-lactamase and Amp resistance, give rise to a
glyphosate-tolerant phenotype. Presumably, this is due to the
exclusion of glyphosate from the cell by the membrane localized
resistance protein. It is also preferred that the selectable marker
be oriented in the same direction as the T7 promoter.
The vector was then cut with HindIII and treated with calf alkaline
phosphatase (CAP) in preparation for cloning. Vector and target
sequences were ligated by combining the following:
TABLE-US-00004 Vector DNA (HindIII/CAP) 3 .mu.g Size fractionated
CP4 HindIII fragments 1.5 .mu.g 10X ligation buffer 2.2 .mu.l T4
DNA ligase (New England Biolabs) (400 U/.mu.l) 1.0 .mu.l
and adding H.sub.2O to 22.0 .mu.l. This mixture was incubated for
18 hours at 16.degree. C. 10X ligation buffer is 250 mM Tris-HCl,
pH 8.0; 100 mM MgCl.sub.2; 100 mM Dithiothreitol; 2 mM Spermidine.
The ligated DNA (5 .mu.l) was packaged into lambda phage particles
(Stratagene; Gigapack Gold) using the manufacturer's procedure.
A sample (200 .mu.l) of E. coli HB101 (Boyer and Rolland-Dussoix,
1973) containing the T7 polymerase expression plasmid pGP1-2 (Tabor
and Richardson, 1985) and grown overnight in L-Broth (with maltose
at 0.2% and kanamycin at 50/.mu.g/ml) was infected with 50 .mu.l of
the packaged DNA. Transformants were selected at 30.degree. C. on
M9 (Miller, 1972) agar containing kanamycin (50 .mu.g/ml),
chloramphenicol (25 .mu.g/ml), L-proline (50 .mu.g/ml), L-leucine
(50 .mu.g/ml) and B1 (5 .mu.g/ml), and with glyphosate at 3.0 mM.
Aliquot samples were also plated on the same media lacking
glyphosate to titer the packaged cosmids. Cosmid transformants were
isolated on this latter medium at a rate of .about.5.times.10.sup.5
per .mu.g CP4 HindIII DNA after 3 days at 30.degree. C. Colonies
arose on the glyphosate agar from day 3 until day 15 with a final
rate of .about.1 per 200 cosmids. DNA was prepared from 14
glyphosate-tolerant clones and, following verification of this
phenotype, was transformed into E. coli GB100/pGP1-2 (E. coli GB100
is an aroA derivative of MM294 [Talmadge and Gilbert, 1980]) and
tested for complementation for growth in the absence of added
aromatic amino acids and aminobenzoic acids. Other aroA strains
such as SR481 (Bachman et al., 1980; Padgette et al., 1987), could
be used and would be suitable for this experiment. The use of GB100
is merely exemplary and should not be viewed in a limiting sense.
This aroA strain usually requires that growth media be supplemented
with L-phenylalanine, L-tyrosine and L-tryptophan each at 100
.mu.g/ml and with para-hydroxybenzoic acid, 2,3-dihydroxybenzoic
acid and para-aminobenzoic acid each at 5 .mu.g/ml for growth in
minimal media. Of the fourteen cosmids tested only one showed
complementation of the aroA- phenotype. Transformants of this
cosmid, pMON17076, showed weak but uniform growth on the
unsupplemented minimal media after 10 days.
The proteins encoded by the cosmids were determined in vivo using a
T7 expression system (Tabor and Richardson, 1985). Cultures of E.
coli containing pGP1-2 (Tabor and Richardson, 1985) and test and
control cosmids were grown at 30.degree. C. in L-broth (2 ml) with
chloramphenicol and kanamycin (25 and 50 .mu.g/ml, respectively) to
a Klett reading of .about.50. An aliquot was removed and the cells
collected by centrifugation, washed with M9 salts (Miller, 1972)
and resuspended in 1 ml M9 medium containing glucose at 0.2%,
thiamine at 20 .mu.g/ml and containing the 18 amino acids at 0.01%
(minus cysteine and methionine). Following incubation at 30.degree.
C. for 90 minutes, the cultures were transferred to a 42.degree. C.
water bath and held there for 15 minutes. Rifampicin (Sigma) was
added to 200 .mu.g/ml and the cultures held at 42.degree. C. for 10
additional minutes and then transferred to 30.degree. C. for 20
minutes. Samples were pulsed with 10 .mu.Ci of .sup.35S-methionine
for 5 minutes at 30.degree. C. The cells were collected by
centrifugation and suspended in 60-120 .mu.l cracking buffer (60 mM
Tris-HCl 6.8, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol, 0.01%
bromophenol blue). Aliquot samples were electrophoresed on 12.5%
SDS-PAGE and following soaking for 60 minutes in 10 volumes of
Acetic Acid-Methanol-water (10:30:60), the gel was soaked in
ENLIGHTNING.TM. (DUPONT) following manufacturer's directions,
dried, and exposed at -70.degree. C. to X-Ray film. Proteins of
about 45 kd in size, labeled with .sup.35S-methionine, were
detected in number of the cosmids, including pMON17076.
Purification of EPSPS from Agrobacterium sp. strain CP4
All protein purification procedures were carried out at
3.degree.-5.degree. C. EPSPS enzyme assays were performed using
either the phosphate release or radioactive HPLC method, as
previously described in Padgette et al., 1987, using 1 mM
phosphoenol pyruvate (PEP, Boehringer) and 2 mM
shikimate-3-phosphate (S3P) substrate concentrations. For
radioactive HPLC assays, .sup.14-CPEP (Amersham) was utilized. S3P
was synthesized as previously described in Wibbenmeyer et al. 1988.
N-terminal amino acid sequencing was performed by loading samples
onto a Polybrene precycled filter in aliquots while drying.
Automated Edman degradation chemistry was used to determine the
N-terminal protein sequence, using an Applied Biosystems Model 470A
gas phase sequencer (Hunkapiller et al., 1983) with an Applied
Biosystems 120A PTH analyzer.
Five 10-liter fermentations were carried out on a spontaneous
"smooth" isolate of strain CP4 that displayed less clumping when
grown in liquid culture. This reduced clumping and smooth colony
morphology may be due to reduced polysaccharide production by this
isolate. In the following section dealing with the purification of
the EPSPS enzyme, CP4 refers to the "smooth" isolate--CP4-S1. The
cells from the three batches showing the highest specific
activities were pooled. Cell paste of Agrobacterium sp. CP4 (300 g)
was washed twice with 0.5 L of 0.9% saline and collected by
centrifugation (30 minutes, 8000 rpm in a GS3 Sorvall rotor). The
cell pellet was suspended in 0.9 L extraction buffer (100 mM
TrisCl, 1 mM EDTA, 1 mM BAM (Benzamidine), 5 mM DTT, 10% glycerol,
pH 7.5) and lysed by 2 passes through a Manton Gaulin cell. The
resulting solution was centrifuged (30 minutes, 8000 rpm) and the
supernatant was treated with 0.21 L of 1.5% protamine sulfate (in
100 mM TrisCl, pH 7.5, 0.2% w/v final protamine sulfate
concentration). After stirring for 1 hour, the mixture was
centrifuged (50 minutes, 8000 rpm) and the resulting supernatant
treated with solid ammonium sulfate to 40% saturation and stirred
for 1 hour. After centrifugation (50 minutes, 8000 rpm), the
resulting supernatant was treated with solid ammonium sulfate to
70% saturation, stirred for 50 minutes, and the insoluble protein
was collected by centrifugation (1 hour, 8000 rpm). This 40-70%
ammonium sulfate fraction was then dissolved in extraction buffer
to give a final volume of 0.2 L, and dialyzed twice (Spectrum
10,000 MW cutoff dialysis tubing) against 2 L of extraction buffer
for a total of 12 hours.
To the resulting dialyzed 40-70% ammonium sulfate fraction (0.29 L)
was added solid ammonium sulfate to give a final concentration of
1M. This material was loaded (2 ml/min) onto a column (5
cm.times.15 cm, 295 ml) packed with phenyl Sepharose CL-4B
(Pharmacia) resin equilibrated with extraction buffer containing 1M
ammonium sulfate, and washed with the same buffer (1.5 L, 2
ml/min). EPSPS was eluted with a linear gradient of extraction
buffer going from 1M to 0.00M ammonium sulfate (total volume of 1.5
L, 2 ml/min). Fractions were collected (20 ml) and assayed for
EPSPS activity by the phosphate release assay. The fractions with
the highest EPSPS activity (fractions 36-50) were pooled and
dialyzed against 3.times.2 L (18 hours) of 10 mM TrisCl, 25 mM KCl,
1 mM EDTA, 5 mM DTT, 10% glycerol, pH 7.8.
The dialyzed EPSPS extract (350 ml) was loaded (5 ml/min) onto a
column (2.4 cm.times.30 cm, 136 ml) packed with Q-Sepharose Fast
Flow (Pharmacia) resin equilibrated with 10 mM TrisCl, 25 mM KCl, 5
mM DTT, 10% glycerol, pH 7.8 (Q Sepharose buffer), and washed with
1 L of the same buffer. EPSPS was eluted with a linear gradient of
Q Sepharose buffer going from 0.025M to 0.40M KCl (total volume of
1.4 L, 5 ml/min). Fractions were collected (15 ml) and assayed for
EPSPS activity by the phosphate release assay. The fractions with
the highest EPSPS activity (fractions 47-60) were pooled and the
protein was precipitated by adding solid ammonium sulfate to 80%
saturation and stirring for 1 hour. The precipitated protein was
collected by centrifugation (20 minutes, 12000 rpm in a GSA Sorvall
rotor), dissolved in Q Sepharose buffer (total volume of 14 ml),
and dialyzed against the same buffer (2.times.1 L, 18 hours).
The resulting dialyzed partially purified EPSPS extract (19 ml) was
loaded (1.7 ml/min) onto a Mono Q 10/10 column (Pharmacia)
equilibrated with Q Sepharose buffer, and washed with the same
buffer (35 ml). EPSPS was eluted with a linear gradient of 0.025M
to 0.35M KCl (total volume of 119 ml, 1.7 ml/min). Fractions were
collected (1.7 ml) and assayed for EPSPS activity by the phosphate
release assayed. The fractions with the highest EPSPS activity
(fractions 30-37) were pooled (6 ml).
The Mono Q pool was made 1M in ammonium sulfate by the addition of
solid ammonium sulfate and 2 ml aliquots were chromatographed on a
Phenyl Superose 5/5 column (Pharmacia) equilibrated with 100 mM
TrisCl, 5 mM DTT, 1M ammonium sulfate, 10% glycerol, pH 7.5 (Phenyl
Superose buffer). Samples were loaded (1 ml/min), washed with
Phenyl Superose buffer (10 ml), and eluted with a linear gradient
of Phenyl Superose buffer going from 1M to 0.00M ammonium sulfate
(total volume of 60 ml, 1 ml/min). Fractions were collected (1 ml)
and assayed for EPSPS activity by the phosphate release assay. The
fractions from each run with the highest EPSPS activity (fractions
.about.36-40) were pooled together (10 ml, 2.5 mg protein). For
N-terminal amino acid sequence determination, a portion of one
fraction (#39 from run 1) was dialyzed against 50 mM NaHCO.sub.3
(2.times.1 L). The resulting pure EPSPS sample (0.9 ml, 77 .mu.g
protein) was found to exhibit a single N-terminal amino acid
sequence of: XH(G)ASSRPATARKSS(G)LX(G)(T)V(R)IPG(D)(K)(M) (SEQ ID
NO:18).
The remaining Phenyl Superose EPSPS pool was dialyzed against 50 mM
TrisCl, 2 mM DTT, 10 mM KCl, 10% glycerol, pH 7.5 (2.times.1 L). An
aliquot (0.55 ml, 0.61 mg protein) was loaded (1 ml/min) onto a
Mono Q 5/5 column (Pharmacia) equilibrated with Q Sepharose buffer,
washed with the same buffer (5 ml), and eluted with a linear
gradient of Q Sepharose buffer going from 0-0.14M KCl in 10
minutes, then holding at 0.14M KCl (1 ml/min). Fractions were
collected (1 ml) and assayed for EPSPS activity by the phosphate
release assay and were subjected to SDS-PAGE (10-15%, Phast System,
Pharmacia, with silver staining) to determine protein purity.
Fractions exhibiting a single band of protein by SDS-PAGE (22-25,
222 .mu.g) were pooled and dialyzed against 100 mM ammonium
bicarbonate, pH 8.1 (2.times.1 L, 9 hours).
Trypsinolysis and peptide sequencing of Agrobacterium sp strain CP4
EPSPS
To the resulting pure Agrobacterium sp. strain CP4 EPSPS (111
.mu.g) was added 3 .mu.g of trypsin (Calbiochem), and the
trypsinolysis reaction was allowed to proceed for 16 hours at
37.degree. C. The tryptic digest was then chromatographed (1
ml/min) on a C18 reverse phase HPLC column (Vydac) as previously
described in Padgette et al., 1988 for E. coli EPSPS. For all
peptide purifications, 0.1% trifluoroacetic acid (TFA, Pierce) was
designated buffer "RP-A" and 0.1% TFA in acetonitrile was buffer
"RP-B". The gradient used for elution of the trypsinized
Agrobacterium sp. CP4 EPSPS was: 0-8 minutes, 0% RP-B; 8-28
minutes, 0-15% RP-B; 28-40 minutes, 15-21% RP-B; 40-68 minutes,
21-49% RP-B; 68-72 minutes, 49-75% RP-B; 72-74 minutes, 75-100%
RP-B. Fractions were collected (1 ml) and, based on the elution
profile at 210 nm, at least 70 distinct peptides were produced from
the trypsinized EPSPS. Fractions 40-70 were evaporated to dryness
and redissolved in 150 .mu.l each of 10% acetonitrile, 0.1%
trifluoroacetic acid.
The fraction 61 peptide was further purified on the C18 column by
the gradient: 0-5 minutes, 0% RP-B; 5-10 minutes, 0-38% RP-B; 10-30
minutes, 38-45% B. Fractions were collected based on the UV signal
at 210 nm. A large peptide peak in fraction 24 eluted at 42% RP-B
and was dried down, resuspended as described above, and
rechromatographed on the C18 column with the gradient: 0-5 minutes,
0% RP-B; 5-12 min, 0-38% RP-B; 12-15 min, 38-39% RP-B; 15-18
minutes, 39% RP-B; 18-20 minutes, 39-41% RP-B; 20-24 minutes, 41%
RP-B; 24-28 minutes, 42% RP-B. The peptide in fraction 25, eluting
at 41% RP-B and designated peptide 61-24-25, was subjected to
N-terminal amino acid sequencing, and the following sequence was
determined: APSM(I)(D)EYPILAV (SEQ ID NO:19) The CP4 EPSPS fraction
53 tryptic peptide was further purified by C18 HPLC by the gradient
0% B (5 minutes), 0-30% B (5-17 minutes), 30-40% B (17-37 minutes).
The peptide in fraction 28, eluting at 34% B and designated peptide
53-28, was subjected to N-terminal amino acid sequencing, and the
following sequence was determined: ITGLLEGEDVINTGK (SEQ ID
NO:20).
In order to verify the CP4 EPSPS cosmid clone, a number of
oligonucleotide probes were designed on the basis of the sequence
of two of the tryptic sequences from the CP4 enzyme (Table III).
The probe identified as MID was very low degeneracy and was used
for initial screening. The probes identified as EDV-C and EDV-T
were based on the same amino acid sequences and differ in one
position (underlined in Table III below) and were used as
confirmatory probes, with a positive to be expected only from one
of these two probes. In the oligonucleotides below, alternate
acceptable nucleotides at a particular position are designated by a
"/" such as A/C/T.
TABLE-US-00005 TABLE III Selected CP4 EPSPS peptide sequences and
DNA probes PEPTIDE 61-24-25 APSM(I)(D)EYPILAV (SEQ ID NO:19) Probe
MID; 17-mer; mixed probe; 24-fold degenerate (SEQ ID NO:21)
ATGATA/C/TGAC/TGAG/ATAC/TCC PEPTIDE 53-28 ITGLLEGEDVINTGK (SEQ ID
NO:20) Probe EDV-C; 17-mer; mixed probe; 48-fold (SEQ ID NO:22)
degenerate GAA/GGAC/TGTA/C/G/TATA/C/TAACAC Probe EDV-T; 17-mer;
mixed probe; 48-fold (SEQ ID NO:23) degenerate
GAA/GGAC/TGTA/C/G/TATA/C/TAATAC
The probes were labeled using gamma-.sup.32P-ATP and polynucleotide
kinase. DNA from fourteen of the cosmids described above was
restricted with EcoRI, transferred to membrane and probed with the
oligonucleotide probes. The conditions used were as follows:
prehybridization was carried out in 6.times. SSC, 10.times.
Denhardt's for 2-18 hour periods at 60.degree. C., and
hybridization was for 48-72 hours in 6.times. SSC, 10.times.
Denhardt's, 100 .mu.g/ml tRNA at 10.degree. C. below the T.sub.d
for the probe. The T.sub.d of the probe was approximated by the
formula 2.degree. C.times.(A+T)+4.degree. C.times.(G+C). The
filters were then washed three times with 6.times. SSC for ten
minutes each at room temperature, dried and autoradiographed. Using
the MID probe, an .about.9.9 kb fragment in the pMON17076 cosmid
gave the only positive signal. This cosmid DNA was then probed with
the EDV-C (SEQ ID NO:22) and EDV-T (SEQ ID NO:23) probes separately
and again this .about.9.9 kb band gave a signal and only with the
EDV-T probe.
The combined data on the glyphosate-tolerant phenotype, the
complementation of the E. coli aroA- phenotype, the expression of a
.about.45 Kd protein, and the hybridization to two probes derived
from the CP4 EPSPS amino acid sequence strongly suggested that the
pMON17076 cosmid contained the EPSPS gene.
Localization and subcloning of the CP4 EPSPS gene
The CP4 EPSPS gene was further localized as follows: a number of
additional Southern analyses were carried out on different
restriction digests of pMON17076 using the MID (SEQ ID NO:21) and
EDV-T (SEQ ID NO:23) probes separately. Based on these analyses and
on subsequent detailed restriction mapping of the pBlueScript
(Stratagene) subclones of the .about.9.9 kb fragment from
pMON17076, a 3.8 kb EcoRI-SalI fragment was identified to which
both probes hybridized. This analysis also showed that MID (SEQ ID
NO:21) and EDV-T (SEQ ID NO:23) probes hybridized to different
sides of BamHI, ClaI, and SacII sites. This 3.8 kb fragment was
cloned in both orientations in pBlueScript to form pMON17081 and
pMON17082. The phenotypes imparted to E. coli by these clones were
then determined. Glyphosate tolerance was determined following
transformation into E. coli MM294 containing pGP1-2 (pBlueScript
also contains a T7 promoter) on M9 agar media containing glyphosate
at 3 mM. Both pMON17081 and pMON17082 showed glyphosate-tolerant
colonies at three days at 30.degree. C. at about half the size of
the controls on the same media lacking glyphosate. This result
suggested that the 3.8 kb fragment contained an intact EPSPS gene.
The apparent lack of orientation-dependence of this phenotype could
be explained by the presence of the T7 promoter at one side of the
cloning sites and the lac promoter at the other. The aroA phenotype
was determined in transformants of E. coli GB100 on M9 agar media
lacking aromatic supplements. In this experiment, carried out with
and without the Plac inducer IPTG, pMON17082 showed much greater
growth than pMON17081, suggesting that the EPSPS gene was expressed
from the SalI site towards the EcoRI site.
Nucleotide sequencing was begun from a number of restriction site
ends, including the BamHI site discussed above. Sequences encoding
protein sequences that closely matched the N-terminus protein
sequence and that for the tryptic fragment 53-28 (SEQ ID NO:20)
(the basis of the EDV-T probe) (SEQ ID NO:23) were localized to the
SalI side of this BamHI site. These data provided conclusive
evidence for the cloning of the CP4 EPSPS gene and for the
direction of transcription of this gene. These data coupled with
the restriction mapping data also indicated that the complete gene
was located on an .about.2.3 kb XhoI fragment and this fragment was
subcloned into pBlueScript. The nucleotide sequence of almost 2 kb
of this fragment was determined by a combination of sequencing from
cloned restriction fragments and by the use of specific primers to
extend the sequence. The nucleotide sequence of the CP4 EPSPS gene
and flanking regions is shown in FIG. 3 (SEQ ID NO:2). The sequence
corresponding to peptide 61-24-25 (SEQ ID NO:19) was also located.
The sequence was determined using both the SEQUENASE.TM. kit from
IBI (International Biotechnologies Inc.) and the T7
sequencing/Deaza Kit from Pharmacia.
That the cloned gene encoded the EPSPS activity purified from the
Agrobacterium sp. strain CP4 was verified in the following manner:
By a series of site directed mutageneses, BglII and NcoI sites were
placed at the N-terminus with the fMet contained within the NcoI
recognition sequence, the first internal NcoI site was removed (the
second internal NcoI site was removed later), and a SacI site was
placed after the stop codons. At a later stage the internal NotI
site was also removed by site-directed mutagenesis. The following
list includes the primers for the site-directed mutagenesis
(addition or removal of restriction sites) of the CP4 EPSPS gene.
Mutagenesis was carried out by the procedures of Kunkel et al.
(1987), essentially as described in Sambrook et al. (1989).
TABLE-US-00006 PRIMER BgNc (addition of BgIII and NcoI sites to
N-terminus) CGTGGATAGATCTAGGAAGACAACCATGGCTCACGGTC (SEQ ID NO:24)
PRIMER Sph2 (addition of SphI site to N-terminus)
GGATAGATTAAGGAAGACGCGCATGCTTCACGGTGCAAGCAGCC (SEQ ID NO:25) PRIMER
S1 (addition of SacI site immediately after stop codons)
GGCTGCCTGATGAGCTCCACAATCGCCATCGATGG (SEQ ID NO:26) PRIMER N1
(removal of internal NotI recognition site)
CGTCGCTCGTCGTGCGTGGCCGCCCTGACGGC (SEQ ID NO:27) PRIMER NcoI
(removal of first internal NcoI recognition site)
CGGGCAAGGCCATGCAGGCTATGGGCGCC (SEQ ID NO:28) PRIMER Nco2 (removal
of second internal NcoI recognition site)
CGGGCTGCCGCCTGACTATGGGCCTCGTCGG (SEQ ID NO:29)
This CP4 EPSPS gene was then cloned as a NcoI-BamHI N-terminal
fragment plus a BamHI-SacI C-terminal fragment into a PrecA-gene10L
expression vector similar to those described (Wong et al., 1988;
Olins et al., 1988) to form pMON17101. The K.sub.m for PEP and the
K.sub.i for glyphosate were determined for the EPSPS activity in
crude lysates of pMON17101/GB100 transformants following induction
with nalidixic acid (Wong et al., 1988) and found to be the same as
that determined for the purified and crude enzyme preparations from
Agrobacterium sp. strain CP4.
Characterization of the EPSPS gene from Achromobacter sp. strain
LBAA and from Pseudomonas sp. strain PG2982
A cosmid bank of partially HindIII-restricted LBAA DNA was
constructed in E. coli MM294 in the vector pHC79 (Hohn and Collins,
1980). This bank was probed with a full length CP4 EPSPS gene probe
by colony hybridization and positive clones were identified at a
rate of .about.1 per 400 cosmids. The LBAA EPSPS gene was further
localized in these cosmids by Southern analysis. The gene was
located on an .about.2.8 kb XhoI fragment and by a series of
sequencing steps, both from restriction fragment ends and by using
the oligonucleotide primers from the sequencing of the CP4 EPSPS
gene, the nucleotide sequence of the LBAA EPSPS gene was completed
and is presented in FIG. 4 (SEQ ID NO:4).
The EPSPS gene from PG2982 was also cloned. The EPSPS protein was
purified, essentially as described for the CP4 enzyme, with the
following differences: Following the Sepharose CL-4B column, the
fractions with the highest EPSPS activity were pooled and the
protein precipitated by adding solid ammonium sulfate to 85%
saturation and stirring for 1 hour. The precipitated protein was
collected by centrifugation, resuspended in Q Sepharose buffer and
following dialysis against the same buffer was loaded onto the
column (as for the CP4 enzyme). After purification on the Q
Sepharose column, .about.40 mg of protein in 100 mM Tris pH 7.8,
10% glycerol, 1 mM EDTA, 1 mM DTT, and 1M ammonium sulfate, was
loaded onto a Phenyl Superose (Pharmacia) column. The column was
eluted at 1.0 ml/minutes with a 40 ml gradient from 1.0M to 0.00M
ammonium sulfate in the above buffer.
Approximately 1.0 mg of protein from the active fractions of the
Phenyl Superose 10/10 column was loaded onto a Pharmacia Mono P
5/10 Chromatofocusing column with a flow rate of 0.75 ml/minutes.
The starting buffer was 25 mM bis-Tris at pH 6.3, and the column
was eluted with 39 ml of Polybuffer 74, pH 4.0. Approximately 50
.mu.g of the peak fraction from the Chromatofocusing column was
dialyzed into 25 mM ammonium bicarbonate. This sample was then used
to determine the N-terminal amino acid sequence.
The N-terminal sequence obtained was: XHSASPKPATARRSE (where X=an
unidentified residue) (SEQ ID NO:30)
A number of degenerate oligonucleotide probes were designed based
on this sequence and used to probe a library of PG2982
partial-HindIII DNA in the cosmid pHC79 (Hohn and Collins, 1980) by
colony hybridization under nonstringent conditions. Final washing
conditions were 15 minutes with 1.times. SSC, 0.1% SDS at
55.degree. C. One probe with the sequence GCGGTBGCSGGYTTSGG (where
B=C, G, or T; S=C or G, and Y=C or T) (SEQ ID NO:31) identified a
set of cosmid clones.
The cosmid set identified in this way was made up of cosmids of
diverse HindIII fragments. However, when this set was probed with
the CP4 EPSPS gene probe, a cosmid containing the PG2982 EPSPS gene
was identified (designated as cosmid 9C1 originally and later as
pMON20107). By a series of restriction mappings and Southern
analysis this gene was localized to a .about.2.8 kb XhoI fragment
and the nucleotide sequence of this gene was determined. This DNA
sequence (SEQ ID NO:6) is shown in FIG. 5. There are no nucleotide
differences between the EPSPS gene sequences from LBAA (SEQ ID
NO:4) and PG2982 (SEQ ID NO:6). The kinetic parameters of the two
enzymes are within the range of experimental error.
A gene from PG2982 that imparts glyphosate tolerance in E. coli has
been sequenced (Fitzgibbon, 1988; Fitzgibbon and Brayruer, 1990).
The sequence of the PG2982 EPSPS Class II gene shows no homology to
the previously reported sequence suggesting that the
glyphosate-tolerant phenotype of the previous work is not related
to EPSPS.
Characterization of the EPSPS from Bacillus subtilis
Bacillus subtilis 1A2 (prototroph) was obtained from the Bacillus
Genetic Stock Center at Ohio State University. Standard EPSPS assay
reactions contained crude bacterial extract with, 1 mM
phosphoenolpyruvate (PEP), 2 mM shikimate-3-phosphate (S3P), 0.1 mM
ammonium molybdate, 5 mM potassium fluoride, and 50 mM HEPES, pH
7.0 at 25.degree. C. One unit (U) of EPSPS activity is defined as
one .mu.mol EPSP formed per minute under these conditions. For
kinetic determinations, reactions contained crude bacterial, 2 mM
S3P, varying concentrations of PEP, and 50 mM HEPES, pH 7.0 at
25.degree. C. The EPSPS specific activity was found to be 0.003
U/mg. When the assays were performed in the presence of 1 mM
glyphosphate, 100% of the EPSPS activity was retained. The
appK.sub.m(PEP) of the B. subtilis EPSPS was determined by
measuring the reaction velocity at varying concentrations of PEP.
The results were analyzed graphically by the hyperbolic,
Lineweaver-Burk and Eadie-Hofstee plots, which yielded
appK.sub.m(PEP) values of 15.3 .mu.M, 10.8 .mu.M and 12.2 .mu.M,
respectively. These three data treatments are in good agreement,
and yield an average value for appK.sub.m(PEP) of 13 .mu.M. The
appK.sub.i(glyphosate) was estimated by determining the reaction
rates of B. subtilis 1A2 EPSPS in the presence of several
concentrations of glyphosphate, at a PEP concentration of 2 .mu.M.
These results were compared to the calculated V.sub.max of the
EPSPS, and making the assumption that glyphosate is a competitive
inhibitor versus PEP for B. subtilis EPSPS, as it is for all other
characterized EPSPSs, an appK.sub.i(glyphosate) was determined
graphically. The appK.sub.i(glyphosate) was found to be 0.44
mM.
The EPSPS expressed from the B. subtilis aroE gene described by
Henner et al. (1986) was also studied. The source of the B.
subtilis aroE (EPSPS) gene was the E. coli plasmid-bearing strain
ECE13 (original code=MM294[p trp100]; Henner, et al., 1984;
obtained from the Bacillus Genetic Stock Center at Ohio State
University; the culture genotype is [pBR322 trp100] Ap [in MM294]
[pBR322::6 kb insert with trpFBA-hisH]). Two strategies were taken
to express the enzyme in E. coli GB100 (aroA-): 1) the gene was
isolated by PCR and cloned into an overexpression vector, and 2)
the gene was subcloned into an overexpression vector. For the PCR
cloning of the B. subtilis aroE from ECE13, two oligonucleotides
were synthesized which incorporated two restriction enzyme
recognition sites (NdeI and EcoRI) to the sequences of the
following oligonucleotides:
TABLE-US-00007 (SEQ ID NO:45) GGAACATATGAAACGAGATAAGGTGCAG (SEQ ID
NO:46) GGAATTCAAACTTCAGGATCTTGAGATAGAAAATG
The other approach to the isolation of the B. subtilis aroE gene,
subcloning from ECE13 into pUC118, was performed as follows: (i)
Cut ECE13 and pUC with XmaI and SphI. (ii) Isolate 1700bp aroE
fragment and 2600bp pUC118 vector fragment. (iii) Ligate fragments
and transform into GB100. The subclone was designated pMON21133 and
the PCR-derived clone was named pMON21132. Clones from both
approaches were first confirmed for complementation of the aroA
mutation in E. coli GB100. The cultures exhibited EPSPS specific
activities of 0.044 U/mg and 0.71 U/mg for the subclone (pMON21133)
and PCR-derived clone (pMON21132) enzymes, respectively. These
specific activities reflect the expected types of expression levels
of the two vectors. The B. subtilis EPSPS was found to be 88% and
100% resistant to inhibition by 1 mM glyphosate under these
conditions for the subcloned (pMON21133) and PCR-derived
(pMON21132) enzymes, respectively. The appK.sub.m (PEP) and the
appK.sub.i(glyphosate) of the subcloned B. subtilis EPSPS
(pMON21133) were determined as described above. The data were
analyzed graphically by the same methods used for the 1A2 isolate,
and the results obtained were comparable to those reported above
for B. subtilis 1A2 culture. Characterization of the EPSPS gene
from Staphylococcus aureus
The kinetic properties of the S. aureus EPSPS expressed in E. coli
were determined, including the specific activity, the
appK.sub.m(PEP), and the appK.sub.i(glyphosate). The S. aureus
EPSPS gene has been previously described (O'Connell et al.,
1993)
The strategy taken for the cloning of the S. aureus EPSPS was
polymerase chain reaction (PCR), utilizing the known nucleotide
sequence of the S. aureus aroA gene encoding EPSPS (O'Cormell et
al., 1993). The S. aureus culture (ATCC 35556) was fermeated in an
M2 facility in three 250 mL shake flasks containing 55 mL of TYE
(tryptone 5 g/L, yeast extract 3 g/L, pH 6.8). The three flasks
were inoculated with 1.5 mL each of a suspension made from freeze
dried ATCC 35556 S. aureus cells in 90 mL of PBS
(phosphate-buffered saline) buffer. Flasks were incubated at
30.degree. C. for 5 days while shaking at 250 rpm. The resulting
cells were lysed (boiled in TE [tris/EDTA] buffer for 8 minutes)
and the DNA utilized for PCR reactions. The EPSPS gene was
amplified using PCR and engineered into an E. coli expression
vector as follows: (i) two oligonucleotides were synthesized which
incorporated two restriction enzyme recognition sites (NcoI and
SacI) to the sequences of the oligonucleotides:
TABLE-US-00008 (SEQ ID NO:47) GGGGCCATGGTAAATGAACAAATCATTG (SEQ ID
NO:48) GGGGGAGCTCATTATCCCTCATTTTGTAAAAGC
(ii) The purified, PCR-amplified aroA gene from S. aureus was
digested using NcoI and SacI enzymes. (iii) DNA of pMON 5723, which
contains a pRecA bacterial promoter and Gene10 leader sequence
(Olins et al., 1988) was digested NcoI and SacI and the 3.5 kb
digestion product was purified. (iv) The S. aureus PCR product and
the NcoI/SacI pMON 5723 fragment were ligated and transformed into
E. coli JM101 competent cells. (v) Two spectinomycin-resistant E.
coli JM101 clones from above (SA#2 and SA#3) were purified and
transformed formed into a competent aroA- E. coli strain, GB100
For complementation experiments SAGB#2 and SAGB#3 were utilized,
which correspond to SA#2 and SA#3, respectively, transformed into
E. coli GB100. In addition, E. coli GB100 (negative control) and
pMON 9563 (wt petunia EPSPS, positive control) were tested for AroA
complementation. The organisms were grown in minimal media plus and
minus aromatic amino acids. Later analyses showed that the SA#2 and
SA#3 clones were identical, and they were assigned the plasmid
identifier pMON21139.
