JPH025872A - Novel compound - Google Patents

Abstract

PURPOSE: To produce an endotoxin protein by forming structural gene containing DNA containing a coded part of a specific amino acid sequence and encoded in endotoxin protein having toxic activity on insect.

CONSTITUTION: An expression vector which is a structural gene containing DNA coding endotoxin and coding an amino acid sequence ranging from 1st amino acid position to 205th amino acid position and expressing the gene in Escherichia coli by activating gene under control of DNA is formed. Then, a cell is transformed or transfected with the expression vector and the resultant cell is cultured to produce the objective endotoxin. The transformed or transfected cell includes a plant cell or a bacterial cell.

COPYRIGHT: (C)1990,JPO

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to Bacillus thuringiensis (Bacillus thuringiensis).
41usthuringiei+5-is) Novel mutants of the active portion of notertarxintot'r are provided. The invention also provides mutant strains effective for producing Bacillus thuringiensis endotoxin-like activity in the form of truncated or full chain length delta-endotoxin. In addition, this invention
Mutations are provided that enhance the insecticidal activity of Bacillus thuringiensis delta-endotoxin.

[Prior Art] Bacillus thuringiensis (B, t) is a spore-forming bacterium that produces protein crystal delta-endotoxin (δ-endotoxin) during the late reproductive phase of growth. This δ
- When ingested by some insects, endotoxins produce toxic effects such as cessation of feeding, gastrointestinal disturbances, dehydration, and ultimately lead to light exposure. - Finally, δ-endo1-xin is 130,000 to 140,000 from plasmid source.
Calabrese, Canadian Research Institute of Microbiology (C.
an, J. Microbiol, ), vol. 26, (1
980), p. 1006] 0 Normally, as a result of the action of intestinal proteolytic enzymes, proteolysis f1, which removes approximately half of the terminal end and perhaps a few amino acids at the N-terminus, occurs in insects. This decomposition process occurs in the intestines of (
J 5 (l OO~70 (l OO is necessary to produce an active toxin with a molecular weight of 70 Daltons
Title et al., Journal of Bacteriology (J
, BaetL-I-iology), volume 145 (1
981), p. 10521j.

A number of B, t, subspecies have been identified. Most of these are Lepidoptera (LepidopLe+-a) insects -1 [
However, toxicity has also been demonstrated to insects belonging to a limited number of pond taxa, such as B. t. israelensis (R. t.).

1sraelcnsis) is a member of the order Diptera.
It is toxic to larvae (mosquitoes and mosquitoes), and recently two subspecies of mosquitoes have been added to the order Coleoptera (ColeoI Tsujipi 1-a).
It was confirmed that it is toxic to larvae [Hofte et al., Nucleitsk Acid Research (Nuc, A
ci+J.

Rt・s, ), vol. 15, (17), (1987), 7
183 pages.”

Although much work has been done to produce truncated toxins and their encoding structural genes, the ability of B, t, δ-endotoxin to resist against point mutations within the active or toxic portion of the endotoxin sequence has been investigated. and the effect of such mutations on the activity of toxin molecules remains unknown.

DESCRIPTION OF THE INVENTION The object of this invention is to provide novel mutants of the active part of B, t, δ-entodoxy.

Another object of this invention is to create mutations that are effective in producing B,t, endotoxin-like activity in both the truncated and full length δ-endotoxin forms. .

Another object of the present invention is to provide mutations that enhance the insecticidal activity of the B,t,δ-todoxin structure.

In this invention, we have randomly generated single point and multiple mutations in a large number of DNA strands encoding large fragments of endotoxin active moieties, representative of which have insecticidal activity against Lepidoptera. Several insects of B, t, and δ-endotoxin exhibited the characteristic insecticidal activity against Lepidoptera that wild-type B, t, and δ-endotoxin originally had.
A δ-endotoxin mutant was isolated and produced by recombinant techniques. It has also been found that upon these discovered point mutations, any other naturally occurring amino acid can be replaced with the encoded codon to produce an active endodon protein. It has also been found that a number of arbitrary mutations result in higher levels of insecticidal activity.

