U.S. patent application number 10/035060 was filed with the patent office on 2003-02-27 for process for altering the host range of bacillus thuringiensis toxins, and novel toxins produced thereby.
Invention is credited to Edwards, David L., Herrnstadt, Corinna, Wilcox, Edward R., Wong, Siu-Yin.
Application Number | 20030040619 10/035060 |
Document ID | / |
Family ID | 27123085 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030040619 |
Kind Code |
A1 |
Edwards, David L. ; et
al. |
February 27, 2003 |
Process for altering the host range of bacillus thuringiensis
toxins, and novel toxins produced thereby
Abstract
The invention concerns an in vitro process for altering the
insect host range (spectrum) of pesticidal toxins. The process
comprises recombining in vitro the variable region(s)
(non-homologous) of two or more genes encoding a pesticidal toxin.
Specifically exemplified is the recombining of the variable regions
of two genes obtained from well-known strains of Bacillius
thuringiensis var. kurstaki. The resulting products are chimeric
toxins which are shown to have an expanded and/or amplified insect
host range as compared to the parent toxins.
Inventors: |
Edwards, David L.; (Del Mar,
CA) ; Herrnstadt, Corinna; (San Diego, CA) ;
Wilcox, Edward R.; (Escondido, CA) ; Wong,
Siu-Yin; (San Diego, CA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Family ID: |
27123085 |
Appl. No.: |
10/035060 |
Filed: |
December 27, 2001 |
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Application
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10035060 |
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09405788 |
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09405788 |
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08580781 |
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08420615 |
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08420615 |
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07980128 |
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07980128 |
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07803920 |
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07803920 |
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07356599 |
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07356599 |
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06904572 |
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06904572 |
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06808129 |
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Current U.S.
Class: |
536/23.7 ;
435/252.31; 435/485 |
Current CPC
Class: |
A01N 63/50 20200101;
C12N 1/205 20210501; C12R 2001/075 20210501; C07K 14/325 20130101;
A01N 63/50 20200101; A01N 63/23 20200101 |
Class at
Publication: |
536/23.7 ;
435/485; 435/252.31 |
International
Class: |
C12N 015/75; C07H
021/04; C12N 001/21 |
Claims
1. A process for altering the host range of Bacillus toxins which
comprises recombining in vitro the variable region of two or more
Bacillus toxin genes.
2. A process, according to claim 1, wherein the Bacillus is a
Bacillius thuringiensis.
3. A process, according to claim 2, wherein variable regions of
Bacillius thuringiensis var. kurstaki HD-1 and Bacillius
thuringiensis var. kurstaki HD-73 are recombined in vitro to give
genes encoding chimeric toxins having altered host ranges.
4. DNA, denoted pEW3, encoding a chimeric toxin having pesticidal
activity, as follows:
19 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGGA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
and equivalent nucleotide sequences coding for toxin EW3 with the
following amino acid sequence:
20 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N Q L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E.
5. DNA, denoted pEW4, encoding a chimeric toxin, having pesticidal
activity, as follows:
21 (start HD-1) ATGG ATAACAATCC GAACATCAAT GAATGCATTC CTTATAATTG
TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 600 TGGAGAAAGA ATAGAAACTG
GTTACACCCC AATCGATATT TCCTTGTCGC TAACGCAATT TCTTTTGAGT GAATTTGTTC
CCGGTGCTGG ATTTGTGTTA 700 GGACTAGTTG ATATAATATG GGGAATTTTT
GGTCCCTCTC AATGGGACGC ATTTCCTGTA CAAATTGAAC AGTTAATTAA CCAAAGAATA
GAAGAATTCG 800 CTAGGAACCA AGCCATTTCT AGATTAGAAG GACTAAGCAA
TCTTTATCAA ATTTACGCAG AATCTTTTAG AGAGTGGGAA GCAGATCCTA CTAATCCAGC
900 ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC AGTGCCCTTA
CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTTTA 1000
TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT TTCAGTGTTT
GGACAAAGGT GGGGATTTGA TGCCGCGACT ATCAATAGTC 1100 GTTATAATGA
TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG CGCTGGTACA ATACGGGATT
AGAGCGTGTA TGGGGACCGG ATTCTAGAGA 1200 TTGGGTAAGG TATAATCAAT
TTAGAAGAGA GCTAACACTT ACTGTATTAG ATATCGTTGC TCTATTCTCA AATTATGATA
GTCGAAGGTA TCCAATTCGA 1300 ACAGTTTCCC AATTAACAAG AGAAATTTAT
ACGAACCCAG TATTAGAAAA TTTTGATGGT AGTTTTCGTG GAATGGCTCA GAGAATAGAA
CAGAATATTA 1400 GGCAACCACA TCTTATGGAT ATCCTTAATA GTATAACCAT
TTATACTGAT GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA CAGCTTCTCC
1500 TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT GGGAATGCGG
GGAATGCAGC TCCACCCGTA CTTGTCTCAT TAACTGGTTT GGGGPTTTTT 1600
AGAACATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCAGGCCC AAATAATCAG
GAACTGTTTG TCCTTGATGG AACGGAGTTT TCTTTTGCCT 1700 CCCTAACGAC
CAACTTGCCT TCCACTATAT ATAGACAAAG GGGTACAGTC GATTCACTAG ATGTAATACC
GCCACAGGAT AATAGTGTAC CACCTCGTGC 1800 GGGATTTAGC CATCGATTGA
GTCATGTTAC AATGCTGAGC CAAGCAGCTG GAGCAGTTTA CACCTTGAGA GCTCAACGT
(stop HD-1) (start HD-73) CCT ATGTTCTCTT GGATACATCG TAGTGCTGAA
TTTAATAATA TAATTGCATC GGATAGTATT 1800 ACTCAAATCC CTGCAGTGAA
GGGAAACTTT CTTTTTAATG GTTCTGTAAT TTCAGGACCA GGATTTACTG GTGGGGACTT
AGTTAGATTA AATAGTAGTG 1900 GAAATAACAT TCAGAATAGA GGGTATATTG
AAGTTCCAAT TCACTTCCCA TCGACATCTA CCAGATATCG AGTTCGTGTA CGGTATGCTT
CTGTAACCCC 2000 GATTCACCTC AACGTTAATT GGGGTAATTC ATCCATTTTT
TCCAATACAG TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG TGATTTTGGT
2100 TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA ATATAGTAGG
TGTTAGAAAT TTTAGTGGGA CTGCAGGAGT GATAATAGAC AGATTTGAAT 2200
TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGAAAGAGCG CAGAAGGCGG
TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 2300 AACAAATGTA
ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT ATTTATCGGA TGAATTTTGT
CTGGATGAAA AGCGAGAATT GTCCGAGAAA 2400 GTCAAACATG CGAAGCGACT
CAGTGATGAA CGCAATTTAC TCCAAGATTC AAATTTCAAA GACATTAATA GGCAACCAGA
ACGTGGGTGG GGCGGAAGTA 2500 CAGGGATTAC CATCCAAGGA GGGGATGACG
TATTTAAAGA AAATTACGTC ACACTATCAG GTACCTTTGA TGAGTGCTAT CCAACATATT
TGTATCAAAA 2600 AATCGATGAA TCAAAATTAA AAGCCTTTAC CCGTTATCAA
TTAAGAGGGT ATATCGAAGA TAGTCAAGAC TTAGAAATCT ATTTAATTCG CTACAATGCA
2700 AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT GGCCGCTTTC
AGCCCAAAGT CCAATCGGAA AGTGTGGAGA GCCGAATCGA TGCGCGCCAC 2800
ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG TGTGCCCATC
ATTCGCATCA TTTCTCCTTA GACATTGATG TAGGATGTAC 2900 AGACTTAAAT
GAGGACCTAG GTGTATGGGT GATCTTTAAG ATTAAGACGC AAGATGGGCA CGCAAGACTA
GGGAATCTAG AGTTTCTCGA AGAGAAACCA 3000 TTAGTAGGAG AAGCGCTAGC
TCGTGTGAAA AGAGCGGAGA AAAAATGGAG AGACAAACGT GAAAAATTGG AATGGGAAAC
AAATATCGTT TATAAAGAGG 3100 CAAAAGAATC TGTAGATGCT TTATTTGTAA
ACTCTCAATA TGATCAATTA CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA
AACGTGTTCA 3200 TAGCATTCGA GAAGCTTATC TGCCTGAGCT GTCTGTGATT
CCGGGTGTCA ATGCGGCTAT TTTTGAAGAA TTAGAAGGGC GTATTTTCAC TGCATTCTCC
3300 CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA ATAATGGCTT
ATCCTGCTGG AACGTGAAAG GGCATGTAGA TGTAGAAGAA CAAAACAACC 3400
AACGTTCGGT CCTTGTTGTT CCGGAATGGG AAGCAGAAGT GTCACAAGAA GTTCGTGTCT
GTCCGGGTCG TGGCTATATC CTTCGTGTCA CAGCGTACAA 3500 GGAGGGATAT
GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA CAGACGAACT GAAGTTTAGC
AACTGCGTAG AAGAGGAAAT CTATCCAAAT 3600 AACACGGTAA CGTGTAATGA
TTATACTGTA AATCAAGAAG AATACGGAGG TGCGTACACT TCTCGTAATC GAGGATATAA
CGAAGCTCCT TCCGTACCAG 3700 CTGATTATGC GTCAGTCTAT GAAGAAAAAT
CGTATACAGA TGGACGAAGA GAGAATCCTT GTGAATTTAA CAGAGGGTAT AGGGATTACA
CGCCACTACC 3800 AGTTGGTTAT GTGACAAAAG AATTAGAATA CTTCCCAGAA
ACCGATAAGG TATGGATTGA GATTGGAGAA ACGGAAGGAA CATTTATCGT GGACAGCGTG
3900 GAATTACTCC TTATGGAGGA A (end HD-73)
and equivalent nucleotide sequences coding for toxin EW4 with the
following amino acid sequence:
22 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F P V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L L A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F S N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G M A Q R I E Q N I R Q P H L M D I L N S I T
I Y T D V H R G F N Y W S G H Q I T A S P V G F S G P E F A F P L F
G N A G N A A P P V L V S L T G L G I F R T L S S P L Y R R I I L G
S G P N N Q E L F V L D G T E F S F A S L T T N L P S T I Y R Q R G
T V D S L D V I P P Q D N S V P P R A G F S H R L S H V T M L S Q A
A G A V Y T L R A Q R P M F S W I H R S A E F N N I I A S D S I T Q
I P A V K G N F L F N G S V I S G P G F T G G D L V R L N S S G N N
I Q N R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H L
N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F G Y F E S
A N A F T S S L G N I V G V R N F S G T A G V I I D R F E F I P V T
A T L E A E Y N L E R A Q K A V N A L F T S T N Q L G L K T N V T D
Y H I D Q V S N L V T Y L S D E F C L D E K R E L S E K V K H A K R
L S D E R N L L Q D S N F K D I N R Q P E R G W G G S T G I T I Q G
G D D V F K E N Y V T L S G T F D E C Y P T Y L Y Q K I D E S K L K
A F T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P
G T G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D
C S C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W
V I F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K
R A E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N
S Q Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S
V I P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D
F N N G L S C W N V K G H V D V E E Q N N Q R S V L V V P E W E A E
V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E
Y G G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G
R R E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D
K V W I E I G E T E G T F I V D S V E L L L M E E.
6. DNA, denoted pACB-l, encoding a chimeric toxin, having
pesticidal activity, as follows:
23 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGAAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG AATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
and equivalent; nucleotide sequences coding for toxin ACB-1 with
the following amino acid sequence:
24 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L N E I P P Q N N N V P P R Q E F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E.
7. DNA, denoted pSYW1, encoding a chimeric toxin, having pesticidal
activity, as follows:
25 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AGGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAAA GGGGAATATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
and equivalent nucleotide sequences coding for toxin SYW1 with the
following amino acid sequence:
26 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E G S I R S P H L M D I L N S I T
I Y T D A H K G E Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A P L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E.
8. A chimeric toxin, EW3, having pesticidal activity, having the
following amino acid sequence:
27 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E
and muteins thereof which do not alter the protein secondary
structure.
9. A chimeric toxin, EW4, having pesticidal activity, having the
following amino acid sequence:
28 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F P V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L L A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F S N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G M A Q R I E Q N I R Q P H L M D I L N S I T
I Y T D V H R G F N Y W S G H Q I T A S P V G F S G P E F A F P L F
G N A G N A A P P V L V S L T G L G I F R T L S S P L Y R R I I L G
S G P N N Q E L F V L D G T E F S F A S L T T N L P S T I Y R Q R G
T V D S L D V I P P Q D N S V P P R A G F S H R L S H V T M L S Q A
A G A V Y T L R A Q R P M F S W I H R S A E F N N I I A S D S I T Q
I P A V K G N F L F N G S V I S G P G F T G G D L V R L N S S G N N
I Q N R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H L
N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F G Y F E S
A N A F T S S L G N I V G V R N F S G T A G V I I D R F E F I P V T
A T L E A E Y N L E R A Q K A V N A L F T S T N Q L G L K T N V T D
Y H I D Q V S N L V T Y L S D E F C L D E K R E L S E K V K H A K R
L S D E R N L L Q D S N F K D I N R Q P E R G W G G S T G I T I Q G
G D D V F K E N Y V T L S G T F D E C Y P T Y L Y Q K I D E S K L K
A F T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P
G T G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D
C S C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W
V I F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K
R A E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N
S Q Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S
V I P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D
F N N G L S C W N V K G H V D V E E Q N N Q R S V L V V P E W E A E
V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E
Y G G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G
R R E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D
K V W I E I G E T E G T F I V D S V E L L L M E E
and muteins thereof which do not alter the protein secondary
structure.
10. A chimeric toxin, ACB-1, having pesticidal activity, having the
following amino acid sequence:
29 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L N E I P P Q N N N V P P R Q E F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E
and muteins thereof which do not alter the protein secondary
structure.
11. A chimeric toxin, SYW1, having pesticidal activity, having the
following amino acid sequence:
30 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E G S I R S P H L M D I L N S I T
I Y T D A H K G E Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E
and muteins thereof which do not alter the protein secondary
structure.
12. A pesticidal composition comprising pesticide-containing
substantially intact cells having prolonged pesticidal activity
when applied to the environment of a target pest, wherein said
pesticide is a chimeric toxin, is intracellular and is produced as
a result of expression of a heterologous gene encoding said
chimeric toxin in said cell.
13. A pesticidal composition according to claim 12, wherein said
cells are killed under protease deactivating or cell wall
strengthening conditions, while retaining pesticidal activity.
14. A pesticidal composition, according to claim 12, wherein said
cells are prokaryotes selected from the group consisting of
Enterobacteriaceae, Bacillaceae, Rhizobiaceae, Spirillaceae,
Lactobacillaceae, Pseudomonadaceae, Azotobacteraceae, and
Nitrobacteraceae; or lower eukaryotes selected from the group
consisting of Phycomycetes, Ascomycetes, and Basidiomycetes.