SAGB#2 in E. coli GB100 (pMON21139) was also grown in M9 minimal
media and induced with nalidixic acid. A negative control, E. coli
GB100, was grown under identical conditions except the media was
supplemented with aromatic amino acids. The cells were harvested,
washed with 0.9% NaCl, and frozen at -80.degree. C., for extraction
and EPSPS analysis.
The frozen pMON21139 E. coli GB100 cell pellet from above was
extracted and assayed for EPSPS activity as previously described.
EPSPS assays were performed using 1 mM phosphoenolpyruvate (PEP), 2
mM shikimate-3-phosphate (S3P), 0. 1 mM ammonium molybdate, 5 mM
potassium fluoride, pH 7.0, 25.degree. C. The total assay volume
was 50.mu.L, which contained 10 .mu.L of the undiluted desalted
extract.
The results indicate that the two clones contain a functional
aroA/EPSPS gene since they were able to grow in minimal media which
contained no aromatic amino acids. As expected, the GB100 culture
did not grow on minimal medium without aromatic amino acids (since
no functional EPSPS is present), and the pMON9563 did confer growth
in minimal media. These results demonstrated the successful cloning
of a functional EPSPS gene from S. aureus. Both clones tested were
identical, and the E. coli expression vector was designated
pMON21139.
The plasmid pMON21139 in E. coli GB100 was grown in M9 minimal
media and was induced with nalidixic acid to induce EPSPS
expression driven from the RecA promoter. A desalted extract of the
intracellular protein was analyzed for EPSPS activity, yielding an
EPSPS specific activity of 0.005 .mu.mol/min mg. Under these assay
conditions, the S. aureus EPSPS activity was completely resistant
to inhibition by 1 mM glyphosate. Previous analysis had shown that
E. coli GB100 is devoid of EPSPS activity.
The appK.sub.m(PEP) of the S. aureus EPSPS was determined by
measuring the reaction velocity of the enzyme (in crude bacterial
extracts) at varying concentrations of PEP. The results were
analyzed graphically using several standard kinetic plotting
methods. Data analysis using the hyperbolic, Lineweaver-Burke, and
Eadie-Hofstee methods yielded appK.sub.m(PEP) constants of 7.5,
4.8, and 4.0 .mu.M, respectively. These three data treatments are
in good agreement, and yield an average value for appK.sub.m(PEP)
of 5 .mu.M.
Further information of the glyphosate tolerance of S. aureus EPSPS
was obtained by determining the reaction rates of the enzyme in the
presence of several concentrations of glyphosate, at a PEP
concentration of 2 .mu.M. These results were compared to the
calculated maximal velocity of the EPSPS, and making the assumption
that glyphosate is a competitive inhibitor versus PEP for S. aureus
EPSPS, as it is for all other characterized EPSPSs, an
appK.sub.i(glyphosate) was determined graphically. The
appK.sub.i(glyphosate) for S. aureus EPSPS estimated using this
method was found to be 0.20 mM.
The EPSPS from S. aureus was found to be glyphosate-tolerant, with
an appK.sub.i(glyphosate) of approximately 0.2 mM. In addition, the
appK.sub.m(PEP) for the enzyme is approximately 5 .mu.M, yielding a
appK.sub.i(glyphosate)/appK.sub.m(PEP) of 40.
Alternative Isolation Protocols for Other Class II EPSPS Structural
Genes
A number of Class II genes have been isolated and described here.
While the cloning of the gene from CP4 was difficult due to the low
degree of similarity between the Class I and Class II enzymes and
genes, the identification of the other genes were greatly
facilitated by the use of this first gene as a probe. In the
cloning of the LBAA EPSPS gene, the CP4 gene probe allowed the
rapid identification of cosmid clones and the localization of the
intact gene to a small restriction fragment and some of the CP4
sequencing primers were also used to sequence the LBAA (and PG2982)
EPSPS gene(s). The CP4 gene probe was also used to confirm the
PG2982 gene clone. The high degree of similarity of the Class II
EPSPS genes may be used to identify and clone additional genes in
much the same way that Class I EPSPS gene probes have been used to
clone other Class I genes. An example of the latter was in the
cloning of the A. thaliana EPSPS gene using the P. hybrida gene as
a probe (Klee et al., 1987).
Glyphosate-tolerant EPSPS activity has been reported previously for
EPSP synthases from a number of sources. These enzymes have not
been characterized to any extent in most cases. The use of Class I
and Class II EPSPS gene probes or antibody probes provide a rapid
means of initially screening for the nature of the EPSPS and
provide tools for the rapid cloning and characterization of the
genes for such enzymes.
Two of the three genes described were isolated from bacteria that
were isolated from a glyphosate treatment facility (Strains CP4 and
LBAA). The third (PG2982) was from a bacterium that had been
isolated from a culture collection strain. This latter isolation
confirms that exposure to glyphosate is not a prerequisite for the
isolation of high glyphosate-tolerant EPSPS enzymes and that the
screening of collections of bacteria could yield additional
isolates. It is possible to enrich for glyphosate degrading or
glyphosate resistant microbial populations (Quinn et al., 1988;
Talbot et al., 1984) in cases where it was felt that enrichment for
such microorganisms would enhance the isolation frequency of Class
II EPSPS microorganisms. Additional bacteria containing class II
EPSPS gene have also been identified. A bacterium called C 12,
isolated from the same treatment column beads as CP4 (see above)
but in a medium in which glyphosate was supplied as both the carbon
and phosphorus source, was shown by Southern analysis to hybridize
with a probe consisting of the CP4 EPSPS coding sequence. This
result, in conjunction with that for strain LBAA, suggests that
this enrichment method facilitates the identification of Class II
EPSPS isolates. New bacterial isolates containing Class II EPSPS
genes have also been identified from environments other than
glyphosate waste treatment facilities. An inoculum was prepared by
extracting soil (from a recently harvested soybean field in
Jerseyville, Ill.) and a population of bacteria selected by growth
at 28.degree. C. in Dworkin-Foster medium containing glyphosate at
10 mM as a source of carbon (and with cycloheximide at 100 .mu.g/ml
to prevent the growth of fungi). Upon plating on L-agar media, five
colony types were identified. Chromosomal DNA was prepared from 2ml
L-broth cultures of these isolates and the presence of a Class II
EPSPS gene was probed using a the CP4 EPSPS coding sequence probe
by Southern analysis under stringent hybridization and washing
conditions. One of the soil isolates, S2, was positive by this
screen.
Class II EPSPS enzymes are identifiable by an elevated Ki for
glyphosate and thus the genes for these will impart a glyphosate
tolerance phenotype in heterologous hosts. Expression of the gene
from recombinant plasmids or phage may be achieved through the use
of a variety of expression promoters and include the T7 promoter
and polymerase. The T7 promoter and polymerase system has been
shown to work in a wide range of bacterial (and mammalian) hosts
and offers the advantage of expression of many proteins that may be
present on large cloned fragments. Tolerance to growth on
glyphosate may be shown on minimal growth media. In some cases,
other genes or conditions that may give glyphosate tolerance have
been observed, including over expression of beta-lactamase, the
igrA gene (Fitzgibbon and Braymer, 1990), or the gene for
glyphosate oxidoreductase (PCT Pub. No. WO92/00377). These are
easily distinguished from Class II EPSPS by the absence of EPSPS
enzyme activity.
The EPSPS protein is expressed from the aroA gene (also called aroE
in some genera, for example, in Bacillus) and mutants in this gene
have been produced in a wide variety of bacteria. Determining the
identity of the donor organism (bacterium) aids in the isolation of
Class II EPSPS gene--such identification may be accomplished by
standard micro-biological methods and could include Gram stain
reaction, growth, color of culture, and gas or acid production on
different substrates, gas chromatography analysis of methylesters
of the fatty acids in the membranes of the microorganism, and
determination of the GC % of the genome. The identity of the donor
provides information that may be used to more easily isolate the
EPSPS gene. An AroA- host more closely related to the donor
organism could be employed to clone the EPSPS gene by
complementation but this is not essential since complementation of
the E. coli AroA mutant by the CP4 EPSPS gene was observed. In
addition, the information on the GC content the genome may be used
in choosing nucleotide probes--donor sources with high GC % would
preferably use the CP4 EPSPS gene or sequences as probes and those
donors with low GC would preferably employ those from Bacillus
subtilis, for example. Relationships between different EPSPS
genes
The deduced amino acid sequences of a number of Class I and the
Class II EPSPS enzymes were compared using the Bestfit computer
program provided in the UWGCG package (Devereux et al. 1984). The
degree of similarity and identity as determined using this program
is reported. The degree of similarity/identity determined within
Class I and Class II protein sequences is remarkably high, for
instance, comparing E. coli with S. typhimurium
(similarity/identity=93%/88%) and even comparing E. coli with a
plant EPSPS (Petunia hybrida; 72%/55%). These data are shown in
Table IV. The comparison of sequences between Class I and Class II,
however, shows a much lower degree of relatedness between the
Classes (similarity/identity=50-53%/23-30%). The display of the
Bestfit analysis for the E. coli (SEQ ID NO:8) and CP4 (SEQ ID
NO:3) sequences shows the positions of the conserved residues and
is presented in FIG. 6. Previous analyses of EPSPS sequences had
noted the high degree of conservation of sequences of the enzymes
and the almost invariance of sequences in two regions--the "20-35"
and "95-107" regions (Gasser et al., 1988; numbered according to
the Petunia EPSPS sequence)--and these regions are less conserved
in the case of CP4 and LBAA when compared to Class I bacterial and
plant EPSPS sequences (see FIG. 6 for a comparison of the E. coli
and CP4 EPSPS sequences with the E. coli sequence appearing as the
top sequence in the Figure). The corresponding sequences in the CP4
Class II EPSPS are:
TABLE-US-00009 PGDKSTSHRSFMGGL (SEQ ID NO:32) and LDFGNAATGCRLT.
(SEQ ID NO:33)
These comparisons show that the overall relatedness of Class I and
Class II is EPSPS proteins is low and that sequences in putative
conserved regions have also diverged considerably.
In the CP4 EPSPS an alanine residue is present at the "glycine101"
position. The replacement of the conserved glycine (from the
"95-107" region) by an alanine results in an elevated K.sub.i for
glyphosate and in an elevation in the K.sub.m for PEP in Class I
EPSPS. In the case of the CP4 EPSPS, which contains an alanine at
this position, the K.sub.m for PEP is in the low range, indicating
that the Class II enzymes differ in many aspects from the EPSPS
enzymes heretofore characterized.
Within the Class II isolates, the degree of similarity/identity is
as high as that noted for that within Class I (Table IVA). FIG. 7
displays the Bestfit computer program alignment of the CP4 (SEQ ID
NO:3) and LBAA (SEQ ID NO:5) EPSPS deduced amino acid sequences
with the CP4 sequence appearing as the top sequence in the Figure.
The symbols used in FIGS. 6 and 7 are the standard symbols used in
the Bestfit computer program to designate degrees of similarity and
identity.
TABLE-US-00010 TABLE IVA.sup.1.2 Comparison of relatedness of EPSPS
protein sequences Comparison between Class I and Class II EPSPS
protein sequences similarity identity S. cerevisiae vs. CP4 54 30
A. nidulans vs. CP4 50 25 B. napus vs. CP4 47 22 A. thaliana vs.
CP4 48 22 N. tabacum vs. CP4 50 24 L. esculentum vs. CP4 50 24 P.
hybrida vs. CP4 50 23 Z. mays vs. CP4 48 24 S. gallinarum vs. CP4
51 25 S. typhimurium vs. CP4 51 25 S. typhi vs. CP4 51 25 K.
pneumoniae vs. CP4 56 28 Y. enterocolitica vs. CP4 53 25 H.
influenzae vs. CP4 53 27 P. multocida vs. CP4 55 30 A. salmonicida
vs. CP4 53 23 B. pertussis vs. CP4 53 27 E. coli vs. CP4 52 26 E.
coli vs. LBAA 52 26 E. coli vs. B. subtilis 55 29 E. coli vs. D.
nodosus 55 32 E. coli vs. S. aureus 55 29 E. coli vs. Synechocystis
sp. PCC6803 53 30 Comparison between Class I EPSPS protein
sequences similarity identity E. coli vs. S. typhimurium 93 88 P.
hybrids vs. E. coli 72 55 Comparison between Class II EPSPS protein
sequences similarity identity D. nodosus vs. CP4 62 43 LBAA vs. CP4
90 83 PG2892 vs. CP4 90 83 S. aureus vs. CP4 58 34 B. subtills vs.
CP4 59 41 Synechocystis sp. PCC6803 vs. CP4 62 45 .sup.1The EPSPS
sequences compared here were obtained from the following
references: E. coli, Rogers et al., 1983; S. typhimurium, Stalker
et al., 1985; Petunia hybrids, Shah et al., 1986; B. pertussis,
Maskell et al., 1988; S. cerevisiae, Duncan et al., 1987,
Synechocystis sp. PCC6803, Dalla Chiesa et al., 1994 and D.
nodosus, Alm et al., 1994. .sup.2"GAP" Program, Genetics Computer
Group, (1991), Program Manual for the GCG Package, Version 7, April
1991, 575 Science Drive, Madison, Wisconsin, USA 53711
The relative locations of the major conserved sequences among Class
II EPSP sythase which distinguishes this group from the Class I
EPSP synthases is listed below in Table IVB.
TABLE-US-00011 TABLE IVB Location of Conserved Sequences in Class
II EPSP Synthases Source Seq. 1.sup.1 Seq. 2.sup.2 Seq. 3.sup.3
Seq. 4.sup.4 CP4 start 200 26 173 271 end 204 29 177 274 LBAA start
200 26 173 271 end 204 29 177 274 PG2982 start 200 26 173 273 end
204 29 177 276 B. subtilis start 190 17 164 257 end 194 20 168 260
S. aureus start 193 21 166 261 end 197 24 170 264 Synechocystis sp.
PCC6803 start 210 34 183 278 end 214 38 187 281 D. nodosus start
195 22 168 261 end 199 25 172 264 min. start 190 17 164 257 max.
end 214 38 187 281 .sup.1-R-X.sub.1-H-X.sub.2-E-(SEQ ID NO:37)
.sup.2-G-D-K-X.sub.3-(SEQ ID NO:38) .sup.3-S-A-Q-X.sub.4-K-(SEQ ID
NO:39) .sup.4-N-X.sub.5-T-E-(SEQ ID NO:40)
The domains of EPSP synthase sequence identified in this
application were determined to be those important for maintenance
of glyphosate resistance and productive binding of PEP. The
information used in indentifying these domains included sequence
alignments of numerous glyphosate-sensitive EPSPS molecules and the
three-dimensional x-ray structures of E. coli EPSPS (Stallings, et
al. 1991) and CP4 EPSPS. The structures are representative of a
glyphosate-sensitive (i.e., Class I) enzyme, and a
naturally-occuring glyphosate-tolerant (i.e., Class II) enzyme of
the present invention. These exemplary molecules were superposed
three-dimensionally and the results displayed on a computer
graphics terminal. Inspection of the display allowed for
structure-based fine-tuning of the sequence alignments of
glyphosate-sensitive and glyphosate-resistant EPSPS molecules. The
new sequence alignments were examined to determine differences
between Class I and Class II EPSPS enzymes. Seven regions were
identified and these regions were located in the x-ray structure of
CP4 EPSPS which also contained a bound analog of the intermediate
which forms catalytically between PEP and S3P.
The structure of the CP4 EPSPS with the bound intermediate analog
was displayed on a computer graphics terminal and the seven
sequence segments were examined. Important residues for glyphosate
binding were identified as well as those residues which stabilized
the conformations of those important residues; adjoining residues
were considered necessary for maintenance of correct
three-dimensional structural motifs in the context of glyphosate-
sensitive EPSPS molecules. Three of the seven domains were
determined not to be important for glyphosate tolerance and
maintenance of productive PEP binding. The following four primary
domains were determined to be characteristic of Class II EPSPS
enzymes of the present invention: -R-XrH-X.sub.2-E(SEQ ID NO:37),
in which X.sub.1 is an uncharged polar or acidic amino acid,
X.sub.2 is serine or threonine, The Arginine (R) reside at position
1 is important because the positive charge of its guanidium group
destabilizes the binding of glyphosate. The Histidine (H) residue
at position 3 stabilizes the Arginine (R) residue at position 4 of
SEQ ID NO:40. The Glutamic Acid (E) residue at position 5
stabilizes the Lysine (K) residue at position 5 of SEQ ID NO:39.
-G-D-K-X.sub.3(SEQ ID NO:38), in which X.sub.3 is serine or
threonine, The Aspartic acid (D) residue at position 2 stabilizes
the Arginine (R) residue at position 4 of SEQ ID NO:40. The Lysine
(K) residue at position 3 is important because for productive PEP
binding. -S-A-Q-X.sub.4-K(SEQ ID NO:39), in which X.sub.4 is any
amino acid, The Alanine (A) residue at position 2 stabilizes the
Arginine (R) residue at position 1 of SEQ ID NO:37. The Serine (S)
residue at position 1 and the Glutamine (Q) residue at position 3
are important for productive S3P binding. -N-X.sub.5-T-R(SEQ ID
NO:40), in which X.sub.5 is any amino acid, The Asparagine (N)
residue at position 1 and the Threonine (T) residue at position 3
stabilize residue X.sub.1 at position 2 of SEQ ID NO:37. The
Arginine (R) residue at position 4 is important because the
positive charge of its guanidium group destabilizes the binding of
glyphosate.
Since the above sequences are only representative of the Class II
EPSPSs which would be included within the generic structure of this
group of EPSP synthases, the above sequences may be found within a
subject EPSP synthase molecule within slightly more expanded
regions. It is believed that the above-described conserved
sequences would likely be found in the following regions of the
mature EPSP synthases molecule: -R-X.sub.1-H-X.sub.2-E-(SEQ ID
NO:37) located between amino acids 175 and 230 of the mature EPSP
synthase sequence; -G-D-K-X.sub.3-(SEQ ID NO:38) located between
amino acids 5 and 55 of the mature EPSP synthase sequence;
-S-A-Q-X.sub.4-K-(SEQ ID NO:39) located between amino acids 150 and
200 of the mature EPSP synthase sequence; and -N-X.sub.5-T-R-(SEQ
ID NO:40) located between amino acids 245 and 295 of the mature
EPSPS synthase sequence.
One difference that may be noted between the deduced amino acid
sequences of the CP4 and LBAA EPSPS proteins is at position 100
where an Alanine is found in the case of the CP4 enzyme and a
Glycine is found in the case of the LBAA enzyme. In the Class I
EPSPS enzymes a Glycine is usually found in the equivalent
position, i.e Glycine96 in E. coli and K. pneumoniae and Glycine101
in Petunia. In the case of these three enzymes it has been reported
that converting that Glycine to an Alanine results in an elevation
of the appKi for glyphosate and a concomitant elevation in the
appKm for PEP (Kishore et al., 1986; Kishore and Shah, 1988; Sost
and Amrhein, 1990), which, as discussed above, makes the enzyme
less efficient especially under conditions of lower PEP
concentrations. The Glycine100 of the LBAA EPSPS was converted to
an Alanine and both the appKm for PEP and the appKi for glyphosate
were determined for the variant. The Glycine100Alanine change was
introduced by mutagenesis using the following primer:
TABLE-US-00012 CGGCAATGCCGCCACCGGCGCGCGCC (SEQ ID NO:34)
and both the wild type and variant genes were expressed in E. coli
in a RecA promoter expression vector (pMON17201 and pMON17264,
respectively) and the appKm's and appKi's determined in crude
lysates. The data indicate that the appKi(glyphosate) for the G100A
variant is elevated about 16-fold (Table V). This result is in
agreement with the observation of the importance of this G-A change
in raising the appKi(glyphosate) in the Class I EPSPS enzymes.
However, in contrast to the results in the Class I G-A variants,
the appKm(PEP) in the Class II (LBAA) G-A variant is unaltered.
This provides yet another distinction between the Class II and
Class I EPSPS enzymes.
TABLE-US-00013 TABLE V sppKi sppKm(PEP) (glyphosate) Lysato
prepared from: E. coli/pMON17201 (wild 5.3 .mu.M 28 .mu.M* type) E.
coli/pMON17264 5.5 .mu.M 459 .mu.M# (G100A variant) @range of PEP;
2-40 .mu.M *range of glyphosate; 0-310 .mu.M; #range of glyphosate;
0-5000 .mu.M.
The LBAA G100A variant, by virtue of its superior kinetic
properties, should be capable of imparting improved in planta
glyphosate tolerance. Modification and Resynthesis of the
Agrobacterium sp. strain CP4 EPSPS Gene Sequence
The EPSPS gene from Agrobacterium sp. strain CP4 contains sequences
that could be inimical to high expression of the gene in plants.
These sequences include potential polyadenylation sites that are
often and A+T rich, a higher G+C % than that frequently found in
plant genes (63% versus .about.50%), concentrated stretches of G
and C residues, and codons that are not used frequently in plant
genes. The high G+C % in the CP4 EPSPS gene has a number of
potential consequences including the following: a higher usage of G
or C than that found in plant genes in the third position in
codons, and the potential to form strong hair-pin structures that
may affect expression or stability of the RNA. The reduction in the
G+C content of the CP4 EPSPS gene, the disruption of stretches of
G's and C's, the elimination of potential polyadenylation
sequences, and improvements in the codon usage to that used more
frequently in plant genes, could result in higher expression of the
CP4 EPSPS gene in plants.
A synthetic CP4 gene was designed to change as completely as
possible those inimical sequences discussed above. In summary, the
gene sequence was redesigned to eliminate as much as possible the
following sequences or sequence features (while avoiding the
introduction of unnecessary restriction sites): stretches of G's
and C's of 5 or greater; and A+T rich regions (predominantly) that
could function as polyadenylation sites or potential RNA
destabilization region. The sequence of this gene is shown in FIG.
8 (SEQ ID NO:9). This coding sequence was expressed in E. coli from
the RecA promoter and assayed for EPSPS activity and compared with
that from the native CP4 EPSPS gene. The apparent Km for PEP for
the native and synthetic genes was 11.8 and 12.7, respectively,
indicating that the enzyme expressed from the synthetic gene was
unaltered. The N-terminus of the coding sequence was mutagenized to
place an SphI site at the ATG to permit the construction of the
CTP2-CP4 synthetic fusion for chloroplast import. The following
primer was used to accomplish this mutagenesis:
TABLE-US-00014 (SEQ ID NO:35)
GGACCGCTGCTTGCACCGTGAAGCATGCTTAAGCTTGGCGTAATCATGG.
Expression of Chloroplast Directed CP4 EPSPS
The glyphosate target in plants, the
5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) enzyme, is
located in the chloroplast. Many chloroplast-localized proteins,
including EPSPS, are expressed from nuclear genes as precursors and
are targeted to the chloroplast by a chloroplast transit peptide
(CTP) that is removed during the import steps. Examples of other
such chloroplast proteins include the small subunit (SSU) of
Ribulose-1,5-bisphosphate carboxylase (RUBISCO), Ferredoxia,
Ferredoxin oxidoreductase, the Light-harvesting-complex protein I
and protein II, and Thioredoxin F. It has been demonstrated in vivo
and in vitro that non-chloroplast proteins may be targeted to the
chloroplast by use of protein fusions with a CTP and that a CTP
sequence is sufficient to target a protein to the chloroplast.
A CTP-CP4 EPSPS fusion was constructed between the Arabidopsis
thaliana EPSPS CTP (Klee et al., 1987) and the CP4 EPSPS coding
sequences. The Arabidopsis CTP was engineered by site-directed
mutagenesis to place a SphI restriction site at the CTP processing
site. This mutagenesis replaced the Glu-Lys at this location with
Cys-Met. The sequence of this CTP, designated as CTP2 (SEQ ID
NO:10), is shown in FIG. 9. The N-terminus of the CP4 EPSPS gene
was modified to place a SphI site that spans the Met codon. The
second codon was converted to one for leucine in this step also.
This change had no apparent effect on the in vivo activity of CP4
EPSPS in E. coli as judged by rate of complementation of the aroA
allele. This modified N-terminus was then combined with the SacI
C-terminus and cloned downstream of the CTP2 sequences. The
CTP2-CP4 EPSPS fusion was cloned into pBlueScript KS(+). This
vector may be transcribed in vitro using the T7 polymerase and the
RNA translated with .sup.35S-Methionine to provide material that
may be evaluated for import into chloroplasts isolated from Lactuca
sativa using the methods described hereinafter (della-Cioppa et
al., 1986, 1987). This template was transcribed in vitro using T7
polymerase and the .sup.35S-methionine-labeled CTP2-CP4 EPSPS
material was shown to import into chloroplasts with an efficiency
comparable to that for the control Petunia EPSPS (control=.sup.35S
labeled PreEPSPS [pMON6140; della-Cioppa et al., 1986]).
In another example the Arabidopsis EPSPS CTP, designated as CTP3,
was fused to the CP4 EPSPS through an EcoRI site. The sequence of
this CTP3 (SEQ ID NO:12) is shown in FIG. 10. An EcoRI site was
introduced into the Arabidopsis EPSPS mature region around amino
acid 27, replacing the sequence -Arg-Ala-Leu-Leu- with
-Arg-Ile-Leu-Leu- in the process. The primer of the following
sequence was used to modify the N-terminus of the CP4 EPSPS gene to
add an EcoRI site to effect the fusion to the
TABLE-US-00015 CTG3: GGAAGACGCCCAGATTCACGGTGCAAGCAGCCGG (the EcoRI
site is underlined) (SEQ ID NO:36)
This CTP3-CP4 EPSPS fusion was also cloned into the pBlueScript
vector and the T7 expressed fusion was found to also import into
chloroplasts with an efficiency comparable to that for the control
Petunia EPSPS (pMON6140).
A related series of CTPs, designated as CTP4 (SphI) and CTP5
(EcoRI), based on the Petunia EPSPS CTP and gene were also fused to
the SphI- and EcoRI-modified CP4 EPSPS gene sequences. The SphI
site was added by site-directed mutagenesis to place this
restriction site (and change the amino acid sequence to -Cys-Met-)
at the chloroplast processing site. All of the CTP-CP4 EPSPS
fusions were shown to import into chloroplasts with approximately
equal efficiency. The CTP4 (SEQ ID NO:14) and CTP5 (SEQ ID NO:16)
sequences are shown in FIGS. 11 and 12.
A CTP2-LBAA EPSPS fusion was also constructed following the
modification of the N-terminus of the LBAA EPSPS gene by the
addition of a SphI site. This fusion was also found to be imported
efficiently into chloroplasts.
By similar approaches, the CTP2-CP4 EPSPS and the CTP4-CP4 EPSPS
fusion have also been shown to import efficiently into chloroplasts
prepared from the leaf sheaths of corn. These results indicate that
these CTP-CP4 fusions could also provide useful genes to impart
glyphosate tolerance in monocot species.
The use of CTP2 or CTP4 is preferred because these transit peptide
constructions yield mature EPSPS enzymes upon import into the
chloroplast which are closer in composition to the native EPSPSs
not containing a transit peptide signal. Those skilled in the art
will recognize that various chimeric constructs can be made which
utilize the functionality of a particular CTP to import a Class II
EPSPS enzyme into the plant cell chloroplast. The chloroplast
import of the Class II EPSPS can be determined using the following
assay.
Chloroplast Uptake Assay
Intact chloroplasts are isolated from lettuce (Latuca sativa, var.
longifolia) by centrifugation in Percoll/ficoll gradients as
modified from Bartlett et al., (1982). The final pellet of intact
chloroplasts is suspended in 0.5 ml of sterile 330 mM sorbitol in
50 mM Hepes-KOH, pH 7.7, assayed for chlorophyll (Arnon, 1949), and
adjusted to the final chlorophyll concentration of 4 mg/ml (using
sorbitol/Hepes). The yield of intact chloroplasts from a single
head of lettuce is 3-6 mg chlorophyll.
A typical 300 .mu.l uptake experiment contained 5 mM ATP, 8.3 mM
unlabeled methionine, 322 mM sorbitol, 58.3 mM Hepes-KOH (pH 8.0),
50 .mu.l reticulocyte lysate translation products, and intact
chloroplasts from L. sativa (200 .mu.g chlorophyll). The uptake
mixture is gently rocked at room temperature (in 10.times.75 mm
glass tubes) directly in front of a fiber optic illuminator set at
maximum light intensity (150 Watt bulb). Aliquot samples of the
uptake mix (about 50 .mu.l) are removed at various times and
fractionated over 100 .mu.l silicone-oil gradients (in 150 .mu.l
polyethylene tubes) by centrifugation at 11,000.times. g for 30
seconds. Under these conditions, the intact chloroplasts form a
pellet under the silicone-oil layer and the incubation medium
(containing the reticulocyte lysate) floats on the surface. After
centrifugation, the silicone-oil gradients are immediately frozen
in dry ice. The chloroplast pellet is then resuspended in 50-100
.mu.l of lysis buffer (10 mM Hepes-KOH pH 7.5, 1 mM PMSF, 1 mM
benzamidine, 5 mM e-amino-n-caproic acid, and 30 .mu.g/ml
aprotinin) and centrifuged at 15,000.times. g for 20 minutes to
pellet the thylakoid membranes. The clear supernatant (stromal
proteins) from this spin, and an aliquot of the reticulocyte lysate
incubation medium from each uptake experiment, are mixed with an
equal volume of 2.times.SDS-PAGE sample buffer for electrophoresis
(Laemmli, 1970).
SDS-PAGE is carried out according to Laemmli (1970) in 3-17% (w/v)
acrylamide slab gels (60 mm.times.1.5 mm) with 3% (w/v) acrylamide
stacking gels (5 mm.times.1.5 mm). The gel is fixed for 20-30 rain
in a solution with 40% methanol and 10% acetic acid. Then, the gel
is soaked in EN.sup.3HANCE.TM. (DuPont) for 20-30 minutes, followed
by drying the gel on a gel dryer. The gel is imaged by
autoradiography, using an intensifying screen and an overnight
exposure to determine whether the CP4 EPSPS is imported into the
isolated chloroplasts.
Plant Transformation
Plants which can be made glyphosate-tolerant by practice of the
present invention include, but are not limited to, soybean, cotton,
corn, canola, oil seed rape, flax, sugarbeet, sunflower, potato,
tobacco, tomato, wheat, rice, alfalfa and lettuce as well as
various tree, nut and vine species.
A double-stranded DNA molecule of the present invention ("chimeric
gene") can be inserted into the genome of a plant by any suitable
method. Suitable plant transformation vectors include those derived
from a Ti plasmid of Agrobacterium tumefaciens, as well as those
disclosed, e.g., by Herrera-Estrella (1983), Beyart (1984), Klee
(1985) and EPO publication 120,516 (Schilperoort et al.). In
addition to plant transformation vectors derived from the Ti or
root-inducing (Ri) plasmids of Agrobacterium, alternative methods
can be used to insert the DNA constructs of this invention into
plant cells. Such methods may involve, for example, the use of
liposomes, electroporation, chemicals that increase free DNA
uptake, free DNA delivery via microprojectile bombardment, and
transformation using viruses or pollen.
Class II EPSPS Plant transformation vectors
Class II EPSPS DNA sequences may be engineered into vectors capable
of transforming plants by using known techniques. The following
description is meant to be illustrative and not to be read in a
limiting sense. One of ordinary skill in the art would know that
other plasmids, vectors, markers, promoters, etc. would be used
with suitable results. The CTP2-CP4 EPSPS fusion was cloned as a
BglII-EcoRI fragment into the plant vector pMON979 (described
below) to form pMON17110, a map of which is presented in FIG. 13.
In this vector the CP4 gene is expressed from the enhanced CaMV35S
promoter (E35S; Kay et al. 1987). A FMV35S promoter construct
(pMON17116) was completed in the following way: The SaII-NotI and
the NotI-BglII fragments from pMON979 containing the
Spc/AAC(3)-III/oriV and the pBR322/Right Border/NOS 3'/CP4 EPSPS
gene segment from pMON17110 were ligated with the XhoI-BglII FMV35S
promoter fragment from pMON981. These vectors were introduced into
tobacco, cotton and canola.
A series of vectors was also completed in the vector pMON977 in
which the CP4 EPSPS gene, the CTP2-CP4 EPSPS fusion, and the
CTP3-CP4 fusion were cloned as BglII-SacI fragments to form
pMON17124, pMON17119, and pMON17120, respectively. These plasmids
were introduced into tobacco. A pMON977 derivative containing the
CTP2-LBAA EPSPS gene was also completed (pMON17206) and introduced
into tobacco.
The pMON979 plant transformation/expression vector was derived from
pMON886 (described below) by replacing the neomycin
phosphotransferase typeII (KAN) gene in pMON886 with the 0.89 kb
fragment containing the bacterial gentamicin-3-N-acetyltransferase
type III (AAC(3)-III) gene (Hayford et al., 1988). The chimeric
P-35S/AA(3)-III/NOS 3' gene encodes gentamicin resistance which
permits selection of transformed plant cells. pMON979 also contains
a 0.95 kb expression cassette consisting of the enhanced CaMV 35S
promoter (Kay et al., 1987), several unique restriction sites, and
the NOS 3' end (P-En-CaMV35SfNOS 3'). The rest of the pMON979 DNA
segments are exactly the same as in pMON886.