Such activity can be observed, for example, in the larva of the giant leaf moth [lleiothisv.
iriscens) or Trichoplusia ni [Trichoprusia ni], and in many cases this increase in activity is at least due to the original endotoxin protein reaching two to several times the activity. It was extremely well written. Multiple mutations (usually 2 or 3 amino acids per mutant DNA strand) were also evaluated individually and in various combinations to identify some of the more effective mutations and combinations thereof. It has also been found that the mutations of this invention reside within amino acid sequence fragments that, with one exception, are highly conserved among a number of wild-type B, t, δ-endotoxins, and are therefore so conserved. It has become clear that the mutations provided by this invention can be broadly applied to a variety of endotoxin structures, such that the region provided with insecticidal B,t, endotoxin.

If we explain in more detail the amino acid sequences in Table A and their number indications [described below, starting with methionine (MET) at position number 1 and ending with the normal N-terminus of representative lepidopteran active endotoxins], other In points.

Like natural n,t, endotoxin protein, it exhibits insecticidal activity against lepidopteran insects.B,t,endotoxin protein has 116 amino acids! In the 2-residue sequence, 9
For the 116 amino acid residue sequences shown from position 0 to position 205 (from +n-1 to m-116 in terms of amino acid positions), the amino acid residue sequence is the same as that shown in Table A or If the active portion contains amino acid residue sequences with substantial homology, it may have one or more of the following amino acid residues shown herein or in equivalent homologous positions (in parentheses): m number is 116 amino P
(i residue sequence not shown): (a) Genetic code encoding any natural amino acid other than Asn at position 94 (position m-5 of 116 amino acid sequence), (b) C at position 95 (11
Genetic code encoding any natural amino acid other than Gin at position m-6 of the 6-amino acid sequence, (C) 10'l
Gl at the position (m-12 position of the 116 amino acid sequence)
Genetic code encoding any natural amino acid other than u,
(d) 105th place T! Genetic code encoding any natural amino acid other than Asn at position (m-16 of the 116-residue sequence), (e) position 116 (m-16 of the 116-residue sequence);
Genetic code encoding any natural amino acid other than Glu at position 27), (f) Gene encoding any natural amino acid other than Ala at position 119 (position m-30 of the 116 residue sequence) code, (g) position 122 (11
Genetic code that encodes any natural amino acid other than Thr at position m-33 of the 6-residue sequence (h) position 123! Genetic code encoding any natural amino acid other than Asn at position (m-34 of the 116-residue sequence), (i) 1
25th place! A at (position m-36 of the '116 residue sequence)! Genetic code encoding any natural amino acid other than Met, (j) Genetic code encoding any natural amino acid other than Met at position 130 (position m-41 of the 116 residue sequence), (k) Genetic code encoding any natural amino acid other than Phe at position 184i (position m-95 of the 116 residue sequence), (1) position 187 (1
Genetic code encoding any natural amino acid other than Ala at position m-98 of the 16-residue sequence), (m) Any natural amino acid other than Thr at position 188 (position m-99 of the 116-residue sequence) Genetic code encoding natural amino acids, (n)
Genetic code encoding any natural amino acid other than Asn at position 194 (position m-105 of the 116'!1 base sequence), (o) position 201 (position m-1 of the 116-residue sequence)
Genetic code that encodes any natural amino acid other than any at position 12).

Furthermore, based on the amino acid residue sequence numbers shown in Table A,
To explain again, this invention is based on amino acid position 4.
of the amino acid Asn by the genetic code encoding any other naturally occurring amino acid.