15. A pesticidal composition, according to claim 14, wherein said
prokaryote is a Bacillus specie selected from a pesticide-Producing
strain of Bacillus thuringiensis, consisting of B. thuringiensis
M-7, B. thuringiensis var. kurstaki, B. thuringiensis var.
finitimus, B. thuringiensis var. alesti, B. thuringiensis var.
sotto, B. thuringiensis var. dendrolimus, B. thuringiensis var.
kenyae B. thuringiensis var. galleriae, B. thuringiensis var.
canadensis, B. thuringiensis var. entomocidus, B. thuringiensis
var. subtoxicus, B. thuringiensis var. aizawai, B. thuringiensis
var. morrisoni, B. thuringiensis var. ostriniae, B. thuringiensis
var. tolworthi, B. thuringiensis var. darmstadiensis, B.
thuringiensis var. toumanoffi, B. thuringiensis var. kyushuensis,
B. thuringiensis var. thompsoni, B. thuringiensis var. pakistani,
B. thuringiensis var. israelensis, B. thuringiensis var. indiana,
B. thuringiensis var. dakota, B. thuringiensis var. tohokuensis, B.
thuringiensis var. kumanotoensis, B. thuringiensis var.
tochigiensis, B. thuringiensis var. colmeri, B. thuringiensis var.
wuhanensis, B. thuringiensis var. tenebrionis, B. thuringiensis
var. thuringiensis, and other Bacillus species selected from B.
cereus, B. moritai, B. popilliae, B. lentimorbus, and B.
sphaericus.
16. A method of protecting plants against pests which comprises
applying to said plants an effective amount of a pesticidal
composition comprising pesticide-containing substantially intact
unicellular microorganisms, wherein said pesticide is a chimeric
toxin, is intracellular, and is produced as a result of expression
of a heterologous gene encoding said chimeric toxin in said
microorganism, and said microorganism is treated under conditions
which prolong the pesticidal activity when said composition is
applied to the environment of a target pest.
17. A method according to claim 16, wherein said microorganisms are
prokaryotes selected from the group consisting of
Enterobacteriaceae, Bacillaceae, Rhizobiaceae, Spirillaceae,
Lactobacillaceae, Pseudomonadaceae, Azotobacteraceae, and
Nitrobacteraceae; or lower eukaryotes, selected from the group
consisting of Phycomycetes, Ascomycetes, and Basidiomycetes.
18. A method according to claim 16, wherein said unicellular
microorganisms are killed under protease deactivating or cell wall
strengthening conditions, while retaining pesticidal activity.
19. Substantially intact unicellular microorganism cells containing
an intracellular chimeric toxin, which toxin is a result of
expression of a heterologous gene encoding said chimeric toxin,
wherein said cells are killed under protease deactivating or cell
wall strengthening conditions, while retaining pesticidal activity
when said cell is applied to the environment of a target pest.
20. Cells according to claim 19, wherein said microorganism is a
Pseudomonad and said toxin is derived from a B. thuringiensis.
21. A pesticidal composition, according to claim 12, wherein said
gene, denoted pEW3, encoding a chimeric toxin, is as follows:
31 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
and equivalent nucleotide sequences coding for toxin EW3 with the
following amino acid sequence:
32 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E.
22. A pesticidal composition, according to claim 12, wherein said
gene, denoted pEW4, encoding a chimeric toxin, is as follows:
33 (start HD-1) ATGG ATAACAATCC GAACATCAAT GAATGCATTC CTTATAATTG
TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 600 TGGAGAAAGA ATAGAAACTG
GTTACACCCC AATCGATATT TCCTTGTCGC TAACGCAATT TCTTTTGAGT GAATTTGTTC
CCGGTGCTGG ATTTGTGTTA 700 GGACTAGTTG ATATAATATG GGGAATTTTT
GGTCCCTCTC AATGGGACGC ATTTCCTGTA CAAATTGAAC AGTTAATTAA CCAAAGAATA
GAAGAATTCG 800 CTAGGAACCA AGCCATTTCT AGATTAGAAG GACTAAGCAA
TCTTTATCAA ATTTACGCAG AATCTTTTAG AGAGTGGGAA GCAGATCCTA CTAATCCAGC
900 ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC AGTGCCCTTA
CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTTTA 1000
TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT TTCAGTGTTT
GGACAAAGGT GGGGATTTGA TGCCGCGACT ATCAATAGTC 1100 GTTATAATGA
TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG CGCTGGTACA ATACGGGATT
AGAGCGTGTA TGGGGACCGG ATTCTAGAGA 1200 TTGGGTAAGG TATAATCAAT
TTAGAAGAGA GCTAACACTT ACTGTATTAG ATATCGTTGC TCTATTCTCA AATTATGATA
GTCGAAGGTA TCCAATTCGA 1300 ACAGTTTCCC AATTAACAAG AGAAATTTAT
ACGAACCCAG TATTAGAAAA TTTTGATGGT AGTTTTCGTG GAATGGCTCA GAGAATAGAA
CAGAATATTA 1400 GGCAACCACA TCTTATGGAT ATCCTTAATA GTATAACCAT
TTATACTGAT GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA CAGCTTCTCC
1500 TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT GGGAATGCGG
GGAATGCAGC TCCACCCGTA CTTGTCTCAT TAACTGGTTT GGGGATTTTT 1600
AGAACATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCAGGCCC AAATAATCAG
GAACTGTTTG TCCTTGATGG AACGGAGTTT TCTTTTGCCT 1700 CCCTAACGAC
CAACTTGCCT TCCACTATAT ATAGACAAAG GGGTACAGTC GATTCACTAG ATGTAATACC
GCCACAGGAT AATAGTGTAC CACCTCGTGC 1800 GGGATTTAGC CATCGATTGA
GTCATGTTAC AATGCTGAGC CAAGCAGCTG GAGCAGTTTA CACCTTGAGA GCTCAACGT
(stop HD-1 (start HD-73) CCT ATGTTCTCTT GGATACATCG TAGTGCTGAA
TTTAATAATA TAATTGCATC GGATAGTATT 1800 ACTCAAATCC CTGCAGTGAA
GGGAAACTTT CTTTTTAATG GTTCTGTAAT TTCAGGACCA GGATTTACTG GTGGGGACTT
AGTTAGATTA AATAGTAGTG 1900 GAAATAACAT TCAGAATAGA GGGTATATTG
AAGTTCCAAT TCACTTCCCA TCGACATCTA CCAGATATCG AGTTCGTGTA CGGTATGCTT
CTGTAACCCC 2000 GATTCACCTC AACGTTAATT GGGGTAATTC ATCCATTTTT
TCCAATACAG TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG TGATTTTGGT
2100 TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA ATATAGTAGG
TGTTAGAAAT TTTAGTGGGA CTGCAGGAGT GATAATAGAC AGATTTGAAT 2200
TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGAAAGAGCG CAGAAGGCGG
TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 2300 AACAAATGTA
ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT ATTTATCGGA TGAATTTTGT
CTGGATGAAA AGCGAGAATT GTCCGAGAAA 2400 GTCAAACATG CGAAGCGACT
CAGTGATGAA CGCAATTTAC TCCAAGATTC AAATTTCAAA GACATTAATA GGCAACCAGA
ACGTGGGTGG GGCGGAAGTA 2500 CAGGGATTAC CATCCAAGGA GGGGATGACG
TATTTAAAGA AAATTACGTC ACACTATCAG GTACCTTTGA TGAGTGCTAT CCAACATATT
TGTATCAAAA 2600 AATCGATGAA TCAAAATTAA AAGCCTTTAC CCGTTATCAA
TTAAGAGGGT ATATCGAAGA TAGTCAAGAC TTAGAAATCT ATTTAATTCG CTACAATGCA
2700 AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT GGCCGCTTTC
AGCCCAAAGT CCAATCGGAA AGTGTGGAGA GCCGAATCGA TGCGCGCCAC 2800
ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG TGTGCCCATC
ATTCGCATCA TTTCTCCTTA GACATTGATG TAGGATGTAC 2900 AGACTTAAAT
GAGGACCTAG GTGTATGGGT GATCTTTAAG ATTAAGACGC AAGATGGGCA CGCAAGACTA
GGGAATCTAG AGTTTCTCGA AGAGAAACCA 3000 TTAGTAGGAG AAGCGCTAGC
TCGTGTGAAA AGAGCGGAGA AAAAATGGAG AGACAAACGT GAAAAATTGG AATGGGAAAC
AAATATCGTT TATAAAGAGG 3100 CAAAAGAATC TGTAGATGCT TTATTTGTAA
ACTCTCAATA TGATCAATTA CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA
AACGTGTTCA 3200 TAGCATTCGA GAAGCTTATC TGCCTGAGCT GTCTGTGATT
CCGGGTGTCA ATGCGGCTAT TTTTGAAGAA TTAGAAGGGC GTATTTTCAC TGCATTCTCC
3300 CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA ATAATGGCTT
ATCCTGCTGG AACGTGAAAG GGCATGTAGA TGTAGAAGAA CAAAACAACC 3400
AACGTTCGGT CCTTGTTGTT CCGGAATGGG AAGCAGAAGT GTCACAAGAA GTTCGTGTCT
GTCCGGGTCG TGGCTATATC CTTCGTGTCA CAGCGTACAA 3500 GGAGGGATAT
GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA CAGACGAACT GAAGTTTAGC
AACTGCGTAG AAGAGGAAAT CTATCCAAAT 3600 AACACGGTAA CGTGTAATGA
TTATACTGTA AATCAAGAAG AATACGGAGG TGCGTACACT TCTCGTAATC GAGGATATAA
CGAAGCTCCT TCCGTACCAG 3700 CTGATTATGC GTCAGTCTAT GAAGAAAAAT
CGTATACAGA TGGACGAAGA GAGAATCCTT GTGAATTTAA CAGAGGGTAT AGGGATTACA
CGCCACTACC 3800 AGTTGGTTAT GTGACAAAAG AATTAGAATA CTTCCCAGAA
ACCGATAAGG TATGGATTGA GATTGGAGAA ACGGAAGGAA CATTTATCGT GGACAGCGTG
3900 GAATTACTCC TTATGGAGGA A (end HD-73)
and equivalent nucleotide sequences coding for toxin EW4 with the
following amino acid sequence:
34 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F P V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L L A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F S N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G M A Q R I E Q N I R Q P H L M D I L N S I T
I Y T D V H R G F N Y W S G H Q I T A S P V G F S G P E F A F P L F
G N A G N A A P P V L V S L T G L G I F R T L S S P L Y R R I I L G
S G P N N Q E L F V L D G T E F S F A S L T T N L P S T I Y R Q R G
T V D S L D V I P P Q D N S V P P R A G F S H R L S H V T M L S Q A
A G A V Y T L R A Q R P M F S W I H R S A E F N N I I A S D S I T Q
I P A V K G N F L F N G S V I S G P G F T G G D L V R L N S S G N N
I Q N R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H L
N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F G Y F E S
A N A F T S S L G N I V G V R N F S G T A G V I I D R F E F I P V T
A T L E A E Y N L E R A Q K A V N A L F T S T N Q L G L K T N V T D
Y H I D Q V S N L V T Y L S D E F C L D E K R E L S E K V K H A K R
L S D E R N L L Q D S N F K D I N R Q P E R G W G G S T G I T I Q G
G D D V F K E N Y V T L S G T F D E C Y P T Y L Y Q K I D E S K L K
A F T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P
G T G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D
C S C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W
V I F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K
R A E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N
S Q Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S
V I P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D
F N N G L S C W N V K G H V D V E E Q N N Q R S V L V V P E W E A E
V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E
Y G G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G
R R E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D
K V W I E I G E T E G T F I V D S V E L L L M E E.
23. A pesticidal composition, according to claim 12, wherein said
gene, denoted pACB-1, encoding a chimeric toxin, is as follows:
35 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGAAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG AATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
and equivalent nucleotide sequences coding for toxin ACB-1 with the
following amino acid sequence:
36 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L N E I P P Q N N N V P P R Q E F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E.
24. A pesticidal composition, according to claim 12, wherein said
gene, denoted pSYW1, encoding a chimeric toxin, is as follows:
37 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AGGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAAA GGGGAATATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
and equivalent nucleotide sequences coding for toxin SYW1 with the
following amino acid sequence:
38 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E G S I R S P H L M D I L N S I T
I Y T D A H K G E Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E.
25. A recombinant DNA transfer vector comprising DNA having the
following nucleotide sequence or equivalent nucleotide sequences
containing bases whose translated region codes for the same amino
acid sequence:
39 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1).
26. A recombinant DNA transfer vector comprising DNA having the
following nucleotide sequence or equivalent nucleotide sequences
containing bases whose translated region codes for the same amino
acid sequence:
40 start HD-1) ATGG ATAACAATCC GAACATCAAT GAATGCATTC CTTATAATTG
TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 600 TGGAGAAAGA ATAGAAACTG
GTTACACCCC AATCGATATT TCCTTGTCGC TAACGCAATT TCTTTTGAGT GAATTTGTTC
CCGGTGCTGG ATTTGTGTTA 700 GGACTAGTTG ATATAATATG GGGAATTTTT
GGTCCCTCTC AATGGGACGC ATTTCCTGTA CAAATTGAAC AGTTAATTAA CCAAAGAATA
GAAGAATTCG 800 CTAGGAACCA AGCCATTTCT AGATTAGAAG GACTAAGCAA
TCTTTATCAA ATTTACGCAG AATCTTTTAG AGAGTGGGAA GCAGATCCTA CTAATCCAGC
900 ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC AGTGCCCTTA
CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTTTA 1000
TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT TTCAGTGTTT
GGACAAAGGT GGGGATTTGA TGCCGCGACT ATCAATAGTC 1100 GTTATAATGA
TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG CGCTGGTACA ATACGGGATT
AGAGCGTGTA TGGGGACCGG ATTCTAGAGA 1200 TTGGGTAAGG TATAATCAAT
TTAGAAGAGA GCTAACACTT ACTGTATTAG ATATCGTTGC TCTATTCTCA AATTATGATA
GTCGAAGGTA TCCAATTCGA 1300 ACAGTTTCCC AATTAACAAG AGAAATTTAT
ACGAACCCAG TATTAGAAAA TTTTGATGGT AGTTTTCGTG GAATGGCTCA GAGAATAGAA
CAGAATATTA 1400 GGCAACCACA TCTTATGGAT ATCCTTAATA GTATAACCAT
TTATACTGAT GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA CAGCTTCTCC
1500 TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT GGGAATGCGG
GGAATGCAGC TCCACCCGTA CTTGTCTCAT TAACTGGTTT GGGGATTTTT 1600
AGAACATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCAGGCCC AAATAATCAG
GAACTGTTTG TCCTTGATGG AACGGAGTTT TCTTTTGCCT 1700 CCCTAACGAC
CAACTTGCCT TCCACTATAT ATAGACAAAG GGGTACAGTC GATTCACTAG ATGTAATACC
GCCACAGGAT AATAGTGTAC CACCTCGTGC 1800 GGGATTTAGC CATCGATTGA
GTCATGTTAC AATGCTGAGC CAAGCAGCTG GAGCAGTTTA CACCTTGAGA GCTCAACGT
(stop HD-1) (start HD-73) CCT ATGTTCTCTT GGATACATCG TAGTGCTGAA
TTTAATAATA TAATTGCATC GGATAGTATT 1800 ACTCAAATCC CTGCAGTGAA
GGGAAACTTT CTTTTTAATG GTTCTGTAAT TTCAGGACCA GGATTTACTG GTGGGGACTT
AGTTAGATTA AATAGTAGTG 1900 GAAATAACAT TCAGAATAGA GGGTATATTG
AAGTTCCAAT TCACTTCCCA TCGACATCTA CCAGATATCG AGTTCGTGTA CGGTATGCTT
CTGTAACCCC 2000 GATTCACCTC AACGTTAATT GGGGTAATTC ATCCATTTTT
TCCAATACAG TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG TGATTTTGGT
2100 TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA ATATAGTAGG
TGTTAGAAAT TTTAGTGGGA CTGCAGGAGT GATAATAGAC AGATTTGAAT 2200
TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGAAAGAGCG CAGAAGGCGG
TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 2300 AACAAATGTA
ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT ATTTATCGGA TGAATTTTGT
CTGGATGAAA AGCGAGAATT GTCCGAGAAA 2400 GTCAAACATG CGAAGCGACT
CAGTGATGAA CGCAATTTAC TCCAAGATTC AAATTTCAAA GACATTAATA GGCAACCAGA
ACGTGGGTGG GGCGGAAGTA 2500 CAGGGATTAC CATCCAAGGA GGGGATGACG
TATTTAAAGA AAATTACGTC ACACTATCAG GTACCTTTGA TGAGTGCTAT CCAACATATT
TGTATCAAAA 2600 AATCGATGAA TCAAAATTAA AAGCCTTTAC CCGTTATCAA
TTAAGAGGGT ATATCGAAGA TAGTCAAGAC TTAGAAATCT ATTTAATTCG CTACAATGCA
2700 AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT GGCCGCTTTC
AGCCCAAAGT CCAATCGGAA AGTGTGGAGA GCCGAATCGA TGCGCGCCAC 2800
ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG TGTGCCCATC
ATTCGCATCA TTTCTCCTTA GACATTGATG TAGGATGTAC 2900 AGACTTAAAT
GAGGACCTAG GTGTATGGGT GATCTTTAAG ATTAAGACGC AAGATGGGCA CGCAAGACTA
GGGAATCTAG AGTTTCTCGA AGAGAAACCA 3000 TTAGTAGGAG AAGCGCTAGC
TCGTGTGAAA AGAGCGGAGA AAAAATGGAG AGACAAACGT GAAAAATTGG AATGGGAAAC
AAATATCGTT TATAAAGAGG 3100 CAAAAGAATC TGTAGATGCT TTATTTGTAA
ACTCTCAATA TGATCAATTA CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA
AACGTGTTCA 3200 TAGCATTCGA GAAGCTTATC TGCCTGAGCT GTCTGTGATT
CCGGGTGTCA ATGCGGCTAT TTTTGAAGAA TTAGAAGGGC GTATTTTCAC TGCATTCTCC
3300 CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA ATAATGGCTT
ATCCTGCTGG AACGTGAAAG GGCATGTAGA TGTAGAAGAA CAAAACAACC 3400
AACGTTCGGT CCTTGTTGTT CCGGAATGGG AAGCAGAAGT GTCACAAGAA GTTCGTGTCT
GTCCGGGTCG TGGCTATATC CTTCGTGTCA CAGCGTACAA 3500 GGAGGGATAT
GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA CAGACGAACT GAAGTTTAGC
AACTGCGTAG AAGAGGAAAT CTATCCAAAT 3600 AACACGGTAA CGTGTAATGA
TTATACTGTA AATCAAGAAG AATACGGAGG TGCGTACACT TCTCGTAATC GAGGATATAA
CGAAGCTCCT TCCGTACCAG 3700 CTGATTATGC GTCAGTCTAT GAAGAAAAAT
CGTATACAGA TGGACGAAGA GAGAATCCTT GTGAATTTAA CAGAGGGTAT AGGGATTACA
CGCCACTACC 3800 AGTTGGTTAT GTGACAAAAG AATTAGAATA CTTCCCAGAA
ACCGATAAGG TATGGATTGA GATTGGAGAA ACGGAAGGAA CATTTATCGT GGACAGCGTG
3900 GAATTACTCC TTATGGAGGA A (end HD-73).
27. A recombinant DNA transfer vector comprising DNA having the
following nucleotide sequence or equivalent nucleotide sequences
containing bases whose translated region codes for the same amino
acid sequence:
41 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGAAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG AATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACACA AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT APTTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1).
28. A recombinant DNA transfer vector comprising DNA having the
following nucleotide sequence or equivalent nucleotide sequences
containing bases whose translated region codes for the same amino
acid sequence:
42 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AGGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAAA GGGGAATATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1).
29. The DNA transfer vector of claim 25 transferred to and
replicated in a prokaryotic or lower eukaryotic microorganism.
30. The DNA transfer vector of claim 26 transferred to and
replicated in a prokaryotic or lower eukaryotic microorganism.
31. The DNA transfer vector of claim 27 transferred to and
replicated in a prokaryotic or lower eukaryotic microorganism.
32. The DNA transfer vector of claim 28 transferred to and
replicated in a prokaryotic or lower eukaryotic microorganism.
33. Plasmid pEW1 as shown in FIG. 1 of the drawings.
34. Plasmid pEW2 as shown in FIG. 2 of the drawings.
35. Plasmid pEW3 as shown in FIG. 3 of the drawings.
36. Plasmid pEW4 as shown in FIG. 4 of the drawings.
37. Plasmid pACB-1, having the construction of plasmid pEW3 except
that the DNA encoding aspartic acid at position 411 is converted to
encode asparagine, and the DNA encoding glycine at position 425 is
converted to encode glutamic acid.
38. Plasmid pSYW1, having the construction of plasmid pEW3 except
that the DNA encoding arginine at position 289 is converted to
encode glycine, the DNA encoding arginine at position 311 is
converted to encode lysine, and the DNA encoding tyrosine at
position 313 is converted to encode glutamate.
39. A microorganism transformed by the transfer vector of claim
25.
40. A microorganism transformed by the transfer vector of claim
26.
41. A microorganism transformed by the transfer vector of claim
27.
42. A microorganism transformed by the transfer vector of claim
28.
43. E. coli (pEW3), a microorganism according to claim 39.
44. E. coli (pEW4), a microorganism according to claim 40.
45. E. coli (pACB-1), a microorganism according to claim 41.
46. E. coli (pSYW1), a microorganism according to claim 42.
47. A process for preparing pesticidal chimeric toxin EW3 having
the following amino acid sequence:
43 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E
which comprises culturing a prokaryotic microbe hosting a
recombinant DNA transfer vector, denoted pEW3, comprising DNA
having the following nucleotide sequence or equivalent nucleotide
sequences containing bases whose translated region codes for the
same amino acid sequence:
44 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1).
48. A process for preparing pesticidal chimeric toxin EW4 having
the following amino acid sequence:
45 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F P V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L L A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F S N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G M A Q R I E Q N I R Q P H L M D I L N S I T
I Y T D V H R G F N Y W S G H Q I T A S P V G F S G P E F A F P L F
G N A G N A A P P V L V S L T G L G I F R T L S S P L Y R R I I L G
S G P N N Q E L F V L D G T E F S F A S L T T N L P S T I Y R Q R G
T V D S L D V I P P Q D N S V P P R A G F S H R L S H V T M L S Q A
A G A V Y T L R A Q R P M F S W I H R S A E F N N I I A S D S I T Q
I P A V K G N F L F N G S V I S G P G F T G G D L V R L N S S G N N
I Q N R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H L
N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F G Y F E S
A N A F T S S L G N I V G V R N F S G T A G V I I D R F E F I P V T
A T L E A E Y N L E R A Q K A V N A L F T S T N Q L G L K T N V T D
Y H I D Q V S N L V T Y L S D E F C L D E K R E L S E K V K H A K R
L S D E R N L L Q D S N F K D I N R Q P E R G W G G S T G I T I Q G
G D D V F K E N Y V T L S G T F D E C Y P T Y L Y Q K I D E S K L K
A F T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P
G T G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D
C S C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W
V I F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K
R A E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N
S Q Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S
V I P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D
F N N G L S C W N V K G H V D V E E Q N N Q R S V L V V P E W E A E
V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E
Y G G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G
R R E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D
K V W I E I G E T E G T F I V D S V E L L L M E E
which comprises culturing a prokaryotic microbe hosting a
recombinant DNA transfer vector, denoted pEW4, comprising DNA
having the following nucleotide sequence or equivalent nucleotide
sequences containing bases whose translated region codes for the
same amino acid sequence:
46 (start HD-1) ATGG ATAACAATCC GAACATCAAT GAATGCATTC CTTATAATTG
TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 600 TGGAGAAAGA ATAGAAACTG
GTTACACCCC AATCGATATT TCCTTGTCGC TAACGCAATT TCTTTTGAGT GAATTTGTTC
CCGGTGCTGG ATTTGTGTTA 700 GGACTAGTTG ATATAATATG GGGAATTTTT
GGTCCCTCTC AATGGGACGC ATTTCCTGTA CAAATTGAAC AGTTAATTAA CCAAAGAATA
GAAGAATTCG 800 CTAGGAACCA AGCCATTTCT AGATTAGAAG GACTAAGCAA
TCTTTATCAA ATTTACGCAG AATCTTTTAG AGAGTGGGAA GCAGATCCTA CTAATCCAGC
900 ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC AGTGCCCTTA
CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTTTA 1000
TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT TTCAGTGTTT
GGACAAAGGT GGGGATTTGA TGCCGCGACT ATCAATAGTC 1100 GTTATAATGA
TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG CGCTGGTACA ATACGGGATT
AGAGCGTGTA TGGGGACCGG ATTCTAGAGA 1200 TTGGGTAAGG TATAATCAAT
TTAGAAGAGA GCTAACACTT ACTGTATTAG ATATCGTTGC TCTATTCTCA AATTATGATA
GTCGAAGGTA TCCAATTCGA 1300 ACAGTTTCCC AATTAACAAG AGAAATTTAT
ACGAACCCAG TATTAGAAAA TTTTGATGGT AGTTTTCGTG GAATGGCTCA GAGAATAGAA
CAGAATATTA 1400 GGCAACCACA TCTTATGGAT ATCCTTAATA GTATAACCAT
TTATACTGAT GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA CAGCTTCTCC
1500 TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT GGGAATGCGG
GGAATGCAGC TCCACCCGTA CTTGTCTCAT TAACTGGTTT GGGGATTTTT 1600
AGAACATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCAGGCCC AAATAATCAG
GAACTGTTTG TCCTTGATGG AACGGAGTTT TCTTTTGCCT 1700 CCCTAACGAC
CAACTTGCCT TCCACTATAT ATAGACAAAG GGGTACAGTC GATTCACTAG ATGTAATACC
GCCACAGGAT AATAGTGTAC CACCTCGTGC 1800 GGGATTTAGC CATCGATTGA
GTCATGTTAC AATGCTGAGC CAAGCAGCTG GAGCAGTTTA CACCTTGAGA GCTCAACGT
(stop HD-1) (start HD-73) CCT ATGTTCTCTT GGATACATCG TAGTGCTGAA
TTTAATAATA TAATTGCATC GGATAGTATT 1800 ACTCAAATCC CTGCAGTGAA
GGGAAACTTT CTTTTTAATG GTTCTGTAAT TTCAGGACCA GGATTTACTG GTGGGGACTT
AGTTAGATTA AATAGTAGTG 1900 GAAATAACAT TCAGAATAGA GGGTATATTG
AAGTTCCAAT TCACTTCCCA TCGACATCTA CCAGATATCG AGTTCGTGTA CGGTATGCTT
CTGTAACCCC 2000 GATTCACCTC AACGTTAATT GGGGTAATTC ATCCATTTTT
TCCAATACAG TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG TGATTTTGGT
2100 TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA ATATAGTAGG
TGTTAGAAAT TTTAGTGGGA CTGCAGGAGT GATAATAGAC AGATTTGAAT 2200
TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGAAAGAGCG CAGAAGGCGG
TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 2300 AACAAATGTA
ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT ATTTATCGGA TGAATTTTGT
CTGGATGAAA AGCGAGAATT GTCCGAGAAA 2400 GTCAAACATG CGAAGCGACT
CAGTGATGAA CGCAATTTAC TCCAAGATTC AAATTTCAAA GACATTAATA GGCAACCAGA
ACGTGGGTGG GGCGGAAGTA 2500 CAGGGATTAC CATCCAAGGA GGGGATGACG
TATTTAAAGA AAATTACGTC ACACTATCAG GTACCTTTGA TGAGTGCTAT CCAACATATT
TGTATCAAAA 2600 AATCGATGAA TCAAAATTAA AAGCCTTTAC CCGTTATCAA
TTAAGAGGGT ATATCGAAGA TAGTCAAGAC TTAGAAATCT ATTTAATTCG CTACAATGCA
2700 AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT GGCCGCTTTC
AGCCCAAAGT CCAATCGGAA AGTGTGGAGA GCCGAATCGA TGCGCGCCAC 2800
ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG TGTGCCCATC
ATTCGCATCA TTTCTCCTTA GACATTGATG TAGGATGTAC 2900 AGACTTAAAT
GAGGACCTAG GTGTATGGGT GATCTTTAAG ATTAAGACGC AAGATGGGCA CGCAAGACTA
GGGAATCTAG AGTTTCTCGA AGAGAAACCA 3000 TTAGTAGGAG AAGCGCTAGC
TCGTGTGAAA AGAGCGGAGA AAAAATGGAG AGACAAACGT GAAAAATTGG AATGGGAAAC
AAATATCGTT TATAAAGAGG 3100 CAAAAGAATC TGTAGATGCT TTATTTGTAA
ACTCTCAATA TGATCAATTA CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA
AACGTGTTCA 3200 TAGCATTCGA GAAGCTTATC TGCCTGAGCT GTCTGTGATT
CCGGGTGTCA ATGCGGCTAT TTTTGAAGAA TTAGAAGGGC GTATTTTCAC TGCATTCTCC
3300 CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA ATAATGGCTT
ATCCTGCTGG AACGTGAAAG GGCATGTAGA TGTAGAAGAA CAAAACAACC 3400
AACGTTCGGT CCTTGTTGTT CCGGAATGGG AAGCAGAAGT GTCACAAGAA GTTCGTGTCT
GTCCGGGTCG TGGCTATATC CTTCGTGTCA CAGCGTACAA 3500 GGAGGGATAT
GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA CAGACGAACT GAAGTTTAGC
AACTGCGTAG AAGAGGAAAT CTATCCAAAT 3600 AACACGGTAA CGTGTAATGA
TTATACTGTA AATCAAGAAG AATACGGAGG TGCGTACACT TCTCGTAATC GAGGATATAA
CGAAGCTCCT TCCGTACCAG 3700 CTGATTATGC GTCAGTCTAT GAAGAAAAAT
CGTATACAGA TGGACGAAGA GAGAATCCTT GTGAATTTAA CAGAGGGTAT AGGGATTACA
CGCCACTACC 3800 AGTTGGTTAT GTGACAAAAG AATTAGAATA CTTCCCAGAA
ACCGATAAGG TATGGATTGA GATTGGAGAA ACGGAAGGAA CATTTATCGT GGACAGCGTG
3900 GAATTACTCC TTATGGAGGA A (end HD-73).
49. A process for preparing pesticidal chimeric toxin ACB-1 having
the following amino acid sequence:
47 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E W E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L N S I T
I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L N E I P P Q N N N V P P R Q E F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E
which comprises culturing a prokaryotic microbe hosting a
recombinant DNA transfer vector, denoted pACB-1, comprising DNA
having the following nucleotide sequence or equivalent nucleotide
sequences containing bases whose translated region codes for the
same amino acid sequence:
48 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGAAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG AATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
50. A process for preparing pesticidal chimeric toxin SYW1 having
the following amino acid sequence:
49 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E T G
Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L V D I I W G
I F G P S Q W D A F L V Q I E Q L I N Q R I E E F A R N Q A I S R L
E G L S N L Y Q I Y A E S F R E Q E A D P T N P A L R E E M R I Q F
N D M N S A L T T A I P L F A V Q N Y Q V P L L S V Y V Q A A N L H
L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L T R L I G N
Y T D Y A V R W Y N T G L E R V W G P D S R D W V R Y N Q F R R E L
T L T V L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N
P V L E N F D G S F R G S A Q G I E G S I R S P H L M D I L N S I T
I Y T D A H K G E Y Y W S G H Q I M A S P V G F S G P E F T F P L Y
G T M G N A A P Q Q R I V A Q L G Q G V Y R T L S S T L Y R R P F N
I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T
V D S L D E I P P Q N N N V P P R Q G F S H R L S H V S M F R S G F
S N S S V S I I R A P T F S W Q H R S A E F N N I I P S S Q I T Q I
P L T K S T N L G S G T S V V K G P G F T G G D I L R R T S P G Q I
S T L R V N I T A P L S Q R Y R V R I R Y A S T T N L Q F H T S I D
G R P I N Q G N F S A T M S S G S N L Q S G S F R T V G F T T P F N
F S N G S S V F T L S A H V F N S G N E V Y I D R I E F V P A E V T
F E A E Y D L E R A Q K A V N E L F T S S N Q I G L K T D V T D Y H
I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K H A K R L S
D E R N L L Q D P N F R G I N R Q L D R G W R G S T D I T I Q G G D
D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y
T R Y Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G T
G S L W P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S
C R D G E K C A H H S H H F S L D I D V G C T D L N E D L G V W V I
F K I K T Q D G H A R L G N L E F L E E K P L V G E A L A R V K R A
E K K W R D K R E K L E W E T N I V Y K E A K E S V D A L F V N S Q
Y D Q L Q A D T N I A M I H A A D K R V H S I R E A Y L P E L S V I
P G V N A A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N
N G L S C W N V K G H V D V E E Q N N Q R S V L V L P E W E A E V S
Q E V P V C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N
N T D E L K F S N C V E E E I Y P N N T V T C N D Y T V N Q E E Y G
G A Y T S R N R G Y N E A P S V P A D Y A S V Y E E K S Y T D G R R
E N P C E F N R G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V
W I E I G E T E G T F I V D S V E L L L M E E
which comprises culturing a prokaryotic microbe hosting a
recombinant DNA transfer vector, denoted pSYW1, comprising DNA
having the following nucleotide sequence or equivalent nucleotide
sequences containing bases whose translated region codes for the
same amino acid sequence:
50 (start HD-73) ATG GATAACAATC 400 CGAACATCAA TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTDTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TGGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AGGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAAA GGGGAATATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGGA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT
(end hd-73) (start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGGCGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGGA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GGTCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGGT TATGTGACAA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1).
51. A chimeric toxin, having the amino acid sequence of toxin EW3,
with changes which can be shown schematically as follows: 5wherein
X is one of the 20 common amino acids except Asp when the amino
acid at position 425 is Gly; Y is one of the 20 common amino acids
except Gly when the amino acid at position 411 is Asp.
52. A chimeric toxin, having the amino acid sequence of toxin EW3,
with changes which can be shown schematically as follows: 6wherein
X is one of the 20 common amino acids except Arg when the amino
acid at position 311 is Arg and the amino acid at position 313 is
Tyr; Y is one of the 20 common amino acids except Arg when the
amino acid at position 289 is Arg and the amino acid at position
313 is Tyr; and Z is one of the 20 common amino acids except Tyr
when the amino acid at position 289 is Arg and the amino acid at
position 311 is Arg.
53. DNA encoding a chimeric toxin as shown in claim 51.
54. DNA encoding a chimeric toxin as shown in claim 52.
55. A recombinant DNA transfer vector comprising DNA encoding a
chimeric toxin as shown in claim 51.
56. A recombinant DNA transfer vector comprising DNA encoding a
chimeric toxin as shown in claim 52.
57. A chimeric toxin comprising the variable region or regions of
two or more Bacillus toxins.
58. A toxin, according to claim 57, wherein the Bacillus toxins are
B. thuringiensis toxins.
59. A toxin, according to claim 58, wherein the B. thuringiensis
toxins are B. thuringiensis var. kurstaki HD-1 toxin and B.
thuringiensis var. kurstaki HD-73 toxin.
60. A toxin, according to claim 58, wherein the B. thuringiensis
toxins are encoded by a pesticide-producing strain of Bacillius
thuringiensis, consisting of B. thuringiensis M-7, B. thuringiensis
var. kurstaki, B. thuringiensis var. finitimus, B. thuringiensis
var. alesti, B. thuringiensis var. sotto, B. thuringiensis var.
dendrolimus, B. thuringiensis var. kenyae, B. thuringiensis var.
galleriae, B. thuringiensis var. canadensis, B. thuringiensis var.
entomocidus, B. thuringiensis var. subtoxicus, B. thuringiensis
var. aizawai, B. thuringiensis var. morrisoni, B. thuringiensis
var. ostriniae, B. thuringiensis var. tolworthi, B. thuringiensis
var. darmstadiensis, B. thuringiensis var. toumanoffi, B.
thuringiensis var. kyushuensis, B. thuringiensis var. thompsoni, B.
thuringiensis var. pakistani, B. thuringiensis var. israelensis, B.
thuringiensis var. indiana, B. thuringiensis var. dakota, B.
thuringiensis var. tohokuensis, B. thuringiensis var.
kumanotoensis, B. thuringiensis var. tochigiensis, B. thuringiensis
var. colmeri, B. thuringiensis var. wuhanensis, B. thuringiensis
var. tenebrionis, B. thuringiensis var. thuringiensis, and other
Bacillus species selected from B. cereus, B. moritai, B. popilliae,
B. lentimorbus, and B. sphaericus.
Description
CROSS REFERENCE TO A RELATED APPLICATION
[0001] This is a continuation-in-part of our copending application
Ser. No. 808,129, filed on Dec. 12, 1985.
BACKGROUND OF THE INVENTION
[0002] The most widely used microbial pesticides are derived from
the bacterium Bacillius thuringiensis. This bacterial agent is used
to control a wide range of leaf-eating caterpillars, Japanese
beetles and mosquitos. Bacillius thuringiensis produces a
proteinaceous paraspore or crystal which is toxic upon ingestion by
a susceptible insect host. For example, B. thuringiensis var.
kurstaki HD-1 produces a crystal called a delta toxin which is
toxic to the larvae of a number of lepidopteran insects. The
cloning and expression of this B.t. crystal protein gene in
Escherichia coli has been described in the published literature
(Schnepf, H. E. and Whiteley, H. R. [1981] Proc. Natl. Acad. Sci.
USA 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No.
4,467,036 both disclose the expression of B.t. crystal protein in
E. coli. In U.S. Pat. No. 4,467,036 B. thuringiensis var. kurstaki
HD-1 is disclosed as being available from the well-known NRRL
culture repository at Peoria, Ill. Its accession number there is
NRRL B-3792. B. thuringiensis var. kurstaki HD-73 is also available
from NRRL. Its accession number is NRRL B-4488.
BRIEF SUMMARY OF THE INVENTION
[0003] The subject invention concerns a novel process for altering
the insect host range of Bacillius thuringiensis toxins, and novel
toxins produced as exemplification of this useful process. This
alteration can result in expansion of the insect host range of the
toxin, and/or, amplification of host toxicity. The process
comprises recombining in vitro the variable region(s) of two or
more .delta.-endotoxin genes. Specifically exemplified is the
recombining of portions of two Bacillius thuringiensis var.
kurstaki DNA sequences, i.e., referred to herein as k-1 and k-73,
to produce chimeric B.t. toxins with expanded host ranges as
compared to the toxins produced by the parent DNA's.
[0004] "Variable regions," as used herein, refers to the
non-homologous regions of two or more DNA sequences. As shown by
the examples presented herein, the recombining of such variable
regions from two different B.t. DNA sequences yields, unexpectedly,
a DNA sequence encoding a .delta.-endotoxin with an expanded insect
host range. In a related example, the recombining of two variable
regions of two different B.t. toxin genes results in the creation
of a chimeric toxin molecule with increased toxicity toward the
target insect. The utility of this discovery by the inventors is
clearly broader than the examples disclosed herein. From this
discovery, it can be expected that a large number of new and useful
toxins will be produced. Thus, though the subject process is
exemplified by construction of chimeric toxin-producing DNA
sequences from two well-known B.t. kurstaki DNA sequences, it
should be understood that the process is not limited to these
starting DNA sequences. The invention process also can be used to
construct chimeric toxins from any B. thuringiensis toxin-producing
DNA sequence.
DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1: A schematic diagram of plasmid pEW1 which contains
the DNA sequence encoding Bacillius thuringiensis toxin k-1.
[0006] FIG. 2: A schematic diagram of plasmid pEW2 which contains
the DNA sequence encoding Bacillius thuringiensis toxin k-73.
[0007] FIG. 3: A schematic diagram of plasmid pEW3 which contains
the DNA sequence encoding Bacillius thuringiensis chimeric toxin
k-73/k-1 (pHY).
[0008] FIG. 4: A schematic diagram of plasmid pEW4 which contains
the DNA sequence encoding Bacillius thuringiensis chimeric toxin
k-1/k-73 (pYH).
DETAILED DISCLOSURE OF THE INVENTION
[0009] Upon recombining in vitro the variable region(s) of two or
more .delta.-endotoxin genes, there is obtained a gene(s) encoding
a chimeric toxin(s) which has an expanded and/or amplified host
toxicity as compared to the toxin produced by the starting genes.
This recombination is done using standard well-known genetic
engineering techniques.
[0010] The restriction enzymes disclosed herein can be purchased
from Bethesda Research Laboratories, Gaithersburg, Md., or New
England Biolabs, Beverly, Mass. The enzymes are used according to
the instructions provided by the supplier.
[0011] The various methods employed in the preparation of the
plasmids and transformation of host organisms are well known in the
art. These procedures are all described in Maniatis, T., Fritsch,
E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within
the skill of those in the genetic engineering art to extract DNA
from microbial cells, perform restriction enzyme digestions,
electrophorese DNA fragments, tail and anneal plasmid and insert
DNA, ligate DNA, transform cells, prepare plasmid DNA,
electrophorese proteins, and sequence DNA.
[0012] Plasmids pEW1, pEW2, pEW3, and pEW4, constructed as
described infra, have been deposited in E. coli hosts in the
permanent collection (to be maintained for at least 30 years) of
the Northern Regional Research Laboratory (NRRL), U.S. Department
of Agriculture, Peoria, Ill., USA. Their accession numbers and
dates of deposit are as follows:
[0013] pEWl--NRRL B-18032; deposited on Nov. 29, 1985
[0014] pEW2--NRRL B-18033; deposited on Nov. 29, 1985
[0015] pEW3--NRRL B-18034; deposited on Nov. 29, 1985
[0016] pEW4--NRRL B-18035; deposited on Nov. 29, 1985
[0017] B. thuringiensis strain MTX-36, NRRL B-18101 was deposited
on Aug. 25, 1986.
[0018] Plasmid pBR322 is a well-known and available plasmid. It is
maintained in the E. coli host ATCC 37017. Purified pBR322 DNA can
be obtained as described in Bolivar, F., Rodriguez, R. L., Greene,
P. J., Betlach, M. C., Heynecker, H. L., Boyer, H. W., Crosa, J. H.
and Falkow, S. (1977) Gene 2:95-113; and Sutcliffe, J. G. (1978)
Nucleic Acids Res. 5:2721-2728.
[0019] NRRL B-18032, NRRL B-18033, NRRL B-18034, NRRL B-18035, and
NRRL B-18101 are available to the public upon the grant of a patent
which discloses these accession numbers in conjunction with the
invention described herein. It should be understood that the
availability of these deposits does not constitute a license to
practice the subject invention in derogation of patent rights
granted for the subject invention by governmental action.
[0020] As disclosed above, any B. thuringiensis toxin-producing DNA
sequence can be used as starting material for the subject
invention. Examples of B. thuringiensis organisms, other than those
previously given, are as follows:
[0021] Bacillius thuringiensis var. israelensis--ATCC 35646
[0022] Bacillius thuringiensis M-7--NRRb B-15939
[0023] Bacillius thuringiensis var. tenebrionis--DSM 2803
[0024] The following B. thuringiensis cultures are available from
the United States Department of Agriculture (USDA) at Brownsville,
Texas. Requests should be made to Joe Garcia, USDA, ARS, Cotton
Insects Research Unit, P.O. Box 1033, Brownsville, Tex. 78520
USA.
1 B. thuringiensis HD2 B. thuringiensis var. finitimus HD3 B.
thuringiensis var. alesti HD4 B. thuringiensis var. kurstaki HD73
B. thuringiensis var. sotto HD770 B. thuringiensis var. dendrolimus
HD7 B. thuringiensis var. kenyae HD5 B. thuringiensis var.
galleriae HD29 B. thuringiensis var. canadensis HD224 B.
thuringiensis var. entomocidus HD9 B. thuringiensis var. subtoxicus
HD109 B. thuringiensis var. aizawai 1-HD11 B. thuringiensis var.
morrisoni HD12 B. thuringiensis var. ostriniae HD501 B.
thuringiensis var. tolworthi HD537 B. thuringiensis var.
darmscadiensis HD146 B. thuringiensis var. tournanoffi HD201 B.
thuringiensis var. kyushuensis HD541 B. thuringiensis var.
thotnosoni HD542 B. thurinpiensis var. pakistani HD395 B.
thuringiensis var. israelensis HD567 B. thuringiensis var. indiana
HD521 B. thuringiensis var. dakota B. thuringiensis var.
tohokuensis HD866 B. thuringiensis var. kumanotoensis HD867 B.
thuringiensis var. cochigiensis HD868 B. thuringiensis var. colmeri
HD847 B. thuringiensis var. wuhanensis HD525
[0025] Though the main thrust of the subject invention is directed
toward a process for altering the host range of B. thuringiensis
toxins, the process is also applicable in the same sense to other
Bacillus toxin-producing microbes. Examples of such Bacillus
organisms which can be used as starting material are as
follows:
2 Bacillus cereus--ATCC 21281 Bacillus moritai--ATGC 21282 Bacillus
popilliae--ATCC 14706 Bacillus lentimorbus--ATCC 14707 Bacillus
sphaericus--ATCC 33203
[0026] Bacillius thuringiensis M-7, exemplified herein, is a
Bacillius thuringiensis isolate which, surprisingly, has activity
against beetles of the order Coleoptera but not against
Trichoplusia ni, Spodoptera exigua or Aedes aegypti. Included in
the Coleoptera are various Diabrotica species (family
Chrysomelidae) that are responsible for large agricultural losses,
for example, D. undecimpunctata (western spotted cucumber beetle),
D. longicornis (northern corn rootworm), D. virgitera (western corn
rootworm), and D. undecimpunctata howardi (southern corn
rootworm).
[0027] B. thuringiensis M-7 is unusual in having a unique
parasporal body (crystal) which under phase contrast microscopy is
dark in appearance with a flat, square configuration.
[0028] The pesticide encoded by the DNA sequence used as starting
material for the invention process can be any toxin produced by a
microbe. For example, it can be a polypeptide which has toxic
activity toward a eukaryotic multicellular pest, such as insects,
e.g., coleoptera, lepidoptera, diptera, hemiptera, dermaptera, and
orthoptera; or arachnids; gastropods; or worms, such as nematodes
and platyhelminths. Various susceptible insects include beetles,
moths, flies, grasshoppers, lice, and earwigs.
[0029] Further, it can be a polypeptide produced in active form or
a precursor or proform requiring further processing For toxin
activity, e.g., the novel crystal toxin of B. thuringiensis var.
kurstaki, which requires processing by the pest.
[0030] The constructs produced by the process of the invention,
containing chimeric toxin-producing DNA sequences, can be
transformed into suitable hosts by using standard procedures.