Plasmid pMON886 is made up of the following segments of DNA. The
first is a 0.93 kb AvaI to engineered-EcoRV fragment isolated from
transposon Tn7 that encodes bacterial spectinomycin/streptomycin
resistance (Spc/Str), which is a determinant for selection in E.
coli and Agrobacterium tumefaciens. This is joined to the 1.61 kb
segment of DNA encoding a chimeric kanamycin resistance which
permits selection of transformed plant cells. The chimeric gene
(P-35S/KANfNOS 3') consists of the cauliflower mosaic virus (CaMV)
35S promoter, the neomycin phosphotransferase typeII (KAN) gene,
and the 3'-nontranslated region of the nopaline synthase gene (NOS
3') (Fraley et al., 1983). The next segment is the 0.75 kb oriV
containing the origin of replication from the RK2 plasmid. It is
joined to the 3.1 kb SalI to PvuI segment of pBR322 (ori322) which
provides the origin of replication for maintenance in E. coli and
the bom site for the conjugational transfer into the Agrobacterium
tumefaciens cells. The next segment is the 0.36 kb PvuI to BclI
from pTiT37 that carries the nopaline-type T-DNA right border
(Fraley et al., 1985).
The pMON977 vector is the same as pMON981 except for the presence
of the P-En-CaMV35S promoter in place of the FMV35S promoter (see
below).
The pMON981 plasmid contains the following DNA segments: the 0.93
kb fragment isolated from transposon Tn7 encoding bacterial
spectinomycin/streptomycin resistance [Spc/Str; a determinant for
selection in E. coli and Agrobacterium tumefaciens (Fling et al.,
1985)]; the chimeric kanamycin resistance gene engineered for plant
expression to allow selection of the transformed tissue, consisting
of the 0.35 kb cauliflower mosaic virus 35S promoter (P-35S) (Odell
et al., 1985), the 0.83 kb neomycin phosphotransferase typeII gene
(KAN), and the 0.26 kb 3'-nontranslated region of the nopaline
synthase gene (NOS 3') (Fraley et al., 1983); the 0.75 kb origin of
replication from the RK2 plasmid (oriV) (Stalker et al., 1981); the
3.1 kb SalI to PvuI segment of pBR322 which provides the origin of
replication for maintenance in E. coli (ori-322) and the bom site
for the conjugational transfer into the Agrobacterium tumefaciens
cells, and the 0.36 kb PvuI to BclI fragment from the pTiT37
plasmid containing the nopaline-type T-DNA right border region
(Fraley et al., 1985). The expression cassette consists of the 0.6
kb 35S promoter from the figwort mosaic virus (P-FMV35S) (Gowda et
al., 1989) and the 0.7 kb 3' non-translated region of the pea
rbcS-E9 gene (E9 3') (Coruzzi et al., 1984, and Morelli et al.,
1985). The 0.6 kb SspI fragment containing the FMV35S promoter
(FIG. 1) was engineered to place suitable cloning sites downstream
of the transcriptional start site. The CTP2-CP4syn gene fusion was
introduced into plant expression vectors (including pMON981, to
form pMON17131; FIG. 14) and transformed into tobacco, canola,
potato, tomato, sugarbeet, cotton, lettuce, cucumber, oil seed
rape, poplar, and Arabidopsis.
The plant vector containing the Class II EPSPS gene may be
mobilized into any suitable Agrobacterium strain for transformation
of the desired plant species. The plant vector may be mobilized
into an ABI Agrobacterium strain. A suitable ABI strain is the A208
Agrobacterium tumefaciens carrying the disarmed Ti plasmid pTiC58
(pMP90RK) (Koncz and Schell, 1986). The Ti plasmid does not carry
the T-DNA phytohormone genes and the strain is therefore unable to
cause the crown gall disease. Mating of the plant vector into ABI
was done by the triparental conjugation system using the helper
plasmid pRK2013 (Ditta et al., 1980). When the plant tissue is
incubated with the ABI::plant vector conjugate, the vector is
transferred to the plant cells by the vir functions encoded by the
disarmed pTiC58 plasmid. The vector opens at the T-DNA right border
region, and the entire plant vector sequence may be inserted into
the host plant chromosome. The pTiC58 Ti plasmid does not transfer
to the plant cells but remains in the Agrobacterium. Class II EPSPS
free DNA vectors
Class II EPSPS genes may also be introduced into plants through
direct delivery methods. A number of direct delivery vectors were
completed for the CP4 EPSPS gene. The vector pMON13640, a map of
which is presented in FIG. 15, is described here. The plasmid
vector is based on a pUC plasmid (Vieira and Messing, 1987)
containing, in this case, the nptII gene (kanamycin resistance;
KAN) from Tn903 to provide a selectable marker in E. coli. The
CTP4-EPSPS gene fusion is expressed from the P-FMV35S promoter and
contains the NOS 3' polyadenylation sequence fragment and from a
second cassette consisting of the E35S promoter, the CTP4-CP4 gene
fusion and the NOS 3' sequences. The scoreable GUS marker gene
(Jefferson et al., 1987) is expressed from the mannopine synthase
promoter (P-MAS; Velten et al., 1984) and the soybean 7S storage
protein gene 3' sequences (Schuler et al., 1982). Similar plasmids
could also be made in which CTP-CP4 EPSPS fusions are expressed
from the enhanced CaMV35S promoter or other plant promoters. Other
vectors could be made that are suitable for free DNA delivery into
plants and such are within the skill of the art and contemplated to
be within the scope of this disclosure.
Plastid transformation:
While transformation of the nuclear genome of plants is much more
developed at this time, a rapidly advancing alternative is the
transformation of plant organelles. The transformation of plastids
of land plants and the regeneration of stable transformants has
been demonstrated (Svab et al., 1990; Maliga et al., 1993).
Transformants are selected, following double cross-over events into
the plastid genome, on the basis of resistance to spectinomycin
conferred through rRNA changes or through the introduction of an
aminoglycoside 3''-adenyltransferase gene (Svab et al., 1990; Svab
and Maliga, 1993), or resistance to kanamycin through the neomycin
phosphotransferase NptII (Carrer et al., 1993). DNA is introduced
by biolistic means (Svab et al, 1990; Maliga et al., 1993) or by
using polyethylene glycol (O'Neill et al., 1993). This
transformation route results in the production of 500-10,000 copies
of the introduced sequence per cell and high levels of expression
of the introduced gene have been reported (Carrer et al., 1993;
Maliga et al., 1993). The use of plastid transformation offers the
advantages of not requiring the chloroplast transit peptide signal
sequence to result in the localization of the heterologous Class II
EPSPS in the chloroplast and the potential to have many copies of
the heterologous plant-expressible Class II EPSPS gene in each
plant cell since at least one copy of the gene would be in each
plastid of the cell.
Plant Regeneration
When expression of the Class II EPSPS gene is achieved in
transformed cells (or protoplasts), the cells (or protoplasts) are
regenerated into whole plants. Choice of methodology for the
generation step is not critical, with suitable protocols being
available for hosts from Leguminosae (alfalfa, soybean, clover,
etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage,
radish, rapeseed, etc.), Cucurbitaceae (melons and cucumber),
Gramineae (wheat, rice, corn, etc.), Solanaceae (potato, tobacco,
tomato, peppers), various floral crops as well as various trees
such as poplar or apple, nut crops or vine plants such as grapes.
See, e.g., Ammirato, 1984; Shimamoto, 1989; Fromm, 1990; Vasil,
1990.
The following examples are provided to better elucidate the
practice of the present invention and should not be intrepreted in
any way to limit the scope of the present invention. Those skilled
in the art will recognize that various modifications, truncations,
etc. can be made to the methods and genes described herein while
not departing from the spirit and scope of the present
invention.
In the examples that follow, EPSPS activity in plants is assayed by
the following method. Tissue samples were collected and immediately
frozen in liquid nitrogen. One gram of young leaf tissue was frozen
in a mortar with liquid nitrogen and ground to a fine powder with a
pestle. The powder was then transferred to a second mortar,
extraction buffer was added (1 ml/gram), and the sample was ground
for an additional 45 seconds. The extraction buffer for canola
consists of 100 mM Tris, 1 mM EDTA, 10% glycerol, 5 mM DTT, 1 mM
BAM, 5 mM ascorbate, 1.0 mg/ml BSA, pH 7.5 (4.degree. C.). The
extraction buffer for tobacco consists of 100 mM Tris, 10 mM EDTA,
35 mM KCl, 20% glycerol, 5 mM DTT, 1 mM BAM, 5 mM ascorbate, 1.0
mg/ml BSA, pH 7.5 (4.degree. C.). The mixture was transferred to a
microfuge tube and centrifuged for 5 minutes. The resulting
supernatants were desalted on spin G-50 (Pharmacia) columns,
previously equilibrated with extraction buffer (without BSA), in
0.25 ml aliquots. The desalted extracts were assayed for EPSP
synthase activity by radioactive HPLC assay. Protein concentrations
in samples were determined by the BioRad microprotein assay with
BSA as the standard.
Protein concentrations were determined using the BioRad
Microprotein method, BSA was used to generate a standard curve
ranging from 2-24 .mu.g. Either 800 .mu.l of standard or diluted
sample was mixed with 200 .mu.l of concentrated BioRad Bradford
reagent. The samples were vortexed and read at A(595) after
.about.5 minutes and compared to the standard curve.
EPSPS enzyme assays contained HEPES (50 mM), shikimate-3-phosphate
(2 mM), NH.sub.4 molybdate (0.1 mM) and KF (5 mM), with or without
glyphosate (0.5 or 1.0 mM). The assay mix (30 .mu.l) and plant
extract (10 .mu.l) were preincubated for 1 minute at 25.degree. C.
and the reactions were initiated by adding .sup.14C-PEP (1 mM). The
reactions were quenched after 3 minutes with 50 .mu.l of 90%
EtOH/0.1M HOAc, pH 4.5. The samples were spun at 6000 rpm and the
resulting supernatants were analyzed for .sup.14C-EPSP production
by HPLC. Percent resistant EPSPS is calculated from the EPSPS
activities with and without glyphosate.
The percent conversion of .sup.14C labeled PEP to .sup.14C EPSP was
determined by HPLC radioassay using a C18 guard column (Brownlee)
and an AX.sub.100 HPLC column (0.4.times.25 cm, Synchropak) with
0.28M isocratic potassium phosphate eluant, pH 6.5, at 1 ml/min.
Initial velocities were calculated by multiplying fractional
turnover per unit time by the initial concentration of the labeled
substrate (1 mM). The assay was linear with time up to .about.3
minutes and 30% turnover to EPSPS. Samples were diluted with 10 mM
Tris, 10% glycerol, 10 mM DTT, pH 7.5 (4.degree. C.) if necessary
to obtain results within the linear range.
In these assays DL-dithiotheitol (DTT), benzamidine (BAM), and
bovine serum albumin (BSA, essentially globulin free) were obtained
from Sigma. Phosphoenolpyruvate (PEP) was from Boehringer Mannheim
and phosphoenol[1-.sup.14C]pyruvate (28 mCi/mmol) was from
Amersham.
EXAMPLES
Example 1
Transformed tobacco plants have been generated with a number of the
Class II EPSPS gene vectors containing the CP4 EPSPS DNA sequence
as described above with suitable expression of the EPSPS. These
transformed plants exhibit glyphosate tolerance imparted by the
Class II CP4 EPSPS.
Transformation of tobacco employs the tobacco leaf disc
transformation protocol which utilizes healthy leaf tissue about 1
month old. After a 15-20 minutes surface sterilization with 10%
Clorox plus a surfactant, the leaves are rinsed 3 times in sterile
water. Using a sterile paper punch, leaf discs are punched and
placed upside down on MS104 media (MS salts 4.3 g/l, sucrose 30
g/l, B5vitamins 500.times.2 ml/l, NAA 0.1 mg/l, and BA 1.0 mg/l)
for a 1 day preculture.
The discs are then inoculated with an overnight culture of a
disarmed Agrobacterium ABI strain containing the subject vector
that had been diluted 1/5 (i.e.: about 0.6 OD). The inoculation is
done by placing the discs in centrifuge tubes with the culture.
After 30 to 60 seconds, the liquid is drained off and the discs
were blotted between sterile filter paper. The discs are then
placed upside down on MS104 feeder plates with a filter disc to
co-culture.
After 2-3 days of co-culture, the discs are transferred, still
upside down, to selection plates with MS104 media. After 2-3 weeks,
callus tissue formed, and individual clumps are separated from the
leaf discs. Shoots are cleanly cut from the callus when they are
large enough to be distinguished from stems. The shoots are placed
on hormone-free rooting media (MSO: MS salts 4.3 g/l, sucrose 30
g/l, and B5 vitamins 500.times.2 ml/l) with selection for the
appropriate antibiotic resistance. Root formation occurred in 1-2
weeks. Any leaf callus assays are preferably done on rooted shoots
while still sterile. Rooted shoots are then placed in soil and kept
in a high humidity environment (i.e.: plastic containers or bags).
The shoots are hardened off by gradually exposing them to ambient
humidity conditions.
Expression of CP4 EPSPS protein in transformed plants
Tobacco cells were transformed with a number of plant vectors
containing the native CP4 EPSPS gene, and using different promoters
and/or CTP's. Preliminary evidence for expression of the gene was
given by the ability of the leaf tissue from antibiotic selected
transformed shoots to recallus on glyphosate. In some cases,
glyphosate-tolerant callus was selected directly following
transformation. The level of expression of the CP4 EPSPS was
determined by the level of glyphosate-tolerant EPSPS activity
(assayed in the presence of 0.5 mM glyphosate) or by Western blot
analysis using a goat anti-CP4 EPSPS antibody. The Western blots
were quantitated by densitometer tracing and comparison to a
standard curve established using purified CP4 EPSPS. These data are
presented as % soluble leaf protein. The data from a number of
transformed plant lines and transformation vectors are presented in
Table VI below.
TABLE-US-00016 TABLE VI Expression of CP4 EPSPS in transformed
tobacco tissue CP4 EPSPS** Vector Plant # (% leaf protein)
pMON17110 25313 0.02 pMON17110 25329 0.04 pMON17116 25095 0.02
pMON17119 25106 0.09 pMON17119 25762 0.09 pMON17119 25767 0.03
**Glyphosate-tolerant EPSPS activity was also demonstrated in leaf
extracts for these plants.
Glyphosate tolerance has also been demonstrated at the whole plant
level in transformed tobacco plants. In tobacco, R.sub.o
transformants of CTP2-CP4 EPSPS were sprayed at 0.4 lb/acre (0.448
kg/hectare), a rate sufficient to kill control non-transformed
tobacco plants corresponding to a rating of 3, 1 and 0 at days 7,
14 and 28, respectively, and were analyzed vegetatively and
reproductively (Table VII).
TABLE-US-00017 TABLE VII Glyphosate tolerance in R.sub.o tobacco
CP4 transformants* Score** Vegetative Vector/Plant # day 7 day 14
day 28 Fertile pMON17110/25313 6 4 2 no pMON17110/25329 9 10 10 yes
pMON17119/25106 9 9 10 yes *Spray rate = 0.4 lb/acre (0.448
kg/hectare) **Plants are evaluated on a numerical scoring system of
0-10 where a vegetative score of 10 represents no damage relative
to nonsprayed controls and 0 represents a dead plant. Reproductive
scores (Fertile) are determined at 28 days after spraying and are
evaluated as to whether or not the plant is fertile.
Example 2A
Canola plants were transformed with the pMON17110, pMON17116, and
pMON17131 vectors and a number of plant lines of the transformed
canola were obtained which exhibit glyphosate tolerance.
Plant Material
Seedlings of Brassica napus cv Westar were established in 2 inch
(.about.5 cm) pots containing Metro Mix 350. They were grown in a
growth chabmer at 24.degree. C., 16/8 hour photoperiod, light
intensity of 400 .mu.Em.sup.-2sec.sup.-1 (HID lamps). They were
fertilized with Peters 20-10-20 General Purpose Special. After 21/2
weeks they were transplanted to 6 inch (.about.15 cm) pots and
grown in a growth chamber at 15.degree./10.degree. C. day/night
temperature, 16/8 hour photoperiod, light intensity of 800
uEm.sup.-2sec.sup.-1 (HID lamps). They were fertilized with Peters
15-30-15 Hi-Phos Special.
Transformation/Selection/Regeneration
Four terminal internodes from plants just prior to bolting or in
the process of bolting but before flowering were removed and
surfaced sterilized in 70% v/v ethanol for 1 minute, 2% w/v sodium
hypochlorite for 20 minutes and rinsed 3 times with sterile
deionized water. Stems with leaves attached could be refrigerated
in moist plastic bags for up to 72 hours prior to sterilization.
Six to seven stem segments were cut into 5 mm discs with a Redco
Vegetable Slicer 200 maintaining orientation of basal end.
The Agrobacterium was grown overnight on a rotator at 24.degree. C.
in 2 mls of Luria Broth containing 50 mg/l kanamycin, 24 mg/l
chloramphenicol and 100 mg/l spectinomycin. A 1:10 dilution was
made in MS (Murashige and Skoog) media giving approximately
9.times.10.sup.8 cells per ml. This was confirmed with optical
density readings at 660 mu. The stem discs (explants) were
inoculated with 1.0 ml of Agrobacterium and the excess was
aspirated from the explants.
The explants were placed basal side down in petri plates containing
1/10.times. standard MS salts, B5 vitamins, 3% sucrose, 0.8% agar,
pH 5.7, 1.0 mg/l 6-benzyladenine (BA). The plates were layered with
1.5 ml of media containing MS salts, B5 vitamins, 3% sucrose, pH
5.7, 4.0 mg/l p-chlorophenoxyacetic acid, 0.005 mg/l kinetin and
covered with sterile filter paper.
Following a 2 to 3 day co-culture, the explants were transferred to
deep dish petri plates containing MS salts, B5 vitamins, 3%
sucrose, 0.8% agar, pH 5.7, 1 mg/l BA, 500 mg/l carbenicillin, 50
mg/l cefotaxime, 200 mg/l kanamycin or 175 mg/l gentamicin for
selection. Seven explants were placed on each plate. After 3 weeks
they were transferred to fresh media, 5 explants per plate. The
explants were cultured in a growth room at 25.degree. C.,
continuous light (Cool White).
Expression Assay
After 3 weeks shoots were excised from the explants. Leaf
recallusing assays were initiated to confirm modification of
R.sub.o shoots. Three tiny pieces of leaf tissue were placed on
recallusing media containing MS salts, B5 vitamins, 3% sucrose,
0.8% agar, pH 5.7, 5.0 mg/l BA, 0.5 mg/l naphthalene acetic acid
(NAA), 500 mg/l carbenicillin, 50 mg/l cefotaxime and 200 mg/l
kanamycin or gentamicin or 0.5 mM glyphosate. The leaf assays were
incubated in a growth room under the same conditions as explant
culture. After 3 weeks the leaf recallusing assays were scored for
herbicide tolerance (callus or green leaf tissue) or sensitivity
(bleaching).
Transplantation
At the time of excision, the shoot stems were dipped in
Rootone.RTM. and placed in 2 inch (.about.5 cm) pots containing
Metro-Mix 350 and placed in a closed humid environment. They were
placed in a growth chamber at 24.degree. C., 16/8 hour photoperiod,
400 uEm.sup.-1sec.sup.-2(HID lamps) for a hardening-off period of
approximately 3 weeks.
The seed harvested from R.sub.o plants is R.sub.1 seed which gives
rise to R.sub.1 plants. To evaluate the glyphosate tolerance of an
R.sub.o plant, its progeny are evaluated. Because an R.sub.o plant
is assumed to be hemizygous at each insert location, selfing
results in maximum genotypic segregation in the R.sub.1. Because
each insert acts as a dominant allele, in the absence of linkage
and assuming only one hemizygous insert is required for tolerance
expression, one insert would segregate 3:1, two inserts, 15:1,
three inserts 63:1, etc. Therefore, relatively few R.sub.1 plants
need be grown to find at least one resistant phenotype.
Seed from an R.sub.o plant is harvested, threshed, and dried before
planting in a glyphosate spray test. Various techniques have been
used to grow the plants for R.sub.1 spray evaluations. Tests are
conducted in both greenhouses and growth chambers. Two planting
systems are used; .about.10 cm pots or plant trays containing 32 or
36 cells. Soil used for planting is either Metro 350 plus three
types of slow release fertilizer or plant Metro 350. Irrigation is
either overhead in greenhouses or sub-irrigation in growth
chambers. Fertilizer is applied as required in irrigation water.
Temperature regimes appropriate for canola were maintained. A
sixteen hour photoperiod was maintained. At the onset of flowering,
plants are transplanted to .about.15 cm pots for seed
production.
A spray "batch" consists of several sets of R.sub.1 progenies all
sprayed on the same date. Some batches may also include evaluations
of other than R.sub.1 plants. Each batch also includes sprayed and
unsprayed non-transgenic genotypes representing the genotypes in
the particular batch which were putatively transformed. Also
included in a batch is one or more non-segregating transformed
genotypes previously identified as having some resistance.
Two-six plants from each individual R.sub.o progeny are not sprayed
and serve as controls to compare and measure the glyphosate
tolerance, as well as to assess any variability not induced by the
glyphosate. When the other plants reach the 2-4 leaf stage, usually
10 to 20 days after planting, glyphosate is applied at rates
varying from 0.28 to 1.12 kg/ha, depending on objectives of the
study. Low rate technology using low volumes has been adopted. A
laboratory track sprayer has been calibrated to deliver a rate
equivalent to field conditions.
A scale of 0 to 10 is used to rate the sprayed plants for
vegetative resistance. The scale is relative to the unsprayed
plants from the same R.sub.o plant. A 0 is death, while a 10
represents no visible difference from the unsprayed plant. A higher
number between 0 and 10 represents progressively less damage as
compared to the unsprayed plant. Plants are scored at 7, 14, and 28
days after treatment (DAT), or until bolting, and a line is given
the average score of the sprayed plants within an R.sub.o plant
family.
Six integers are used to qualitatively describe the degree of
reproductive damage from glyphosate:
0: No floral bud development
2: Floral buds present, but aborted prior to opening
4: Flowers open, but no anthers, or anthers fail to extrude past
petals
6: Sterile anthers
8: Partially sterile anthers
10: Fully fertile flowers
Plants are scored using this scale at or shortly after initiation
of flowering, depending on the rate of floral structure
development.
Expression of EPSPS in Canola
After the 3 week period, the transformed canola plants were assayed
for the presence of glyphosate-tolerant EPSPS activity (assayed in
the presence of glyphosate at 0.5 mM). The results are shown in
Table VIII.
TABLE-US-00018 TABLE VIII Expression of CP4 EPSPS in transformed
Canola plants % resistant EPSPS activity of Leaf extract Plant #
(at 0.5 mM glyphosate) Vector Control 0 pMON17110 41 47 pMON17110
52 28 pMON17110 71 82 pMON17110 104 75 pMON17110 172 84 pMON17110
177 85 pMON17110 252 29* pMON17110 350 49 pMON17116 40 25 pMON17116
99 87 pMON17116 175 94 pMON17116 178 43 pMON17116 182 18 pMON17116
252 69 pMON17116 298 44* pMON17116 332 89 pMON17116 383 97
pMON17116 395 52 *assayed in the presence of 1.0 mM glyphosate
R.sub.1 transformants of canola were then grown in a growth chamber
and sprayed with glyphosate at 0.56 kg/ha (kilogram/hectare) and
rated vegetatively. These results are shown in Table IXA-IXC. It is
to be noted that expression of glyphosate resistant EPSPS in all
tissues is preferred to observe optimal glyphosate tolerance
phenotype in these transgenic plants. In the Tables below, only
expression results obtained with leaf tissue are described.
TABLE-US-00019 TABLE IXA Glyphosate tolerance in Class II EPSPS
canola R.sub.1 transformants (pMON17110 = P-E35S; pMON17116 =
P-FMV35S; R1 plants; Spray rate = 0.56 kg/ha) Vegetative %
resistant Score** Vector/Plant No. EPSPS* day 7 day 14 Control
Westar 0 5 3 pMON17110/41 47 6 7 pMON17110/71 82 6 7 pMON17110/177
85 9 10 pMON17116/40 25 9 9 pMON17116/99 87 9 10 pMON17116/175 94 9
10 pMON17116/178 43 6 3 pMON17116/182 18 9 10 pMON17116/383 97 9
10
TABLE-US-00020 TABLE IXB Glyphosate tolerance in Class II EPSPS
canola R.sub.1 transformants (pMON17131 = P-FWV35S; R1 plants;
Spray rate = 0.84 kg/ha) Vegetative score** Reproductive score
Vector/Plant No. day 14 day 28 17131/78 10 10 17131/102 9 10
17131/115 9 10 17131/116 9 10 17131/157 9 10 17131/169 10 10
17131/255 10 10 control Westar 1 0
TABLE-US-00021 TABLE IXC Glyphosate tolerance in Class I EPSPS
canola transformants (P-E35S; R2 Plants; Spray rate = 0.28 kg/ha)
Vegetative % resistant Score** Vector/Plant No. EPSPS* day 7 day 14
Control Westar 0 4 2 pMON899/715 96 5 6 pMON899/744 95 8 8
pMON899/794 86 6 4 pMON899/818 81 7 8 pMON899/885 57 7 6 *%
resistant EPSPS activity in the presence of 0.5 mM glyphosate **A
vegetative score of 10 indicates no damage, a score of 0 is given
to a dead plant.
The data obtained for the Class II EPSPS transformants may be
compared to glyphosate-tolerant Class I EPSP transformants in which
the same promoter is used to express the EPSPS genes and in which
the level of glyphosate-tolerant EPSPS activity was comparable for
the two types of transformants. A comparison of the data of
pMON17110 [in Table IXA] and pMON17131 [Table IXB] with that for
pMON899 [in Table IXC; the Class I gene in pMON899 is that from A.
thaliana {Klee et al., 1987} in which the glycine at position 101
was changed to an alanine] illustrates that the Class II EPSPS is
at least as good as that of the Class I EPSPS. An improvement in
vegetative tolerance of Class II EPSPS is apparent when one takes
into account that the Class II plants were sprayed at twice the
rate and were tested as R.sub.1 plants.
Example 2B
The construction of two plant transformation vectors and the
transformation procedures used to produce glyphosate-tolerant
canola plants are described in this example The vectors, pMON17209
and pMON17237, were used to generate transgenic glyphosate-tolerant
canola lines. The vectors each contain the gene encoding the
5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) from
Agrobacterium sp. strain CP4. The vectors also contain either the
gox gene encoding the glyphosate oxidoreductase enzyme (GOX) from
Achromobacter sp. strain LBAA (Barry et al., 1992) or the gene
encoding a variant of GOX (GOX v.247) which displays improved
catalytic properties. These enzymes convert glyphosate to
aminomethylphosphonic acid and glyoxylate and protect the plant
from damage by the metabolic inactivation of glyphosate. The
combined result of providing an alternative, resistant EPSPS enzyme
and the metabolism of glyphosate produces transgenic plants with
enhanced tolerance to glyphosate
Molecular biology techniques. In general, standard molecular
biology and microbial genetics approaches were employed (Maniatis
et al., 1982). Site-directed mutageneses were carried out as
described by Kunkel et al. (1987). Plant-preferred genes were
synthesized and the sequence confirmed.
Plant transformation vectors. The following describes the general
features of the plant transformation vectors that were modified to
form vectors pMON17209 and pMON17237. The Agrobacterium mediated
plant transformation vectors contain the following
well-characterized DNA segments which are required for replication
and function of the plasmids (Rogers and Klee, 1987; Klee and
Rogers, 1989). The first segment is the 0.45 kb ClaI-DraI fragment
from the pTi15955 octopine Ti plasmid which contains the T-DNA left
border region (Barker et al., 1983). It is joined to the 0.75 kb
origin of replication (oriV) derived from the broad-host range
plasmid RK2 (Stalker et al., 1981). The next segment is the 3.1 kb
SalI-PvuI segment of pBR.sub.322 which provides the origin of
replication for maintenance in E. coli and the born site for the
conjugational transfer into the Agrobacterium turnefaciens cells
(Bolivar et al., 1977). This is fused to the 0.93 kb fragment
isolated from transposon Tn7 which encodes bacterial spectinomycin
and streptomycin resistance (Fling et al., 1985), a determinant for
the selection of the plasmids in E. coli and Agrobacterium. It is
fused to the 0.36 kb PvuI-BclI fragment from the pTiT37 plasmid
which contains the nopaline-type T-DNA right border region (Fraley
et al., 1985). Several chimeric genes engineered for plant
expression can be introduced between the Ti right and left border
regions of the vector. In addition to the elements described above,
this vector also includes the 35S promoter/NPTII/NOS 3' cassette to
enable selection of transformed plant tissues on kanamycin (Klee
and Rogers, 1989; Fraley et al., 1983; and Odell, et al., 1985)
within the borders. An "empty" expression cassette is also present
between the borders and consists of the enhanced E35S promoter (Kay
et al., 1987), the 3' region from the small subunit of RUBP
carboxylase of pea (E9) (Coruzzi et al., 1984; Morelli et al.,
1986), and a number of restriction enzyme sites that may be used
for the cloning of DNA sequences for expression in plants. The
plant transformation system based on Agrobacterium tumefaciens
delivery has been reviewed (Klee and Rogers, 1989; Fraley et al.,
1986). The Agrobacterium mediated transfer and integration of the
vector T-DNA into the plant chromosome results in the expression of
the chimeric genes conferring the desired phenotype in plants.
Bacterial Inoculum. The binary vectors are mobilized into
Agrobacterium tumefaciens strain ABI by the triparental conjugation
system using the helper plasmid pRK2013 (Ditta et al., 1980). The
ABI strain contains the disarmed pTiC58 plasmid pMP90RK (Koncz and
Schell, 1986) in the chloramphenicol resistant derivative of the
Agrobacterium tumefaciens strain A208.
Transformation procedure. Agrobacterium inocula were grown
overnight at 28.degree. C. in 2 ml of LBSCK (LBSCK is made as
follows: LB liquid medium [1 liter volume]=10 g NaCl; 5 g Yeast
Extract; 10 g tryptone; pH 7.0, and autoclave for 22 minutes. After
autoclaving, add spectinomycin (50 mg/ml stock)--2 ml, kanamycin
(50 mg/ml stock)--1 ml, and chloramphenicol (25 mg/ml stock)--1
ml.). One day prior to inoculation, the Agrobacterium was
subcultured by inoculating 200 .mu.l into 2 ml of fresh LBSCK and
grown overnight. For inoculation of plant material, the culture was
diluted with MSO liquid medium to an A.sub.660 range of
0.2-0.4.
Seedlings of Brassica napus cv. Westar were grown in Metro Mix 350
(Huminert Seed Co., St. Louis, Mo.) in a growth chamber with a
day/night temperature of 15.degree./10.degree. C., relative
humidity of 50%, 16h/8h photoperiod, and at a light intensity of
500 .mu.mol m.sup.-2 sec.sup.-1. The plants were watered daily (via
sub-irrigation) and fertilized every other day with Peter's
15:30:15 (Fogelsville, Pa.).
In general, all media recipes and the transformation protocol
follow those in Fry et al. (1987). Five to six week-old Westar
plants were harvested when the plants had bolted (but prior to
flowering), the leaves and buds were removed, and the 4-5 inches of
stem below the flower buds were used as the explant tissue source.
Following sterilization with 70% ethanol for 1 min and 38% Clorox
for 20 min, the stems were rinsed three times with sterile water
and cut into 5 mm-long segments (the orientation of the basal end
of the stem segments was noted). The plant material was incubated
for 5 minutes with the diluted Agrobacterium culture at a rate of 5
ml of culture per 5 stems. The suspension of bacteria was removed
by aspiration and the explants were placed basal side down--for an
optimal shoot regeneration response--onto co-culture plates ( 1/10
MSO solid medium with a 1.5 ml TXD (tobacco xanthi diploid) liquid
medium overlay and covered with a sterile 8.5 cm filter paper).
Fifty-to-sixty stem explants were placed onto each co-culture
plate.
After a 2 day co-culture period, stem explants were moved onto MS
medium containing 750 mg/l carbenicillin, 50 mg/l cefotaxime, and 1
mg/l BAP (benzylaminopurine) for 3 days. The stem explants were
then placed for two periods of three weeks each, again basal side
down and with 5 explants per plate, onto an MS/0.1 mM glyphosate,
selection medium (also containing carbenicillin, cefotaxime, and
BAP (The glyphosate stock [0.5M] is prepared as described in the
following: 8.45 g glyphosate [analytical grade] is dissolved in 50
ml deionized water, adding KOH pellets to dissolve the glyphosate,
and the volume is brought to 100 ml following adjusting the pH to
5.7. The solution is filter-sterilized and stored at 4.degree. C.).
After 6 weeks on this glyphosate selection medium, green, normally
developing shoots were excised from the stem explants and were
placed onto fresh MS medium containing 750 mg/l carbenicillin, 50
mg/l cefotaxime, and 1 mg/l BAP, for further shoot development.
When the shoots were 2-3 inches tall, a fresh cut at the end of the
stem was made, the cut end was dipped in Root-tone, and the shoot
was placed in Metro Mix 350 soil and allowed to harden-off for 2-3
weeks.