Regarding the amino acid residues within the 116-residue sequence provided by this invention, this invention provides all mature sequences listed in Table A (
(i.e., its 116 residue sequence), further
Preferably characterized by one or more of the following amino acids at the indicated position: (a) 94
ys at the position (position 5 of the 116 residue sequence), (b)
95th place! (position 6 of the 116-residue sequence) Lys, (
C) L at position 101 (position 12 of the 116 residue sequence)
ys, (d) Tyr at position 105 (position 16 of the 116 residue sequence), (e) Lys or Arg at position 116 (position 27 of the 116 residue sequence), - layer preferably A
rg, (f) at position 119 (position 30 of the 116 residue sequence) and Thr (g) at position 122 (position 3 of the 116 residue sequence).
I le at position 3), (h) position 123 (116
Tyr at position 34 (W) of the residue sequence, (i) Val at position 125 (position 36 of the 116 residue sequence), (j>13
Ile at position 0 (position 41 of the 116 residue sequence), (
k) I at position 184 (position 1 of 95 of the 116 residue sequence)
le, (1) Thr at position 187 (position 98 T! of 116 residue sequence), (m) position 188! (5erun at position 99 of 116 residue sequence) W at position 194 (116
Lys at position 105 of the residue sequence) and (o)20
Asp0 at position 1 (position 112 of the 116 residue sequence)
When changing amino 1Asn at position 4, Ty+-
It is preferable to change to

Table A, listed near the end of the specification, shows natural B,t.

Figure 2 shows the nucleotide sequence and amino acid sequence deduced therefrom related to the δ-endotoxin product. With one exception, this nucleotide sequence is B,
T. wuhanensis (B. t. wuhanensis)
It was obtained from the δ-endotoxin producing plasmid discovered in ). In particular, the complete structural gene (which actually encoded endotoxin itself) is B.

T. Uhanensis, with one exception, the sequence preceding the methionine (Met) at the beginning of the structural gene is B.
T, kurstaki (B, t, kurstaki)
HD-1 (so-called HD-1 5.3K b Hindu
It is derived from the endotoxin-producing gene discovered in the primary plasmid), and the upstream sequence includes B, t,
Contains the natural liposome binding site (RBS) from the Kurstachyendotoxin HD-1 production plasmid. B
The upstream sequence, which contains the natural liposome binding site as found in B. t. kurstakii, is slightly different from that of B. t. The nucleotide and amino acid sequences of the B, t, uhanensis and B, t, kurstaki HD-1t14 synthetic genes are identical from the beginning of the endotoxin sequence to the entire active portion up to at least the Kpn 1 site shown in Table A. There it is,
This Kpn 1 site is a protoxin moiety. Therefore, the active toxin moieties generated by cleavage after ingestion by insects are
In Table A, the amino acids of the endotoxin protein produced by the expression of the tilf 3n gene are listed in parentheses with positive numbers from 1 to 1181 below the amino acids. It was shown to.

Amino acids contained in the untranslated region upstream of 1-Met are shown in negative numbers (counting by amino acid position) in the opposite direction, i.e. upstream, and the nucleotides in the structure 3nl are aligned. Number the top of the column (
(without parentheses), with the last digit of the number directly above the nucleotide corresponding to that number. Nucleotides in the untranslated region containing the liposome binding site are counted backwards from the start ATG codon (1-Met) and are indicated by negative numbers. Some of the above numbered sequences are used to illustrate in detail the highly conserved regions in which most of the mutations provided by this invention are found to be localized. In addition, two or three fl+l+ restriction enzyme cleavage sites related to the nucleotide sequences in Table A, where the amino acids are numbered 11-1 to m-116 and the nucleotides corresponding to the amino acids are numbered n-1 to n-348. is indicated by a line above the nucleotide contained in the restriction enzyme site.

The specific parts are indicated with footnotes.

The art-recognized toxic portion of Endoxy〉' shown in Table A contains the amino acid sequence starting at amino acid position 1 (Met) and spanning amino acid position '1610 (Th r ). In view of the nature of the entire DNA sequence presented, and to make the following description easier to understand, the Kpn sequence starting in the untranslated portion of column 1 of Table A at approximately amino acid positions 724-725 is used herein.
B, t, Kurstaki HD-1 (5,3K
b) It can be cited as derived from the IindI class 1 lasmid), so it will be cited as such.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the truncated B,t endotoxin derived from Fl,t, Kurstaki HD-1, used in the mild stage related to this invention. encrypted D
Figure 3 is a map of the hybrid plasmids prAK and p1-AK-3 containing NA and having insecticidal activity against Lepidoptera.