Illustrative host cells may include either prokaryotes or
eukaryotes, normally being limited to those cells which do not
produce substances toxic to higher organisms, such as mammals.
However, organisms which produce substances toxic to higher
organisms could be used, where the toxin is unstable or the level
of application sufficiently low as to avoid any possibility of
toxicity to a mammalian host. As hosts, of particular interest will
be the prokaryotes and lower eukaryotes, such as fungi.
Illustrative prokaryotes, both Gram-negative and -positive, include
Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,
Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as
Rhizobium; Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and
Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among
eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which
includes yeast, such as Saccharomyces and Schizosaccharomyces; and
Basidiomycetes yeast, such as Rhodotorula, Aureobasidium,
Sporobolomyces, and the like.
[0031] Characteristics of particular interest in selecting a host
cell for purposes of production include ease of introducing the
chimeric toxin-producing gene into the host, availability of
expression systems, efficiency of expression, stability of the
pesticide in the host, and the presence of auxiliary genetic
capabilities. Characteristics of interest for use as a pesticide
microcapsule include protective qualities for the pesticide, such
as thick cell walls, pigmentation, and intracellular packaging or
formation of inclusion bodies; leaf affinity; lack of mammalian
toxicity; attractiveness to pests for ingestion; ease of killing
and fixing without damage to the toxin; and the like. Other
considerations include ease of formulation and handling, economics,
storage stability, and the like.
[0032] Host organisms of particular interest include yeast, such as
Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and
Sporobolomyces sp.; phylloplane organisms such Pseudomonas sp.,
Erwinia sp. and Flavobacterium sp.; or such other organisms as
Escherichia, Lactobacillus sp., Bacillus sp., and the like.
Specific organisms include Pseudomonas aeruginosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,
Escherichia coli, Bacillus subtilis, and the like.
[0033] The chimeric toxin-producing gene(s) can be introduced into
the host in any convenient manner, either providing for
extrachromosomal maintenance or integration into the host
genome.
[0034] Various constructs may be used, which include replication
systems from plasmids, viruses, or centromeres in combination with
an autonomous replicating segment (ars) for stable maintenance.
Where only integration is desired, constructs can be used which may
provide for replication, and are either transposons or have
transposon-like insertion activity or provide for homology with the
genome of the host. DNA sequences can be employed having the
chimeric toxin-producing gene between sequences which are
homologous with sequences in the genome of the host, either
chromosomal or plasmid. Desirably, the chimeric toxin-producing
gene(s) will be present in multiple copies. See for example, U.S.
Pat. No. 4,399,216. Thus, conjugation, transduction, transfection
and transformation may be employed for introduction of the
gene.
[0035] A large number of vectors are presently available which
depend upon eukaryotic and prokaryotic replication systems, such as
Co1E1, P-1 incompatibility plasmids, e.g., pRK290, yeast 2m .mu.
plasmid, lambda, and the like.
[0036] Where an extrachromosomal element is employed, the DNA
construct will desirably include a marker which allows for a
selection of those host cells containing the construct. The marker
is commonly one which provides for biocide resistance, e.g.,
antibiotic resistance or heavy metal resistance, complementation
providing prototrophy to an auxotrophic host, or the like. The
replication systems can provide special properties, such as runaway
replication, can involve cos cells, or other special feature.
[0037] Where the chimeric toxin-producing gene(s) has
transcriptional and translational initiation and termination
regulatory signals recognized by the host cell, it will frequently
be satisfactory to employ those regulatory features in conjunction
with the gene. However, in those situations where the chimeric
toxin-producing gene is modified, as for example, removing a leader
sequence or providing a sequence which codes for the mature form of
the pesticide, where the entire gene encodes for a precursor, it
will frequently be necessary to manipulate the DNA sequence, so
that a transcriptional initiation regulatory sequence may be
provided which is different from the natural one.
[0038] A wide variety of transcriptional initiation sequences exist
for a wide variety of hosts. The sequence can provide for
constitutive expression of the pesticide or regulated expression,
where the regulation may be inducible by a chemical, e.g., a
metabolite, by temperature, or by a regulatable repressor. See for
example, U.S. Pat. No. 4,374,927. The particular choice of the
promoter will depend on a number of factors, the strength of the
promoter, the interference of the promoter with the viability of
the cells, the effect of regulatory mechanisms endogenous to the
cell on the promoter, and the like. A large number of promoters are
available from a variety of sources, including commercial
sources.
[0039] The cellular host containing the chimeric toxin-producing
pesticidal gene may be grown in any convenient nutrient medium,
where-the DNA construct provides a selective advantage, providing
for a selective medium so that substantially all or all of the
cells retain the chimeric toxin-producing gene. These cells may
then be harvested in accordance with conventional ways and modified
in the various manners described above. Alternatively, the cells
can be fixed prior to harvesting.
[0040] Host cells transformed to contain chimeric toxin-producing
DNA sequences can be treated to prolong pesticidal activity when
the cells are applied to the environment of a target pest. This
treatment can involve the killing of the host cells under protease
deactivating or cell wall strengthening conditions, while retaining
pesticidal activity.
[0041] The cells may be inhibited from proliferation in a variety
of ways, so long as the technique does not deleteriously affect the
properties of the pesticide, nor diminish the cellular capability
in protecting the pesticide. The techniques may involve physical
treatment, chemical treatment, changing the physical character of
the cell or leaving the physical character of the cell
substantially intact, or the like.
[0042] Various techniques for inactivating the host cells include
heat, usually 50.degree. C. to 70.degree. C.; freezing; UV
irradiation; lyophilization; toxins, e.g., antibiotics; phenols;
anilides, e.g., carbanilide and salicylanilide; hydroxyurea;
quaternaries; alcohols; antibacterial dyes; EDTA and amidines;
non-specific organic and inorganic chemicals, such as halogenating
agents, e.g., chlorinating, brominating or iodinating agents;
aldehydes, e.g., glutaraldehyde or formaldehyde; toxic gases, such
as ozone and ethylene oxide; peroxide; psoralens; desiccating
agents; or the like, which may be used individually or in
combination. The choice of agent will depend upon the particular
pesticide, the nature of the host cell, the nature of the
modification of the cellular structure, such as fixing and
preserving the cell wall with crosslinking agents, or the like.
[0043] The cells generally will have enhanced structural stability
which will enhance resistance to environmental degradation in the
field. Where the pesticide is in a proform, the method of
inactivation should be selected so as not to inhibit processing of
the proform to the mature form of the pesticide by the target pest
pathogen. For example, formaldehyde will crosslink proteins and
could inhibit processing of the proform of a polypeptide pesticide.
The method of inactivation or killing retains at least a
substantial portion of the bioavailability or bioactivity of the
toxin.
[0044] The method of treating the organism can fulfill a number of
functions. First, it may enhance structural integrity. Second, it
may provide for enhanced proteolytic stability of the toxin, by
modifying the toxin so as to reduce its susceptibility to
proteolytic degradation and/or by reducing the proteolytic activity
of proteases naturally present in the cell. The cells are
preferably modified at an intact stage and when there has been a
substantial build-up of the toxin protein. These modifications can
be achieved in a variety of ways, such as by using chemical
reagents having a broad spectrum of chemical reactivity. The intact
cells can be combined with a liquid reagent medium containing the
chemical reagents, with or without agitation at temperatures in the
range of about -10 to 60.degree. C. The reaction time may be
determined empirically and will vary widely with the reagents and
reaction conditions. Cell concentrations will vary from about 10E2
to 10E10 per ml.
[0045] Of particular interest as chemical reagents are halogenating
agents, particularly halogens of atomic no. 17-80. More
particularly, iodine can be used under mild conditions and for
sufficient time to achieve the desired results. Other suitable
techniques include treatment with aldehydes, such as formaldehyde
and glutaraldehyde; anti-infectives, such as zephiran chloride and
cetylpyridinium chloride; alcohols, such as isopropyl and ethanol;
various histologic fixatives, such as Bouin's fixative and Helly's
fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W.H.
Freeman and Company, 1967); or a combination of physical (heat) and
chemical agents that prolong the activity of the toxin produced in
the cell when the cell is applied to the environment of the target
pest(s).
[0046] For halogenation with iodine, temperatures will generally
range from about 0 to 50.degree. C., but the reaction can be
conveniently carried out at room temperature. Conveniently, the
iodination may be performed using triiodide or iodine at 0.5 to 5%
in an acidic aqueous medium, particularly an aqueous carboxylic
acid solution that may vary from about 0.5-5M. Conveniently, acetic
acid may be used, although other carboxylic acids, generally of
from about 1 to 4 carbon atoms, may also be employed. The time for
the reaction will generally range from less than a minute to about
24 hrs, usually from about 1 to 6 hrs. Any residual iodine may be
removed by reaction with a reducing agent, such as dithionite,
sodium thiosulfate, or other reducing agent compatible with
ultimate usage in the field. In addition, the modified cells may be
subjected to further treatment, such as washing to remove all of
the reaction medium, isolation in dry form, and formulation with
typical stickers, spreaders, and adjuvants generally utilized in
agricultural applications, as is well known to those skilled in the
art.
[0047] Of particular interest are reagents capable of crosslinking
the cell wall. A number of reagents are known in the art for this
purpose. The treatment should result in enhanced stability of the
pesticide. That is, there should be enhanced persistence or
residual activity of the pesticide under field conditions. Thus,
under conditions where the pesticidal activity of untreated cells
diminishes, the activity of treated cells remains for periods of
from 1 to 3 times longer.
[0048] The cells can be formulated for use in the environment in a
variety of ways. They can be employed as wettable powders,
granules, or dusts, by mixing with various inert materials, such as
inorganic minerals (phyllosilicates, carbonates, sulfates, or
phosphates) or botanical materials (powdered corncobs, rice hulls,
or walnut shells). The formulations can include spreader/sticker
adjuvants, stabilizing agents, other pesticidal additives, or
surfactants. Liquid formulations can be aqueous-based or
non-aqueous and employed as foams, gels, suspensions, emulsifiable
concentrates, and the like. The ingredients can include Theological
agents, surfactants, emulsifiers, dispersants, polymers, and the
like.
[0049] The pesticidal concentration will vary depending upon the
nature of the particular formulation, e.g., whether it is a
concentrate or to be used undiluted. The pesticide will generally
be present at a concentration of at least about 1% by weight, but
can be up to 100% by weight. The dry formulations will have from
about 1 to 95% by weight of the pesticide, while the liquid
formulations will generally be from about 1 to 60% by weight of the
solids in the liquid phase. The formulations will generally have
from about 1E2 to 1E8 cells/mg.
[0050] The formulations can be applied to the environment of the
pest(s), e.g., plants, soil or water, by spraying, dusting,
sprinkling, or the like. These formulations can be administered at
about 2 oz (liquid or dry) to 2 or more pounds Der hectare, as
required.
[0051] Following are examples which illustrate procedures,
including the best mode, for practicing the invention. These
examples should not be construed as limiting. All percentages are
by weight and all solvent mixture proportions are by volume unless
otherwise noted.
EXAMPLE 1
Construction of Plasmid pEW1
[0052] The k-1 gene is the hd-l gene described by Schnepf et al.
(J. Biol. Chem. 260:6264-6272 1985). The k-1 gene was resected from
the 5' end with Bal31 up to position 504. To this position was
added a SalI linker (5'GTCGACC3'). The 3' end of the gene was
cleaved at position 4211 with the enzyme NdeI and blunt ended with
the Klenow fragment of DNA polymerase.
[0053] The cloning vector pUC8 (Messing, J. and Vieira, J. [1982]
Gene 19:269-276) which can be purchased from Pharmacia, Piscataway,
N.J., was cleaved with SalI and EcoRI and cloned into plasmid
pBR322 which had been cut with the same enzymes. The trp promoter
(Genblock, available from Pharmacia) was blunt ended at the 5' end
with Klenow and inserted into this hybrid vector by blunt end
ligation of the 5' end to the SmaI site of the vector, and by
insertion of the 3' end at the SalI site of the vector. The k-1
gene was then inserted using the SalI site at the 5' end and by
blunt end ligation of the 3' end to the PvuII site of the vector. A
schematic drawing of this construct, called pEW1, is shown in FIG.
1 of the drawings.
[0054] Plasmid pEW1 contains the DNA sequence encoding Bacillius
thuringiensis toxin k-1.
EXAMPLE 2
Construction of Plasmid pEW2
[0055] The k-73 gene is the HD-73 gene described by Adang et al.
(Gene 36:289-300 1985). The k-73 gene was cleaved at position 176
with NsiI. The sequence was then cleaved at position 3212 with
HindIII and the 3036 base fragment consisting of residues 176-3212
was isolated by agarose gel electrophoresis.
[0056] Plasmid pEW1, prepared as described in Example 1, was also
cleaved with HindIII (position 3345 in Table 1) and partially
digested with NsiI (position 556 in Table 1). The 3036 base
fragment from k-73, disclosed above, was inserted into the NsiI to
HindIII region of pEW1 replacing the comparable fragment of the k-1
gene, and creating plasmid pEW2. A schematic diagram of pEW2 is
shown in FIG. 2 of the drawings.
[0057] Plasmid pEW2 contains the DNA sequence encoding Bacillius
thuringiensis toxin k-73.
EXAMPLE 3
Construction of Plasmid pEW3
[0058] The k-1 gene was cut with SacI at position 1873. The gene
was then submitted to partial digestion with HindIII and the 1427
base fragment consisting of residues 1873 to 3345 was isolated by
agarose gel electrophoresis. Plasmid pEW2 was cut with SacI and
HindIII and the large fragment representing the entire plasmid
minus the SacI to HindIII fragment of the k-2 gene was isolated by
agarose gel electrophoresis. The 1427 base fragment from the k-1
gene was then ligated into the SacI to HindIII region of pEW2,
creating plasmid pEW3. A schematic diagram of pEW3 is shown in FIG.
3 of the drawings.
[0059] Plasmid pEW3 contains the DNA sequence encoding Bacillius
thuringiensis chimeric toxin k-73/k-1 (pHY).
[0060] The nucleotide sequence encoding the chimeric toxin is shown
in Table 1. The deduced amino acid sequence is shown in Table
1A.
EXAMPLE 4
Construction of Plasmid pEW4
[0061] The k-1 gene was cut at position 556 with NsiI. The gene was
then cut with SacI at position 1873 and the 1317 base fragment from
NsiI to SacI was isolated by agarose gel electrophoresis. Plasmid
pEW2 was cut with SacI and then submitted to partial digestion with
NsiI. The large fragment representing the entire plasmid; minus the
NsiI to SacI region of the k-73 gene, was isolated by agarose gel
electrophoresis. The 1317 base NsiI to SacI fragment of gene k-1
was then ligated into NsiI to SacI region of pEW2 to create plasmid
pEW4. A schematic diagram of pEW4 is shown in FIG. 4 of the
drawings.
[0062] The nucleotide sequence encoding the chimeric toxin is shown
in Table 2. The deduced amino acid sequence is shown in Table
2A.
[0063] Plasmid pEW4 contains the DNA sequence encoding Bacillius
thuringiensis chimeric toxin k-1/k-73 (PYH).
EXAMPLE 5
Insertion of Chimeric Toxin Genes into Plants
[0064] Genes coding for chimeric insecticidal toxins, as disclosed
herein, can be inserted into plant cells using the Ti plasmid from
Agrobacter tumefaciens. Plant cells can then be caused to
regenerate into plants (Zambryski, P., Joos, H., Gentello, C.,
Leemans, J., Van Montague, M. and Schell, J. [1983] EMBO J.