Construction of Canola transformation vector pMON17209. The EPSPS
gene was isolated originally from Agrobacterium sp. strain CP4 and
expresses a highly tolerant enzyme. The original gene contains
sequences that could be inimical to high expression of the gene in
some plants. These sequences include potential polyadenylation
sites that are often A+T rich, a higher G+C % than that frequently
found in dicotyledonous plant genes (63% versus .about.50%),
concentrated stretches of G and C residues, and codons that may not
used frequently in dicotyledonous plant genes. The high G+C % in
the CP4 EPSPS gene could also result in the formation of strong
hairpin structures that may affect expression or stability of the
RNA. A plant preferred version of the gene was synthesized and used
for these vectors. This coding sequence was expressed in E. coli
from a PRecA-gene10L vector (Olins et al., 1988) and the EPSPS
activity was compared with that from the native CP4 EPSPS gene. The
appK.sub.m for PEP for the native and synthetic genes was 11.8
.mu.M and 12.7 .mu.M, respectively, indicating that the enzyme
expressed from the synthetic gene was unaltered. The N-terminus of
the coding sequence was then mutagenized to place an SphI site
(GCATGC) at the ATG to permit the construction of the CTP2-CP4
synthetic fusion for chloroplast import. This change had no
apparent effect on the in vivo activity of CP4 EPSPS in E. coli as
judged by complementation of the aroA mutant. A CTP-CP4 EPSPS
fusion was constructed between the Arabidopsis thaliana EPSPS CTP
(Klee et al., 1987) and the CP4 EPSPS coding sequences. The
Arabidopsis CTP was engineered by site-directed mutagenesis to
place a SphI restriction site at the CTP processing site. This
mutagenesis replaced the Glu-Lys at this location with Cys-Met. The
CTP2-CP4 EPSPS fusion was tested for import into chloroplasts
isolated from Lactuca sativa using the methods described previously
(della-Cioppa et al., 1986; 1987).
The GOX gene that encodes the glyphosate metabolizing enzyme
glyphosate oxidoreductase (GOX) was cloned originally from
Achromobacter sp. strain LBAA (Hallas et al., 1988; Barry et al.,
1992). The gox gene from strain LBAA was also resynthesized in a
plant-preferred sequence version and in which many of the
restriction sites were removed (PCT Appln. No. WO 92/00377). The
GOX protein is targeted to the plastids by a fusion between the
C-terminus of a CTP and the N-terminus of GOX. A CTP, derived from
the SSU1A gene from Arabidopsis thaliana (Timko et al., 1988) was
used. This CTP (CTP1) was constructed by a combination of
site-directed mutageneses. The CTP1 is made up of the SSU1A CTP
(amino acids 1-55), the first 23 amino acids of the mature SSU1A
protein (56-78), a serine residue (amino acid 79), a new segment
that repeats amino acids 50 to 56 from the CTP and the first two
from the mature protein (amino acids 80-87), and an alanine and
methionine residue (amino acid 88 and 89). An NcoI restriction site
if located at the 3' end (spans the Met89 codon) to facilitate the
construction of precise fusions to the 5' of GOX. At a later stage,
a BglII site was introduced upstream of the N-terminus of the SSU1A
sequences to facilitate the introduction of the fusions into plant
transformation vectors. A fusion was assembled between CTP1 and the
synthetic GOX gene.
The CP4 EPSPS and GOX genes were combined to form pMON17209 as
described in the following. The CTP2-CP4 EPSPS fusion was assembled
and inserted between the constitutive FMV35S promoter (Gowda et
al., 1989; Richins et al., 1987) and the E9 3' region (Coruzzi et
al., 1984; Morelli et al., 1985) in a pUC vector (Yannisch-Perron
et al., 1985; Vieira and Messing, 1987) to form pMON17190; this
completed element may then be moved easily as a NotI-NotI fragment
to other vectors. The CTP1-GOX fusion was also assembled in a pUC
vector with the FMV35S promoter. This element was then moved as a
HindIII-BamHI fragment into the plant transformation vector
pMON10098 and joined to the E9 3' region in the process. The
resultant vector pMON17193 has a single NotI site into which the
FMV 35S/CTP2-CP4 EPSPS/E9 3' element from pMON17190 was cloned to
form pMON17194. The kanamycin plant transformation selection
cassette (Fraley et al., 1985) was then deleted from pMON17194, by
cutting with XhoI and re-ligating, to form the pMON17209 vector
(FIG. 24).
Construction of Canola transformation vector pMON17237. The GOX
enzyme has an apparent Km for glyphosate [appK.sub.m(glyphosate)]
of .about.25 mM. In an effort to improve the effectiveness of the
glyphosate metabolic rate in plants, a variant of GOX has been
identified in which the appK.sub.m(glyphosate) has been reduced
approximately 10-fold; this variant is referred to as GOX v.247 and
the sequence differences between it and the original
plant-preferred GOX are illustrated in PCT Appln. No. WO 92/00377.
The GOX v.247 coding sequence was combined with CTP1 and assembled
with the FMV35S promoter and the E9 3' by cloning into the
pMON17227 plant transformation vector to form pMON17241. In this
vector, effectively, the CP4 EPSPS was replaced by GOX v.247. The
pMON17227 vector had been constructed by replacing the CTP1-GOX
sequence in pMON17193 with those for the CTP2-CP4 EPSPS, to form
pMON17199 and followed by deleting the kanamycin cassette (as
described above for pMON17209). The pMON17237 vector (FIG. 25) was
then completed by cloning the FMV35S/CTP2-CP4 EPSPS/E9 3' element
as a NotI-NotI fragment into pMON17241.
Example 3
Soybean plants were transformed with the pMON13640 (FIG. 15) vector
and a number of plant lines of the transformed soybean were
obtained which exhibit glyphosate tolerance.
Soybean plants are transformed with pMON13640 by the method of
microprojectile injection using particle gun technology as
described in Christou et al. (1988). The seed harvested from
R.sub.o plants is R.sub.1 seed which gives rise to R.sub.1 plants.
To evaluate the glyphosate tolerance of an R.sub.o plant, its
progeny are evaluated. Because an R.sub.o plant is assumed to be
hemizygous at each insert location, selfing results in maximum
genotypic segregation in the R.sub.1. Because each insert acts as a
dominant allele, in the absence of linkage and assuming only one
hemizygous insert is required for tolerance expression, one insert
would segregate 3:1, two inserts, 15:1, three inserts 63:1, etc.
Therefore, relatively few R.sub.1 plants need be grown to find at
least one resistant phenotype.
Seed from an R.sub.o soybean plant is harvested, and dried before
planting in a glyphosate spray test. Seeds are planted into 4 inch
(.about.5 cm) square pots containing Metro 350. Twenty seedlings
from each R.sub.o plant is considered adequate for testing. Plants
are maintained and grown in a greenhouse environment. A 12.5-14
hour photoperiod and temperatures of 30.degree. C. day and
24.degree. C. night is regulated. Water soluble Peters Pete Lite
fertilizer is applied as needed.
A spray "batch" consists of several sets of R.sub.1 progenies all
sprayed on the same date. Some batches may also include evaluations
of other than R.sub.1 plants. Each batch also includes sprayed and
unsprayed non-transgenic genotypes representing the genotypes in
the particular batch which were putatively transformed. Also
included in a batch is one or more non-segregating transformed
genotypes previously identified as having some resistance.
One to two plants from each individual R.sub.o progeny are not
sprayed and serve as controls to compare and measure the glyphosate
tolerance, as well as to assess any variability not induced by the
glyphosate. When the other plants reach the first trifoliate leaf
stage, usually 2-3 weeks after planting, glyphosate is applied at a
rate equivalent of 128 oz./acre (8.895 kg/ha) of Roundup.RTM.. A
laboratory track sprayer has been calibrated to deliver a rate
equivalent to those conditions.
A vegetative score of 0 to 10 is used. The score is relative to the
unsprayed progenies from the same R.sub.o plant. A 0 is death,
while a 10 represents no visible difference from the unsprayed
plant. A higher number between 0 and 10 represents progressively
less damage as compared to the unsprayed plant. Plants are scored
at 7, 14, and 28 days after treatment (DAT). The data from the
analysis of one set of transformed and control soybean plants are
described on Table X and show that the CP4 EPSPS gene imparts
glyphosate tolerance in soybean also.
TABLE-US-00022 TABLE X Glyphosate tolerance in Class II EPSPS
soybean transformants (P-H35S, P-FMV35S; R0 plants; Spray rate =
128 oz./acre) Vegetative score Vector/Plant No. day 7 day 14 day 28
13640/40-11 5 6 7 13640/40-3 9 10 10 13640/40-7 4 7 7 control A5403
2 1 0 control A5403 1 1 0
Example 4
The CP4 EPSPS gene may be used to select transformed plant material
directly on media containing glyphosate. The ability to select and
to identify transformed plant material depends, in most cases, on
the use of a dominant selectable marker gene to enable the
preferential and continued growth of the transformed tissues in the
presence of a normally inhibitory substance. Antibiotic resistance
and herbicide tolerance genes have been used almost exclusively as
such dominant selectable marker genes in the presence of the
corresponding antibiotic or herbicide. The nptII/kanamycin
selection scheme is probably the most frequently used. It has been
demonstrated that CP4 EPSPS is also a useful and perhaps superior
selectable marker/selection scheme for producing and identifying
transformed plants.
A plant transformation vector that may be used in this scheme is
pMON17227 (FIG. 16). This plasmid resembles many of the other
plasmids described infra and is essentially composed of the
previously described bacterial replicon system that enables this
plasmid to replicate in E. coli and to be introduced into and to
replicate in Agrobacterium, the bacterial selectable marker gene
(Spc/Str), and located between the T-DNA right border and left
border is the CTP2-CP4 synthetic gene in the FMV35S promoter-E9 3'
cassette. This plasmid also has single sites for a number of
restriction enzymes, located within the borders and outside of the
expression cassette. This makes it possible to easily add other
genes and genetic elements to the vector for introduction into
plants.
The protocol for direct selection of transformed plants on
glyphosate is outlined for tobacco. Explants are prepared for
pre-culture as in the standard procedure as described in Example 1:
surface sterilization of leaves from 1 month old tobacco plants (15
minutes in 10% clorox+surfactant; 3.times.dH.sub.2O washes);
explants are cut in 0.5.times.0.5 cm squares, removing leaf edges,
mid-rib, tip, and petiole end for uniform tissue type; explants are
placed in single layer, upside down, on MS104 plates+2 ml 4COO5K
media to moisten surface; pre-culture 1-2 days. Explants are
inoculated using overnight culture of Agrobacterium containing the
plant transformation plasmid that is adjusted to a titer of
1.2.times.10.sup.9 bacteria/ml with 4COO5K media. Explants are
placed into a centrifuge tube, the Agrobacterium suspension is
added and the mixture of bacteria and explants is "Vortexed" on
maximum setting for 25 seconds to ensure even penetration of
bacteria. The bacteria are poured off and the explants are blotted
between layers of dry sterile filter paper to remove excess
bacteria. The blotted explants are placed upside down on MS104
plates+2 ml 4COO5K media+filter disc. Co-culture is 2-3 days. The
explants are transferred to MS104+Carbenicillin 1000
mg/l+cefotaxime 100 mg/l for 3 days (delayed phase). The explants
are then transferred to MS104+glyphosate 0.05 mM+Carbenicillin 1000
mg/l+cefotaxime 100 mg/l for selection phase. At 4-6 weeks shoots
are cut from callus and placed on MSO+Carbenicillin 500 mg/l
rooting media. Roots form in 3-5 days, at which time leaf pieces
can be taken from rooted plates to confirm glyphosate tolerance and
that the material is transformed.
The presence of the CP4 EPSPS protein in these transformed tissues
has been confirmed by immunoblot analysis of leaf discs. The data
from one experiment with pMON17227 is presented in the following:
139 shoots formed on glyphosate from 400 explants inoculated with
Agrobacterium ABI/pMON17227; 97 of these were positive on
recallusing on glyphosate. These data indicate a transformation
rate of 24 per 100 explants, which makes this a highly efficient
and time saving transformation procedure for plants. Similar
transformation frequencies have been obtained with pMON17131 and
direct selection of transformants on glyphosate with the CP4 EPSPS
genes has also been shown in other plant species, including,
Arabidopsis, soybean, corn, wheat, potato, tomato, cotton, lettuce,
and sugarbeet.
The pMON17227 plasmid contains single restriction enzyme
recognition cleavage sites (NotI, XhoI, and BstXI) between the CP4
glyphosate selection region and the left border of the vector for
the cloning of additional genes and to facilitate the introduction
of these genes into plants.
EXAMPLE 5A
The CP4 EPSPS gene has also been introduced into Black Mexican
Sweet (BMS) corn cells with expression of the protein and
glyphosate resistance detected in callus.
The backbone for this plasmid was a derivative of the high copy
plasmid pUC119 (Viera and Messing, 1987). The 1.3 Kb FspI-DraI
pUC119 fragment containing the origin of replication was fused to
the 1.3 Kb SmaI-HindIII filled fragment from pKC7 (Rao and Rogers,
1979) which contains the neomycin phosphotransferase type II gene
to confer bacterial kanamycin resistance. This plasmid was used to
construct a monocot expression cassette vector containing the 0.6
kb cauliflower mosaic virus (CaMV) 35S RNA promoter with a
duplication of the -90 to -300 region (Kay et al., 1987), an 0.8 kb
fragment containing an intron from a maize gene in the 5'
untranslated leader region, followed by a polylinker and the 3'
termination sequences from the nopaline synthase (NOS) gene (Fraley
et al., 1983). A 1.7 Kb fragment containing the 300 bp chIoroplast
transit peptide from the Arabidopsis EPSP synthase fused in a frame
to the 1.4 Kb coding sequence for the bacterial CP4 EPSP synthase
was inserted into the monocot expression cassette in the polylinker
between the intron and the NOS termination sequence to form the
plasmid pMON19653 (FIG. 17).
pMON19653 DNA was introduced into Black Mexican Sweet (BMS) cells
by co-bombardment with EC9, a plasmid containing a
sulfonylurea-resistant form of the maize acetolactate synthase
gene. 2.5 mg of each plasmid was coated onto tungsten particles and
introduced into log-phase BMS cells using a PDS-1000 particle gun
essentially as described (Klein et al., 1989). Transformants are
selected on MS medium containing 20 ppb chlorsulfuron. After
initial selection on chlorsulfuron, the calli can be assayed
directly by Western blot. Glyphosate tolerance can be assessed by
transferring the calli to medium containing 5mM glyphosate. As
shown in Table XI, CP4 EPSPS confers glyphosate tolerance to corn
callus.
TABLE-US-00023 TABLE XI Expression of CP4 in BMS Corn Callus-pMON
19653 CP4 expression Line (% extract protein) 284 0.006% 287 0.036
290 0.061 295 0.073 299 0.113 309 0.042 313 0.003
To measure CP4 EPSPS expression in corn callus, the following
procedure was used: BMS callus (3 g wet weight) was dried on filter
paper (Whatman#1) under vacuum, reweighed, and extraction buffer
(500 .mu.l/g dry weight; 100 mM Tris, 1 mM EDTA, 10% glycerol) was
added. The tissue was homogenized with a Wheaton overhead stirrer
for 30 seconds at 2.8 power setting. After centrifugation (3
minutes, Eppendorf microfuge), the supernatant was removed and the
protein was quantitated (BioRad Protein Assay). Samples (50
.mu.g/well) were loaded on an SDS PAGE gel (Jule, 3-17%) along with
CP4 EPSPS standard (10 ng), electrophoresed, and transferred to
nitrocellulose similarly to a previously described method
(Padgette, 1987). The nitrocellulose blot was probed with goat
anti-CP4 EPSPS IgG, and developed with I-125 Protein G. The
radioactive blot was visualized by autoradiography. Results were
quantitated by densitometry on an LKB UltraScan XL laser densitomer
and are tabulated below in Table X.
TABLE-US-00024 TABLE XII Glyphosate resistance in BMS Corn Callus
using pMON 19653 # chlorosulfuron- # cross-resistant Vector
Experiment resistant lines to Glyphosate 19653 253 120 81/120 =
67.5% 19653 254 80 37/80 = 46% EC9 control 253/254 8 0/8 = 0%
Improvements in the expression of Class II EPSPS could also be
achieved by expressing the gene using stronger plant promoters,
using better 3' polyadenylation signal sequences, optimizing the
sequences around the initiation codon for ribosome loading and
translation initiation, or by combination of these or other
expression or regulatory sequences or factors.
Example 5B
The plant-expressible genes encoding the CP4 EPSPS and a glyphosate
oxidoreductasease enzyme (PCT Pub. No. WO92/00377) were introduced
into embryogenic corn callus through particle bombardment. Plasmid
DNA was prepared using standard procedures (Ausubel et al., 1987),
cesium-chloride purified, and re-suspended at 1 mg/ml in TE buffer.
DNA was precipitated onto M10 tungsten or 1.0 .mu.g gold particles
(BioRad) using a calcium chloride/spermidine precipitation
protocol, essentially as described by Klein et al. (1987). The
PDS1000.RTM. gunpowder gun (BioRad) was used. Callus tissue was
obtained by isolating 1-2 mm long immature embryos from the "Hi-II"
genotype (Armstrong et al., 1991), or Hi-II X B73 crosses, onto a
modified N6 medium (Armstrong and Green, 1985; Songstad et al.,
1991). Embryogenic callus ("type-II"; Armstrong and Green, 1985)
initiated from these embryos was maintained by subculturing at two
week intervals, and was bombarded when less than two months old.
Each plate of callus tissue was bombarded from 1 to 3 times with
either tungsten or gold particles coated with the plasmid DNA(s) of
interest. Callus was transferred to a modified N6 medium containing
an appropriate selective agent (either glyphosate, or one or more
of the antibiotics kanamycin, G418, or paromomycin) 1-8 days
following bombardment, and then re-transferred to fresh selection
media at 2-3 week intervals. Glyphosate-resistant calli first
appeared approximately 6-12 weeks post-bombardment. These resistant
calli were propagated on selection medium, and samples were taken
for assays gene expression. Plant regeneration from resistant calli
was accomplished essentially as described by Petersen et al.
(1992).
In some cases, both gene(s) were covalently linked together on the
same plasmid DNA molecule. In other instances, the genes were
present on separate plasmids, but were introduced into the same
plant through a process termed "co-transformation". The 1 mg/ml
plasmid preparations of interest were mixed together in an equal
ratio, by volume, and then precipitated onto the tungsten or gold
particles. At a high frequency, as described in the literature
(e.g., Schocher et al., 1986), the different plasmid molecules
integrate into the genome of the same plant cell. Generally the
integration is into the same chromosomal location in the plant
cell, presumably due to recombination of the plasmids prior to
integration. Less frequently, the different plasmids integrate into
separate chromosomal locations. In either case, there is
integration of both DNA molecules into the same plant cell, and any
plants produced from that cell.
Transgenic corn plants were produced as described above which
contained a plant-expressible CP4 gene and a plant-expressible gene
encoding a glyphosate oxidoreductase enzyme.
The plant-expressible CP4 gene comprised a structural DNA sequence
encoding a CTP2/CP4 EPSPS fusion protein. The CTP2/CP4 EPSPS is a
gene fusion composed of the N-terminal 0.23 Kb chloroplast transit
peptide sequence from the Arabidopsis thaliana EPSPS gene (Klee et
al. 1987, referred to herein as CTP2), and the C-terminal 1.36 Kb
5-enolpyruvylshikimate-3-phosphate synthase gene (CP4) from an
Agrobacterium species. Plant expression of the gene fusion produces
a pre-protein which is rapidly imported into chloroplasts where the
CTP is cleaved and degraded (della-Cioppa et al., 1986) releasing
the mature CP4 protein.
The plant-expressible gene expressing a glyphosate oxidoreductase
enzyme comprised a structural DNA sequence comprising CTP1/GOXsyn
gene fusion composed of the N-terminal 0.26 Kb chloroplast transit
peptide sequence derived from the Arabidopsis thaliana SSU 1a gene
(Timko et al., 1988 referred to herein as CTP1), and the C-terminal
1.3 Kb synthetic gene sequence encoding a glyphosate oxidoreductase
enzyme (GOXsyn, as described in PCT Pub. No. WO92/00377 previously
incorporated by reference. The GOXsyn gene encodes the enzyme
glyphosate oxidoreductase from an Achromobacter sp. strain LBAA
which catalyzes the conversion of glyphosate to herbicidally
inactive products, aminomethylphosphonate and glyoxylate. Plant
expression of the gene fusion produces a pre-protein which is
rapidly imported into chloroplasts where the CTP is cleaved and
degraded (della-Cioppa et al., 1986) releasing the mature GOX
protein.
Both of the above described genes also include the following
regulatory sequences for plant expression: (i) a promoter region
comprising a 0.6 Kb 35S cauliflower mosaic virus (CaMV) promoter
(Odell et al., 1985) with the duplicated enhancer region (Kay et
al., 1987) which also contains a 0.8 Kb fragment containing the
first intron from the maize heat shock protein 70 gene (Shah et
al., 1985 and PCT Pub. No. WO93/19189, the disclosure of which is
hereby incorporated by reference); and (ii) a 3' non-translated
region comprising a 0.3 Kb fragment of the 3' non-translated region
of the nopaline synthase gene (Fraley et al., 1983 and Depicker, et
al., 1982) which functions to direct polyadenylation of the
mRNA.
The above described transgenic corn plants exhibit tolerance to
glyphosate herbicide in greenhouse and field trials.
Example 6
The LBAA Class II EPSPS gene has been introduced into plants and
also imparts glyphosate tolerance. Data on tobacco transformed with
pMON17206 (infra) are presented in Table XIII.
TABLE-US-00025 TABLE XIII Tobacco Glyphosate Spray Test (pMON17206;
E35S-CTP2-LBAA EPSPS; 0/4 lbs/ac) Line 7 Day Rating 33358 9 34586 9
33328 9 34606 9 33377 9 34611 10 34607 10 34601 9 34589 9 Samsun
(Control) 4
From the foregoing, it will be recognized that this invention is
one well adapted to attain all the ends and objects hereinabove set
forth together with advantages which are obvious and which are
inherent to the invention. It will be further understood that
certain features and subcombinations are to utility and may be
employed without reference to other features and subcombinations.
This is contemplated by and is within the scope of the claims.
Since many possible embodiments may be made of the invention
without departing from the scope thereof, it is to be understood
that all matter herein set forth or shown in the accompanying
drawings is to be interpreted as illustrative and not in a limiting
sense.
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SEQUENCE LISTINGS
1
701597DNAFigwort mosaic virus 1tcatcaaaat atttagcagc attccagatt
gggttcaatc aacaaggtac gagccatatc 60actttattca aattggtatc gccaaaacca
agaaggaact cccatcctca aaggtttgta 120aggaagaatt ctcagtccaa
agcctcaaca aggtcagggt acagagtctc caaaccatta 180gccaaaagct
acaggagatc aatgaagaat cttcaatcaa agtaaactac tgttccagca
240catgcatcat ggtcagtaag tttcagaaaa agacatccac cgaagactta