Figure 2 shows DNA that encodes B, t, and endotoxin protein, which has a slightly longer chain length than that encoding p r A K, and contains DNA that encodes the endotoxin protein, as well as Fl, t, Kuru, and Stachy HD − 1 to 26 led to Lepidoptera "C"
A map of the plasmid p B 8 r It with insecticidal activity is shown.

Figures 3a and 3b show two representative examples that were synthesized to perform the desired B, t, endotoxin DNA coding sequence and are useful for conveniently introducing desired mutations into the endotoxin DNA coding sequence. This shows the front of the double-stacked DNA.

Part 1 shows a map of the 1 lasmid p, B T 210, which contains DNA encoding a full-length natural endotoxin derived from B. t. B, t, Kurstaki H encoded in p r A K and pB8rl+
It has the same amino acid sequence as the endotoxin derived from r)-1 in its active portion.

FIG. 5 is an abbreviated map of plasmid prAK-9 together with an enlarged view of its partial fragment;
9 is otherwise identical in detail to plasmid prAK-3, but this section and further subsections of the circular plasmid are useful for performing codon spin experiments or for mutations provided by this invention. Useful for introduction.

1 Lasmid prAK was published in the Agricultural Research Culture Collection (February 19, 1988).
Agricultural Re5earch Cu1
tureCollec, tion), (NRRL), Peoria (eeoria), Illinois (llinoi)
Escherichia coli (E. coli)
JM 103, preservation number NRRL B-
It was accepted in 18329.

Plasmid pB8rll was deposited on February 19, 1988 at the Agricultural Research Culture Collection (NRRL), Peoria, Illinois, and stored in Escherichia coli JM103, with storage number NRRL B-18331. was accepted.

Plasmid pBT210 was deposited with the National Agricultural Collection (NRRL), Veolia, Illinois on February 19, 1988 and stored in Escherichia coli JM103.
It was accepted under archive number NRRL B-1833077).

Essentially, the point mutations of this invention can be applied to endotoxin protein sequences produced by Bacillus thuringiensis sp.
Natural full-length or substantially full-length protein endotoxin sequences, and downstream protoxins or inactive portions thereof, as long as they are conserved sequences consisting of amino acids or sequences that are highly homologous or essentially equivalent thereto. sequences that are truncated by removing all or part of the normal C-terminus upstream and up to the active region), even when containing the truncated sequences Insecticidal active against larvae. P! As is clear from 1, endotoxins derived from B, t, kurstaki and [3,t uhane] have the same 116 amino acid (Tf) spectrum, and other endotoxins also have the same The 116 amino acid sequence seems to be extremely homologous to the gold-containing isogi, or can be expected to have 9 such as Bt, soft (B,t).

s+:+ttu), B, t Kurstaki H
D, -73 Co., Ltd. and B, t Galleriae (B, T
, Galleriae) have an identical 116 amino acid sequence in some of them, at least to some extent and in extreme cases even though they differ both in the remainder of the toxic part of the entodoxy and in the fragment of the tox. It is already known to produce endotoxin.

B, t, , Kurstaki) (D -1 dibel (D
ipel) (commercial substrains), there is a 1 amino acid change in the above 116 amino acid sequence (m ~ 59 is encoded by T T U and Leu).
and other changes/deletions/additions in other sequence parts. Even in the case of this stock and that pond stock;
In strains that are known to have at least about 70% amino acid homology to the 116 amino acid sequence, even if they have multiple changes, especially if the amino acids to be changed are If we '4f' 32, preferably 4, of the other amino acids that match and correspond to the 116 amino acid sequence, we will create an amino acid within the sequence that matches and corresponds to the amino acids in said 116 amino acid non-mutated sequence. One or more of the mutant changes of the invention may be made (9"). The mutations of the invention may also primarily result in deletions or additions such that the sequence itself is shorter or longer than the 116 amino acid sequence shown. In such cases, deletions that occur in the sequence to be changed are counted as if they actually existed, and Additions to the sequence to be changed are simply not counted, keeping the numbers used for the 116 amino acid sequence +l as a reference.Thus, the amino acid configuration hlm is used to represent deletions or additions in such sequences. It can be said that it should be performed regardless of addition.