2:2143-2150; Bartok, K., Binns, A., Matzke, A. and Chilton, M-D.
[1983] Cell 32:1033-1043). A particularly useful vector in this
regard is pEND4K (Klee, H. J., Yanofsky, M. F. and Nester, E. W.
[1985] Bio/Technology 3:637-642). This plasmid can replicate both
in plant cells and in bacteria and has multiple cloning sites for
passenger genes. Toxin genes, for example, can be inserted into the
BamHI site of pEND4K, propagated in E. coli, and transformed into
appropriate plant cells.
EXAMPLE 6
Cloning of B. thuringiensis Genes into Baculoviruses
[0065] Genes coding for Bacillus thuringiensis chimeric toxins, as
disclosed herein, can be cloned into baculoviruses such as
Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids
can be constructed that contain the AcNPV genome cloned into a
commercial cloning vector such as pUC8. The AcNPV genome is
modified so that the coding region of the polyhedrin gene is
removed and a unique cloning site for a passenger gene is placed
directly behind the polyhedrin promoter. Examples of such vectors
are DGP-B6874, described by Pennock et al. (Pennock, G. D.,
Shoemaker, C. and Miller, L. K. [19841 Mol. Cell. Biol. 4:399-406),
and pAC380, described by Smith et al. (Smith, G. E., Summers, M. D.
and Fraser, M. J. [1983] Mol. Cell. Biol. 3:2156-2165). The genes
coding for k-1, k-73, k-73/k-1, k-1/k-73, or other B.t. genes can
be modified with BamHI linkers at appropriate regions both upstream
and downstream from the coding regions and inserted into the
passenger site of one of the AcNPV vectors.
EXAMPLE 7
Chimeric Toxin Denoted ACB-1
[0066] Enhanced toxicity against all three insects tested was shown
by a toxin denoted ACB-1. The toxin ACB-1 (Table 3A) is encoded by
plasmid pACB-1 (Table 3). The insecticidal activity encoded by
pACB-1, in comparison with pEW3 (Example 3), is as follows:
3 LC.sub.50 (O.D..sub.575/ml) Clone T. ni H. zea S. exigua pEW3 4.3
23.0 12.3 pACB-1 1.2 3.9 1.2
[0067] The above test was conducted using the conditions described
previously.
[0068] The above results show that the ACB-1 toxin has the best
composite activity as compared to the other toxins tested herein
against all three insects.
[0069] Plasmid pACB-l was constructed between the variable region
of MTX-36, a wild B. thuringiensis strain, having the deposit
accession number NRRL B-18101, and the variable region of HD-73 as
follows: MTX-36; N-terminal to SacI site. HD-73; SacI site to
C-terminal.
[0070] Total plasmid DNA was prepared from strain MTX-36 by
standard procedures. The DNA was submitted to complete digestion by
restriction enzymes SpeI and DraI. The digest was separated
according to size by agarose gel electrophoresis and a 1962 bp
fragment was purified by electroelution using standard
procedures.
[0071] Plasmid pEW2 was purified and digested completely with SpeI
and then submitted to partial digestion with DraI. The digest was
submitted to agarose gel electrophoresis and a 4,138 bp fragment
was purified by electroelution as above.
[0072] The two fragments (1962 bp from MTX-36 and 4138 bp from pEW2
were ligated together to form construct pACB.
[0073] Plasmid DNA was prepared from pACB, digested completely with
SacI and NdeI and a 3760 bp fragment was isolated by electroelution
following agarose gel electrophoresis.
[0074] Plasmid pEW1 was digested completely with SacI and NdeI and
a 2340 bp fragment was isolated by electroelution following agarose
gel electrophoresis.
[0075] The two fragments (3760 bp from pACB and 2340 from pEW1)
were ligated together to form construct pACB-1.
[0076] The complete nucleotide sequence of the ACB-1 gene was
determined and the deduced amino acid sequence of the toxin was
compared with that determined for the toxin encoded by pEW3 (EW3).
The result was that the deduced amino acid sequence of the ACB-1
toxin was identical to that of EW3 with two exceptions: (1)
Aspartic acid residue 411 in EW3 was changed to asparagine in ACB-1
and (2) glycine residue 425 in EW3 was changed to glutamic acid in
ACB-1. These two amino acid changes account for all of the changes
in insect toxicity between these strains. The amino acid sequence
of the EW3 toxin is as reported in Table 1. A schematic
representation of these two toxins is as follows: 1
[0077] The above disclosure is further exemplification of the
subject invention process for altering the host range of Bacillus
toxins which comprises recombining in vitro the variable region of
two or more toxin genes. Once a chimeric toxin is produced, the
gene encoding the same can be sequenced by standard procedures, as
disclosed above. The sequencing data can be used to alter other DNA
by known molecular biology procedures to obtain the desired novel
toxin. For example, the above-noted changes in the ACB-1 gene from
HD-73, makes it possible to construct the ACB-1 gene as
follows:
[0078] Plasmid pEW3, NRRL B-18034, was modified by altering the
coding sequence for the toxin. The 151 bp DNA fragment bounded by
the AccI restriction site at nucleotide residue 1199 in the coding
sequence, and the SacI restriction site at residue 1350 were
removed by digestion with the indicated restriction endonucleases
using standard procedures. The removed 151 bp DNA fragment was
replaced with the following synthetic DNA oligomer by standard
procedures:
4 A TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG AAT GAA ATA CCG CCA CAG
AAT AAC AAC GTG CCC CCG AGG CAA GAA TTT AGT CAT CGA TTA AGC CAT GTT
TCA ATG TTT AGA TCT GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA AGA
GCT
[0079] The net result of this change is that the aspartic residue
at position 411 in the toxin encoded by pEW3 (Table 1A) is
converted to asparagine, and the glycine residue at position 425 is
converted to a glutamic residue. All other amino acids encoded by
these genes are identical.
[0080] The changes made at positions 411 and 425, discussed above,
clearly illustrate the sensitivity of these two positions in toxin
EW3. Accordingly, the scope of the invention is not limited to the
particular amino acids depicted as participating in the changes.
The scope of the invention includes substitution of all 19 other
amino acids at these positions. This can be shown by the following
schematic: 2
[0081] wherein X is one of the 20 common amino acids except Asp
when the amino acid at position 425 is Gly; Y is one of the 20
common amino acids except Gly when the amino acid at position 411
is Asp. The 20 common amino acids are as follows: alanine,
arginine, asparagine, aspartate, cysteine, glutamine, glutamate,
glycine, histidine, isoleucine, leucine, lysine, methionine,
pheniylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine.
EXAMPLE 8
Chimeric Toxin Denoted SYW1
[0082] Enhanced toxicity against tested insects was shown by a
toxin denoted SYW1. The toxin SYW1 (Table 4A) is encoded by plasmid
pSYW1 (Table 4). The insecticidal activity encoded by pSYW1, in
comparison with pEW1 (Example 1) and pEW2 (Example 2), is as
follows:
5 LC.sub.50 (O.D..sub.575/ml) Clone T. ni H. zea S. exigua pEW1 3.5
12.3 18.8 pEW2 1.4 52.3 5.9 pSYW1 0.7 1.9 12.0
[0083] The above test was conducted using the conditions described
previously.
[0084] Plasmid pSYW1 was constructed as follows:
[0085] Plasmid DNA from pEW2 was prepared by standard procedures
and submitted to complete digestion with restriction enzyme AsuII
followed by partial digestion with EcoRI. A 5878 bp fragment was
purified by electroelution following agarose gel electrophoresis of
the digest by standard procedures.
[0086] Plasmid DNA from strain HD-1 was prepared and submitted to
complete digestion with restriction enzymes AsuII and EcoRI. A 222
bp fragment was purified by electroelution following agarose gel
electrophoresis of the digest.
[0087] The two fragments (5878 bp from pEW2 and 222 bp from HD-1)
were ligated together, by standard procedures, to form construct
pSYW1.
[0088] The amino acid changes (3) in toxin SYW1 from EW3 are as
follows: (1) Arginine residue 289 in EW3 was changed to glycine in
SYW1, (2) arginine residue 311 in EW3 was changed to lysine in
SYW1, and (3) the tyrosine residue 313 was changed to glycine in
SYW1. A schematic representation of these two toxins is as follows:
3
[0089] The changes made at positions 289, 311, and 313, discussed
above, clearly illustrate the sensitivity of these three positions
in toxin EW3. Accordingly, the scope of the invention is not
limited to the particular amino acids depicted as participating in
the changes. The scope of the invention includes substitution of
all the common amino acids at these positions. This can be shown by
the following schematic: 4
[0090] wherein X is one of the 20 common amino acids except Arg
when the amino acid at position 311 is Arg and the amino acid at
position 313 is Tyr; Y is one of the 20 common amino acids except
Arg when the amino acid at position 289 is Arg and the amino acid
at position 313 is Tyr; and Z is one of the 20 common amino acids
except Tyr when the amino acid at position 289 is Arg and the amino
acid at position 311 is Arg.
[0091] Construction of the SYW1 gene can be carried out by
procedures disclosed above for the construction of the ACB-1 gene
from plasmid pEW3 with appropriate changes in the synthetic DNA
oligomer.
[0092] As is well known in the art, the amino acid sequence of a
protein is determined by the nucleotide sequence of the DNA.
Because of the redundancy of the genetic code, i.e., more than one
coding nucleotide triplet (codon) can be used for most of the amino
acids used to make proteins, different nucleotide sequences can
code for a particular amino acid. Thus, the genetic code can be
depicted as follows:
6 Phenylalanine (Phe) TTK Histidine (His) CAK Leucine (Leu) XTY
Glutamine (Gln) CAJ Isoleucine (Ile) ATM Asparagine (Asn) AAK
Methionine (Met) ATG Lysine (Lys) AAJ Valine (Val) GTL Aspartic
acid (Asp) GAK Serine (Ser) QRS Glutamic acid (Glu) GAJ Proline
(Pro) CCL Cysteine (Cys) TGK Threonine (Thr) ACL Tryptophan (Trp)
TGG Alanine (Ala) GCL Arginine (Arg) WGZ Tyrosine (Tyr) TAK Glycine
(Gly) GGL Termination signal TAJ
[0093] Key: Each 3-letter deoxynucleotide triplet corresponds to a
trinucleotide of mRNA, having a 5'-end on the left and a 3'-end on
the right. All DNA sequences given herein are those of the strand
whose sequence corresponds to the mRNA sequence, with thymine
substituted for uracil. The letters stand for the purine or
pyrimidine bases forming the deoxynucleotide sequence.
[0094] A=adenine
[0095] G=guanine
[0096] C=cytosine
[0097] T=thymine
[0098] X=T or C if Y is A or G
[0099] X=C if Y is C or T
[0100] Y=A, G, C or T if X is C
[0101] Y=A or G if X is T
[0102] W=C or A if Z is A or G
[0103] W=C if Z is C or T
[0104] Z=A, G, C or T if W is C
[0105] Z=A or G if W is A
[0106] QR=TC if S is A, G, C or T; alternatively QR=AG if S is T or
C
[0107] J=A or G
[0108] K=T or C
[0109] L=A, T, C or G
[0110] M=A, C or T
[0111] The above shows that the novel amino acid sequence of the
chimeric toxins, and other useful proteins, can be prepared by
equivalent nucleotide sequences encoding the same amino acid
sequence of the proteins. Accordingly, the subject invention
includes such equivalent nucleotide sequences. In addition it has
been shown that proteins of identified structure and function may
be constructed by changing the amino acid sequence if such changes
do not alter the protein secondary structure (Kaiser, E. T. and
Kezdy, F. J. [1984] Science 223:249-255). Thus, the subject
invention includes muteins of the amino acid sequences depicted
herein which do not alter the protein secondary structure.
[0112] The one-letter symbol for the amino acids used in Tables 1A
and 2A is well known in the art. For convenience, the relationship
of the three-letter abbreviation and the one-letter symbol for
amino acids is as follows:
7 Ala A Arg R Asn N Asp D Cys C Gln Q Glu E Gly G His H Ile I Leu L
Lys K Met M Phe F Pro P Ser S Thr T Trp W Tyr Y Val V
[0113] The work described herein was all done in conformity with
physical and biological containment requirements specified in the
NIH Guidelines.
8 CHART A Bioassay of Chimeric Toxins Against Various Insects LC50
(O.D. 575/ml diet) Plasmid Toxin T. ni S. exigua H. zea pEW1 k-1
3.5 12.3 18.8 pEW2 k-73 1.4 52.3 5.9 pEW3 k-73/k-1 5.7 9.6 10.4
pEW4 k-1/k-73 0.8 30.4 2.2 Recombinant E. coli cells containing the
above plasmids were grown overnight in L-broth.* The cells were
pelleted and resuspended on 0.85% NaCl. The optical density at 575
nm was determined for these cell suspensions and appropriate
dilutions were made in 0.85% NaCl. Three ml of each dilution were
added to 27 ml of USDA diet (Dulmage, H.D., Martinez, A.J. and
Pena, T [1976] USDA Agricultural Research Service Technical
Bulletin No. 1528, U.S. Government Printing Office, Washington,
D.C.). The diet/toxin mixture was then dispensed into 24 wells in a
plastic tissue culture tray (1.0 ml/well). Single neonate larvae
from either Trichoplusia ni, Spodoptera exigua, or Heliothis zea
were then added to each well. The trays were then covered with
Mylar and punctured with small holes for air exchange. The larvae
were observed after 7 days and LC50 values were calculated using
the method of probit analysis (Finney, D.J. [1971] Probit Analysis
3rd ed. Cambridge University Press, Cambridge). *L-broth is 5 g/l
NaCl, 10 g/l bactotryptone, 5 g/l yeast extract.
[0114]
9 CHART B Assay of Toxins Against CF-1 Cells in Culture Live Cells
(% of Control) Plasmid Toxin Expt. 1 Expt. 2 pEW1 k-1 106% 108%
pEW2 k-73 44% 46% pEW3 k-73/k-1 105% 97% pEW4 k-1/k-73 53% 58%
Overnight cultures of E. coli containing the various plasmids were
centrifuged and resuspended in 0.85% NaCl containing 1 mM
EDTA.sup.1, 0.2 mM PMSF.sup.2, 0.2 mM TPCK.sup.3 and 100 mM NaCH.
Cells were broken in a bead beater (Biospec Products, Bartlesville,
OK), centrifuged and the supernatant dialyzed against 20 mM
Tris-glycine pH 8.5. Toxin was activated with 0.7% trypsin. Assays
were carried out on Choristoxieura fumiferana cell line CF-1.
Approximately 100 .mu.g of activated toxin extract was added to 3.2
.times. 10.sup.5 cells in a volume of 1.0 ml. ATP levels were
determined after 30 min incubation and the percentage of live cells
remaining in the suspension was determined from standard curves.
.sup.1ethylenediaminete- traacetic acid .sup.2phenylmethylsulfonyl
fluoride .sup.31-tosylamide-2-phenylethylchloromethyl ketone
[0115]
10 CHART C Facile Comparison of Constructions of Plasmids pEW3 and
pEW4 5' Asu II Sac I Xho I k-73 3' Asu II Sac I Xho I* k-1 pEW3
+++++++++++++++++++++---------------------------------------- pEW4
---------------------++++++++++++++++++++++++++++++++++++++++ ----=
sequences from k-1 ++++= sequences from k-73 Xho I* means that this
restriction site found in k-73 no longer exists in k-1 and will
have to be recreated by site specific mutagenesis (it involves
changing two base pairs in k-1).