aagttagtgg 300gcatctttga aagtaatctt gtcaacatcg agcagctggc
ttgtggggac cagacaaaaa 360aggaatggtg cagaattgtt aggcgcacct
accaaaagca tctttgcctt tattgcaaag 420ataaagcaga ttcctctagt
acaagtgggg aacaaaataa cgtggaaaag agctgtcctg 480acagcccact
cactaatgcg tatgacgaac gcagtgacga ccacaaaaga attccctcta
540tataagaagg cattcattcc catttgaagg atcatcagat actaaccaat atttctc
59721982DNAAgrobacterium sp.CDS(62)..(1426) 2aagcccgcgt tctctccggc
gctccgcccg gagagccgtg gatagattaa ggaagacgcc 60c atg tcg cac ggt gca
agc agc cgg ccc gca acc gcc cgc aaa tcc tct 109 Met Ser His Gly Ala
Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser 1 5 10 15ggc ctt tcc
gga acc gtc cgc att ccc ggc gac aag tcg atc tcc cac 157Gly Leu Ser
Gly Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 20 25 30cgg tcc
ttc atg ttc ggc ggt ctc gcg agc ggt gaa acg cgc atc acc 205Arg Ser
Phe Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45ggc
ctt ctg gaa ggc gag gac gtc atc aat acg ggc aag gcc atg cag 253Gly
Leu Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 50 55
60gcc atg ggc gcc agg atc cgt aag gaa ggc gac acc tgg atc atc gat
301Ala Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile
Asp65 70 75 80ggc gtc ggc aat ggc ggc ctc ctg gcg cct gag gcg ccg
ctc gat ttc 349Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro
Leu Asp Phe 85 90 95ggc aat gcc gcc acg ggc tgc cgc ctg acc atg ggc
ctc gtc ggg gtc 397Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly
Leu Val Gly Val 100 105 110tac gat ttc gac agc acc ttc atc ggc gac
gcc tcg ctc aca aag cgc 445Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp
Ala Ser Leu Thr Lys Arg 115 120 125ccg atg ggc cgc gtg ttg aac ccg
ctg cgc gaa atg ggc gtg cag gtg 493Pro Met Gly Arg Val Leu Asn Pro
Leu Arg Glu Met Gly Val Gln Val 130 135 140aaa tcg gaa gac ggt gac
cgt ctt ccc gtt acc ttg cgc ggg ccg aag 541Lys Ser Glu Asp Gly Asp
Arg Leu Pro Val Thr Leu Arg Gly Pro Lys145 150 155 160acg ccg acg
ccg atc acc tac cgc gtg ccg atg gcc tcc gca cag gtg 589Thr Pro Thr
Pro Ile Thr Tyr Arg Val Pro Met Ala Ser Ala Gln Val 165 170 175aag
tcc gcc gtg ctg ctc gcc ggc ctc aac acg ccc ggc atc acg acg 637Lys
Ser Ala Val Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 180 185
190gtc atc gag ccg atc atg acg cgc gat cat acg gaa aag atg ctg cag
685Val Ile Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln
195 200 205ggc ttt ggc gcc aac ctt acc gtc gag acg gat gcg gac ggc
gtg cgc 733Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly
Val Arg 210 215 220acc atc cgc ctg gaa ggc cgc ggc aag ctc acc ggc
caa gtc atc gac 781Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly
Gln Val Ile Asp225 230 235 240gtg ccg ggc gac ccg tcc tcg acg gcc
ttc ccg ctg gtt gcg gcc ctg 829Val Pro Gly Asp Pro Ser Ser Thr Ala
Phe Pro Leu Val Ala Ala Leu 245 250 255ctt gtt ccg ggc tcc gac gtc
acc atc ctc aac gtg ctg atg aac ccc 877Leu Val Pro Gly Ser Asp Val
Thr Ile Leu Asn Val Leu Met Asn Pro 260 265 270acc cgc acc ggc ctc
atc ctg acg ctg cag gaa atg ggc gcc gac atc 925Thr Arg Thr Gly Leu
Ile Leu Thr Leu Gln Glu Met Gly Ala Asp Ile 275 280 285gaa gtc atc
aac ccg cgc ctt gcc ggc ggc gaa gac gtg gcg gac ctg 973Glu Val Ile
Asn Pro Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 290 295 300cgc
gtt cgc tcc tcc acg ctg aag ggc gtc acg gtg ccg gaa gac cgc 1021Arg
Val Arg Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg305 310
315 320gcg cct tcg atg atc gac gaa tat ccg att ctc gct gtc gcc gcc
gcc 1069Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala
Ala 325 330 335ttc gcg gaa ggg gcg acc gtg atg aac ggt ctg gaa gaa
ctc cgc gtc 1117Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu
Leu Arg Val 340 345 350aag gaa agc gac cgc ctc tcg gcc gtc gcc aat
ggc ctc aag ctc aat 1165Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn
Gly Leu Lys Leu Asn 355 360 365ggc gtg gat tgc gat gag ggc gag acg
tcg ctc gtc gtg cgc ggc cgc 1213Gly Val Asp Cys Asp Glu Gly Glu Thr
Ser Leu Val Val Arg Gly Arg 370 375 380cct gac ggc aag ggg ctc ggc
aac gcc tcg ggc gcc gcc gtc gcc acc 1261Pro Asp Gly Lys Gly Leu Gly
Asn Ala Ser Gly Ala Ala Val Ala Thr385 390 395 400cat ctc gat cac
cgc atc gcc atg agc ttc ctc gtc atg ggc ctc gtg 1309His Leu Asp His
Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 405 410 415tcg gaa
aac cct gtc acg gtg gac gat gcc acg atg atc gcc acg agc 1357Ser Glu
Asn Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 420 425
430ttc ccg gag ttc atg gac ctg atg gcc ggg ctg ggc gcg aag atc gaa
1405Phe Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu
435 440 445ctc tcc gat acg aag gct gcc tgatgacctt cacaatcgcc
atcgatggtc 1456Leu Ser Asp Thr Lys Ala Ala 450 455ccgctgcggc
cggcaagggg acgctctcgc gccgtatcgc ggaggtctat ggctttcatc
1516atctcgatac gggcctgacc tatcgcgcca cggccaaagc gctgctcgat
cgcggcctgt 1576cgcttgatga cgaggcggtt gcggccgatg tcgcccgcaa
tctcgatctt gccgggctcg 1636accggtcggt gctgtcggcc catgccatcg
gcgaggcggc ttcgaagatc gcggtcatgc 1696cctcggtgcg gcgggcgctg
gtcgaggcgc agcgcagctt tgcggcgcgt gagccgggca 1756cggtgctgga
tggacgcgat atcggcacgg tggtctgccc ggatgcgccg gtgaagctct
1816atgtcaccgc gtcaccggaa gtgcgcgcga aacgccgcta tgacgaaatc
ctcggcaatg 1876gcgggttggc cgattacggg acgatcctcg aggatatccg
ccgccgcgac gagcgggaca 1936tgggtcgggc ggacagtcct ttgaagcccg
ccgacgatgc gcactt 19823455PRTAgrobacterium sp. 3Met Ser His Gly Ala
Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser1 5 10 15Gly Leu Ser Gly
Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 20 25 30Arg Ser Phe
Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45Gly Leu
Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 50 55 60Ala
Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp65 70 75
80Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe
85 90 95Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly
Val 100 105 110Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu
Thr Lys Arg 115 120 125Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu
Met Gly Val Gln Val 130 135 140Lys Ser Glu Asp Gly Asp Arg Leu Pro
Val Thr Leu Arg Gly Pro Lys145 150 155 160Thr Pro Thr Pro Ile Thr
Tyr Arg Val Pro Met Ala Ser Ala Gln Val 165 170 175Lys Ser Ala Val
Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 180 185 190Val Ile
Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 195 200
205Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg
210 215 220Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly Gln Val
Ile Asp225 230 235 240Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro
Leu Val Ala Ala Leu 245 250 255Leu Val Pro Gly Ser Asp Val Thr Ile
Leu Asn Val Leu Met Asn Pro 260 265 270Thr Arg Thr Gly Leu Ile Leu
Thr Leu Gln Glu Met Gly Ala Asp Ile 275 280 285Glu Val Ile Asn Pro
Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 290 295 300Arg Val Arg
Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg305 310 315
320Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala
325 330 335Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu Leu
Arg Val 340 345 350Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly
Leu Lys Leu Asn 355 360 365Gly Val Asp Cys Asp Glu Gly Glu Thr Ser
Leu Val Val Arg Gly Arg 370 375 380Pro Asp Gly Lys Gly Leu Gly Asn
Ala Ser Gly Ala Ala Val Ala Thr385 390 395 400His Leu Asp His Arg
Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 405 410 415Ser Glu Asn
Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 420 425 430Phe
Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 435 440
445Leu Ser Asp Thr Lys Ala Ala 450 45541673DNAAgrobacterium
sp.CDS(86)..(1432) 4gtagccacac ataattacta tagctaggaa gcccgctatc
tctcaatccc gcgtgatcgc 60gccaaaatgt gactgtgaaa aatcc atg tcc cat tct
gca tcc ccg aaa cca 112 Met Ser His Ser Ala Ser Pro Lys Pro 1 5gca
acc gcc cgc cgc tcg gag gca ctc acg ggc gaa atc cgc att ccg 160Ala
Thr Ala Arg Arg Ser Glu Ala Leu Thr Gly Glu Ile Arg Ile Pro10 15 20
25ggc gac aag tcc atc tcg cat cgc tcc ttc atg ttt ggc ggt ctc gca
208Gly Asp Lys Ser Ile Ser His Arg Ser Phe Met Phe Gly Gly Leu Ala
30 35 40tcg ggc gaa acc cgc atc acc ggc ctt ctg gaa ggc gag gac gtc
atc 256Ser Gly Glu Thr Arg Ile Thr Gly Leu Leu Glu Gly Glu Asp Val
Ile 45 50 55aat aca ggc cgc gcc atg cag gcc atg ggc gcg aaa atc cgt
aaa gag 304Asn Thr Gly Arg Ala Met Gln Ala Met Gly Ala Lys Ile Arg
Lys Glu 60 65 70ggc gat gtc tgg atc atc aac ggc gtc ggc aat ggc tgc
ctg ttg cag 352Gly Asp Val Trp Ile Ile Asn Gly Val Gly Asn Gly Cys
Leu Leu Gln 75 80 85ccc gaa gct gcg ctc gat ttc ggc aat gcc gga acc
ggc gcg cgc ctc 400Pro Glu Ala Ala Leu Asp Phe Gly Asn Ala Gly Thr
Gly Ala Arg Leu90 95 100 105acc atg ggc ctt gtc ggc acc tat gac atg
aag acc tcc ttt atc ggc 448Thr Met Gly Leu Val Gly Thr Tyr Asp Met
Lys Thr Ser Phe Ile Gly 110 115 120gac gcc tcg ctg tcg aag cgc ccg
atg ggc cgc gtg ctg aac ccg ttg 496Asp Ala Ser Leu Ser Lys Arg Pro
Met Gly Arg Val Leu Asn Pro Leu 125 130 135cgc gaa atg ggc gtt cag
gtg gaa gca gcc gat ggc gac cgc atg ccg 544Arg Glu Met Gly Val Gln
Val Glu Ala Ala Asp Gly Asp Arg Met Pro 140 145 150ctg acg ctg atc
ggc ccg aag acg gcc aat ccg atc acc tat cgc gtg 592Leu Thr Leu Ile
Gly Pro Lys Thr Ala Asn Pro Ile Thr Tyr Arg Val 155 160 165ccg atg
gcc tcc gcg cag gta aaa tcc gcc gtg ctg ctc gcc ggt ctc 640Pro Met
Ala Ser Ala Gln Val Lys Ser Ala Val Leu Leu Ala Gly Leu170 175 180
185aac acg ccg ggc gtc acc acc gtc atc gag ccg gtc atg acc cgc gac
688Asn Thr Pro Gly Val Thr Thr Val Ile Glu Pro Val Met Thr Arg Asp
190 195 200cac acc gaa aag atg ctg cag ggc ttt ggc gcc gac ctc acg
gtc gag 736His Thr Glu Lys Met Leu Gln Gly Phe Gly Ala Asp Leu Thr
Val Glu 205 210 215acc gac aag gat ggc gtg cgc cat atc cgc atc acc
ggc cag ggc aag 784Thr Asp Lys Asp Gly Val Arg His Ile Arg Ile Thr
Gly Gln Gly Lys 220 225 230ctt gtc ggc cag acc atc gac gtg ccg ggc
gat ccg tca tcg acc gcc 832Leu Val Gly Gln Thr Ile Asp Val Pro Gly
Asp Pro Ser Ser Thr Ala 235 240 245ttc ccg ctc gtt gcc gcc ctt ctg
gtg gaa ggt tcc gac gtc acc atc 880Phe Pro Leu Val Ala Ala Leu Leu
Val Glu Gly Ser Asp Val Thr Ile250 255 260 265cgc aac gtg ctg atg
aac ccg acc cgt acc ggc ctc atc ctc acc ttg 928Arg Asn Val Leu Met
Asn Pro Thr Arg Thr Gly Leu Ile Leu Thr Leu 270 275 280cag gaa atg
ggc gcc gat atc gaa gtg ctc aat gcc cgt ctt gca ggc 976Gln Glu Met
Gly Ala Asp Ile Glu Val Leu Asn Ala Arg Leu Ala Gly 285 290 295ggc
gaa gac gtc gcc gat ctg cgc gtc agg gct tcg aag ctc aag ggc 1024Gly
Glu Asp Val Ala Asp Leu Arg Val Arg Ala Ser Lys Leu Lys Gly 300 305
310gtc gtc gtt ccg ccg gaa cgt gcg ccg tcg atg atc gac gaa tat ccg
1072Val Val Val Pro Pro Glu Arg Ala Pro Ser Met Ile Asp Glu Tyr Pro
315 320 325gtc ctg gcg att gcc gcc tcc ttc gcg gaa ggc gaa acc gtg
atg gac 1120Val Leu Ala Ile Ala Ala Ser Phe Ala Glu Gly Glu Thr Val
Met Asp330 335 340 345ggg ctc gac gaa ctg cgc gtc aag gaa tcg gat
cgt ctg gca gcg gtc 1168Gly Leu Asp Glu Leu Arg Val Lys Glu Ser Asp
Arg Leu Ala Ala Val 350 355 360gca cgc ggc ctt gaa gcc aac ggc gtc
gat tgc acc gaa ggc gag atg 1216Ala Arg Gly Leu Glu Ala Asn Gly Val
Asp Cys Thr Glu Gly Glu Met 365 370 375tcg ctg acg gtt cgc ggc cgc
ccc gac ggc aag gga ctg ggc ggc ggc 1264Ser Leu Thr Val Arg Gly Arg
Pro Asp Gly Lys Gly Leu Gly Gly Gly 380 385 390acg gtt gca acc cat
ctc gat cat cgt atc gcg atg agc ttc ctc gtg 1312Thr Val Ala Thr His
Leu Asp His Arg Ile Ala Met Ser Phe Leu Val 395 400 405atg ggc ctt
gcg gcg gaa aag ccg gtg acg gtt gac gac agt aac atg 1360Met Gly Leu
Ala Ala Glu Lys Pro Val Thr Val Asp Asp Ser Asn Met410 415 420
425atc gcc acg tcc ttc ccc gaa ttc atg gac atg atg ccg gga ttg ggc
1408Ile Ala Thr Ser Phe Pro Glu Phe Met Asp Met Met Pro Gly Leu Gly
430 435 440gca aag atc gag ttg agc ata ctc tagtcactcg acagcgaaaa
tattatttgc 1462Ala Lys Ile Glu Leu Ser Ile Leu 445gagattgggc
attattaccg gttggtctca gcgggggttt aatgtccaat cttccatacg
1522taacagcatc aggaaatatc aaaaaagctt tagaaggaat tgctagagca
gcgacgccgc 1582ctaagctttc tcaagacttc gttaaaactg tactgaaatc
ccggggggtc cggggatcaa 1642atgacttcat ttctgagaaa ttggcctcgc a
16735449PRTAgrobacterium sp. 5Met Ser His Ser Ala Ser Pro Lys Pro
Ala Thr Ala Arg Arg Ser Glu1 5 10 15Ala Leu Thr Gly Glu Ile Arg Ile
Pro Gly Asp Lys Ser Ile Ser His 20 25 30Arg Ser Phe Met Phe Gly Gly
Leu Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45Gly Leu Leu Glu Gly Glu
Asp Val Ile Asn Thr Gly Arg Ala Met Gln 50 55 60Ala Met Gly Ala Lys
Ile Arg Lys Glu Gly Asp Val Trp Ile Ile Asn65 70 75 80Gly Val Gly
Asn Gly Cys Leu Leu Gln Pro Glu Ala Ala Leu Asp Phe 85 90 95Gly Asn
Ala Gly Thr Gly Ala Arg Leu Thr Met Gly Leu Val Gly Thr 100 105
110Tyr Asp Met Lys Thr Ser Phe Ile Gly Asp Ala Ser Leu Ser Lys Arg
115 120 125Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val
Gln Val 130 135 140Glu Ala Ala Asp Gly Asp Arg Met Pro Leu Thr Leu
Ile Gly Pro Lys145 150 155 160Thr Ala Asn Pro Ile Thr Tyr Arg Val
Pro Met Ala Ser Ala Gln Val 165 170 175Lys Ser Ala Val Leu Leu Ala
Gly Leu Asn Thr Pro Gly Val Thr Thr 180 185 190Val Ile Glu Pro Val
Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 195 200 205Gly Phe Gly
Ala Asp Leu Thr Val Glu Thr Asp Lys Asp Gly Val Arg 210 215 220His
Ile Arg Ile Thr Gly Gln Gly Lys Leu Val Gly Gln Thr Ile Asp225 230
235 240Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala
Leu 245 250 255Leu Val Glu Gly Ser Asp Val Thr Ile Arg Asn Val Leu
Met Asn Pro 260 265 270Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu
Met Gly Ala Asp Ile
275 280 285Glu Val Leu Asn Ala Arg Leu Ala Gly Gly Glu Asp Val Ala
Asp Leu 290 295 300Arg Val Arg Ala Ser Lys Leu Lys Gly Val Val Val
Pro Pro Glu Arg305 310 315 320Ala Pro Ser Met Ile Asp Glu Tyr Pro
Val Leu Ala Ile Ala Ala Ser 325 330 335Phe Ala Glu Gly Glu Thr Val
Met Asp Gly Leu Asp Glu Leu Arg Val 340 345 350Lys Glu Ser Asp Arg
Leu Ala Ala Val Ala Arg Gly Leu Glu Ala Asn 355 360 365Gly Val Asp
Cys Thr Glu Gly Glu Met Ser Leu Thr Val Arg Gly Arg 370 375 380Pro
Asp Gly Lys Gly Leu Gly Gly Gly Thr Val Ala Thr His Leu Asp385 390
395 400His Arg Ile Ala Met Ser Phe Leu Val Met Gly Leu Ala Ala Glu
Lys 405 410 415Pro Val Thr Val Asp Asp Ser Asn Met Ile Ala Thr Ser
Phe Pro Glu 420 425 430Phe Met Asp Met Met Pro Gly Leu Gly Ala Lys
Ile Glu Leu Ser Ile 435 440 445Leu61500DNAPseudomonas
sp.CDS(34)..(1380) 6gtgatcgcgc caaaatgtga ctgtgaaaaa tcc atg tcc
cat tct gca tcc ccg 54 Met Ser His Ser Ala Ser Pro 1 5aaa cca gca
acc gcc cgc cgc tcg gag gca ctc acg ggc gaa atc cgc 102Lys Pro Ala
Thr Ala Arg Arg Ser Glu Ala Leu Thr Gly Glu Ile Arg 10 15 20att ccg
ggc gac aag tcc atc tcg cat cgc tcc ttc atg ttt ggc ggt 150Ile Pro
Gly Asp Lys Ser Ile Ser His Arg Ser Phe Met Phe Gly Gly 25 30 35ctc
gca tcg ggc gaa acc cgc atc acc ggc ctt ctg gaa ggc gag gac 198Leu
Ala Ser Gly Glu Thr Arg Ile Thr Gly Leu Leu Glu Gly Glu Asp40 45 50
55gtc atc aat aca ggc cgc gcc atg cag gcc atg ggc gcg aaa atc cgt
246Val Ile Asn Thr Gly Arg Ala Met Gln Ala Met Gly Ala Lys Ile Arg
60 65 70aaa gag ggc gat gtc tgg atc atc aac ggc gtc ggc aat ggc tgc
ctg 294Lys Glu Gly Asp Val Trp Ile Ile Asn Gly Val Gly Asn Gly Cys
Leu 75 80 85ttg cag ccc gaa gct gcg ctc gat ttc ggc aat gcc gga acc
ggc gcg 342Leu Gln Pro Glu Ala Ala Leu Asp Phe Gly Asn Ala Gly Thr
Gly Ala 90 95 100cgc ctc acc atg ggc ctt gtc ggc acc tat gac atg
aag acc tcc ttt 390Arg Leu Thr Met Gly Leu Val Gly Thr Tyr Asp Met
Lys Thr Ser Phe 105 110 115atc ggc gac gcc tcg ctg tcg aag cgc ccg
atg ggc cgc gtg ctg aac 438Ile Gly Asp Ala Ser Leu Ser Lys Arg Pro
Met Gly Arg Val Leu Asn120 125 130 135ccg ttg cgc gaa atg ggc gtt
cag gtg gaa gca gcc gat ggc gac cgc 486Pro Leu Arg Glu Met Gly Val
Gln Val Glu Ala Ala Asp Gly Asp Arg 140 145 150atg ccg ctg acg ctg
atc ggc ccg aag acg gcc aat ccg atc acc tat 534Met Pro Leu Thr Leu
Ile Gly Pro Lys Thr Ala Asn Pro Ile Thr Tyr 155 160 165cgc gtg ccg
atg gcc tcc gcg cag gta aaa tcc gcc gtg ctg ctc gcc 582Arg Val Pro
Met Ala Ser Ala Gln Val Lys Ser Ala Val Leu Leu Ala 170 175 180ggt
ctc aac acg ccg ggc gtc acc acc gtc atc gag ccg gtc atg acc 630Gly
Leu Asn Thr Pro Gly Val Thr Thr Val Ile Glu Pro Val Met Thr 185 190
195cgc gac cac acc gaa aag atg ctg cag ggc ttt ggc gcc gac ctc acg
678Arg Asp His Thr Glu Lys Met Leu Gln Gly Phe Gly Ala Asp Leu
Thr200 205 210 215gtc gag acc gac aag gat ggc gtg cgc cat atc cgc
atc acc ggc cag 726Val Glu Thr Asp Lys Asp Gly Val Arg His Ile Arg
Ile Thr Gly Gln 220 225 230ggc aag ctt gtc ggc cag acc atc gac gtg
ccg ggc gat ccg tca tcg 774Gly Lys Leu Val Gly Gln Thr Ile Asp Val
Pro Gly Asp Pro Ser Ser 235 240 245acc gcc ttc ccg ctc gtt gcc gcc
ctt ctg gtg gaa ggt tcc gac gtc 822Thr Ala Phe Pro Leu Val Ala Ala
Leu Leu Val Glu Gly Ser Asp Val 250 255 260acc atc cgc aac gtg ctg
atg aac ccg acc cgt acc ggc ctc atc ctc 870Thr Ile Arg Asn Val Leu
Met Asn Pro Thr Arg Thr Gly Leu Ile Leu 265 270 275acc ttg cag gaa
atg ggc gcc gat atc gaa gtg ctc aat gcc cgt ctt 918Thr Leu Gln Glu
Met Gly Ala Asp Ile Glu Val Leu Asn Ala Arg Leu280 285 290 295gca
ggc ggc gaa gac gtc gcc gat ctg cgc gtc agg gct tcg aag ctc 966Ala
Gly Gly Glu Asp Val Ala Asp Leu Arg Val Arg Ala Ser Lys Leu 300 305
310aag ggc gtc gtc gtt ccg ccg gaa cgt gcg ccg tcg atg atc gac gaa
1014Lys Gly Val Val Val Pro Pro Glu Arg Ala Pro Ser Met Ile Asp Glu
315 320 325tat ccg gtc ctg gcg att gcc gcc tcc ttc gcg gaa ggc gaa
acc gtg 1062Tyr Pro Val Leu Ala Ile Ala Ala Ser Phe Ala Glu Gly Glu
Thr Val 330 335 340atg gac ggg ctc gac gaa ctg cgc gtc aag gaa tcg
gat cgt ctg gca 1110Met Asp Gly Leu Asp Glu Leu Arg Val Lys Glu Ser
Asp Arg Leu Ala 345 350 355gcg gtc gca cgc ggc ctt gaa gcc aac ggc
gtc gat tgc acc gaa ggc 1158Ala Val Ala Arg Gly Leu Glu Ala Asn Gly
Val Asp Cys Thr Glu Gly360 365 370 375gag atg tcg ctg acg gtt cgc
ggc cgc ccc gac ggc aag gga ctg ggc 1206Glu Met Ser Leu Thr Val Arg
Gly Arg Pro Asp Gly Lys Gly Leu Gly 380 385 390ggc ggc acg gtt gca
acc cat ctc gat cat cgt atc gcg atg agc ttc 1254Gly Gly Thr Val Ala
Thr His Leu Asp His Arg Ile Ala Met Ser Phe 395 400 405ctc gtg atg
ggc ctt gcg gcg gaa aag ccg gtg acg gtt gac gac agt 1302Leu Val Met
Gly Leu Ala Ala Glu Lys Pro Val Thr Val Asp Asp Ser 410 415 420aac
atg atc gcc acg tcc ttc ccc gaa ttc atg gac atg atg ccg gga 1350Asn
Met Ile Ala Thr Ser Phe Pro Glu Phe Met Asp Met Met Pro Gly 425 430
435ttg ggc gca aag atc gag ttg agc ata ctc tagtcactcg acagcgaaaa
1400Leu Gly Ala Lys Ile Glu Leu Ser Ile Leu440 445tattatttgc
gagattgggc attattaccg gttggtctca gcgggggttt aatgtccaat
1460cttccatacg taacagcatc aggaaatatc aaaaaagctt
15007449PRTPseudomonas sp. 7Met Ser His Ser Ala Ser Pro Lys Pro Ala
Thr Ala Arg Arg Ser Glu1 5 10 15Ala Leu Thr Gly Glu Ile Arg Ile Pro
Gly Asp Lys Ser Ile Ser His 20 25 30Arg Ser Phe Met Phe Gly Gly Leu
Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45Gly Leu Leu Glu Gly Glu Asp
Val Ile Asn Thr Gly Arg Ala Met Gln 50 55 60Ala Met Gly Ala Lys Ile
Arg Lys Glu Gly Asp Val Trp Ile Ile Asn65 70 75 80Gly Val Gly Asn
Gly Cys Leu Leu Gln Pro Glu Ala Ala Leu Asp Phe 85 90 95Gly Asn Ala
Gly Thr Gly Ala Arg Leu Thr Met Gly Leu Val Gly Thr 100 105 110Tyr
Asp Met Lys Thr Ser Phe Ile Gly Asp Ala Ser Leu Ser Lys Arg 115 120
125Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu Met Gly Val Gln Val
130 135 140Glu Ala Ala Asp Gly Asp Arg Met Pro Leu Thr Leu Ile Gly
Pro Lys145 150 155 160Thr Ala Asn Pro Ile Thr Tyr Arg Val Pro Met
Ala Ser Ala Gln Val 165 170 175Lys Ser Ala Val Leu Leu Ala Gly Leu
Asn Thr Pro Gly Val Thr Thr 180 185 190Val Ile Glu Pro Val Met Thr
Arg Asp His Thr Glu Lys Met Leu Gln 195 200 205Gly Phe Gly Ala Asp
Leu Thr Val Glu Thr Asp Lys Asp Gly Val Arg 210 215 220His Ile Arg
Ile Thr Gly Gln Gly Lys Leu Val Gly Gln Thr Ile Asp225 230 235
240Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro Leu Val Ala Ala Leu
245 250 255Leu Val Glu Gly Ser Asp Val Thr Ile Arg Asn Val Leu Met
Asn Pro 260 265 270Thr Arg Thr Gly Leu Ile Leu Thr Leu Gln Glu Met
Gly Ala Asp Ile 275 280 285Glu Val Leu Asn Ala Arg Leu Ala Gly Gly
Glu Asp Val Ala Asp Leu 290 295 300Arg Val Arg Ala Ser Lys Leu Lys
Gly Val Val Val Pro Pro Glu Arg305 310 315 320Ala Pro Ser Met Ile
Asp Glu Tyr Pro Val Leu Ala Ile Ala Ala Ser 325 330 335Phe Ala Glu
Gly Glu Thr Val Met Asp Gly Leu Asp Glu Leu Arg Val 340 345 350Lys
Glu Ser Asp Arg Leu Ala Ala Val Ala Arg Gly Leu Glu Ala Asn 355 360
365Gly Val Asp Cys Thr Glu Gly Glu Met Ser Leu Thr Val Arg Gly Arg
370 375 380Pro Asp Gly Lys Gly Leu Gly Gly Gly Thr Val Ala Thr His
Leu Asp385 390 395 400His Arg Ile Ala Met Ser Phe Leu Val Met Gly
Leu Ala Ala Glu Lys 405 410 415Pro Val Thr Val Asp Asp Ser Asn Met
Ile Ala Thr Ser Phe Pro Glu 420 425 430Phe Met Asp Met Met Pro Gly
Leu Gly Ala Lys Ile Glu Leu Ser Ile 435 440
445Leu8423PRTEscherichia coli 8Ser Leu Thr Leu Gln Pro Ile Ala Arg
Val Asp Gly Thr Ile Asn Leu1 5 10 15Pro Gly Ser Lys Thr Val Ser Asn
Arg Ala Leu Leu Leu Ala Ala Leu 20 25 30Ala His Gly Lys Thr Val Leu
Thr Asn Leu Leu Asp Ser Asp Asp Val 35 40 45Arg His Met Leu Asn Ala
Leu Thr Ala Leu Gly Val Ser Tyr Thr Leu 50 55 60Ser Ala Asp Arg Thr
Arg Cys Glu Ile Ile Gly Asn Gly Gly Pro Leu65 70 75 80His Ala Glu
Gly Ala Leu Glu Leu Phe Leu Gly Asn Ala Gly Thr Ala 85 90 95Met Arg
Pro Leu Ala Ala Ala Leu Cys Leu Gly Ser Asn Asp Ile Val 100 105
110Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His Leu Val
115 120 125Asp Ala Leu Arg Leu Gly Gly Ala Lys Ile Thr Tyr Leu Glu
Gln Glu 130 135 140Asn Tyr Pro Pro Leu Arg Leu Gln Gly Gly Phe Thr
Gly Gly Asn Val145 150 155 160Asp Val Asp Gly Ser Val Ser Ser Gln
Phe Leu Thr Ala Leu Leu Met 165 170 175Thr Ala Pro Leu Ala Pro Glu
Asp Thr Val Ile Arg Ile Lys Gly Asp 180 185 190Leu Val Ser Lys Pro
Tyr Ile Asp Ile Thr Leu Asn Leu Met Lys Thr 195 200 205Phe Gly Val
Glu Ile Glu Asn Gln His Tyr Gln Gln Phe Val Val Lys 210 215 220Gly
Gly Gln Ser Tyr Gln Ser Pro Gly Thr Tyr Leu Val Glu Gly Asp225 230
235 240Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Ala Ala Ile Lys Gly
Gly 245 250 255Thr Val Lys Val Thr Gly Ile Gly Arg Asn Ser Met Gln
Gly Asp Ile 260 265 270Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala
Thr Ile Cys Trp Gly 275 280 285Asp Asp Tyr Ile Ser Cys Thr Arg Gly
Glu Leu Asn Ala Ile Asp Met 290 295 300Asp Met Asn His Ile Pro Asp
Ala Ala Met Thr Ile Ala Thr Ala Ala305 310 315 320Leu Phe Ala Lys
Gly Thr Thr Arg Leu Arg Asn Ile Tyr Asn Trp Arg 325 330 335Val Lys
Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu Arg Lys 340 345
350Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile Thr Pro
355 360 365Pro Glu Lys Leu Asn Phe Ala Glu Ile Ala Thr Tyr Asn Asp
His Arg 370 375 380Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp
Thr Pro Val Thr385 390 395 400Ile Leu Asp Pro Lys Cys Thr Ala Lys
Thr Phe Pro Asp Tyr Phe Glu 405 410 415Gln Leu Ala Arg Ile Ser Gln
42091377DNAArtificial sequenceSynthetic 9ccatggctca cggtgcaagc
agccgtccag caactgctcg taagtcctct ggtctttctg 60gaaccgtccg tattccaggt
gacaagtcta tctcccacag gtccttcatg tttggaggtc 120tcgctagcgg
tgaaactcgt atcaccggtc ttttggaagg tgaagatgtt atcaacactg
180gtaaggctat gcaagctatg ggtgccagaa tccgtaagga aggtgatact
tggatcattg 240atggtgttgg taacggtgga ctccttgctc ctgaggctcc
tctcgatttc ggtaacgctg 300caactggttg ccgtttgact atgggtcttg
ttggtgttta cgatttcgat agcactttca 360ttggtgacgc ttctctcact
aagcgtccaa tgggtcgtgt gttgaaccca cttcgcgaaa 420tgggtgtgca
ggtgaagtct gaagacggtg atcgtcttcc agttaccttg cgtggaccaa
480agactccaac gccaatcacc tacagggtac ctatggcttc cgctcaagtg
aagtccgctg 540ttctgcttgc tggtctcaac accccaggta tcaccactgt
tatcgagcca atcatgactc 600gtgaccacac tgaaaagatg cttcaaggtt
ttggtgctaa ccttaccgtt gagactgatg 660ctgacggtgt gcgtaccatc
cgtcttgaag gtcgtggtaa gctcaccggt caagtgattg 720atgttccagg
tgatccatcc tctactgctt tcccattggt tgctgccttg cttgttccag
780gttccgacgt caccatcctt aacgttttga tgaacccaac ccgtactggt
ctcatcttga 840ctctgcagga aatgggtgcc gacatcgaag tgatcaaccc
acgtcttgct ggtggagaag 900acgtggctga cttgcgtgtt cgttcttcta
ctttgaaggg tgttactgtt ccagaagacc 960gtgctccttc tatgatcgac
gagtatccaa ttctcgctgt tgcagctgca ttcgctgaag 1020gtgctaccgt