The homologous amino acid sequence that can be directly replaced with the mutant change of this invention preferably has amino acids corresponding to the amino acids of the 116 amino acid sequence in which the homologous sequence to be mutated has nine amino acids, and the corresponding amino acids in the reference sequence. encoded by the same codon (n-1 in Table A, if encoded)
(either the sense or antisense strand or two strands of DNA starting at position n-348)
is a sequence encoded by DNA that hybridizes under stringent hybridization conditions.

Methods for preparing such linked hybridization probes are known in the art. Conditions for tight hybridization were 2.5X saline citric acid <5SC) tyJ
11, which was diluted so as not to affect the resulting hybridization.
This is carried out by rinsing with Sakuragai solution at 37°C.

Preferably against a 116 amino acid reference sequence.

Strain S nosa mutations are performed on amino acid sequences η11 with no more than 1.2 or 3 7' amino acid differences, and most preferably on sequences that are identical to the reference sequence.

It is clear in the art that the 116 amino acid reference sequence may substantially modify or form part of a protein sequence that differs in one respect from the Endoxy protein sequence having insecticidal activity against Lepidoptera larvae. For other modifications outside the reference sequence, please refer to j
VO: It is supposed to be revealed as the technical knowledge acquired. Therefore, the sequences bordering the 116 amino acid reference portion and the necessary mutated sequence portions may be varied to a considerable extent to be sufficient to provide an insecticidal endotoxin protein that can be demonstrated by its insecticidal activity against, for example, the larvae of the giant thorn moth. That's all you need.

The amino acid sequence upstream of the mutated portion may therefore be shortened or lengthened compared to the sequence shown in Table A, or may itself be mutated, but generally begins with methionene, preferably starting with methionene, and optionally It is clear that position 4 may contain the preferred mutations provided by this invention, but is most preferably highly homologous (7096) or identical to the sequence shown in Table A. Similarly, the portion of the sequence downstream of the required mutations may be widely varied and may be shortened or lengthened compared to the remainder of the sequence shown in Table A up to the point of cleavage in the insect intestine; Upon cleavage in the intestine, it may be further elongated to form a protoxin or an inactive portion that provides the insecticidal active protein toxin, or may not be elongated.

DNA containing a sequence encoding a mutant endotoxin provided by this invention is introduced into a plasmid under the control of suitable regulatory sequences and transformed or transfected into cells to form an expression vector that produces endotoxin. do.

In contrast, endotoxin production by recombinant biotechnology techniques, such as those used to produce drugs with such techniques, may involve lysis of cells that have produced endotoxin, with little or no treatment intended to produce endotoxin. , which simply uses the entire contents of a culture or fermentation system used in the past to produce the desired product, after drying by conventional means such as low temperature spray drying known in the B.t. endoxy industry. Depending on the product, there are various known methods to improve product stability! lIJIrl may be added to provide sufficient .IIJIrl to the fermentation system. If Jl does not exist,
Again, protein vA materials such as soy n powder or defatted soy powder can be added independently to improve stability, as is also known.

Although endotoxin can be produced biotechnically in a variety of transformed or transfected cell lines, it is generally preferred to transform or transfect bacterial cells of either the Durham-negative or Durham-positive type. One preferred type of Durham-negative Il bacterium is the one for which considerable biotechnological experience has already been achieved, and for which it is suitable and operationally II! A large number of Escherichia coli functional 1 lasmid and transfer expression vector systems are well known and available. Pseudomonas fluoresceae
・eseudomouas rluorI-sce
ns) represents another type of granonegative bacteria into which a plasmid carrying an endotoxin sequence has been introduced. As already mentioned, the endotoxin-producing gene is expressed in B. t. In particular, regulatory sequences, such as binding site sequences, can be found, especially those with their origin in the livonoids. This fact is particularly within the scope of this invention, since Bacillus m bacteria provides a more natural environment for the mutant endotoxins of this invention (1), thereby encoding the mutant endotoxins of this invention. DNA
It is expected that Bacillus-type bacteria can be transformed or transfected with an expression vector containing . Examples of such Bacillus cells of particular interest include Bat. cell,
B. cereus #H vesicles and B. subtilis (B. sul+tilts)
) Cells can be given.