[0116]
11TABLE 1 Nucleotide Sequence of Plasmid pEW3 Encoding Chimeric
Toxin Numbering of the nucleotide bases is the same as Schnepf et
al. (J. Biol. Chem. 260:6264-6272 [1985]) for HD-1 and Adang et al.
(Gene 36:289-300 [1985]) for HD-73. Only protein coding sequences
are shown. (start HD-73) ATG GATAACAATC 400 CGAACATCAT TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TTGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAATTTACAA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGAA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AGAGCT
(end hd-73) (start HD-1) CCACGT TTTCTTGGCA GCATCGCAGT 1800
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGATAATGA TTTAAGAAGA GCAACAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGCGGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGAATGGAGA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACASACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGFA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTTC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GATCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCATTCGTG TCACAGCGTA CAGGAAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCTAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGAT TATGTGACTA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
[0117]
12TABLE 1A Deduced Amino Acid Sequence of Chimeric Toxin Produced
by Plasmid pEW N G N N P N T N E C I P Y N C L S N P E V E V L G G
E R I E T G Y T P T G T S L S L T Q F L L S E F V P G A G F V L G L
V D I I W G I F G P S Q W D A F L V Q T E Q L T N Q R I E E F A R N
Q A I S R L E G L S N L Y Q T Y A E S F R E W E A D P T N P A L R E
E N R I Q F N D M N S A L T T A I A L F A V Q N Y Q V P L L S V Y V
Q A A N L H L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L
T R L T G N Y T D Y A V R W Y N T G L E R V W G A D S R D W V R Y N
G F R R E L T L T V L D I V A L F P N Y D S R R Y A T R T V S Q L T
R E T Y T N P V L E N F D G S F R G S A G G T E R S T R S A H L N D
I L N S I T I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E
F T F P L Y G T N M N A A A P Q R T V A Q L G Q G V Y R T L S S T L
Y R R P F N I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V
Y R K S G T V D S L D E T P P Q N N N V P P R Q G F S H R L S H V S
M F R S G F S N S S V S I I R A P T F S W Q H R S A E F N N I I P S
S Q T T Q T A L T K S T N L G S G T S V V K G P G F T G G D T L R R
T S P G Q I S T L R V N I T A P L S Q R Y R V R T R Y A S T T N L Q
F H T S I D G R P I N Q G N F S P T N S S G S N L G S G S F R T V G
F T T P F N F S N G S S V F T L S P H V F N S G N E V Y T D R I E F
V P P E V T F E P E Y D L E R P Q K A V N E L F T S S N Q T G L K T
D V T D Y H I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K
H P K P L S D E P N L L Q D P N F R G I N P Q L D R G W R G S T D T
T T Q G G D D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K T D E
S K L K A Y T R Y Q L R G Y T E D S Q D L E T Y L T P Y N P K H E T
V N V P G T G S L W P L S A Q S P I G K C G E P N R C A P H L E W N
P D L D C S C R D G E K C A N H S H H F S L D I D V G C T D L N E D
L G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G E P L
P P V K R P E K K W F D K R E K L E W E T N T V Y K E P K E S V D P
L F V N S Q Y D Q L Q P G T N T P N T H P P D K P V H S T P E P Y L
P E L S V T P G V N P P T F E E L E G P T F T P F S L Y D P P N V T
K N G D F N N G L S C W N V K G H V D V E E P N N Q R S V L V L P E
W E P E V S Q E V R V C P G P G Y T L R V T P Y K E G Y G E G C V T
T H E T E N N T D E L K F S N C V E E E T Y P N N T V T C N D Y T V
N Q E E Y G G A Y I S P N P G Y N E A P S V P A D Y P S V Y E E K S
Y T D G R P E N P C E F N P G Y R D Y T P L P V G Y V T K E L E Y F
P E T D K V W T E T G E T E G T F T V D S V E L L L M E E
[0118]
13TABLE 2 Nucleotide Sequence of Plasmid pEW4 Encoding Chimeric
Toxin Numbering of nucleotide bases is the same as Schnepf et al.
(J. Biol. Chem. 260:6264-6272 [1985]) for HD-1 and Adang et al.
(Gene 36:289-300 [1985]) for HD-7. Only protein coding sequences
are shown. (start HD-1) ATGG ATACATCC GACATCAAT GAATGCATTC
CTTAATATTG TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 600 TGGAGAAAGA
ATAGAAACTG GTTACACCCC AATCGATATT TCCTTGTCGC TAACGCAATT TCTTTTGAGT
GAATTTGTTC CCGGTGCTGG ATTTGTGTTA 700 GGACTAGTTG ATATAATATG
GGGAATTTTT GGTCCCTCTC AATGGGACGC ATTTCCTGTA CAAATTGAAC AGTTAATTAA
CCAAAGAATA GAAGAATTCG 800 CTAGGAACCA AGCCATTTCT AGATTAGAAG
GACTAAGCAA TCTTTATCAA ATTTACGCAG AATCTTTTAG AGAGTGGGAA GCAGATCCTA
CTAATCCAGC 900 ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC
AGTGCCCTTA CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTATA
1000 TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT
TTCAGTGTTT GGACAAAGGT GGGGATTTGA TGCCGCGACT ATCAATAGTC 1100
GTTATAATGA TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG CGCTGGTACA
ATACGGGATT AGAGCGTGTA TGTGGACCGG ATTCTAGAGA 1200 TTGGGTAAGG
TATAATCAAT TTAGAAGAGA GCTAACACTT ACTGTATTAG ATATCGTTGC TCTATTCTCA
AATTATGATA GTCGAAGGTA TCCAATTCGA 1300 ACAGTTTCCC AATTAACAAG
AGAAATTTAT ACGAACCCAG TATTAGAAAA TTTTGATGTT AGTTTTCGTG GAATGGCTCA
GAGAATAGAA CAGAATATTA 1400 GGCAACCACA TCTTATGGAT ATCCTTATTA
GTATAACCAT TTATACTGAT GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA
CATCTTCTCC 1500 TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT
GGGAATGCGA GGAATGCAGC TCCACCCATA CTTGTCTCAT TAACTGGTTT GGGGATTTTT
1600 AGACAATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCATGCCC
AAATAATCAG GAACTGTTTG TCCTTGATGT AACGGAGTTT TCTTTTGCCT 1700
CCCTAACGAC CAACTTGCCT TCCACTATAT ATAGACAAAG GGGTACAGTC GATTCACTAG
ATGTAATACC GCCACAGGAT AATAGTGTAC CACCTCGTGC 1800 GGGATTTAGC
CATCGATTGA GTCATGTTAC AATGCTGAGC CAGCAAGCTG GAGCAGTTTA CACCTTGAGA
GCTCACGT (stop HD-1) (start HD-7) CCT ATGTTCTCTT GGATACATCG
TAGTGCTGAA TTTAATAATA TAATTGCATC GGATAGTATT 1800 ACTCAAATCC
CTGCAGTGAA GGGAAACTTT CTTTTTAATG GTTCTGTAAT TTCAGGACCA GGATTTACTT
GTGGGGACTT AGTTAGATTA AATAGTAGTG 1900 GAAATAACAT TCAGAATAGA
GGGTATATTG AAGTATATTG TCACTTCCCA TCGACATCTA CCAGATATCG AGTTCGTGTA
CGGTATGCTT CTGTAACCCC 2000 GATTCACCTC AACGTTAATT GGGGTAATTC
ATCCATTTTT TCCAATACAG TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG
TGATTTTGGT 2100 TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA
ATATAGTAGG TGTTAGAAAT TTTAGTGGGA CTGCAGGAGT GATAATAGAC AGATTTGAAT
2200 TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGAAAGAGCG
CAGAAGGCGG TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 2300
AACAAATGTA ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT ATTTATCGGA
TGAATTTTGT CTGGATGAAA AGCGAGAATT GTCCGAGAAA 2400 GTCAAACATG
CGAAGCGACT CAGTGATGAA CGCAATTTAC TCCAAGATTC AAATTTCAAA GACATTAATA
ACGCACCAGA ACGTGGGTGG GGCGGAAGTA 2500 CAGGGATTAC CATCCAAGGA
GGGGATGACG TATTTAAAGA AAATTACGTC ACACTATCAG GTACCTTTGA TGAGTGCTAT
CCAACATATT TGTATCAAAA 2600 AATCGATGAA TCAAATTATA AAGCCTTTAC
CCGTTATCAA TTAAGAGGGT ATATCGAAGA TAGTCAAGAC TTAGAAATCT ATTTAATTCG
CTACAATGCA 2700 AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT
GGCCGCTTTC AGCCCAAAGT CCAATCGGAA AGTGTGGAGA GCCGAATCGA TGCGCGCCAC
2800 ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG
TTTGCCCATC ATTCGCATCA TTTCTCCTTA GACATTGATG TAGGATGTAC 2900
AGACTTAAAT GAGGACCTAG ATGTATGGGT GATCTTTAAG ATTAAGACGC AAGATGGGCA
CGCAAGACTA GGGAATCTAG AGTTTCTCGA AGAGAAACCA 3000 TTAGTAGGAG
AAGCGCTAGC TCGTGTGAAA AGAGCGGAGA AAAAATGGAG AGACAAACGT GAAAAATTGG
AATGGGAAAC AAATATCGTT TATAAAGAGG 3100 CAAAAGAATC TGTAGATGCT
TTATTTGTAA ACTCTCAATA TGATCAATTA CAAGCGGATA CGAATATTGC CATGATTCAT
GCGGCAGATA AACGTGTTCA 3200 TAGCATTCGA GAAGCTTATC TGCCTGAGCT
GTCTGTGATT CCGGGTGTCA ATGCGGCTAT TTTTGAAGAA TTAGAAGGGC GTATTTTCAC
TGCATTCTCC 3300 CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA
ATAATGGCTT ATCCTGCTGG AACGTGAAAG GGCATGTAGA TGTAGAAGAA CAAAACAACC
3400 AACGTTCGGT CCTTATTGTT CCGGAATGGG AAGCAGAAGT GTCACAAGAA
GTTCGTGTCT GTCCGGGTCG TGGCTATATC CTTCGTGTCA CAGCGTACAA 3500
GGAGGGATAT GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA CAGACGAACT
GAGTTTAAGC AACTGCGTAG AAGAGGAAAT CTATCCAAAT 3600 AACACGGTAA
CGTGTAATGA TTATACTGTA AATCAAGAAG AATACGGAGG TGCGTACACT TCTCGTAATC
GAGGATATAA CGAAGCTCCT TCCGTACCAG 3700 CTGATTATTC GTCAGTCTAT
GAAGAAAAAT CGTATACAGA TGGACGAAGA GAGAATCCTT GTGAATTTAA CAGAGGGTAT
AGGGATTACA CGCCACTACC 3800 AGTTTGTTAT GTACACAAAA AATTAGAATA
CTTCCCAGAA ACCGATAAGG TATGGATTGA GATTGGAGAA ACGGAAGGAA CATTTATCGT
GGACAGCGTG 3900 GAATTACTCC TTATGGAGGA A (end HD-7)
[0119]
14TABLE 2A Deduced Amino Acid Sequence of Chimeric Toxin Produced
by Plasmid pEW4 N G N N P N T N E C I P Y N C L S N P E V E V L G G
E R I E T G Y T P T G T S L S L T Q F L L S E F V P G A G F V L G L
V D I I W G I F G P S Q W D A F L V Q T E Q L T N Q R I E E F A R N
Q A I S R L E G L S N L Y Q T Y A E S F R E W E A D P T N P A L R E
E N R I Q F N D M N S A L T T A I A L F A V Q N Y Q V P L L S V Y V
Q A A N L H L S V L R D V S V F G Q R W G F D A A T I N S R Y N D L
T R L T G N Y T D Y A V R W Y N T G L E R V W G A D S R D W V R Y N
G F R R E L T L T V L D I V A L F P N Y D S R R Y A T R T V S Q L T
R E T Y T N P V L E N F D G S F R G S A G G T E R S T R S A H L N D
I L N S I T I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G P E
F T F P L Y G T N M N A A A P Q R T V A Q L G Q G V Y R T L S S T L
Y R R P F N I G I N N Q Q L S V L D G T E F A Y G T S S N L P S A V
Y R K S G T V D S L D E T P P Q N N N V P P R Q G F S H R L S H V S
M F R S G F S N S S V S I I R A P T F S W Q H R S A E F N N I I P S
S Q T T Q T A L T K S T N L G S G T S V V K G P G F T G G D T L R R
T S P G Q I S T L R V N I T A P L S Q R Y R V R T R Y A S T T N L Q
F H T S I D G R P I N Q G N F S P T N S S G S N L G S G S F R T V G
F T T P F N F S N G S S V F T L S P H V F N S G N E V Y T D R I E F
V P P E V T F E P E Y D L E R P Q K A V N E L F T S S N Q T G L K T
D V T D Y H I D Q V S N L V E C L S D E F C L D E K Q E L S E K V K
H P K P L S D E P N L L Q D P N F R G I N P Q L D R G W R G S T D T
T T Q G G D D V F K E N Y V T L L G T F D E C Y P T Y L Y Q K T D E
S K L K A Y T R Y Q L R G Y T E D S Q D L E T Y L T P Y N P K H E T
V N V P G T G S L W P L S A Q S P I G K C G E P N R C A P H L E W N
P D L D C S C R D G E K C A N H S H H F S L D I D V G C T D L N E D
L G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G E P L
P P V K R P E K K W F D K R E K L E W E T N T V Y K E P K E S V D P
L F V N S Q Y D Q L Q P G T N T P N T H P P D K P V H S T P E P Y L
P E L S V T P G V N P P T F E E L E G P T F T P F S L Y D P P N V T
K N G D F N N G L S C W N V K G H V D V E E P N N Q R S V L V L P E
W E P E V S Q E V R V C P G P G Y T L R V T P Y K E G Y G E G C V T
T H E T E N N T D E L K F S N C V E E E T Y P N N T V T C N D Y T V
N Q E E Y G G A Y I S P N P G Y N E A P S V P A D Y P S V Y E E K S
Y T D G R P E N P C E F N P G Y R D Y T P L P V G Y V T K E L E Y F
P E T D K V W T E T G E T E G T F T V D S V E L L L M E E
[0120]
15TABLE 3 Nucleotide Sequence of Plasmid pACG-1 Encoding Chimeric
Toxin ACG-1 The nucleotide differences as compared to the sequence
shown in TABLE 1 are underlined at positions 1618 and 1661 and code
for amino acid changes at positions 411 and 425 as shown in TABLE
A. (start HD-73) ATG GATAACAATC 400 CGAACATCAT TGAATGCATT
CCTTATAATT GTTTAAGTAA CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT
GGTTACACCC CAATCGATAT 500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG
TGAATTTGTT CCCGGTGCTG GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT
TTGTCCCTCT 600 CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA
ACCAAAGAAT AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA
700 ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT
ATCAGTATAT GTTCAAGCTG CAATTTACAA TTTATCAGTT 900 TTGAGAGATG
TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG
ATTTAACTAG GCTTATTGGC AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC
AATACGGGAT TAGAACGTGT ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA
TTTAGAAGAG AATTAACACT 1100 AACTGTATTA GATATCGTTG CTCTGTTCCC
GAATTATGAT AGTAGAAGAT ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA
TACAAACCCA 1200 GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC
AGGGCATAGA AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA
1300 TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT
GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500 AATATAGGGA
TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT TGCTTATGAA ACCTCCTCAA
ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT
GAAATACCGC CACAGAATAA CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC
CATGTTTCAA TGTTTCGTTC 1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AGAGCT
(end hd-73) (start HD-1) CCACGT TTTCTTGGCA GCATCGCAGT 1800
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT
ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG
AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC
TGCACCATTA TCACAAAGAT ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA
CAAATTTACA ATTCCATACA TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG
CAACTATGAG TAGTGGGAGT 2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA
GGTTTTACTA CTCCGTTTAA CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT
GTCTTCAATT 2300 CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC
GGCAGAAGTA ACCTTTGAGG CAGATAATGA TTTAAGAAGA GCAACAAAGG CGGTGAATGA
2400 GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 2500
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG ACTTAGTGAT
GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT
AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA GGAGCGGATG ACGTATTCAA
AGAGAATTAC GTTACGCTAT TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT
ATTTATATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG
GGTATATCGA AGATAGTCAA 2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT
GCAAAACATG AAACAGTAAA TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA
AGTCCAATCG 2900 GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA
ATGGAATCCT GACTTAGATT GTTCGTGTAG GGAATGGAGA AAGTGTGCCC ATCATTCGCA
3000 TCATTTCTCC TTAGACATTG ATGTAGGATG TACASACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG
AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGFA
AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA
ATATGATCAA TTACAAGCGG ATACGAATAT 3300 TGCCATGATT CATGCGGCAG
ATAAACGTGT TCATAGCATT CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG
TCAATGCGGC TATTTTTGAA 3400 GAATTAGAAG GGCGTATTTT CACTGCATTC
TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTTC
TGGAACGTGA 3500 AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC
GATCCTTGTT CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG
3600 TCGTGGCTAT ATCATTCGTG TCACAGCGTA CAGGAAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT
GTAAATCTAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA
TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC
AGATGGACGA AGAGAGAATC CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT
ACACGCCACT ACCAGTTGAT TATGTGACTA AAGAATTAGA ATACTTCCCA GAAACCGATA
AGGTATGGAT TGAGATTGGA 4000 GAAACGGAAG GAACATTTAT CGTGGACAGC
GTGGAATTAC TCCTTATGGA GGAA (end HD-1)
[0121]
16TABLE 3A Deduced Amino Acid Sequence of Chimneric Toxin ACG-1 M D
N N P N T N E C I P Y N C L S N P E V E V L G G E R I E T G Y T P T
G T S L S L T Q F L L S E F V P G A G F V L G L V D I I W G I F G P
S Q W D A F L V Q T E Q L T N Q R I E E F A R N Q A I S R L E G L S
N L Y Q T Y A E S F R E W E A D P T N P A L R E E N R I Q F N D M N
S A L T T A I A L F A V Q N Y Q V P L L S V Y V Q A A N L H L S V L
R D V S V F G Q R W G F D A A T I N S R Y N D L T R L T G N Y T D Y
A V R W Y N T G L E R V W G A D S R D W V R Y N G F R R E L T L T V
L D I V A L F P N Y D S R R Y A T R T V S Q L T R E T Y T N P V L E
N F D G S F R G S A G G T E R S T R S A H L N D I L N S I T I Y T D
A H R G Y Y Y W S G H Q I M A S P V G F S G P E F T F P L Y G T N M
N A A A P Q R T V A Q L G Q G V Y R T L S S T L Y R R P F N I G I N
N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T V D S L
N E I P P Q N N N V P P R Q E F S H R L S H V S M F R S G F S N S S
V S I I R A P T F S W Q H R S A E F N N I I P S S Q T T Q T A L T K
S T N L G S G T S V V K G P G F T G G D T L R R T S P G Q I S T L R
V N I T A P L S Q R Y R V R T R Y A S T T N L Q F H T S I D G R P I
N Q G N F S P T N S S G S N L G S G S F R T V G F T T P F N F S N G
S S V F T L S P H V F N S G N E V Y T D R I E F V P P E V T F E P E
Y D L E R P Q K A V N E L F T S S N Q T G L K T D V T D Y H I D Q V
S N L V E C L S D E F C L D E K Q E L S E K V K H P K P L S D E P N
L L Q D P N F R G I N P Q L D R G W R G S T D T T T Q G G D D V F K
E N Y V T L L G T F D E C Y P T Y L Y Q K T D E S K L K A Y T R Y Q
L R G Y T E D S Q D L E T Y L T P Y N P K H E T V N V P G T G S L W
P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S C R D G
E K C A N H S H H F S L D I D V G C T D L N E D L G V W V I F K I K
T Q D G H A R L G N L E F L E E K P L V G E P L P P V K R P E K K W
F D K R E K L E W E T N T V Y K E P K E S V D P L F V N S Q Y D Q L
Q P G T N T P N T H P P D K P V H S T P E P Y L P E L S V T P G V N
P P T F E E L E G P T F T P F S L Y D P P N V T K N G D F N N G L S
C W N V K G H V D V E E P N N Q R S V L V L P E W E P E V S Q E V R
V C P G P G Y T L R V T P Y K E G Y G E G C V T T H E T E N N T D E
L K F S N C V E E E T Y P N N T V T C N D Y T V N Q E E Y G G A Y I
S P N P G Y N E A P S V P A D Y P S V Y E E K S Y T D G R P E N P C
E F N P G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V W T E T
G E T E G T F T V D S V E L L L M E E
[0122]
17TABLE 4 Nucleotide Sequence of Alasmid pSYW1 Encoding Chimeric
Toxin SYW1 The nucleotide differences as compared to the sequence
shown in TABLE 1 are underlined at positions 1252, 1319, 1320,
1323, 1324, and 1326; and code for amino acid changes at positions
289, 311, and 313, as shown in TABLE 4A. (start HD-73) ATG
GATAACAATC 400 CGAACATCAT TGAATGCATT CCTTATAATT GTTTAAGTAA
CCCTGAAGTA GAAGTATTAG GTGGAGAAAG AATAGAAACT GGTTACACCC CAATCGATAT
500 TTCCTTGTCG CTAACGCAAT TTCTTTTGAG TGAATTTGTT CCCGGTGCTG
GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT TTGTCCCTCT 600
CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA ACCAAAGAAT AGAAGAATTC
GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA 700 ATCTTTATCA
AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT ACTAATCCAG CATTAAGAGA
AGAGATGCGT ATTCAATTCA ATGACATGAA 800 CAGTGCCCTT ACAACCGCTA
TTCCTCTTTT TGCAGTTCAA AATTATCAAG TTCCTCTTTT ATCAGTATAT GTTCAAGCTG
CAATTTACAA TTTATCAGTT 900 TTGAGAGATG TTTCAGTGTT TGGACAAAGG
TGGGGATTTG ATGCCGCGAC TATCAATAGT CGTTATAATG ATTTAACTAG GCTTATTGGC
AACTATACAG 1000 ATTATGCTGT ACGCTGGTAC AATACGGGAT TAGAACGTGT
ATGGGGACCG GATTCTAGAG ATTGGGTAAG GTATAATCAA TTTAGAAGAG AATTAACACT
1100 AACTGTATTA GATATCGTTG CTCTGTTCCC GAATTATGAT AGTAGAAGAT
ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA TACAAACCCA 1200
GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC AGGGCATAGA AAGAAGTATT
AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA 1300 TCTATACGGA
TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA ATGGCTTCTC CTGTAGGGTT
TTCGGGGCCA GAATTCACTT TTCCGCTATA 1400 TGGAACTATG GGAAATGCAG
CTCCACAACA ACGTATTGTT GCTCAACTAG GTCAGGGCGT GTATAGAACA TTATCGTCCA
CTTTATATAG AAGACCTTTT 1500 AATATAGGGA TAAATAATCA ACAACTATCT
GTTCTTGACG GGACAGAATT TGCTTATGAA ACCTCCTCAA ATTTGCCATC CGCTGTATAC
AGAAAAAGCG 1600 GAACGGTAGA TTCGCTGGAT GAAATACCGC CACAGAATAA
CAACGTGCCA CCTAGGCAAG GATTTAGTCA TCGATTAAGC CATGTTTCAA TGTTTCGTTC
1700 AGGCTTTAGT AATAGTAGTG TAAGTATAAT AGAGCT (end hd-73) (start
HD-1) CCACGT TTTCTTGGCA GCATCGCAGT 1800 GCTGAATTTA ATAATATAAT
TCCTTCATCA CAAATTACAC AAATACCTTT AACAAAATCT ACTAATCTTG GCTCTGGAAC
TTCTGTCGTT AAAGGACCAG 2000 GATTTACAGG AGGAGATATT CTTCGAAGAA
CTTCACCTGG CCAGATTTCA ACCTTAAGAG TAAATATTAC TGCACCATTA TCACAAAGAT
ATCGGGTAAG 2100 AATTCGCTAC GCTTCTACTA CAAATTTACA ATTCCATACA
TCAATTGACG GAAGACCTAT TAATCAGGGT AATTTTTCAG CAACTATGAG TAGTGGGAGT
2200 AATTTACAGT CCGGAAGCTT TAGGACTGTA GGTTTTACTA CTCCGTTTAA
CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT GTCTTCAATT 2300
CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC GGCAGAAGTA ACCTTTGAGG
CAGATAATGA TTTAAGAAGA GCAACAAAGG CGGTGAATGA 2400 GCTGTTTACT
TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT ATCATATTGA TCAAGTATCC
AATTTAGTTG AGTGTTTATC AGATGAATTT 2500 TGTCTGGATG AAAAACAAGA
ATTGTCCGAG AAAGTCAAA ATGCGAAGCG ACTTAGTGAT GAGCGGAATT TACTTCAAGA
TCCAAACTTC AGAGGGATCA 2600 ATAGACAACT AGACCGTGGC TGGAGAGGAA
GTACGGATAT TACCATCCAA GGAGCGGATG ACGTATTCAA AGAGAATTAC GTTACGCTAT
TGGGTACCTT 2700 TGATGAGTGC TATCCAACGT ATTTATATCA AAAAATAGAT
GAGTCGAAAT TAAAAGCCTA TACCCGTTAT CAATTAAGAG GGTATATCGA AGATAGTCAA
2800 GACTTAGAAA TCTATTTAAT TCGCTACAAT GCAAAACATG AAACAGTAAA
TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA AGTCCAATCG 2900
GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA ATGGAATCCT GACTTAGATT
GTTCGTGTAG GGAATGGAGA AAGTGTGCCC ATCATTCGCA 3000 TCATTTCTCC
TTAGACATTG ATGTAGGATG TACASACTTA AATGAGGACC TAGGTGTATG GGTGATCTTT
AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100 CTAGGGAATC TAGAGTTTCT
CGAAGAGAAA CCATTAGTAG GAGAAGCGCT AGCTCGTGTG AAAAGAGCGG AGAAAAAATG
GAGAGACAAA CGTGAAAAAT 3200 TGGAATGGFA AACAAATATC GTTTATAAAG
AGGCAAAAGA ATCTGTAGAT GCTTTATTTG TAAACTCTCA ATATGATCAA TTACAAGCGG
ATACGAATAT 3300 TGCCATGATT CATGCGGCAG ATAAACGTGT TCATAGCATT
CGAGAAGCTT ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG TCAATGCGGC TATTTTTGAA
3400 GAATTAGAAG GGCGTATTTT CACTGCATTC TCCCTATATG ATGCGAGAAA
TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTTC TGGAACGTGA 3500
AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC GATCCTTGTT CTTCCGGAAT
GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG 3600 TCGTGGCTAT
ATCATTCGTG TCACAGCGTA CAGGAAGGGA TATGGAGAAG GTTGCGTAAC CATTCATGAG
ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700 AGCAACTGCG TAGAAGAGGA
AATCTATCCA AATAACACGG TAACGTGTAA TGATTATACT GTAAATCTAG AAGAATACGG
AGGTGCGTAC ACTTCTCGTA 3800 ATCGAGGATA TAACGAAGCT CCTTCCGTAC
CAGCTGATTA TGCGTCAGTC TATGAAGAAA AATCGTATAC AGATGGACGA AGAGAGAATC
CTTGTGAATT 3900 TAACAGAGGG TATAGGGATT ACACGCCACT ACCAGTTGAT
TATGTGACTA AAGAATTAGA ATACTTCCCA GAAACCGATA AGGTATGGAT TGAGATTGGA
4000 GAAACGGAAG GAACATTTAT CGTGGACAGC GTGGAATTAC TCCTTATGGA GGAA
(end HD-1)
[0123]
18TABLE 4A Deduced Amino Acid Sequence of Chirneric Toxin SYW1 N G
N N P N T N E C I P Y N C L S N P E V E V L G G E R I E T G Y T P T
G T S L S L T Q F L L S E F V P G A G F V L G L V D I I W G I F G P
S Q W D A F L V Q T E Q L T N Q R I E E F A R N Q A I S R L E G L S
N L Y Q T Y A E S F R E W E A D P T N P A L R E E N R I Q F N D M N
S A L T T A I A L F A V Q N Y Q V P L L S V Y V Q A A N L H L S V L
R D V S V F G Q R W G F D A A T I N S R Y N D L T R L T G N Y T D Y
A V R W Y N T G L E R V W G A D S R D W V R Y N G F R R E L T L T V
L D I V A L F P N Y D S R R Y A T R T V S Q L T R E T Y T N P V L E
N F D G S F R G S A Q G I E G S T R S A H L N D I L N S I T I Y T D
A K R G E Y Y W S G H Q I M A S P V G F S G P E F T F P L Y G T N M
N A A A P Q R T V A Q L G Q G V Y R T L S S T L Y R R P F N I G I N
N Q Q L S V L D G T E F A Y G T S S N L P S A V Y R K S G T V D S L
D E T P P Q N N N V P P R Q G F S H R L S H V S M F R S G F S N S S
V S I I R A P T F S W Q H R S A E F N N I I P S S Q T T Q T A L T K
S T N L G S G T S V V K G P G F T G G D T L R R T S P G Q I S T L R
V N I T A P L S Q R Y R V R T R Y A S T T N L Q F H T S I D G R P I
N Q G N F S P T N S S G S N L G S G S F R T V G F T T P F N F S N G
S S V F T L S P H V F N S G N E V Y T D R I E F V P P E V T F E P E
Y D L E R P Q K A V N E L F T S S N Q T G L K T D V T D Y H I D Q V
S N L V E C L S D E F C L D E K Q E L S E K V K H P K P L S D E P N
L L Q D P N F R G I N P Q L D R G W R G S T D T T T Q G G D D V F K
E N Y V T L L G T F D E C Y P T Y L Y Q K T D E S K L K A Y T R Y Q
L R G Y T E D S Q D L E T Y L T P Y N P K H E T V N V P G T G S L W
P L S A Q S P I G K C G E P N R C A P H L E W N P D L D C S C R D G
E K C A N H S H H F S L D I D V G C T D L N E D L G V W V I F K I K
T Q D G H A R L G N L E F L E E K P L V G E P L P P V K R P E K K W
F D K R E K L E W E T N T V Y K E P K E S V D P L F V N S Q Y D Q L
Q P G T N T P N T H P P D K P V H S T P E P Y L P E L S V T P G V N
P P T F E E L E G P T F T P F S L Y D P P N V T K N G D F N N G L S
C W N V K G H V D V E E P N N Q R S V L V L P E W E P E V S Q E V R
V C P G P G Y T L R V T P Y K E G Y G E G C V T T H E T E N N T D E
L K F S N C V E E E T Y P N N T V T C N D Y T V N Q E E Y G G A Y I
S P N P G Y N E A P S V P A D Y P S V Y E E K S Y T D G R P E N P C
E F N P G Y R D Y T P L P V G Y V T K E L E Y F P E T D K V W T E T
G E T E G T F T V D S V E L L L M E E
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