tatgaacggt ttggaagaac tccgtgttaa ggaaagcgac cgtctttctg
1080ctgtcgcaaa cggtctcaag ctcaacggtg ttgattgcga tgaaggtgag
acttctctcg 1140tcgtgcgtgg tcgtcctgac ggtaagggtc tcggtaacgc
ttctggagca gctgtcgcta 1200cccacctcga tcaccgtatc gctatgagct
tcctcgttat gggtctcgtt tctgaaaacc 1260ctgttactgt tgatgatgct
actatgatcg ctactagctt cccagagttc atggatttga 1320tggctggtct
tggagctaag atcgaactct ccgacactaa ggctgcttga tgagctc
137710318DNAArabidopsis thalianaCDS(87)..(317) 10agatctatcg
ataagcttga tgtaattgga ggaagatcaa aattttcaat ccccattctt 60cgattgcttc
aattgaagtt tctccg atg gcg caa gtt agc aga atc tgc aat 113 Met Ala
Gln Val Ser Arg Ile Cys Asn 1 5ggt gtg cag aac cca tct ctt atc tcc
aat ctc tcg aaa tcc agt caa 161Gly Val Gln Asn Pro Ser Leu Ile Ser
Asn Leu Ser Lys Ser Ser Gln10 15 20 25cgc aaa tct ccc tta tcg gtt
tct ctg aag acg cag cag cat cca cga 209Arg Lys Ser Pro Leu Ser Val
Ser Leu Lys Thr Gln Gln His Pro Arg 30 35 40gct tat ccg att tcg tcg
tcg tgg gga ttg aag aag agt ggg atg acg 257Ala Tyr Pro Ile Ser Ser
Ser Trp Gly Leu Lys Lys Ser Gly Met Thr 45 50 55tta att ggc tct gag
ctt cgt cct ctt aag gtc atg tct tct gtt tcc 305Leu Ile Gly Ser Glu
Leu Arg Pro Leu Lys Val Met Ser Ser Val Ser 60 65 70acg gcg tgc atg
c 318Thr Ala Cys Met 751177PRTArabidopsis thaliana 11Met Ala Gln
Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu1 5 10 15Ile Ser
Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20 25 30Ser
Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35 40
45Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg
50 55 60Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met65 70
7512402DNAArabidopsis thalianaCDS(87)..(401) 12agatctatcg
ataagcttga tgtaattgga ggaagatcaa aattttcaat ccccattctt 60cgattgcttc
aattgaagtt tctccg atg gcg caa gtt agc aga atc tgc aat 113 Met Ala
Gln Val Ser Arg Ile Cys Asn 1 5ggt gtg cag aac cca tct ctt atc tcc
aat ctc tcg aaa tcc agt caa 161Gly Val Gln Asn Pro Ser Leu Ile Ser
Asn Leu Ser Lys Ser Ser Gln10 15 20 25cgc aaa tct ccc tta tcg gtt
tct ctg aag acg cag cag cat cca cga 209Arg Lys Ser Pro Leu Ser Val
Ser Leu Lys Thr Gln Gln His Pro Arg 30 35 40gct tat ccg att tcg tcg
tcg tgg gga ttg aag aag agt ggg atg acg 257Ala Tyr Pro Ile Ser Ser
Ser Trp Gly Leu Lys Lys Ser Gly Met Thr 45 50 55tta att ggc tct gag
ctt cgt cct ctt aag gtc atg tct tct gtt tcc 305Leu Ile Gly Ser Glu
Leu Arg Pro Leu Lys Val Met Ser Ser Val Ser 60 65 70acg gcg gag aaa
gcg tcg gag att gta ctt caa ccc att aga gaa atc 353Thr Ala Glu Lys
Ala Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile 75 80 85tcc ggt ctt
att aag ttg cct ggc tcc aag tct cta tca aat aga att c 402Ser Gly
Leu Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile90 95
100
10513105PRTArabidopsis thaliana 13Met Ala Gln Val Ser Arg Ile Cys
Asn Gly Val Gln Asn Pro Ser Leu1 5 10 15Ile Ser Asn Leu Ser Lys Ser
Ser Gln Arg Lys Ser Pro Leu Ser Val 20 25 30Ser Leu Lys Thr Gln Gln
His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35 40 45Trp Gly Leu Lys Lys
Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50 55 60Pro Leu Lys Val
Met Ser Ser Val Ser Thr Ala Glu Lys Ala Ser Glu65 70 75 80Ile Val
Leu Gln Pro Ile Arg Glu Ile Ser Gly Leu Ile Lys Leu Pro 85 90 95Gly
Ser Lys Ser Leu Ser Asn Arg Ile 100 10514233DNAPetunia x
hybridaCDS(14)..(232) 14agatctttca aga atg gca caa att aac aac atg
gct caa ggg ata caa 49 Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile
Gln 1 5 10acc ctt aat ccc aat tcc aat ttc cat aaa ccc caa gtt cct
aaa tct 97Thr Leu Asn Pro Asn Ser Asn Phe His Lys Pro Gln Val Pro
Lys Ser 15 20 25tca agt ttt ctt gtt ttt gga tct aaa aaa ctg aaa aat
tca gca aat 145Ser Ser Phe Leu Val Phe Gly Ser Lys Lys Leu Lys Asn
Ser Ala Asn 30 35 40tct atg ttg gtt ttg aaa aaa gat tca att ttt atg
caa aag ttt tgt 193Ser Met Leu Val Leu Lys Lys Asp Ser Ile Phe Met
Gln Lys Phe Cys45 50 55 60tcc ttt agg att tca gca tca gtg gct aca
gcc tgc atg c 233Ser Phe Arg Ile Ser Ala Ser Val Ala Thr Ala Cys
Met 65 701573PRTPetunia x hybrida 15Met Ala Gln Ile Asn Asn Met Ala
Gln Gly Ile Gln Thr Leu Asn Pro1 5 10 15Asn Ser Asn Phe His Lys Pro
Gln Val Pro Lys Ser Ser Ser Phe Leu 20 25 30Val Phe Gly Ser Lys Lys
Leu Lys Asn Ser Ala Asn Ser Met Leu Val 35 40 45Leu Lys Lys Asp Ser
Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 50 55 60Ser Ala Ser Val
Ala Thr Ala Cys Met65 7016352DNAPetunia x hybridaCDS(49)..(351)
16agatctgcta gaaataattt tgtttaactt taagaaggag atatatcc atg gca caa
57 Met Ala Gln 1att aac aac atg gct caa ggg ata caa acc ctt aat ccc
aat tcc aat 105Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro
Asn Ser Asn 5 10 15ttc cat aaa ccc caa gtt cct aaa tct tca agt ttt
ctt gtt ttt gga 153Phe His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe
Leu Val Phe Gly20 25 30 35tct aaa aaa ctg aaa aat tca gca aat tct
atg ttg gtt ttg aaa aaa 201Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser
Met Leu Val Leu Lys Lys 40 45 50gat tca att ttt atg caa aag ttt tgt
tcc ttt agg att tca gca tca 249Asp Ser Ile Phe Met Gln Lys Phe Cys
Ser Phe Arg Ile Ser Ala Ser 55 60 65gtg gct aca gca cag aag cct tct
gag ata gtg ttg caa ccc att aaa 297Val Ala Thr Ala Gln Lys Pro Ser
Glu Ile Val Leu Gln Pro Ile Lys 70 75 80gag att tca ggc act gtt aaa
ttg cct ggc tct aaa tca tta tct aat 345Glu Ile Ser Gly Thr Val Lys
Leu Pro Gly Ser Lys Ser Leu Ser Asn 85 90 95aga att c 352Arg
Ile10017101PRTPetunia x hybrida 17Met Ala Gln Ile Asn Asn Met Ala
Gln Gly Ile Gln Thr Leu Asn Pro1 5 10 15Asn Ser Asn Phe His Lys Pro
Gln Val Pro Lys Ser Ser Ser Phe Leu 20 25 30Val Phe Gly Ser Lys Lys
Leu Lys Asn Ser Ala Asn Ser Met Leu Val 35 40 45Leu Lys Lys Asp Ser
Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 50 55 60Ser Ala Ser Val
Ala Thr Ala Gln Lys Pro Ser Glu Ile Val Leu Gln65 70 75 80Pro Ile
Lys Glu Ile Ser Gly Thr Val Lys Leu Pro Gly Ser Lys Ser 85 90 95Leu
Ser Asn Arg Ile 1001828PRTAgrobacterium sp.UNSURE(1)..(18)Xaa =
Unknown 18Xaa His Gly Ala Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser
Ser Gly1 5 10 15Leu Xaa Gly Thr Val Arg Ile Pro Gly Asp Lys Met 20
251913PRTAgrobacterium sp. 19Ala Pro Ser Met Ile Asp Glu Tyr Pro
Ile Leu Ala Val1 5 102015PRTAgrobacterium sp. 20Ile Thr Gly Leu Leu
Glu Gly Glu Asp Val Ile Asn Thr Gly Lys1 5 10 152117DNAArtificial
sequenceSynthetic 21atgathgayg artaycc 172217DNAArtificial
sequenceSynthetic 22gargaygtna thaacac 172317DNAArtificial
sequenceSynthetic 23gargaygtna thaatac 172438DNAArtificial
sequenceOligonucleotide 24cgtggataga tctaggaaga caaccatggc tcacggtc
382544DNAArtificial sequenceOligonucleotide 25ggatagatta aggaagacgc
gcatgcttca cggtgcaagc agcc 442635DNAArtificial
sequenceOligonucleotide 26ggctgcctga tgagctccac aatcgccatc gatgg
352732DNAArtificial sequenceOligonucleotide 27cgtcgctcgt cgtgcgtggc
cgccctgacg gc 322829DNAArtificial sequenceOligonucleotide
28cgggcaaggc catgcaggct atgggcgcc 292931DNAArtificial
sequenceOligonucleotide 29cgggctgccg cctgactatg ggcctcgtcg g
313015PRTPseudomonas sp.NON_CONS(1)..(1)Xaa = unknown 30Xaa His Ser
Ala Ser Pro Lys Pro Ala Thr Ala Arg Arg Ser Glu1 5 10
153117DNAArtificial sequenceOligonucleotide 31gcggtbgcsg gyttsgg
173216PRTArtificial sequenceSynthetic 32Pro Gly Asp Lys Ser Ile Ser
His Arg Ser Phe Met Phe Gly Gly Leu1 5 10 153313PRTArtificial
sequenceOligonucleotide 33Leu Asp Phe Gly Asn Ala Ala Thr Gly Cys
Arg Leu Thr1 5 103426DNAArtificial sequenceOligonucleotide
34cggcaatgcc gccaccggcg cgcgcc 263549DNAArtificial
sequenceOligonucleotide 35ggacggctgc ttgcaccgtg aagcatgctt
aagcttggcg taatcatgg 493635DNAArtificial sequenceOligonucleotide
36ggaagacgcc cagaattcac ggtgcaagca gccgg 35375PRTArtificial
sequenceSynthetic 37Arg Xaa His Xaa Glu1 5384PRTArtificial
sequenceSynthetic 38Gly Asp Lys Xaa1395PRTArtificial
sequenceSynthetic 39Ser Ala Gln Xaa Lys1 5404PRTArtificial
sequenceSynthetic 40Asn Xaa Thr Arg1411287DNABacillus
subtilisCDS(1)..(1287) 41atg aaa cga gat aag gtg cag acc tta cat
gga gaa ata cat att ccc 48Met Lys Arg Asp Lys Val Gln Thr Leu His
Gly Glu Ile His Ile Pro1 5 10 15ggt gat aaa tcc att tct cac cgc tct
gtt atg ttt ggc gcg cta gcg 96Gly Asp Lys Ser Ile Ser His Arg Ser
Val Met Phe Gly Ala Leu Ala 20 25 30gca ggc aca aca aca gtt aaa aac
ttt ctg ccg gga gca gat tgt ctg 144Ala Gly Thr Thr Thr Val Lys Asn
Phe Leu Pro Gly Ala Asp Cys Leu 35 40 45agc acg atc gat tgc ttt aga
aaa atg ggt gtt cac att gag caa agc 192Ser Thr Ile Asp Cys Phe Arg
Lys Met Gly Val His Ile Glu Gln Ser 50 55 60agc agc gat gtc gtg att
cac gga aaa gga atc gat gcc ctg aaa gag 240Ser Ser Asp Val Val Ile
His Gly Lys Gly Ile Asp Ala Leu Lys Glu65 70 75 80cca gaa agc ctt
tta gat gtc gga aat tca ggt aca acg att cgc ctg 288Pro Glu Ser Leu
Leu Asp Val Gly Asn Ser Gly Thr Thr Ile Arg Leu 85 90 95atg ctc gga
ata ttg gcg ggc cgt cct ttt tac agc gcg gta gcc gga 336Met Leu Gly
Ile Leu Ala Gly Arg Pro Phe Tyr Ser Ala Val Ala Gly 100 105 110gat
gag agc att gcg aaa cgc cca atg aag cgt gtg act gag cct ttg 384Asp
Glu Ser Ile Ala Lys Arg Pro Met Lys Arg Val Thr Glu Pro Leu 115 120
125aaa aaa atg ggg gct aaa atc gac ggc aga gcc ggc gga gag ttt aca
432Lys Lys Met Gly Ala Lys Ile Asp Gly Arg Ala Gly Gly Glu Phe Thr
130 135 140ccg ctg tca gtg agc ggc gct tca tta aaa gga att gat tat
gta tca 480Pro Leu Ser Val Ser Gly Ala Ser Leu Lys Gly Ile Asp Tyr
Val Ser145 150 155 160cct gtt gca agc gcg caa att aaa tct gct gtt
ttg ctg gcc gga tta 528Pro Val Ala Ser Ala Gln Ile Lys Ser Ala Val
Leu Leu Ala Gly Leu 165 170 175cag gct gag ggc aca aca act gta aca
gag ccc cat aaa tct cgg gac 576Gln Ala Glu Gly Thr Thr Thr Val Thr
Glu Pro His Lys Ser Arg Asp 180 185 190cac act gag cgg atg ctt tct
gct ttt ggc gtt aag ctt tct gaa gat 624His Thr Glu Arg Met Leu Ser
Ala Phe Gly Val Lys Leu Ser Glu Asp 195 200 205caa acg agt gtt tcc
att gct ggt ggc cag aaa ctg aca gct gct gat 672Gln Thr Ser Val Ser
Ile Ala Gly Gly Gln Lys Leu Thr Ala Ala Asp 210 215 220att ttt gtt
cct gga gac att tct tca gcc gcg ttt ttc ctt gct gct 720Ile Phe Val
Pro Gly Asp Ile Ser Ser Ala Ala Phe Phe Leu Ala Ala225 230 235
240ggc gcg atg gtt cca aac agc aga att gta ttg aaa aac gta ggt tta
768Gly Ala Met Val Pro Asn Ser Arg Ile Val Leu Lys Asn Val Gly Leu
245 250 255aat ccg act cgg aca ggt att att gat gtc ctt caa aac atg
ggg gca 816Asn Pro Thr Arg Thr Gly Ile Ile Asp Val Leu Gln Asn Met
Gly Ala 260 265 270aaa ctt gaa atc aaa cca tct gct gat agc ggt gca
gag cct tat gga 864Lys Leu Glu Ile Lys Pro Ser Ala Asp Ser Gly Ala
Glu Pro Tyr Gly 275 280 285gat ttg att ata gaa acg tca tct cta aag
gca gtt gaa atc gga gga 912Asp Leu Ile Ile Glu Thr Ser Ser Leu Lys
Ala Val Glu Ile Gly Gly 290 295 300gat atc att ccg cgt tta att gat
gag atc cct atc atc gcg ctt ctt 960Asp Ile Ile Pro Arg Leu Ile Asp
Glu Ile Pro Ile Ile Ala Leu Leu305 310 315 320gcg act cag gcg gaa
gga acc acc gtt att aag gac gcg gca gag cta 1008Ala Thr Gln Ala Glu
Gly Thr Thr Val Ile Lys Asp Ala Ala Glu Leu 325 330 335aaa gtg aaa
gaa aca aac cgt att gat act gtt gtt tct gag ctt cgc 1056Lys Val Lys
Glu Thr Asn Arg Ile Asp Thr Val Val Ser Glu Leu Arg 340 345 350aag
ctg ggt gct gaa att gaa ccg aca gca gat gga atg aag gtt tat 1104Lys
Leu Gly Ala Glu Ile Glu Pro Thr Ala Asp Gly Met Lys Val Tyr 355 360
365ggc aaa caa acg ttg aaa ggc ggc gct gca gtg tcc agc cac gga gat
1152Gly Lys Gln Thr Leu Lys Gly Gly Ala Ala Val Ser Ser His Gly Asp
370 375 380cat cga atc gga atg atg ctt ggt att gct tcc tgt ata acg
gag gag 1200His Arg Ile Gly Met Met Leu Gly Ile Ala Ser Cys Ile Thr
Glu Glu385 390 395 400ccg att gaa atc gag cac acg gat gcc att cac
gtt tct tat cca acc 1248Pro Ile Glu Ile Glu His Thr Asp Ala Ile His
Val Ser Tyr Pro Thr 405 410 415ttc ttc gag cat tta aat aag ctt tcg
aaa aaa tcc tga 1287Phe Phe Glu His Leu Asn Lys Leu Ser Lys Lys Ser
420 42542428PRTBacillus subtilis 42Met Lys Arg Asp Lys Val Gln Thr
Leu His Gly Glu Ile His Ile Pro1 5 10 15Gly Asp Lys Ser Ile Ser His
Arg Ser Val Met Phe Gly Ala Leu Ala 20 25 30Ala Gly Thr Thr Thr Val
Lys Asn Phe Leu Pro Gly Ala Asp Cys Leu 35 40 45Ser Thr Ile Asp Cys
Phe Arg Lys Met Gly Val His Ile Glu Gln Ser 50 55 60Ser Ser Asp Val
Val Ile His Gly Lys Gly Ile Asp Ala Leu Lys Glu65 70 75 80Pro Glu
Ser Leu Leu Asp Val Gly Asn Ser Gly Thr Thr Ile Arg Leu 85 90 95Met
Leu Gly Ile Leu Ala Gly Arg Pro Phe Tyr Ser Ala Val Ala Gly 100 105
110Asp Glu Ser Ile Ala Lys Arg Pro Met Lys Arg Val Thr Glu Pro Leu
115 120 125Lys Lys Met Gly Ala Lys Ile Asp Gly Arg Ala Gly Gly Glu
Phe Thr 130 135 140Pro Leu Ser Val Ser Gly Ala Ser Leu Lys Gly Ile
Asp Tyr Val Ser145 150 155 160Pro Val Ala Ser Ala Gln Ile Lys Ser
Ala Val Leu Leu Ala Gly Leu 165 170 175Gln Ala Glu Gly Thr Thr Thr
Val Thr Glu Pro His Lys Ser Arg Asp 180 185 190His Thr Glu Arg Met
Leu Ser Ala Phe Gly Val Lys Leu Ser Glu Asp 195 200 205Gln Thr Ser
Val Ser Ile Ala Gly Gly Gln Lys Leu Thr Ala Ala Asp 210 215 220Ile
Phe Val Pro Gly Asp Ile Ser Ser Ala Ala Phe Phe Leu Ala Ala225 230
235 240Gly Ala Met Val Pro Asn Ser Arg Ile Val Leu Lys Asn Val Gly
Leu 245 250 255Asn Pro Thr Arg Thr Gly Ile Ile Asp Val Leu Gln Asn
Met Gly Ala 260 265 270Lys Leu Glu Ile Lys Pro Ser Ala Asp Ser Gly
Ala Glu Pro Tyr Gly 275 280 285Asp Leu Ile Ile Glu Thr Ser Ser Leu
Lys Ala Val Glu Ile Gly Gly 290 295 300Asp Ile Ile Pro Arg Leu Ile
Asp Glu Ile Pro Ile Ile Ala Leu Leu305 310 315 320Ala Thr Gln Ala
Glu Gly Thr Thr Val Ile Lys Asp Ala Ala Glu Leu 325 330 335Lys Val
Lys Glu Thr Asn Arg Ile Asp Thr Val Val Ser Glu Leu Arg 340 345
350Lys Leu Gly Ala Glu Ile Glu Pro Thr Ala Asp Gly Met Lys Val Tyr
355 360 365Gly Lys Gln Thr Leu Lys Gly Gly Ala Ala Val Ser Ser His
Gly Asp 370 375 380His Arg Ile Gly Met Met Leu Gly Ile Ala Ser Cys
Ile Thr Glu Glu385 390 395 400Pro Ile Glu Ile Glu His Thr Asp Ala
Ile His Val Ser Tyr Pro Thr 405 410 415Phe Phe Glu His Leu Asn Lys
Leu Ser Lys Lys Ser 420 425431293DNAStaphylococcus
aureusCDS(1)..(1293) 43atg gta aat gaa caa atc att gat att tca ggt
ccg tta aag ggc gaa 48Met Val Asn Glu Gln Ile Ile Asp Ile Ser Gly
Pro Leu Lys Gly Glu1 5 10 15ata gaa gtg ccg ggc gat aag tca atg aca
cac cgt gca atc atg ttg 96Ile Glu Val Pro Gly Asp Lys Ser Met Thr
His Arg Ala Ile Met Leu 20 25 30gcg tcg cta gct gaa ggt gta tct act
ata tat aag cca cta ctt ggc 144Ala Ser Leu Ala Glu Gly Val Ser Thr
Ile Tyr Lys Pro Leu Leu Gly 35 40 45gaa gat tgt cgt cgt acg atg gac
att ttc cga cac tta ggt gta gaa 192Glu Asp Cys Arg Arg Thr Met Asp
Ile Phe Arg His Leu Gly Val Glu 50 55 60atc aaa gaa gat gat gaa aaa
tta gtt gtg act tcc cca gga tat caa 240Ile Lys Glu Asp Asp Glu Lys
Leu Val Val Thr Ser Pro Gly Tyr Gln65 70 75 80gtt aac acg cca cat
caa gta ttg tat aca ggt aat tct ggt acg aca 288Val Asn Thr Pro His
Gln Val Leu Tyr Thr Gly Asn Ser Gly Thr Thr 85 90 95aca cga tta ttg
gca ggt ttg tta agt ggt tta ggt aat gaa agt gtt 336Thr Arg Leu Leu
Ala Gly Leu Leu Ser Gly Leu Gly Asn Glu Ser Val 100 105 110ttg tct
ggc gat gtt tca att ggt aaa agg cca atg gat cgt gtc ttg 384Leu Ser
Gly Asp Val Ser Ile Gly Lys Arg Pro Met Asp Arg Val Leu 115 120
125aga cca ttg aaa ctt atg gat gcg aat att gaa ggt att gaa gat aat
432Arg Pro Leu Lys Leu Met Asp Ala Asn Ile Glu Gly Ile Glu Asp Asn
130 135 140tat aca cca tta att att aag cca tct gtc ata aaa ggt ata
aat tat 480Tyr Thr Pro Leu Ile Ile Lys Pro Ser Val Ile Lys Gly Ile
Asn Tyr145 150 155 160caa atg gaa gtt gca agt gca caa gta aaa agt
gcc att tta ttt gca 528Gln Met Glu Val Ala Ser Ala Gln Val Lys Ser
Ala Ile Leu Phe Ala 165 170 175agt ttg ttt tct aag gaa ccg acc atc
att aaa gaa tta gat gta agt 576Ser Leu Phe Ser Lys Glu Pro Thr Ile
Ile Lys Glu Leu Asp Val Ser 180 185 190cga aat cat act gag acg atg
ttc aaa cat ttt aat att cca att gaa 624Arg Asn His Thr Glu Thr Met
Phe Lys His Phe Asn Ile Pro Ile Glu 195 200 205gca gaa ggg tta tca
att aat aca acc cct gaa gca att cga tac att 672Ala Glu Gly Leu Ser
Ile Asn
Thr Thr Pro Glu Ala Ile Arg Tyr Ile 210 215 220aaa cct gca gat ttt
cat gtt cct ggc gat att tca tct gca gcg ttc 720Lys Pro Ala Asp Phe
His Val Pro Gly Asp Ile Ser Ser Ala Ala Phe225 230 235 240ttt att
gtt gca gca ctt atc aca cca gga agt gat gta aca att cat 768Phe Ile
Val Ala Ala Leu Ile Thr Pro Gly Ser Asp Val Thr Ile His 245 250
255aat gtt gga atc aat caa aca cgt tca ggt att att gat att gtt gaa
816Asn Val Gly Ile Asn Gln Thr Arg Ser Gly Ile Ile Asp Ile Val Glu
260 265 270aaa atg ggc ggt aat atc caa ctt ttc aat caa aca act ggt
gct gaa 864Lys Met Gly Gly Asn Ile Gln Leu Phe Asn Gln Thr Thr Gly
Ala Glu 275 280 285cct act gct tct att cgt att caa tac aca cca atg
ctt caa cca ata 912Pro Thr Ala Ser Ile Arg Ile Gln Tyr Thr Pro Met
Leu Gln Pro Ile 290 295 300aca atc gaa gga gaa tta gtt cca aaa gca
att gat gaa ctg cct gta 960Thr Ile Glu Gly Glu Leu Val Pro Lys Ala
Ile Asp Glu Leu Pro Val305 310 315 320ata gca tta ctt tgt aca caa
gca gtt ggc acg agt aca att aaa gat 1008Ile Ala Leu Leu Cys Thr Gln
Ala Val Gly Thr Ser Thr Ile Lys Asp 325 330 335gcc gag gaa tta aaa
gta aaa gaa aca aat aga att gat aca acg gct 1056Ala Glu Glu Leu Lys
Val Lys Glu Thr Asn Arg Ile Asp Thr Thr Ala 340 345 350gat atg tta
aac ttg tta ggg ttt gaa tta caa cca act aat gat gga 1104Asp Met Leu
Asn Leu Leu Gly Phe Glu Leu Gln Pro Thr Asn Asp Gly 355 360 365ttg
att att cat ccg tca gaa ttt aaa aca aat gca aca gat att tta 1152Leu
Ile Ile His Pro Ser Glu Phe Lys Thr Asn Ala Thr Asp Ile Leu 370 375
380act gat cat cga ata gga atg atg ctt gca gtt gct tgt gta ctt tca
1200Thr Asp His Arg Ile Gly Met Met Leu Ala Val Ala Cys Val Leu
Ser385 390 395 400agc gag cct gtc aaa atc aaa caa ttt gat gct gta
aat gta tca ttt 1248Ser Glu Pro Val Lys Ile Lys Gln Phe Asp Ala Val
Asn Val Ser Phe 405 410 415cca gga ttt tta cca aaa cta aag ctt tta
caa aat gag gga taa 1293Pro Gly Phe Leu Pro Lys Leu Lys Leu Leu Gln
Asn Glu Gly 420 425 43044430PRTStaphylococcus aureus 44Met Val Asn
Glu Gln Ile Ile Asp Ile Ser Gly Pro Leu Lys Gly Glu1 5 10 15Ile Glu
Val Pro Gly Asp Lys Ser Met Thr His Arg Ala Ile Met Leu 20 25 30Ala
Ser Leu Ala Glu Gly Val Ser Thr Ile Tyr Lys Pro Leu Leu Gly 35 40
45Glu Asp Cys Arg Arg Thr Met Asp Ile Phe Arg His Leu Gly Val Glu
50 55 60Ile Lys Glu Asp Asp Glu Lys Leu Val Val Thr Ser Pro Gly Tyr
Gln65 70 75 80Val Asn Thr Pro His Gln Val Leu Tyr Thr Gly Asn Ser
Gly Thr Thr 85 90 95Thr Arg Leu Leu Ala Gly Leu Leu Ser Gly Leu Gly
Asn Glu Ser Val 100 105 110Leu Ser Gly Asp Val Ser Ile Gly Lys Arg
Pro Met Asp Arg Val Leu 115 120 125Arg Pro Leu Lys Leu Met Asp Ala
Asn Ile Glu Gly Ile Glu Asp Asn 130 135 140Tyr Thr Pro Leu Ile Ile
Lys Pro Ser Val Ile Lys Gly Ile Asn Tyr145 150 155 160Gln Met Glu
Val Ala Ser Ala Gln Val Lys Ser Ala Ile Leu Phe Ala 165 170 175Ser
Leu Phe Ser Lys Glu Pro Thr Ile Ile Lys Glu Leu Asp Val Ser 180 185
190Arg Asn His Thr Glu Thr Met Phe Lys His Phe Asn Ile Pro Ile Glu
195 200 205Ala Glu Gly Leu Ser Ile Asn Thr Thr Pro Glu Ala Ile Arg
Tyr Ile 210 215 220Lys Pro Ala Asp Phe His Val Pro Gly Asp Ile Ser
Ser Ala Ala Phe225 230 235 240Phe Ile Val Ala Ala Leu Ile Thr Pro
Gly Ser Asp Val Thr Ile His 245 250 255Asn Val Gly Ile Asn Gln Thr
Arg Ser Gly Ile Ile Asp Ile Val Glu 260 265 270Lys Met Gly Gly Asn
Ile Gln Leu Phe Asn Gln Thr Thr Gly Ala Glu 275 280 285Pro Thr Ala
Ser Ile Arg Ile Gln Tyr Thr Pro Met Leu Gln Pro Ile 290 295 300Thr
Ile Glu Gly Glu Leu Val Pro Lys Ala Ile Asp Glu Leu Pro Val305 310
315 320Ile Ala Leu Leu Cys Thr Gln Ala Val Gly Thr Ser Thr Ile Lys
Asp 325 330 335Ala Glu Glu Leu Lys Val Lys Glu Thr Asn Arg Ile Asp
Thr Thr Ala 340 345 350Asp Met Leu Asn Leu Leu Gly Phe Glu Leu Gln
Pro Thr Asn Asp Gly 355 360 365Leu Ile Ile His Pro Ser Glu Phe Lys
Thr Asn Ala Thr Asp Ile Leu 370 375 380Thr Asp His Arg Ile Gly Met
Met Leu Ala Val Ala Cys Val Leu Ser385 390 395 400Ser Glu Pro Val
Lys Ile Lys Gln Phe Asp Ala Val Asn Val Ser Phe 405 410 415Pro Gly
Phe Leu Pro Lys Leu Lys Leu Leu Gln Asn Glu Gly 420 425
4304528DNAArtificial sequenceOligonucleotide 45ggaacatatg
aaacgagata aggtgcag 284635DNAArtificial sequenceOligonucleotide
46ggaattcaaa cttcaggatc ttgagataga aaatg 354728DNAArtificial
sequenceOligonucleotide 47ggggccatgg taaatgaaca aatcattg
284833DNAArtificial sequenceOligonucleotide 48gggggagctc attatccctc
attttgtaaa agc 3349480PRTSaccharomyces cerevisiae 49Leu Thr Asp Glu
Thr Leu Val Tyr Pro Phe Lys Asp Ile Pro Ala Asp1 5 10 15Gln Gln Lys
Val Val Ile Pro Pro Gly Ser Lys Ser Ile Ser Asn Arg 20 25 30Ala Leu
Ile Leu Ala Ala Leu Gly Glu Gly Gln Cys Lys Ile Lys Asn 35 40 45Leu
Leu His Ser Asp Asp Thr Lys His Met Leu Thr Ala Val His Glu 50 55
60Leu Lys Gly Ala Thr Ile Ser Trp Glu Asp Asn Gly Glu Thr Val Val65
70 75 80Val Glu Gly His Gly Gly Ser Thr Leu Ser Ala Cys Ala Asp Pro
Leu 85 90 95Tyr Leu Gly Asn Ala Gly Thr Ala Ser Arg Phe Leu Thr Ser
Leu Ala 100 105 110Ala Leu Val Asn Ser Thr Ser Ser Gln Lys Tyr Ile
Val Leu Thr Gly 115 120 125Asn Ala Arg Met Gln Gln Arg Pro Ile Ala
Pro Leu Val Asp Ser Leu 130 135 140Arg Ala Asn Gly Thr Lys Ile Glu
Tyr Leu Asn Asn Glu Gly Ser Leu145 150 155 160Pro Ile Lys Val Tyr
Thr Asp Ser Val Phe Lys Gly Gly Arg Ile Glu 165 170 175Leu Ala Ala
Thr Val Ser Ser Gln Tyr Val Ser Ser Ile Leu Met Cys 180 185 190Ala
Pro Tyr Ala Glu Glu Pro Val Thr Leu Ala Leu Val Gly Gly Lys 195 200
205Pro Ile Ser Lys Leu Tyr Val Asp Met Thr Ile Lys Met Met Glu Lys
210 215 220Phe Gly Ile Asn Val Glu Thr Ser Thr Thr Glu Pro Tyr Thr
Tyr Tyr225 230 235 240Ile Pro Lys Gly His Tyr Ile Asn Pro Ser Glu
Tyr Val Ile Glu Ser 245 250 255Asp Ala Ser Ser Ala Thr Tyr Pro Leu
Ala Phe Ala Ala Met Thr Gly 260 265 270Thr Thr Val Thr Val Pro Asn
Ile Gly Phe Glu Ser Leu Gln Gly Asp 275 280 285Ala Arg Phe Ala Arg
Asp Val Leu Lys Pro Met Gly Cys Lys Ile Thr 290 295 300Gln Thr Ala
Thr Ser Thr Thr Val Ser Gly Pro Pro Val Gly Thr Leu305 310 315
320Lys Pro Leu Lys His Val Asp Met Glu Pro Met Thr Asp Ala Phe Leu
325 330 335Thr Ala Cys Val Val Ala Ala Ile Ser His Asp Ser Asp Pro
Asn Ser 340 345 350Ala Asn Thr Thr Thr Ile Glu Gly Ile Ala Asn Gln
Arg Val Lys Glu 355 360 365Cys Asn Arg Ile Leu Ala Met Ala Thr Glu
Leu Ala Lys Phe Gly Val 370 375 380Lys Thr Thr Glu Leu Pro Asp Gly
Ile Gln Val His Gly Leu Asn Ser385 390 395 400Ile Lys Asp Leu Lys
Val Pro Ser Asp Ser Ser Gly Pro Val Gly Val 405 410 415Cys Thr Tyr
Asp Asp His Arg Val Ala Met Ser Phe Ser Leu Leu Ala 420 425 430Gly
Met Val Asn Ser Gln Asn Glu Arg Asp Glu Val Ala Asn Pro Val 435 440
445Arg Ile Leu Glu Arg His Cys Thr Gly Lys Thr Trp Pro Gly Trp Trp
450 455 460Asp Val Leu His Ser Glu Leu Gly Ala Lys Leu Asp Gly Ala
Glu Pro465 470 475 48050460PRTAspergillus ridulaus 50Leu Ala Pro
Ser Ile Glu Val His Pro Gly Val Ala His Ser Ser Asn1 5 10 15Val Ile
Cys Ala Pro Pro Gly Ser Lys Ser Ile Ser Asn Arg Ala Leu 20 25 30Val
Leu Ala Ala Leu Gly Ser Gly Thr Cys Arg Ile Lys Asn Leu Leu 35 40
45His Ser Asp Asp Thr Glu Val Met Leu Asn Ala Leu Glu Arg Leu Gly
50 55 60Ala Ala Thr Phe Ser Trp Glu Glu Glu Gly Glu Val Leu Val Val
Asn65 70 75 80Gly Lys Gly Gly Asn Leu Gln Ala Ser Ser Ser Pro Leu
Tyr Leu Gly 85 90 95Asn Ala Gly Thr Ala Ser Arg Phe Leu Thr Thr Val
Ala Thr Leu Ala 100 105 110Asn Ser Ser Thr Val Asp Ser Ser Val Leu
Thr Gly Asn Asn Arg Met 115 120 125Lys Gln Arg Pro Ile Gly Asp Leu
Val Asp Ala Leu Thr Ala Asn Val 130 135 140Leu Pro Leu Asn Thr Ser
Lys Gly Arg Ala Ser Leu Pro Leu Lys Ile145 150 155 160Ala Ala Ser
Gly Gly Phe Ala Gly Gly Asn Ile Asn Leu Ala Ala Lys 165 170 175Val
Ser Ser Gln Tyr Val Ser Ser Leu Leu Met Cys Ala Pro Tyr Ala 180 185
190Lys Glu Pro Val Thr Leu Arg Leu Val Gly Gly Lys Pro Ile Ser Gln
195 200 205Pro Tyr Ile Asp Met Thr Thr Ala Met Met Arg Ser Phe Gly
Ile Asp 210 215 220Val Gln Lys Ser Thr Thr Glu Glu His Thr Tyr His
Ile Pro Gln Gly225 230 235 240Arg Tyr Val Asn Pro Ala Glu Tyr Val
Ile Glu Ser Asp Ala Ser Cys 245 250 255Ala Thr Tyr Pro Leu Ala Val
Ala Ala Val Thr Gly Thr Thr Cys Thr 260 265 270Val Pro Asn Ile Gly
Ser Ala Ser Leu Gln Gly Asp Ala Arg Phe Ala 275 280 285Val Glu Val
Leu Arg Pro Met Gly Cys Thr Val Glu Gln Thr Glu Thr 290 295 300Ser
Thr Thr Val Thr Gly Pro Ser Asp Gly Ile Leu Arg Ala Thr Ser305 310
315 320Lys Arg Gly Tyr Gly Thr Asn Asp Arg Cys Val Pro Arg Cys Phe