Bacillus cells, especially B. t. A significantly improved method of transforming cells has recently been discovered and is reported in currently pending UK Patent Application No. 8,729,726. B. t. Kurstaki Fly (Lt. ku)
rstaki cry) B-cell-like fly-minus type and native B. cerevisiae harboring an endo-1 hexine-producing 1 lasmid. t. A wild-type Bacillus cell like B. Plasmids suitable for introduction into Bacillus cells such as tJII cells include, for example, plasmid pBc16, which can be made to carry the mutant endotoxin coding sequence of the invention using or modified by conventional recombinant techniques. .1 [Klett et al., Molecular and General Genetics (Mulec)
．． Gen. Genet. ), 1978, 162
Vol., p. 59] and its parent plasmid are known.

As is known in the art, Bacillus cells characteristically produce the desired amount of endotoxin only during the sporulation phase and, therefore, the growth into which they best obtain products useful as insecticides. It will be done. Accordingly, a plasmid or expression vector provided by the present invention and carrying a mutated endotoxin DNA may lack an endotoxin-producing plasmid, or may already contain one or more such plasmids. Although the mutant endotoxin of this invention, which may be introduced into any Bacillus cell containing the mutant endotoxin, has characteristic activity against Lepidoptera, the endotoxin activity against other classes of insects is similar to that of the unmutated parent. In any case, whether it is present in endodoxy or can be obtained as a result of another permissible sequence variation, endo1-xin provides a broader range of insecticidal activity during sporulation. Even in the case of cells carrying a plasmid encoding an endotoxin that is substantially ineffective against Lepidoptera, the scope of the invention includes transforming Bacillus cells with the Lepidoptera-toxic endotoxin-producing plasmid of this invention. For example, a plasmid carrying a mutated endotoxin coding sequence is virtually ineffective against Lepidoptera.

israeliensis (B, t, 1sraeliens
is) or B, t, tenebrionis (B, t, te
nebrionis).

Although this invention has described truncated and full-length native endotoxin proteins, it is even more desirable to use or produce full-length sequences when using mutant endotoxins directly as insecticides, as described above. Preferably, such a full-length sequence has at least 6 folding abilities similar to that of the native endotoxin protein or is the same as the native form in a laziness that achieves an improved full-length folding effect. or can closely resemble it.

The mutant DNA of this invention can also be inserted into the iris genome. In such cases, any suitable method may be conveniently used to insert the endotoxin sequence into the host plant genome, although full length sequences are also preferred, but truncated mutant sequences are preferred; For example, Agrobacterium tumefaciens (Agroba
cte+-ium tumefac-ens)
via plasmid, electroporation, electro) run transformation, microinjection, viral transfection or free D
Methods such as the use of chemicals that induce or increase NA uptake may be employed. Such methods and their use in the transformation of plants are well known to those skilled in the art. Preferably, the DNA sequence encoding the mutant endotoxin can be inserted in its entirety into an expression vector, including suitable regulatory sequences, such as an operator and three regulatory sequences. Combine with. After transformation of such plant cells, the mutant endotoxin DNA is transmitted from one generation to the next through mitosis and meiosis by regeneration into a whole plant with +J cell propagation, and through expression. It can be a stable and permanent part of the plant genome that can generate insect toxic proteins and confer genetic insect resistance to VL products.

In order to provide a source of B, t, δ-endotoxin sequences for mutagenesis and a carrier for the production and evaluation of B, t, endo I toxin mutants, two A very similar vector (prA
K and prAK-3) were used. Plasmid prA
For K and prAK-3, prA
The plasmids prAK and prA, ni-3 are essentially fully competent expression vectors for Escherichia coli. and have origins of replication and operator sequences, including the ampicillin resistance gene and the Escherichia cot promoter, respectively. Wild type B, t in proper reading frame coordination with the promoter.
, contains the DNA sequence found in Kurstaki strain HD-1. The dark bold line part is the amino site H1 (M
et> to 610 IjlH (T h r ) and further extended to 10 toxin moieties ending at amino acid 723 (Leu), t
Represents the mature sequence truncated to encode δ-entodoxy〉'. The small part indicated by a square frame surrounded by a thick line at the downstream end of the thick line in Figure 1 is 723-Leu.
This sequence, consisting of a total length of 57 base pairs (including the stop signal), represents a short DNA sequence of 54 base pairs that immediately leads to a stop signal, followed by a triple base pair encoding ρr A K. It originates from the well-known plasmid pBR322 used. Thus, the expression vector 1) I-A K is a truncated B, t, with essentially a total of 741 amino acids, consisting of the 610 amino acids of the native protoxin portion and 18 amino acids derived from pBR322. A δ-endo1-xy>' fusion protein is encoded and produced in Escherichia coli. B, t with this tip cut off.