Arg 325 330 335Thr Gly Ser His Arg Pro Met Glu Lys Ser Gln Thr Thr
Pro Pro Val 340 345 350Ser Ser Gly Ile Ala Asn Gln Arg Val Lys Glu
Cys Asn Arg Ile Lys 355 360 365Ala Met Lys Asp Glu Leu Ala Lys Phe
Gly Val Ile Cys Arg Glu His 370 375 380Asp Asp Gly Leu Glu Ile Asp
Gly Ile Asp Arg Ser Asn Leu Arg Gln385 390 395 400Pro Val Gly Gly
Val Phe Cys Tyr Asp Asp His Arg Val Ala Phe Ser 405 410 415Phe Ser
Val Leu Ser Leu Val Thr Pro Gln Pro Thr Leu Ile Leu Glu 420 425
430Lys Glu Cys Val Gly Lys Thr Trp Pro Gly Trp Trp Asp Thr Leu Arg
435 440 445Gln Leu Phe Lys Val Lys Leu Glu Gly Lys Glu Leu 450 455
46051444PRTBrassica napus 51Lys Ala Ser Glu Ile Val Leu Gln Pro Ile
Arg Glu Ile Ser Gly Leu1 5 10 15Ile Lys Leu Pro Gly Ser Lys Ser Leu
Ser Asn Arg Ile Leu Leu Leu 20 25 30Ala Ala Leu Ser Glu Gly Thr Thr
Val Val Asp Asn Leu Leu Asn Ser 35 40 45Asp Asp Ile Asn Tyr Met Leu
Asp Ala Leu Lys Lys Leu Gly Leu Asn 50 55 60Val Glu Arg Asp Ser Val
Asn Asn Arg Ala Val Val Glu Gly Cys Gly65 70 75 80Gly Ile Phe Pro
Ala Ser Leu Asp Ser Lys Ser Asp Ile Glu Leu Tyr 85 90 95Leu Gly Asn
Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 100 105 110Ala
Ala Gly Gly Asn Ala Ser Tyr Val Leu Asp Gly Val Pro Arg Met 115 120
125Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu Gly
130 135 140Ala Asp Val Glu Cys Thr Leu Gly Thr Asn Cys Pro Pro Val
Arg Val145 150 155 160Asn Ala Asn Gly Gly Leu Pro Gly Gly Lys Val
Lys Leu Ser Gly Ser 165 170 175Ile Ser Ser Gln Tyr Leu Thr Ala Leu
Leu Met Ala Ala Pro Leu Ala 180 185 190Leu Gly Asp Val Glu Ile Glu
Ile Ile Asp Lys Leu Ile Ser Val Pro 195 200 205Tyr Val Glu Met Thr
Leu Lys Leu Met Glu Arg Phe Gly Val Ser Ala 210 215 220Glu His Ser
Asp Ser Trp Asp Arg Phe Phe Val Lys Gly Gly Gln Lys225 230 235
240Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala
245 250 255Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Glu Thr Val
Thr Val 260 265 270Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val
Lys Phe Ala Glu 275 280 285Val Leu Glu Lys Met Gly Cys Lys Val Ser
Trp Thr Glu Asn Ser Val 290 295 300Thr Val Thr Gly Pro Ser Arg Asp
Ala Phe Gly Met Arg His Leu Arg305 310 315 320Ala Val Asp Val Asn
Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 325 330 335Ala Val Val
Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp Val 340 345 350Ala
Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 355 360
365Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Ser Asp Tyr Cys
370 375 380Val Ile Thr Pro Pro Ala Lys Val Lys Pro Ala Glu Ile Asp
Thr Tyr385 390 395 400Asp Asp His Arg Met Ala Met Ala Phe Ser Leu
Ala Ala Cys Ala Asp 405 410 415Val Pro Val Thr Ile Lys Asp Pro Gly
Cys Thr Arg Lys Thr Phe Pro 420 425 430Asp Tyr Phe Gln Val Leu Glu
Ser Ile Thr Lys His 435 44052444PRTArabidopsis thaliana 52Lys Ala
Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly Leu1 5 10 15Ile
Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 20 25
30Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn Ser
35 40 45Asp Asp Ile Asn Tyr Met Leu Asp Ala Leu Lys Arg Leu Gly Leu
Asn 50 55 60Val Glu Thr Asp Ser Glu Asn Asn Arg Ala Val Val Glu Gly
Cys Gly65 70 75 80Gly Ile Phe Pro Ala Ser Ile Asp Ser Lys Ser Asp
Ile Glu Leu Tyr 85 90 95Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu
Thr Ala Ala Val Thr 100 105 110Ala Ala Gly Gly Asn Ala Ser Tyr Val
Leu Asp Gly Val Pro Arg Met 115 120 125Arg Glu Arg Pro Ile Gly Asp
Leu Val Val Gly Leu Lys Gln Leu Gly 130 135 140Ala Asp Val Glu Cys
Thr Leu Gly Thr Asn Cys Pro Pro Val Arg Val145 150 155 160Asn Ala
Asn Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 165 170
175Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ser Ala Pro Leu Ala
180 185 190Leu Gly Asp Val Glu Ile Glu Ile Val Asp Lys Leu Ile Ser
Val Pro 195 200 205Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe
Gly Val Ser Val 210 215 220Glu His Ser Asp Ser Trp Asp Arg Phe Phe
Val Lys
Gly Gly Gln Lys225 230 235 240Tyr Lys Ser Pro Gly Asn Ala Tyr Val
Glu Gly Asp Ala Ser Ser Ala 245 250 255Cys Tyr Phe Leu Ala Gly Ala
Ala Ile Thr Gly Glu Thr Val Thr Val 260 265 270Glu Gly Cys Gly Thr
Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu 275 280 285Val Leu Glu
Lys Met Gly Cys Lys Val Ser Trp Thr Glu Asn Ser Val 290 295 300Thr
Val Thr Gly Pro Pro Arg Asp Ala Phe Gly Met Arg His Leu Arg305 310
315 320Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr
Leu 325 330 335Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile
Arg Asp Val 340 345 350Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met
Ile Ala Ile Cys Thr 355 360 365Glu Leu Arg Lys Leu Gly Ala Thr Val
Glu Glu Gly Ser Asp Tyr Cys 370 375 380Val Ile Thr Pro Pro Lys Lys
Val Lys Thr Ala Glu Ile Asp Thr Tyr385 390 395 400Asp Asp His Arg
Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Asp 405 410 415Val Pro
Ile Thr Ile Asn Asp Ser Gly Cys Thr Arg Lys Thr Phe Pro 420 425
430Asp Tyr Phe Gln Val Leu Glu Arg Ile Thr Lys His 435
44053444PRTNicotiana tabacum 53Lys Pro Asn Glu Ile Val Leu Gln Pro
Ile Lys Asp Ile Ser Gly Thr1 5 10 15Val Lys Leu Pro Gly Ser Lys Ser
Leu Ser Asn Arg Ile Leu Leu Leu 20 25 30Ala Ala Leu Ser Lys Gly Arg
Thr Val Val Asp Asn Leu Leu Ser Ser 35 40 45Asp Asp Ile His Tyr Met
Leu Gly Ala Leu Lys Thr Leu Gly Leu His 50 55 60Val Glu Asp Asp Asn
Glu Asn Gln Arg Ala Ile Val Glu Gly Cys Gly65 70 75 80Gly Gln Phe
Pro Val Gly Lys Lys Ser Glu Glu Glu Ile Gln Leu Phe 85 90 95Leu Gly
Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 100 105
110Val Ala Gly Gly His Ser Arg Tyr Val Leu Asp Gly Val Pro Arg Met
115 120 125Arg Glu Arg Pro Ile Gly Asp Leu Val Asp Gly Leu Lys Gln
Leu Gly 130 135 140Ala Glu Val Asp Cys Phe Leu Gly Thr Asn Cys Pro
Pro Val Arg Ile145 150 155 160Val Ser Lys Gly Gly Leu Pro Gly Gly
Lys Val Lys Leu Ser Gly Ser 165 170 175Ile Ser Ser Gln Tyr Leu Thr
Ala Leu Leu Met Ala Ala Pro Leu Ala 180 185 190Leu Gly Asp Val Glu
Ile Glu Ile Ile Asp Lys Leu Ile Ser Val Pro 195 200 205Tyr Val Glu
Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser Val 210 215 220Glu
His Thr Ser Ser Trp Asp Lys Phe Leu Val Arg Gly Gly Gln Lys225 230
235 240Tyr Lys Ser Pro Gly Lys Ala Tyr Val Glu Gly Asp Ala Ser Ser
Ala 245 250 255Ser Tyr Phe Leu Ala Gly Ala Ala Val Thr Gly Gly Thr
Val Thr Val 260 265 270Glu Gly Cys Gly Thr Ser Ser Leu Gln Gly Asp
Val Lys Phe Ala Glu 275 280 285Val Leu Glu Lys Met Gly Ala Glu Val
Thr Trp Thr Glu Asn Ser Val 290 295 300Thr Val Lys Gly Pro Pro Arg
Asn Ser Ser Gly Met Lys His Leu Arg305 310 315 320Ala Val Asp Val
Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 325 330 335Ala Val
Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val 340 345
350Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr
355 360 365Glu Leu Arg Lys Leu Gly Ala Thr Val Val Glu Gly Ser Asp
Tyr Cys 370 375 380Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Glu
Ile Asp Thr Tyr385 390 395 400Asp Asp His Arg Met Ala Met Ala Phe
Ser Leu Ala Ala Cys Ala Asp 405 410 415Val Pro Val Thr Ile Lys Asp
Pro Gly Cys Thr Arg Lys Thr Phe Pro 420 425 430Asn Tyr Phe Asp Val
Leu Gln Gln Tyr Ser Lys His 435 44054444PRTLycopersicon
esculentumUNSURE(1)..(444)Xaa = any 54Lys Pro His Glu Ile Val Leu
Xaa Pro Ile Lys Asp Ile Ser Gly Thr1 5 10 15Val Lys Leu Pro Gly Ser
Lys Ser Leu Ser Asn Arg Ile Leu Leu Leu 20 25 30Ala Ala Leu Ser Glu
Gly Arg Thr Val Val Asp Asn Leu Leu Ser Ser 35 40 45Asp Asp Ile His
Tyr Met Leu Gly Ala Leu Lys Thr Leu Gly Leu His 50 55 60Val Glu Asp
Asp Asn Glu Asn Gln Arg Ala Ile Val Glu Gly Cys Gly65 70 75 80Gly
Gln Phe Pro Val Gly Lys Lys Ser Glu Glu Glu Ile Gln Leu Phe 85 90
95Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr
100 105 110Val Ala Gly Gly His Ser Arg Tyr Val Leu Asp Gly Val Pro
Arg Met 115 120 125Arg Glu Arg Pro Ile Gly Asp Leu Val Asp Gly Leu
Lys Gln Leu Gly 130 135 140Ala Glu Val Asp Cys Ser Leu Gly Thr Asn
Cys Pro Pro Val Arg Ile145 150 155 160Val Ser Lys Gly Gly Leu Pro
Gly Gly Lys Val Lys Leu Ser Gly Ser 165 170 175Ile Ser Ser Gln Tyr
Leu Thr Ala Leu Leu Met Ala Ala Pro Leu Ala 180 185 190Leu Gly Asp
Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Val Pro 195 200 205Tyr
Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Phe Val 210 215
220Glu His Ser Ser Gly Trp Asp Arg Phe Leu Val Lys Gly Gly Gln
Lys225 230 235 240Tyr Lys Ser Pro Gly Lys Ala Phe Val Glu Gly Asp
Ala Ser Ser Ala 245 250 255Ser Tyr Phe Leu Ala Gly Ala Ala Val Thr
Gly Gly Thr Val Thr Val 260 265 270Glu Gly Cys Gly Thr Ser Ser Leu
Gln Gly Asp Val Lys Phe Ala Glu 275 280 285Val Leu Glu Lys Met Gly
Ala Glu Val Thr Trp Thr Glu Asn Ser Val 290 295 300Thr Val Lys Gly
Pro Pro Arg Asn Ser Ser Gly Met Lys His Leu Arg305 310 315 320Ala
Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 325 330
335Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp Val
340 345 350Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile
Cys Thr 355 360 365Glu Leu Arg Lys Leu Gly Ala Thr Val Val Glu Gly
Ser Asp Tyr Cys 370 375 380Ile Ile Thr Pro Pro Glu Lys Leu Asn Val
Thr Glu Ile Asp Thr Tyr385 390 395 400Asp Asp His Arg Met Ala Met
Ala Phe Ser Leu Ala Ala Cys Ala Asp 405 410 415Val Pro Val Thr Ile
Lys Asn Pro Gly Cys Thr Arg Lys Thr Phe Pro 420 425 430Asp Tyr Phe
Glu Val Leu Gln Lys Tyr Ser Lys His 435 44055444PRTPetunia x
hybrida 55Lys Pro Ser Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser
Gly Thr1 5 10 15Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile
Leu Leu Leu 20 25 30Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn
Leu Leu Ser Ser 35 40 45Asp Asp Ile His Tyr Met Leu Gly Ala Leu Lys
Thr Leu Gly Leu His 50 55 60Val Glu Glu Asp Ser Ala Asn Gln Arg Ala
Val Val Glu Gly Cys Gly65 70 75 80Gly Leu Phe Pro Val Gly Lys Glu
Ser Lys Glu Glu Ile Gln Leu Phe 85 90 95Leu Gly Asn Ala Gly Thr Ala
Met Arg Pro Leu Thr Ala Ala Val Thr 100 105 110Val Ala Gly Gly Asn
Ser Arg Tyr Val Leu Asp Gly Val Pro Arg Met 115 120 125Arg Glu Arg
Pro Ile Ser Asp Leu Val Asp Gly Leu Lys Gln Leu Gly 130 135 140Ala
Glu Val Asp Cys Phe Leu Gly Thr Lys Cys Pro Pro Val Arg Ile145 150
155 160Val Ser Lys Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly
Ser 165 170 175Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala
Pro Leu Ala 180 185 190Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys
Leu Ile Ser Val Pro 195 200 205Tyr Val Glu Met Thr Leu Lys Leu Met
Glu Arg Phe Gly Ile Ser Val 210 215 220Glu His Ser Ser Ser Trp Asp
Arg Phe Phe Val Arg Gly Gly Gln Lys225 230 235 240Tyr Lys Ser Pro
Gly Lys Ala Phe Val Glu Gly Asp Ala Ser Ser Ala 245 250 255Ser Tyr
Phe Leu Ala Gly Ala Ala Val Thr Gly Gly Thr Ile Thr Val 260 265
270Glu Gly Cys Gly Thr Asn Ser Leu Gln Gly Asp Val Lys Phe Ala Glu
275 280 285Val Leu Glu Lys Met Gly Ala Glu Val Thr Trp Thr Glu Asn
Ser Val 290 295 300Thr Val Lys Gly Pro Pro Arg Ser Ser Ser Gly Arg
Lys His Leu Arg305 310 315 320Ala Ile Asp Val Asn Met Asn Lys Met
Pro Asp Val Ala Met Thr Leu 325 330 335Ala Val Val Ala Leu Tyr Ala
Asp Gly Pro Thr Ala Ile Arg Asp Val 340 345 350Ala Ser Trp Arg Val
Lys Glu Thr Glu Arg Met Ile Ala Ile Cys Thr 355 360 365Glu Leu Arg
Lys Leu Gly Ala Thr Val Glu Glu Gly Pro Asp Tyr Cys 370 375 380Ile
Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Asp Ile Asp Thr Tyr385 390
395 400Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala
Asp 405 410 415Val Pro Val Thr Ile Asn Asp Pro Gly Cys Thr Arg Lys
Thr Phe Pro 420 425 430Asn Tyr Phe Asp Val Leu Gln Gln Tyr Ser Lys
His 435 44056444PRTZea mays 56Ala Gly Ala Glu Glu Ile Val Leu Gln
Pro Ile Lys Glu Ile Ser Gly1 5 10 15Thr Val Lys Leu Pro Gly Ser Lys
Ser Leu Ser Asn Arg Ile Leu Leu 20 25 30Leu Ala Ala Leu Ser Glu Gly
Thr Thr Val Val Asp Asn Leu Leu Asn 35 40 45Ser Glu Asp Val His Tyr
Met Leu Gly Ala Leu Arg Thr Leu Gly Leu 50 55 60Ser Val Glu Ala Asp
Lys Ala Ala Lys Arg Ala Val Val Val Gly Cys65 70 75 80Gly Gly Lys
Phe Pro Val Glu Asp Ala Lys Glu Glu Val Gln Leu Phe 85 90 95Leu Gly
Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val Thr 100 105
110Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg Met
115 120 125Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln
Leu Gly 130 135 140Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro
Pro Val Arg Val145 150 155 160Asn Gly Ile Gly Gly Leu Pro Gly Gly
Lys Val Lys Leu Ser Gly Ser 165 170 175Ile Ser Ser Gln Tyr Leu Ser
Ala Leu Leu Met Ala Ala Pro Leu Pro 180 185 190Leu Gly Asp Val Glu
Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile Pro 195 200 205Tyr Val Glu
Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala 210 215 220Glu
His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys225 230
235 240Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser
Ala 245 250 255Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr
Val Thr Val 260 265 270Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp
Val Lys Phe Ala Glu 275 280 285Val Leu Glu Met Met Gly Ala Lys Val
Thr Trp Thr Glu Thr Ser Val 290 295 300Thr Val Thr Gly Pro Pro Arg
Glu Pro Phe Gly Arg Lys His Leu Lys305 310 315 320Ala Ile Asp Val
Asn Met Asn Lys Met Pro Asp Val Ala Met Thr Leu 325 330 335Ala Val
Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp Val 340 345
350Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg Thr
355 360 365Glu Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp
Tyr Cys 370 375 380Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala
Ile Asp Thr Tyr385 390 395 400Asp Asp His Arg Met Ala Met Ala Phe
Ser Leu Ala Ala Cys Ala Glu 405 410 415Val Pro Val Thr Ile Arg Asp
Pro Gly Cys Thr Arg Lys Thr Phe Pro 420 425 430Asp Tyr Phe Asp Val
Leu Ser Thr Phe Val Lys Asn 435 44057427PRTSalmonella gallinarum
57Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp Gly Ala Ile1
5 10 15Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu
Ala 20 25 30Ala Leu Ala Cys Gly Lys Thr Val Leu Thr Asn Leu Leu Asp
Ser Asp 35 40 45Asp Val Arg His Met Leu Asn Ala Leu Ser Ala Leu Gly
Ile Asn Tyr 50 55 60Thr Leu Ser Ala Asp Arg Thr Arg Cys Asp Ile Thr
Gly Asn Gly Gly65 70 75 80Pro Leu Arg Ala Pro Gly Ala Leu Glu Leu
Phe Leu Gly Asn Ala Gly 85 90 95Thr Ala Met Arg Pro Leu Ala Ala Ala
Leu Cys Leu Gly Gln Asn Glu 100 105 110Ile Val Leu Thr Gly Glu Pro
Arg Met Lys Glu Arg Pro Ile Gly His 115 120 125Leu Val Asp Ser Leu
Arg Gln Gly Gly Ala Asn Ile Asp Tyr Leu Glu 130 135 140Gln Glu Asn
Tyr Pro Pro Leu Arg Leu Arg Gly Gly Phe Ile Gly Gly145 150 155
160Asp Ile Glu Val Asp Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu
165 170 175Leu Met Thr Ala Pro Leu Ala Pro Lys Asp Thr Ile Ile Arg
Val Lys 180 185 190Gly Glu Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr
Leu Asn Leu Met 195 200 205Lys Thr Phe Gly Val Glu Ile Ala Asn His
His Tyr Gln Gln Phe Val 210 215 220Val Lys Gly Gly Gln Gln Tyr His
Ser Pro Gly Arg Tyr Leu Val Glu225 230 235 240Gly Asp Ala Ser Ser
Ala Ser Tyr Phe Leu Ala Ala Gly Ala Ile Lys 245 250 255Gly Gly Thr
Val Lys Val Thr Gly Ile Gly Arg Lys Ser Met Gln Gly 260 265 270Asp
Ile Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala Thr Ile Thr 275 280
285Trp Gly Asp Asp Phe Ile Ala Cys Thr Arg Gly Glu Leu His Ala Ile
290 295 300Asp Met Asp Met Asn His Ile Pro Asp Ala Ala Met Thr Ile
Ala Thr305 310 315 320Thr Ala Leu Phe Ala Lys Gly Thr Thr Thr Leu
Arg Asn Ile Tyr Asn 325 330 335Trp Arg Val Lys Glu Thr Asp Arg Leu
Phe Ala Met Ala Thr Glu Leu 340 345 350Arg Lys Val Gly Ala Glu Val
Glu Glu Gly His Asp Tyr Ile Arg Ile 355 360 365Thr Pro Pro Ala Lys
Leu Gln His Ala Asp Ile Gly Thr Tyr Asn Asp 370 375 380His Arg Met
Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr Pro385 390 395
400Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr
405 410 415Phe Glu Gln Leu Ala Arg Met Ser Thr Pro Ala 420
42558427PRTSalmonella typhimurium 58Met Glu Ser Leu Thr Leu Gln Pro
Ile Ala Arg Val Asp Gly Ala Ile1 5 10 15Asn Leu Pro Gly Ser Lys Ser
Val Ser Asn Arg Ala Leu Leu Leu Ala 20 25 30Ala Leu Ala Cys
Gly Lys Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 35 40 45Asp Val Arg
His Met Leu Asn Ala Leu Ser Ala Leu Gly Ile Asn Tyr 50 55 60Thr Leu
Ser Ala Asp Arg Thr Arg Cys Asp Ile Thr Gly Asn Gly Gly65 70 75
80Pro Leu Arg Ala Ser Gly Thr Leu Glu Leu Phe Leu Gly Asn Ala Gly
85 90 95Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Gln Asn
Glu 100 105 110Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro
Ile Gly His 115 120 125Leu Val Asp Ser Leu Arg Gln Gly Gly Ala Asn
Ile Asp Tyr Leu Glu 130 135 140Gln Glu Asn Tyr Pro Pro Leu Arg Leu
Arg Gly Gly Phe Ile Gly Gly145 150 155 160Asp Ile Glu Val Asp Gly
Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 165 170 175Leu Met Thr Ala
Pro Leu Ala Pro Glu Asp Thr Ile Ile Arg Val Lys 180 185 190Gly Glu
Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met 195 200
205Lys Thr Phe Gly Val Glu Ile Ala Asn His His Tyr Gln Gln Phe Val
210 215 220Val Lys Gly Gly Gln Gln Tyr His Ser Pro Gly Arg Tyr Leu
Val Glu225 230 235 240Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala
Ala Gly Gly Ile Lys 245 250 255Gly Gly Thr Val Lys Val Thr Gly Ile
Gly Gly Lys Ser Met Gln Gly 260 265 270Asp Ile Arg Phe Ala Asp Val
Leu His Lys Met Gly Ala Thr Ile Thr 275 280 285Trp Gly Asp Asp Phe
Ile Ala Cys Thr Arg Gly Glu Leu His Ala Ile 290 295 300Asp Met Asp
Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr305 310 315
320Thr Ala Leu Phe Ala Lys Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn
325 330 335Trp Arg Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr
Glu Leu 340 345 350Arg Lys Val Gly Ala Glu Val Glu Glu Gly His Asp
Tyr Ile Arg Ile 355 360 365Thr Pro Pro Ala Lys Leu Gln His Ala Asp
Ile Gly Thr Tyr Asn Asp 370 375 380His Arg Met Ala Met Cys Phe Ser
Leu Val Ala Leu Ser Asp Thr Pro385 390 395 400Val Thr Ile Leu Asp
Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 405 410 415Phe Glu Gln
Leu Ala Arg Met Ser Thr Pro Ala 420 42559427PRTKlebsiella
pneumoniae 59Met Glu Ser Leu Thr Leu Gln Pro Ile Ala Arg Val Asp
Gly Thr Val1 5 10 15Asn Leu Pro Gly Ser Lys Ser Val Ser Asn Arg Ala
Leu Leu Leu Ala 20 25 30Ala Leu Ala Arg Gly Thr Thr Val Leu Thr Asn
Leu Leu Asp Ser Asp 35 40 45Asp Val Arg His Met Leu Asn Ala Leu Ser
Ala Leu Gly Val His Tyr 50 55 60Val Leu Ser Ser Asp Arg Thr Arg Cys
Glu Val Thr Gly Thr Gly Gly65 70 75 80Pro Leu Gln Ala Gly Ser Ala
Leu Glu Leu Phe Leu Gly Asn Ala Gly 85 90 95Thr Ala Met Arg Pro Leu
Ala Ala Ala Leu Cys Leu Gly Ser Asn Asp 100 105 110Ile Val Leu Thr
Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His 115 120 125Leu Val
Asp Ala Leu Arg Gln Gly Gly Ala Gln Ile Asp Tyr Leu Glu 130 135
140Gln Glu Asn Tyr Pro Pro Leu Arg Leu Arg Gly Gly Phe Thr Gly
Gly145 150 155 160Asp Val Glu Val Asp Gly Ser Val Ser Ser Gln Phe
Leu Thr Ala Leu 165 170 175Leu Met Ala Ser Pro Leu Ala Pro Gln Asp
Thr Val Ile Ala Ile Lys 180 185 190Gly Glu Leu Val Ser Arg Pro Tyr
Ile Asp Ile Thr Leu His Leu Met 195 200 205Lys Thr Phe Gly Val Glu
Val Glu Asn Gln Ala Tyr Gln Arg Phe Ile 210 215 220Val Arg Gly Asn
Gln Gln Tyr Gln Ser Pro Gly Asp Tyr Leu Val Glu225 230 235 240Gly
Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Ala Ile Lys 245 250
255Gly Gly Thr Val Lys Val Thr Gly Ile Gly Arg Asn Ser Val Gln Gly
260 265 270Asp Ile Arg Phe Ala Asp Val Leu Glu Lys Met Gly Ala Thr
Val Thr 275 280 285Trp Gly Glu Asp Tyr Ile Ala Cys Thr Arg Gly Glu
Leu Asn Ala Ile 290 295 300Asp Met Asp Met Asn His Ile Pro Asp Ala
Ala Met Thr Ile Ala Thr305 310 315 320Ala Ala Leu Phe Ala Arg Gly
Thr Thr Thr Leu Arg Asn Ile Tyr Asn 325 330 335Trp Arg Val Lys Glu
Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 340 345 350Arg Lys Val
Gly Ala Glu Val Glu Glu Gly Glu Asp Tyr Ile Arg Ile 355 360 365Thr
Pro Pro Leu Thr Leu Gln Phe Ala Glu Ile Gly Thr Tyr Asn Asp 370 375
380His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu Ser Asp Thr
Pro385 390 395 400Val Thr Ile Leu Asp Pro Lys Cys Thr Ala Lys Thr
Phe Pro Asp Tyr 405 410 415Phe Gly Gln Leu Ala Arg Ile Ser Thr Leu
Ala 420 42560427PRTYersinia enterocolitica 60Met Leu Glu Ser Leu
Thr Leu His Pro Ile Ala Leu Ile Asn Gly Thr1 5 10 15Val Asn Leu Pro
Gly Ser Lys Ser Val Ser Asn Arg Ala Leu Leu Leu 20 25 30Ala Ala Leu
Ala Glu Gly Thr Thr Gln Leu Asn Asn Leu Leu Asp Ser 35 40 45Asp Asp
Ile Arg His Met Leu Asn Ala Leu Gln Ala Leu Gly Val Lys 50 55 60Tyr
Arg Leu Ser Ala Asp Arg Thr Arg Cys Glu Val Asp Gly Leu Gly65 70 75
80Gly Lys Leu Val Ala Glu Gln Pro Leu Glu Leu Phe Leu Gly Asn Ala
85 90 95Gly Thr Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Lys
Asn 100 105 110Asp Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg
Pro Ile Gly 115 120 125His Leu Val Asp Ala Leu Arg Gln Gly Gly Ala
Gln Ile Asp Tyr Leu 130 135 140Glu Gln Glu Asn Tyr Arg Arg Cys Ile
Ala Gly Gly Phe Arg Gly Gly145 150 155 160Lys Leu Thr Val Asp Gly
Ser Val Ser Ser Gln Phe Leu Thr Ala Leu 165 170 175Leu Met Thr Ala
Pro Leu Ala Glu Gln Asp Thr Glu Ile Gln Ile Gln 180 185 190Gly Glu
Leu Val Ser Lys Pro Tyr Ile Asp Ile Thr Leu His Leu Met 195 200
205Lys Ala Phe Gly Val Asp Val Val His Glu Asn Tyr Gln Ile Phe His
210 215 220Ile Lys Gly Gly Gln Thr Tyr Arg Ser Pro Gly Ile Tyr Leu
Val Glu225 230 235 240Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala
Ala Ala Ala Ile Lys 245 250 255Gly Gly Thr Val Arg Val Thr Gly Ile
Gly Lys Gln Ser Val Gln Gly 260 265 270Asp Thr Lys Phe Ala Asp Val
Leu Glu Lys Met Gly Ala Lys Ile Ser 275 280 285Trp Gly Asp Asp Tyr
Ile Glu Cys Ser Arg Gly Glu Leu Gln Gly Ile 290 295 300Asp Met Asp
Met Asn His Ile Pro Asp Ala Ala Met Thr Ile Ala Thr305 310 315
320Thr Ala Leu Phe Ala Asp Gly Pro Thr Val Ile Arg Asn Ile Tyr Asn
325 330 335Trp Arg Val Lys Glu Thr Asp Arg Leu Ser Ala Met Ala Thr
Glu Leu 340 345 350Arg Lys Val Gly Ala Glu Val Glu Glu Gly Gln Asp
Tyr Ile Arg Val 355 360 365Val Pro Pro Ala Gln Leu Ile Ala Ala Glu
Ile Gly Thr Tyr Asn Asp 370 375 380His Arg Met Ala Met Cys Phe Ser
Leu Val Ala Leu Ser Asp Thr Pro385 390 395 400Val Thr Ile Leu Asp
Pro Lys Cys Thr Ala Lys Thr Phe Pro Asp Tyr 405 410 415Phe Glu Gln
Leu Ala Arg Leu Ser Gln Ile Ala 420 42561432PRTHaemophilus
influenzae 61Met Glu Lys Ile Thr Leu Ala Pro Ile Ser Ala Val Glu
Gly Thr Ile1 5 10 15Asn Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ala
Leu Leu Leu Ala 20 25 30Ala Leu Ala Lys Gly Thr Thr Lys Val Thr Asn
Leu Leu Asp Ser Asp 35 40 45Asp Ile Arg His Met Leu Asn Ala Leu Lys
Ala Leu Gly Val Arg Tyr 50 55 60Gln Leu Ser Asp Asp Lys Thr Ile Cys
Glu Ile Glu Gly Leu Gly Gly65 70 75 80Ala Phe Asn Ile Gln Asp Asn
Leu Ser Leu Phe Leu Gly Asn Ala Gly 85 90 95Thr Ala Met Arg Pro Leu
Thr Ala Ala Leu Cys Leu Lys Gly Asn His 100 105 110Glu Val Glu Ile
Ile Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro 115 120 125Ile Leu
His Leu Val Asp Ala Leu Arg Gln Ala Gly Ala Asp Ile Arg 130 135
140Tyr Leu Glu Asn Glu Gly Tyr Pro Pro Leu Ala Ile Arg Asn Lys
Gly145 150 155 160Ile Lys Gly Gly Lys Val Lys Ile Asp Gly Ser Ile
Ser Ser Gln Phe 165 170 175Leu Thr Ala Leu Leu Met Ser Ala Pro Leu
Ala Glu Asn Asp Thr Glu 180 185 190Ile Glu Ile Ile Gly Glu Leu Val
Ser Lys Pro Tyr Ile Asp Ile Thr 195 200 205Leu Ala Met Met Arg Asp
Phe Gly Val Lys Val Glu Asn His His Tyr 210 215 220Gln Lys Phe Gln
Val Lys Gly Asn Gln Ser Tyr Ile Ser Pro Asn Lys225 230 235 240Tyr
Leu Val Glu Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala 245 250
255Gly Ala Ile Lys Gly Lys Val Lys Val Thr Gly Ile Gly Lys Asn Ser
260 265 270Ile Gln Gly Asp Arg Leu Phe Ala Asp Val Leu Glu Lys Met
Gly Ala 275 280 285Lys Ile Thr Trp Gly Glu Asp Phe Ile Gln Ala Glu
His Ala Glu Leu 290 295 300Asn Gly Ile Asp Met Asp Met Asn His Ile
Pro Asp Ala Ala Met Thr305 310 315 320Ile Ala Thr Thr Ala Leu Phe
Ser Asn Gly Glu Thr Val Ile Arg Asn 325 330 335Ile Tyr Asn Trp Arg
Val Lys Glu Thr Asp Arg Leu Thr Ala Met Ala 340 345 350Thr Glu Leu
Arg Lys Val Gly Ala Glu Val Glu Glu Gly Glu Asp Phe 355 360 365Ile
Arg Ile Gln Pro Leu Ala Leu Asn Gln Phe Lys His Ala Asn Ile 370 375
380Glu Thr Tyr Asn Asp His Arg Met Ala Met Cys Phe Ser Leu Ile
Ala385 390 395 400Leu Ser Asn Thr Pro Val Thr Ile Leu Asp Pro Lys
Cys Thr Ala Lys 405 410 415Thr Phe Pro Thr Phe Phe Asn Glu Phe Glu
Lys Ile Cys Leu Lys Asn 420 425 43062441PRTPasteurella multocida
62Val Ile Lys Asp Ala Thr Ala Ile Thr Leu Asn Pro Ile Ser Tyr Ile1
5 10 15Glu Gly Glu Val Arg Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg
Ala 20 25 30Leu Leu Leu Ser Ala Leu Ala Lys Gly Lys Thr Thr Leu Thr
Asn Leu 35 40 45Leu Asp Ser Asp Asp Val Arg His Met Leu Asn Ala Leu
Lys Glu Leu 50 55 60Gly Val Thr Tyr Gln Leu Ser Glu Asp Lys Ser Val
Cys Glu Ile Glu65 70 75 80Gly Leu Gly Arg Ala Phe Glu Trp Gln Ser
Gly Leu Ala Leu Phe Leu 85 90 95Gly Asn Ala Gly Thr Ala Met Arg Pro
Leu Thr Ala Ala Leu Cys Leu 100 105 110Ser Thr Pro Asn Arg Glu Gly
Lys Asn Glu Ile Val Leu Thr Gly Glu 115 120 125Pro Arg Met Lys Glu
Arg Pro Ile Gln His Leu Val Asp Ala Leu Cys 130 135 140Gln Ala Gly
Ala Glu Ile Gln Tyr Leu Glu Gln Glu Gly Tyr Pro Pro145 150 155
160Ile Ala Ile Arg Asn Thr Gly Leu Lys Gly Gly Arg Ile Gln Ile Asp
165 170 175Gly Ser Val Ser Ser Gln Phe Leu Thr Ala Leu Leu Met Ala
Ala Pro 180 185 190Met Ala Glu Ala Asp Thr Glu Ile Glu Ile Ile Gly
Glu Leu Val Ser 195 200 205Lys Pro Tyr Ile Asp Ile Thr Leu Lys Met
Met Gln Thr Phe Gly Val 210 215 220Glu Val Glu Asn Gln Ala Tyr Gln
Arg Phe Leu Val Lys Gly His Gln225 230 235 240Gln Tyr Gln Ser Pro
His Arg Phe Leu Val Glu Gly Asp Ala Ser Ser 245 250 255Ala Ser Tyr
Phe Leu Ala Ala Ala Ala Ile Lys Gly Lys Val Lys Val 260 265 270Thr
Gly Val Gly Lys Asn Ser Ile Gln Gly Asp Arg Leu Phe Ala Asp 275 280
285Val Leu Glu Lys Met Gly Ala His Ile Thr Trp Gly Asp Asp Phe Ile
290 295 300Gln Val Glu Lys Gly Asn Leu Lys Gly Ile Asp Met Asp Met
Asn His305 310 315 320Ile Pro Asp Ala Ala Met Thr Ile Ala Thr Thr
Ala Leu Phe Ala Glu 325 330 335Gly Glu Thr Val Ile Arg Asn Ile Tyr
Asn Trp Arg Val Lys Glu Thr 340 345 350Asp Arg Leu Thr Ala Met Ala
Thr Glu Leu Arg Lys Val Gly Ala Glu 355 360 365Val Glu Glu Gly Glu
Asp Phe Ile Arg Ile Gln Pro Leu Asn Leu Ala 370 375 380Gln Phe Gln
His Ala Glu Leu Asn Ile His Asp His Arg Met Ala Met385 390 395
400Cys Phe Ala Leu Ile Ala Leu Ser Lys Thr Ser Val Thr Ile Leu Asp
405 410 415Pro Ser Cys Thr Ala Lys Thr Phe Pro Thr Phe Leu Ile Leu
Phe Thr 420 425 430Leu Asn Thr Arg Glu Val Ala Tyr Arg 435
44063426PRTAeromonas salmonicida 63Asn Ser Leu Arg Leu Glu Pro Ile
Ser Arg Val Ala Gly Glu Val Asn1 5 10 15Leu Pro Gly Ser Lys Ser Val
Ser Asn Arg Ala Leu Leu Leu Ala Ala 20 25 30Leu Ala Arg Gly Thr Thr
Arg Leu Thr Asn Leu Leu Asp Ser Asp Asp 35 40 45Ile Arg His Met Leu
Ala Ala Leu Thr Gln Leu Gly Val Lys Tyr Lys 50 55 60Leu Ser Ala Asp
Lys Thr Glu Cys Thr Val His Gly Leu Gly Arg Ser65 70 75 80Phe Ala
Val Ser Ala Pro Val Asn Leu Phe Leu Gly Asn Ala Gly Thr 85 90 95Ala
Met Arg Pro Leu Cys Ala Ala Leu Cys Leu Gly Ser Gly Glu Tyr 100 105
110Met Leu Gly Gly Glu Pro Arg Met Glu Glu Arg Pro Ile Gly His Leu
115 120 125Val Asp Cys Leu Ala Leu Lys Gly Ala His Ile Gln Tyr Leu
Lys Lys 130 135 140Asp Gly Tyr Pro Pro Leu Val Val Asp Ala Lys Gly
Leu Trp Gly Gly145 150 155 160Asp Val His Val Asp Gly Ser Val Ser
Ser Gln Phe Leu Thr Ala Phe 165 170 175Leu Met Ala Ala Pro Ala Met
Ala Pro Val Ile Pro Arg Ile His Ile 180 185 190Lys Gly Glu Leu Val
Ser Lys Pro Tyr Ile Asp Ile Thr Leu His Ile 195 200 205Met Asn Ser
Ser Gly Val Val Ile Glu His Asp Asn Tyr Lys Leu Phe 210 215 220Tyr
Ile Lys Gly Asn Gln Ser Ile Val Ser Pro Gly Asp Phe Leu Val225 230
235 240Glu Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Ala
Ile 245 250 255Lys Gly Lys Val Arg Val Thr Gly Ile Gly Lys His Ser
Ile Gly Asp 260 265 270Ile His Phe Ala Asp Val Leu Glu Arg Met Gly
Ala Arg Ile Thr Trp 275 280 285Gly Asp Asp Phe Ile Glu Ala Glu Gln
Gly Pro Leu His Gly Val Asp 290 295 300Met Asp Met Asn His Ile Pro
Asp Val Gly His Asp His Ser Gly Gln305 310 315 320Ser His Cys Leu
Pro Arg Val Pro Pro His Ser Gln His Leu Gln Leu
325 330 335Ala Val Arg Asp Asp Arg Cys Thr Pro Cys Thr His Gly His
Arg Arg 340 345 350Ala Gln Ala Gly Val Ser Glu Glu Gly Thr Thr Phe
Ile Thr Arg Asp 355 360 365Ala Ala Asp Pro Ala Gln Ala Arg Arg Asp
Arg His Leu Gln Arg Ser 370 375 380Arg Ile Ala Met Cys Phe Ser Leu
Val Ala Leu Ser Asp Ile Ala Val385 390 395 400Thr Ile Asn Asp Pro
Gly Cys Thr Ser Lys Thr Phe Pro Asp Tyr Phe 405 410 415Asp Lys Leu
Ala Ser Val Ser Gln Ala Val 420 42564442PRTBacillus pertussis 64Met
Ser Gly Leu Ala Tyr Leu Asp Leu Pro Ala Ala Arg Leu Ala Arg1 5 10
15Gly Glu Val Ala Leu Pro Gly Ser Lys Ser Ile Ser Asn Arg Val Leu
20 25 30Leu Leu Ala Ala Leu Ala Glu Gly Ser Thr Glu Ile Thr Gly Leu
Leu 35 40 45Asp Ser Asp Asp Thr Arg Val Met Leu Ala Ala Leu Arg Gln
Leu Gly 50 55 60Val Ser Val Gly Glu Val Ala Asp Gly Cys Val Thr Ile
Glu Gly Val65 70 75 80Ala Arg Phe Pro Thr Glu Gln Ala Glu Leu Phe
Leu Gly Asn Ala Gly 85 90 95Thr Ala Phe Arg Pro Leu Thr Ala Ala Leu
Ala Leu Met Gly Gly Asp 100 105 110Tyr Arg Leu Ser Gly Val Pro Arg
Met His Glu Arg Pro Ile Gly Asp 115 120 125Leu Val Asp Ala Leu Arg
Gln Phe Gly Ala Gly Ile Glu Tyr Leu Gly 130 135 140Gln Ala Gly Tyr
Pro Pro Leu Arg Ile Gly Gly Gly Ser Ile Arg Val145 150 155 160Asp
Gly Pro Val Arg Val Glu Gly Ser Val Ser Ser Gln Phe Leu Thr 165 170
175Ala Leu Leu Met Ala Ala Pro Val Leu Ala Arg Arg Ser Gly Gln Asp
180 185 190Ile Thr Ile Glu Val Val Gly Glu Leu Ile Ser Lys Pro Tyr
Ile Glu 195 200 205Ile Thr Leu Asn Leu Met Ala Arg Phe Gly Val Ser
Val Arg Arg Asp 210 215 220Gly Trp Arg Ala Phe Thr Ile Ala Arg Asp
Ala Val Tyr Arg Gly Pro225 230 235 240Gly Arg Met Ala Ile Glu Gly
Asp Ala Ser Thr Ala Ser Tyr Phe Leu 245 250 255Ala Leu Gly Ala Ile
Gly Gly Gly Pro Val Arg Val Thr Gly Val Gly 260 265 270Glu Asp Ser
Ile Gln Gly Asp Val Ala Phe Ala Ala Thr Leu Ala Ala 275 280 285Met
Gly Ala Asp Val Arg Tyr Gly Pro Gly Trp Ile Glu Thr Arg Gly 290 295
300Val Arg Val Ala Glu Gly Gly Arg Leu Lys Ala Phe Asp Ala Asp
Phe305 310 315 320Asn Leu Ile Pro Asp Ala Ala Met Thr Ala Ala Thr
Leu Ala Leu Tyr 325 330 335Ala Asp Gly Pro Cys Arg Leu Arg Asn Ile
Gly Ser Trp Arg Val Lys 340 345 350Glu Thr Asp Arg Ile His Ala Met
His Thr Glu Leu Glu Lys Leu Gly 355 360 365Ala Gly Val Gln Ser Gly
Ala Asp Trp Leu Glu Val Ala Pro Pro Glu 370 375 380Pro Gly Gly Trp
Arg Asp Ala His Ile Gly Thr Trp Asp Asp His Arg385 390 395 400Met
Ala Met Cys Phe Leu Leu Ala Ala Phe Gly Pro Ala Ala Val Arg 405 410
415Ile Leu Asp Pro Gly Cys Val Ser Lys Thr Phe Pro Asp Tyr Phe Asp
420 425 430Val Tyr Ala Gly Leu Leu Ala Ala Arg Asp 435
44065427PRTSalmonella typhimurium 65Met Glu Ser Leu Thr Leu Gln Pro
Ile Ala Arg Val Asp Gly Ala Ile1 5 10 15Asn Leu Pro Gly Ser Lys Ser
Val Ser Asn Arg Ala Leu Leu Leu Ala 20 25 30Ala Leu Ala Cys Gly Lys
Thr Val Leu Thr Asn Leu Leu Asp Ser Asp 35 40 45Asp Val Arg His Met
Leu Asn Ala Leu Ser Ala Leu Gly Ile Asn Tyr 50 55 60Thr Leu Ser Ala
Asp Arg Thr Arg Cys Asp Ile Thr Gly Asn Gly Gly65 70 75 80Pro Leu
Arg Ala Ser Gly Thr Leu Glu Leu Phe Leu Gly Asn Ala Gly 85 90 95Thr
Ala Met Arg Pro Leu Ala Ala Ala Leu Cys Leu Gly Gln Asn Glu 100 105
110Ile Val Leu Thr Gly Glu Pro Arg Met Lys Glu Arg Pro Ile Gly His
115 120 125Leu Val Asp Ser Leu Arg Gln Gly Gly Ala Asn Ile Asp Tyr
Leu Glu 130 135 140Gln Glu Asn Tyr Pro Pro Leu Arg Leu Arg Gly Gly
Phe Ile Gly Gly145 150 155 160Asp Ile Glu Val Asp Gly Ser Val Ser
Ser Gln Phe Leu Thr Ala Leu 165 170 175Leu Met Thr Ala Pro Leu Ala
Pro Glu Asp Thr Ile Ile Arg Val Lys 180 185 190Gly Glu Leu Val Ser
Lys Pro Tyr Ile Asp Ile Thr Leu Asn Leu Met 195 200 205Lys Thr Phe
Gly Val Glu Ile Ala Asn His His Tyr Gln Gln Phe Val 210 215 220Val
Lys Gly Gly Gln Gln Tyr His Ser Pro Gly Arg Tyr Leu Val Glu225 230
235 240Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Ala Gly Gly Ile
Lys 245 250 255Gly Gly Thr Val Lys Val Thr Gly Ile Gly Gly Lys Ser
Met Gln Gly 260 265 270Asp Ile Arg Phe Ala Asp Val Leu His Lys Met
Gly Ala Thr Ile Thr 275 280 285Trp Gly Asp Asp Phe Ile Ala Cys Thr
Arg Gly Glu Leu His Ala Ile 290 295 300Asp Met Asp Met Asn His Ile
Pro Asp Ala Ala Met Thr Ile Ala Thr305 310 315 320Thr Ala Leu Phe
Ala Lys Gly Thr Thr Thr Leu Arg Asn Ile Tyr Asn 325 330 335Trp Arg
Val Lys Glu Thr Asp Arg Leu Phe Ala Met Ala Thr Glu Leu 340 345
350Arg Lys Val Gly Ala Glu Val Glu Glu Gly His Asp Tyr Ile Arg Ile
355 360 365Thr Pro Pro Ala Lys Leu Gln His Ala Asp Ile Gly Thr Tyr
Asn Asp 370 375 380His Arg Met Ala Met Cys Phe Ser Leu Val Ala Leu
Ser Asp Thr Pro385 390 395 400Val Thr Ile Leu Asp Pro Lys Cys Thr
Ala Lys Thr Phe Pro Asp Tyr 405 410 415Phe Glu Gln Leu Ala Arg Met
Ser Thr Pro Ala 420 425661894DNASynechocystis sp.CDS(275)..(1618)
66acgggctgta acggtagtag gggtcccgag cacaaaagcg gtgccggcaa gcagaactaa
60tttccatggg gaataatggt atttcattgg tttggcctct ggtctggcaa tggttgctag
120gcgatcgcct gttgaaatta acaaactgtc gcccttccac tgaccatggt
aacgatgttt 180tttacttcct tgactaaccg aggaaaattt ggcggggggc
agaaatgcca atacaattta 240gcttggtctt ccctgcccct aatttgtccc ctcc atg
gcc ttg ctt tcc ctc aac 295 Met Ala Leu Leu Ser Leu Asn 1 5aat cat
caa tcc cat caa cgc tta act gtt aat ccc cct gcc caa ggg 343Asn His
Gln Ser His Gln Arg Leu Thr Val Asn Pro Pro Ala Gln Gly 10 15 20gtc
gct ttg act ggc cgc cta agg gtg ccg ggg gat aaa tcc att tcc 391Val
Ala Leu Thr Gly Arg Leu Arg Val Pro Gly Asp Lys Ser Ile Ser 25 30
35cat cgg gcc ttg atg ttg ggg gcg atc gcc acc ggg gaa acc att atc
439His Arg Ala Leu Met Leu Gly Ala Ile Ala Thr Gly Glu Thr Ile
Ile40 45 50 55gaa ggg cta ctg ttg ggg gaa gat ccc cgt agt acg gcc
cat tgc ttt 487Glu Gly Leu Leu Leu Gly Glu Asp Pro Arg Ser Thr Ala
His Cys Phe 60 65 70cgg gcc atg gga gca gaa atc agc gaa cta aat tca
gaa aaa atc atc 535Arg Ala Met Gly Ala Glu Ile Ser Glu Leu Asn Ser
Glu Lys Ile Ile 75 80 85gtt cag ggt cgg ggt ctg gga cag ttg cag gaa
ccc agt acc gtt ttg 583Val Gln Gly Arg Gly Leu Gly Gln Leu Gln Glu
Pro Ser Thr Val Leu 90 95 100gat gcg ggg aac tct ggc acc acc atg
cgc tta atg ttg ggc ttg cta 631Asp Ala Gly Asn Ser Gly Thr Thr Met
Arg Leu Met Leu Gly Leu Leu 105 110 115gcc ggg caa aaa gat tgt tta
ttc acc gtc acc ggc gat gat tcc ctc 679Ala Gly Gln Lys Asp Cys Leu
Phe Thr Val Thr Gly Asp Asp Ser Leu120 125 130 135cgt cac cgc ccc
atg tcc cgg gta att caa ccc ttg caa caa atg ggg 727Arg His Arg Pro
Met Ser Arg Val Ile Gln Pro Leu Gln Gln Met Gly 140 145 150gca aaa
att tgg gcc cgg agt aac ggc aag ttt gcg ccg ctg gca gtc 775Ala Lys
Ile Trp Ala Arg Ser Asn Gly Lys Phe Ala Pro Leu Ala Val 155 160
165cag ggt agc caa tta aaa ccg atc cat tac cat tcc ccc att gct tca
823Gln Gly Ser Gln Leu Lys Pro Ile His Tyr His Ser Pro Ile Ala Ser
170 175 180gcc cag gta aag tcc tgc ctg ttg cta gcg ggg tta acc acc
gag ggg 871Ala Gln Val Lys Ser Cys Leu Leu Leu Ala Gly Leu Thr Thr
Glu Gly 185 190 195gac acc acg gtt aca gaa cca gct cta tcc cgg gat
cat agc gaa cgc 919Asp Thr Thr Val Thr Glu Pro Ala Leu Ser Arg Asp
His Ser Glu Arg200 205 210 215atg ttg cag gcc ttt gga gcc aaa tta
acc att gat cca gta acc cat 967Met Leu Gln Ala Phe Gly Ala Lys Leu
Thr Ile Asp Pro Val Thr His 220 225 230agc gtc act gtc cat ggc ccg
gcc cat tta acg ggg caa cgg gtg gtg 1015Ser Val Thr Val His Gly Pro
Ala His Leu Thr Gly Gln Arg Val Val 235 240 245gtg cca ggg gac atc
agc tcg gcg gcc ttt tgg tta gtg gcg gca tcc 1063Val Pro Gly Asp Ile
Ser Ser Ala Ala Phe Trp Leu Val Ala Ala Ser 250 255 260att ttg cct
gga tca gaa ttg ttg gtg gaa aat gta ggc att aac ccc 1111Ile Leu Pro
Gly Ser Glu Leu Leu Val Glu Asn Val Gly Ile Asn Pro 265 270 275acc
agg aca ggg gtg ttg gaa gtg ttg gcc cag atg ggg gcg gac att 1159Thr
Arg Thr Gly Val Leu Glu Val Leu Ala Gln Met Gly Ala Asp Ile280 285
290 295acc ccg gag aat gaa cga ttg gta acg ggg gaa ccg gta gca gat
ctg 1207Thr Pro Glu Asn Glu Arg Leu Val Thr Gly Glu Pro Val Ala Asp
Leu 300 305 310cgg gtt agg gca agc cat ctc cag ggt tgc acc ttc ggc
ggc gaa att 1255Arg Val Arg Ala Ser His Leu Gln Gly Cys Thr Phe Gly
Gly Glu Ile 315 320 325att ccc cga ctg att gat gaa att ccc att ttg
gca gtg gcg gcg gcc 1303Ile Pro Arg Leu Ile Asp Glu Ile Pro Ile Leu
Ala Val Ala Ala Ala 330 335 340ttt gca gag ggc act acc cgc att gaa
gat gcc gca gaa ctg agg gtt 1351Phe Ala Glu Gly Thr Thr Arg Ile Glu
Asp Ala Ala Glu Leu Arg Val 345 350 355aaa gaa agc gat cgc ctg gcg
gcc att gct tcg gag ttg ggc aaa atg 1399Lys Glu Ser Asp Arg Leu Ala
Ala Ile Ala Ser Glu Leu Gly Lys Met360 365 370 375ggg gcc aaa gtc
acc gaa ttt gat gat ggc ctg gaa att caa ggg gga 1447Gly Ala Lys Val
Thr Glu Phe Asp Asp Gly Leu Glu Ile Gln Gly Gly 380 385 390agc ccg
tta caa ggg gcc gag gtg gat agc ttg acg gat cat cgc att 1495Ser Pro
Leu Gln Gly Ala Glu Val Asp Ser Leu Thr Asp His Arg Ile 395 400
405gcc atg gcg ttg gcg atc gcc gct tta ggt agt ggg ggg caa aca att
1543Ala Met Ala Leu Ala Ile Ala Ala Leu Gly Ser Gly Gly Gln Thr Ile
410 415 420att aac cgg gcg gaa gcg gcc gcc att tcc tat cca gaa ttt
ttt ggc 1591Ile Asn Arg Ala Glu Ala Ala Ala Ile Ser Tyr Pro Glu Phe
Phe Gly 425 430 435acg cta ggg caa gtt gcc caa gga taa agttagaaaa
actcctgggc 1638Thr Leu Gly Gln Val Ala Gln Gly440 445ggtttgtaaa
tgttttacca aggtagtttg gggtaaaggc cccagcaagt gctgccaggg
1698taatttatcc gcaattgacc aatcggcatg gaccgtatcg ttcaaactgg
gtaattctcc 1758ctttaattcc ttaaaagctc gcttaaaact gcccaacgta
tctccgtaat ggcgagtgag 1818tagaagtaat ggggccaaac ggcgatcgcc
acgggaaatt aaagcctgca tcactgacca 1878cttataactt tcggga
189467447PRTSynechocystis sp. 67Met Ala Leu Leu Ser Leu Asn Asn His
Gln Ser His Gln Arg Leu Thr1 5 10 15Val Asn Pro Pro Ala Gln Gly Val
Ala Leu Thr Gly Arg Leu Arg Val 20 25 30Pro Gly Asp Lys Ser Ile Ser
His Arg Ala Leu Met Leu Gly Ala Ile 35 40 45Ala Thr Gly Glu Thr Ile
Ile Glu Gly Leu Leu Leu Gly Glu Asp Pro 50 55 60Arg Ser Thr Ala His
Cys Phe Arg Ala Met Gly Ala Glu Ile Ser Glu65 70 75 80Leu Asn Ser
Glu Lys Ile Ile Val Gln Gly Arg Gly Leu Gly Gln Leu 85 90 95Gln Glu
Pro Ser Thr Val Leu Asp Ala Gly Asn Ser Gly Thr Thr Met 100 105
110Arg Leu Met Leu Gly Leu Leu Ala Gly Gln Lys Asp Cys Leu Phe Thr
115 120 125Val Thr Gly Asp Asp Ser Leu Arg His Arg Pro Met Ser Arg
Val Ile 130 135 140Gln Pro Leu Gln Gln Met Gly Ala Lys Ile Trp Ala
Arg Ser Asn Gly145 150 155 160Lys Phe Ala Pro Leu Ala Val Gln Gly
Ser Gln Leu Lys Pro Ile His 165 170 175Tyr His Ser Pro Ile Ala Ser
Ala Gln Val Lys Ser Cys Leu Leu Leu 180 185 190Ala Gly Leu Thr Thr
Glu Gly Asp Thr Thr Val Thr Glu Pro Ala Leu 195 200 205Ser Arg Asp
His Ser Glu Arg Met Leu Gln Ala Phe Gly Ala Lys Leu 210 215 220Thr
Ile Asp Pro Val Thr His Ser Val Thr Val His Gly Pro Ala His225 230
235 240Leu Thr Gly Gln Arg Val Val Val Pro Gly Asp Ile Ser Ser Ala
Ala 245 250 255Phe Trp Leu Val Ala Ala Ser Ile Leu Pro Gly Ser Glu
Leu Leu Val 260 265 270Glu Asn Val Gly Ile Asn Pro Thr Arg Thr Gly
Val Leu Glu Val Leu 275 280 285Ala Gln Met Gly Ala Asp Ile Thr Pro
Glu Asn Glu Arg Leu Val Thr 290 295 300Gly Glu Pro Val Ala Asp Leu
Arg Val Arg Ala Ser His Leu Gln Gly305 310 315 320Cys Thr Phe Gly
Gly Glu Ile Ile Pro Arg Leu Ile Asp Glu Ile Pro 325 330 335Ile Leu
Ala Val Ala Ala Ala Phe Ala Glu Gly Thr Thr Arg Ile Glu 340 345
350Asp Ala Ala Glu Leu Arg Val Lys Glu Ser Asp Arg Leu Ala Ala Ile
355 360 365Ala Ser Glu Leu Gly Lys Met Gly Ala Lys Val Thr Glu Phe
Asp Asp 370 375 380Gly Leu Glu Ile Gln Gly Gly Ser Pro Leu Gln Gly
Ala Glu Val Asp385 390 395 400Ser Leu Thr Asp His Arg Ile Ala Met
Ala Leu Ala Ile Ala Ala Leu 405 410 415Gly Ser Gly Gly Gln Thr Ile
Ile Asn Arg Ala Glu Ala Ala Ala Ile 420 425 430Ser Tyr Pro Glu Phe
Phe Gly Thr Leu Gly Gln Val Ala Gln Gly 435 440
445681479DNADichelobacter nodosusCDS(107)..(1438) 68tttaaaaaca
atgagttaaa aaattatttt tctggcacac gcgctttttt tgcatttttt 60ctcccatttt
tccggcacaa taacgttggt tttataaaag gaaatg atg atg acg 115 Met Met Thr
1aat ata tgg cac acc gcg ccc gtc tct gcg ctt tcc ggc gaa ata acg
163Asn Ile Trp His Thr Ala Pro Val Ser Ala Leu Ser Gly Glu Ile Thr
5 10 15ata tgc ggc gat aaa tca atg tcg cat cgc gcc tta tta tta gca
gcg 211Ile Cys Gly Asp Lys Ser Met Ser His Arg Ala Leu Leu Leu Ala
Ala20 25 30 35tta gca gaa gga caa acg gaa atc cgc ggc ttt tta gcg
tgc gcg gat 259Leu Ala Glu Gly Gln Thr Glu Ile Arg Gly Phe Leu Ala
Cys Ala Asp 40 45 50tgt ttg gcg acg cgg caa gca ttg cgc gca tta ggc
gtt gat att caa 307Cys Leu Ala Thr Arg Gln Ala Leu Arg Ala Leu Gly
Val Asp Ile Gln 55 60 65aga gaa aaa gaa ata gtg acg att cgc ggt gtg
gga ttt ctg ggt ttg 355Arg Glu Lys Glu Ile Val Thr Ile Arg Gly Val
Gly Phe Leu Gly Leu 70 75 80cag ccg ccg aaa gca ccg tta aat atg caa
aac agt ggc act agc atg 403Gln Pro Pro Lys Ala Pro Leu Asn Met Gln
Asn Ser Gly Thr Ser Met 85 90 95cgt tta ttg gca gga att ttg gca gcg
cag cgc ttt gag agc gtg tta 451Arg Leu Leu Ala Gly Ile Leu Ala Ala
Gln Arg Phe Glu Ser Val Leu100 105
110 115tgc ggc gat gaa tca tta gaa aaa cgt ccg atg cag cgc att att
acg 499Cys Gly Asp Glu Ser Leu Glu Lys Arg Pro Met Gln Arg Ile Ile
Thr 120 125 130ccg ctt gtg caa atg ggg gca aaa att gtc agt cac agc
aat ttt acg 547Pro Leu Val Gln Met Gly Ala Lys Ile Val Ser His Ser
Asn Phe Thr 135 140 145gcg ccg tta cat att tca gga cgc ccg ctg acc
ggc att gat tac gcg 595Ala Pro Leu His Ile Ser Gly Arg Pro Leu Thr
Gly Ile Asp Tyr Ala 150 155 160tta ccg ctt ccc agc gcg caa tta aaa
agt tgc ctt att ttg gca gga 643Leu Pro Leu Pro Ser Ala Gln Leu Lys
Ser Cys Leu Ile Leu Ala Gly 165 170 175tta ttg gct gac ggt acc acg
cgg ctg cat act tgc ggc atc agt cgc 691Leu Leu Ala Asp Gly Thr Thr
Arg Leu His Thr Cys Gly Ile Ser Arg180 185 190 195gac cac acg gaa
cgc atg ttg ccg ctt ttt ggt ggc gca ctt gag atc 739Asp His Thr Glu
Arg Met Leu Pro Leu Phe Gly Gly Ala Leu Glu Ile 200 205 210aag aaa
gag caa ata atc gtc acc ggt gga caa aaa ttg cac ggt tgc 787Lys Lys
Glu Gln Ile Ile Val Thr Gly Gly Gln Lys Leu His Gly Cys 215 220
225gtg ctt gat att gtc ggc gat ttg tcg gcg gcg gcg ttt ttt atg gtt
835Val Leu Asp Ile Val Gly Asp Leu Ser Ala Ala Ala Phe Phe Met Val
230 235 240gcg gct ttg att gcg ccg cgc gcg gaa gtc gtt att cgt aat
gtc ggc 883Ala Ala Leu Ile Ala Pro Arg Ala Glu Val Val Ile Arg Asn
Val Gly 245 250 255att aat ccg acg cgg gcg gca atc att act ttg ttg
caa aaa atg ggc 931Ile Asn Pro Thr Arg Ala Ala Ile Ile Thr Leu Leu
Gln Lys Met Gly260 265 270 275gga cgg att gaa ttg cat cat cag cgc
ttt tgg ggc gcc gaa ccg gtg 979Gly Arg Ile Glu Leu His His Gln Arg
Phe Trp Gly Ala Glu Pro Val 280 285 290gca gat att gtt gtt tat cat
tca aaa ttg cgc ggc att acg gtg gcg 1027Ala Asp Ile Val Val Tyr His
Ser Lys Leu Arg Gly Ile Thr Val Ala 295 300 305ccg gaa tgg att gcc
aac gcg att gat gaa ttg ccg att ttt ttt att 1075Pro Glu Trp Ile Ala
Asn Ala Ile Asp Glu Leu Pro Ile Phe Phe Ile 310 315 320gcg gca gct
tgc gcg gaa ggg acg act ttt gtg ggc aat ttg tca gaa 1123Ala Ala Ala
Cys Ala Glu Gly Thr Thr Phe Val Gly Asn Leu Ser Glu 325 330 335ttg
cgt gtg aaa gaa tcg gat cgt tta gcg gcg atg gcg caa aat tta 1171Leu
Arg Val Lys Glu Ser Asp Arg Leu Ala Ala Met Ala Gln Asn Leu340 345
350 355caa act ttg ggc gtg gcg tgc gac gtt ggc gcc gat ttt att cat
ata 1219Gln Thr Leu Gly Val Ala Cys Asp Val Gly Ala Asp Phe Ile His
Ile 360 365 370tat gga aga agc gat cgg caa ttt tta ccg gcg cgg gtg
aac agt ttt 1267Tyr Gly Arg Ser Asp Arg Gln Phe Leu Pro Ala Arg Val
Asn Ser Phe 375 380 385ggc gat cat cgg att gcg atg agt ttg gcg gtg
gca ggt gtg cgc gcg 1315Gly Asp His Arg Ile Ala Met Ser Leu Ala Val
Ala Gly Val Arg Ala 390 395 400gca ggt gaa tta ttg att gat gac ggc
gcg gtg gcg gcg gtt tct atg 1363Ala Gly Glu Leu Leu Ile Asp Asp Gly
Ala Val Ala Ala Val Ser Met 405 410 415ccg caa ttt cgc gat ttt gcc
gcc gca att ggt atg aat gta gga gaa 1411Pro Gln Phe Arg Asp Phe Ala
Ala Ala Ile Gly Met Asn Val Gly Glu420 425 430 435aaa gat gcg aaa
aat tgt cac gat tga tggtcctagc ggtgttggaa 1458Lys Asp Ala Lys Asn
Cys His Asp 440aaggcacggt ggcgcaagct t 147969443PRTDichelobacter
nodosus 69Met Met Thr Asn Ile Trp His Thr Ala Pro Val Ser Ala Leu
Ser Gly1 5 10 15Glu Ile Thr Ile Cys Gly Asp Lys Ser Met Ser His Arg
Ala Leu Leu 20 25 30Leu Ala Ala Leu Ala Glu Gly Gln Thr Glu Ile Arg
Gly Phe Leu Ala 35 40 45Cys Ala Asp Cys Leu Ala Thr Arg Gln Ala Leu
Arg Ala Leu Gly Val 50 55 60Asp Ile Gln Arg Glu Lys Glu Ile Val Thr
Ile Arg Gly Val Gly Phe65 70 75 80Leu Gly Leu Gln Pro Pro Lys Ala
Pro Leu Asn Met Gln Asn Ser Gly 85 90 95Thr Ser Met Arg Leu Leu Ala
Gly Ile Leu Ala Ala Gln Arg Phe Glu 100 105 110Ser Val Leu Cys Gly
Asp Glu Ser Leu Glu Lys Arg Pro Met Gln Arg 115 120 125Ile Ile Thr
Pro Leu Val Gln Met Gly Ala Lys Ile Val Ser His Ser 130 135 140Asn
Phe Thr Ala Pro Leu His Ile Ser Gly Arg Pro Leu Thr Gly Ile145 150
155 160Asp Tyr Ala Leu Pro Leu Pro Ser Ala Gln Leu Lys Ser Cys Leu
Ile 165 170 175Leu Ala Gly Leu Leu Ala Asp Gly Thr Thr Arg Leu His
Thr Cys Gly 180 185 190Ile Ser Arg Asp His Thr Glu Arg Met Leu Pro
Leu Phe Gly Gly Ala 195 200 205Leu Glu Ile Lys Lys Glu Gln Ile Ile
Val Thr Gly Gly Gln Lys Leu 210 215 220His Gly Cys Val Leu Asp Ile
Val Gly Asp Leu Ser Ala Ala Ala Phe225 230 235 240Phe Met Val Ala
Ala Leu Ile Ala Pro Arg Ala Glu Val Val Ile Arg 245 250 255Asn Val
Gly Ile Asn Pro Thr Arg Ala Ala Ile Ile Thr Leu Leu Gln 260 265
270Lys Met Gly Gly Arg Ile Glu Leu His His Gln Arg Phe Trp Gly Ala
275 280 285Glu Pro Val Ala Asp Ile Val Val Tyr His Ser Lys Leu Arg
Gly Ile 290 295 300Thr Val Ala Pro Glu Trp Ile Ala Asn Ala Ile Asp
Glu Leu Pro Ile305 310 315 320Phe Phe Ile Ala Ala Ala Cys Ala Glu
Gly Thr Thr Phe Val Gly Asn 325 330 335Leu Ser Glu Leu Arg Val Lys
Glu Ser Asp Arg Leu Ala Ala Met Ala 340 345 350Gln Asn Leu Gln Thr
Leu Gly Val Ala Cys Asp Val Gly Ala Asp Phe 355 360 365Ile His Ile
Tyr Gly Arg Ser Asp Arg Gln Phe Leu Pro Ala Arg Val 370 375 380Asn
Ser Phe Gly Asp His Arg Ile Ala Met Ser Leu Ala Val Ala Gly385 390
395 400Val Arg Ala Ala Gly Glu Leu Leu Ile Asp Asp Gly Ala Val Ala
Ala 405 410 415Val Ser Met Pro Gln Phe Arg Asp Phe Ala Ala Ala Ile
Gly Met Asn 420 425 430Val Gly Glu Lys Asp Ala Lys Asn Cys His Asp
435 44070455PRTArtificial sequenceSynthetic 70Met Leu His Gly Ala
Ser Ser Arg Pro Ala Thr Ala Arg Lys Ser Ser1 5 10 15Gly Leu Ser Gly
Thr Val Arg Ile Pro Gly Asp Lys Ser Ile Ser His 20 25 30Arg Ser Phe
Met Phe Gly Gly Leu Ala Ser Gly Glu Thr Arg Ile Thr 35 40 45Gly Leu
Leu Glu Gly Glu Asp Val Ile Asn Thr Gly Lys Ala Met Gln 50 55 60Ala
Met Gly Ala Arg Ile Arg Lys Glu Gly Asp Thr Trp Ile Ile Asp65 70 75
80Gly Val Gly Asn Gly Gly Leu Leu Ala Pro Glu Ala Pro Leu Asp Phe
85 90 95Gly Asn Ala Ala Thr Gly Cys Arg Leu Thr Met Gly Leu Val Gly
Val 100 105 110Tyr Asp Phe Asp Ser Thr Phe Ile Gly Asp Ala Ser Leu
Thr Lys Arg 115 120 125Pro Met Gly Arg Val Leu Asn Pro Leu Arg Glu
Met Gly Val Gln Val 130 135 140Lys Ser Glu Asp Gly Asp Arg Leu Pro
Val Thr Leu Arg Gly Pro Lys145 150 155 160Thr Pro Thr Pro Ile Thr
Tyr Arg Val Pro Met Ala Ser Ala Gln Val 165 170 175Lys Ser Ala Val
Leu Leu Ala Gly Leu Asn Thr Pro Gly Ile Thr Thr 180 185 190Val Ile
Glu Pro Ile Met Thr Arg Asp His Thr Glu Lys Met Leu Gln 195 200
205Gly Phe Gly Ala Asn Leu Thr Val Glu Thr Asp Ala Asp Gly Val Arg
210 215 220Thr Ile Arg Leu Glu Gly Arg Gly Lys Leu Thr Gly Gln Val
Ile Asp225 230 235 240Val Pro Gly Asp Pro Ser Ser Thr Ala Phe Pro
Leu Val Ala Ala Leu 245 250 255Leu Val Pro Gly Ser Asp Val Thr Ile
Leu Asn Val Leu Met Asn Pro 260 265 270Thr Arg Thr Gly Leu Ile Leu
Thr Leu Gln Glu Met Gly Ala Asp Ile 275 280 285Glu Val Ile Asn Pro
Arg Leu Ala Gly Gly Glu Asp Val Ala Asp Leu 290 295 300Arg Val Arg
Ser Ser Thr Leu Lys Gly Val Thr Val Pro Glu Asp Arg305 310 315
320Ala Pro Ser Met Ile Asp Glu Tyr Pro Ile Leu Ala Val Ala Ala Ala
325 330 335Phe Ala Glu Gly Ala Thr Val Met Asn Gly Leu Glu Glu Leu
Arg Val 340 345 350Lys Glu Ser Asp Arg Leu Ser Ala Val Ala Asn Gly
Leu Lys Leu Asn 355 360 365Gly Val Asp Cys Asp Glu Gly Glu Thr Ser
Leu Val Val Arg Gly Arg 370 375 380Pro Asp Gly Lys Gly Leu Gly Asn
Ala Ser Gly Ala Ala Val Ala Thr385 390 395 400His Leu Asp His Arg
Ile Ala Met Ser Phe Leu Val Met Gly Leu Val 405 410 415Ser Glu Asn
Pro Val Thr Val Asp Asp Ala Thr Met Ile Ala Thr Ser 420 425 430Phe
Pro Glu Phe Met Asp Leu Met Ala Gly Leu Gly Ala Lys Ile Glu 435 440
445Leu Ser Asp Thr Lys Ala Ala 450 455
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