The endotoxin fusion protein has a high insecticidal activity of 2 (1) and was used for the purpose of evaluating its mutants produced in the research of this invention.
Completely truncated B, t, δ containing only 610 amino acids of 1 to 610 amines 111F)
- could not be distinguished from the endotoxin protein.

The truncated B,t, dark bold line that represents most of the sequence encoding the endotoxin fusion protein is its upstream end in Figure 1, which contains the B,t, liposome binding site (RBS). It is concatenated with a filled square box representing the array. This part (first and second columns of Table A) contains RBS.

It has approximately 47 nucleotides in front of its upstream terminal origin starting with the Ban H1 site (inserted into the preceding plasmid to link this portion to a portion of the Escherichia coli promoter (indicated by the arrow in FIG. 1)). The promoter and R83 portion are arranged and linked in suitable reading frame cooperation with the endotoxin coding sequence. Therefore, both the dark thick line part and the two boxes are B, t
, represents DNA originating from C. kurstaki HD-1.

A number of other restriction enzyme sites shown in FIG. 1 are relevant to conventional strategies for removing and reinserting DNA segments for mutation experiments. These other restriction enzyme sites are the Nsi site (starting approximately 26 nucleotides after the origin of the endotoxin coding sequence), the two Xba1 sites (see below, but related to prAK-3), the Sst1 site, and the Hindl1 site. .

As shown above, prAK-3 differs from prAK in only one very small respect.

This difference is t! 292 in Table A using Aat-like techniques
Xba 1 site (TC”) at nucleotide position ~297
! 'AGA)': TCG changed to CGA, thereby defining the Nru1 site. This change results in no change in the encoded amino acid sequence, p.
The production of rAK-3 is reported in step (a) of the performed PAl. In addition, ρrAK-3 is derived from other plasmids prAK
-7 and the first intermediate in producing prAK-9.

Conventional methods such as Crake [Biotechniques (1)
1iotechniques), January/February issue (198
5 years), pp. 12-19, [Use of Oligonucleotides for Sight-Specific Mutagenesis]
s forSite-3specific Mutage
prAK and prAK-3 were derived from single-stranded DNA fragments of different strand length (including both sense and antisense strands) mutated as described in For convenience, the DNA section
Denoted as M-1 and M-2, M-1 is prAK 2
375 base pair intercept between Xba1 sites, M-2 is ρr
It is defined as the section between the Ba...H+ and Xba 1 sites of AK-3 (see Figure 1).

The mutations of this invention found between the two Xba 1 sites can be found in the plasmids prAK-7, disclosed in this document.
prAK-8 or prAK-9 and Example 2
It can be easily obtained using synthetic double-stranded oligonucleotides assembled in a manner similar to that described in .

After mutation, in the case of the 0M-1 mutant, the mutated single-stranded portion is made double-stranded by normal means, prAK is used as a mutation propagator, and the resulting double-stranded mutant plasmid is transformed into Escherichia Cori, JM] 03'\Introduced, 5
YT agar containing 0 μg/ml Ambicily was inoculated onto YT agar containing 3 and incubated overnight.
Groups of colonies were obtained that had a number of different mutations in their plasmids that were identical to K.

For the M-2 mutation, the mutated double-stranded region between the B) m old and XL+a 1 sites of the mutant propagator was isolated using B) m1 and Each was cut and ligated into the prAK-3 vector digested with the same two enzymes. The prAK-3 plasmid containing the resulting M-2 region mutant was introduced into Escherichia coli JM103, and 50J
After inoculating onto YT agar containing 1.g/ml ampicillin and incubating overnight, another group of colonies containing a number of different mutations were obtained.

Tests to demonstrate the activity of mutated endotoxin sequences include, for example, Escherichia coli J M 10
3 or Escherichia coli SG4044 [this bacterium is available from the Agricultural Research Culture Collection (NRRL, ), Veolia, Illinois under stock number B-15969]. and Tobacco budworm, as reported in Example A.
T B W ) test or Trichobrusia bis [T,
ni, Nettle Kinuwaba] tests were performed using one or both of the following tests.

The DNA of the mutant that showed increased activity over the standard non-mutant endotoxin was sequenced in the mutation-associated region to determine mutations in the DNA and associated protein sequence. In this way M-1 or M-
Over 6000 different colonies with plasmids containing either of the mutated fragments of 2 were evaluated, and each mutation experiment uniquely resulted in 1 to 3 amino acid changes.

Table B below shows the amino acid positions where each mutation occurred by comparing the up-mutants directly recovered as a result of the mutation experiment with the position numbers returned in Table A.
The codons mutated in each variant and the amino acid changes that occurred at the indicated positions as a result of the codon changes are identified.

In Table B, minus or negative amino acid position numbers are:
B) Shows changes between the m1l1 site and the Met at position 1, which is the origin of the endodoxy>sequence; therefore, such changes do not affect the endotoxin sequence encoded by the mutant sequence. No.

The mutants identified in Table B were tested in all three phases of the TBW assay and the results obtained are reported in Table B-1 below. These various mutants were also evaluated by Tni test, and the results are shown in Table B-2 below.

[Table B] Mutation M-1p26-3 M~1 48a14 48c5 M-■ 36a65 95a76 p95a8 et al. 98c1 1) 99C62 ``'2 1) +07C22 11-2p107c25 M -2pl +4a30 Amino acid end)~Change in xin region n9 +01 n6 +25 +23 OCA-ACA ATG -ATA GGC-GAC GAA →AAA GAG -AAG CGT→CAT GAG -AAG GCG -ACG ACT→A゛口” GCA -GTA T-TAT ACT→TCT ACT→TC TM Yu → ACT AAT -TAT AAT →TAT AAT -AAA AAC-AAA m→A゛口口'Ci -TAG CAA -IAAA TAT -TAA Ala-Thr Yazuet → l1e G1y→Asp Glu→Lys Glu -Lys Arg -* His Glu→Lys Ala -”Thr Thr” 1le Ala -Val 11→Tyr Tln--→Ser η+r -5er Tbr -Tln- Asn→Tyr Asn -Tyr ksn-Lys 11→Lys Phe-→1ie Gin→Ly s In the toxicity column of Table B-1 in section F, the lower the toxicity score, the higher the activity level (see explanation of toxicity score in Example A).The control was Escherichia that was not transformed with the plasmid. - Contains equal amounts of E. coli JM10311 and Escherichia coli SG4044m cells.

It is noted that all mutants exhibit substantially higher activity than the truncated native endotoxin produced by an equal amount of cells containing plasmid prAK.

[Person B-13 Mutant Ill shown in Table B 26-3 1) 48 a 14 48c5 p 3 () a 65 p 95 a 76 95a86 ) 1198c 1 99c62 Toxicity score (average toxicity) 2.75 2.25 2.63 1.50 2.50 1.30 1.33 1.83 1) 107 c 22 p 1 14 a 30 Control (JM103 cells) ρr A K vs. ff1 (SG40444a cells) [Table B-2] Table B Mutants shown in Table B-2 above, the LD of standard pB7301
,. The activity ratio was set to 100 (-layer complete description is given in Example A).
), therefore any value of activity ratio higher than this indicates increased toxicity caused by the mutation.

The invention further analyzed certain mutant DNA containing multiple mutations as shown in Table B above and determined the effects of individual and paired mutations on such multiple mutated segments. .

Subcloning of such individual mutations and paired mutations was performed using a strategy that involved isolating targeted multiple mutant fragments and then cutting at restriction enzyme sites internal to the fragments to determine the mutated We located it between two codons. The two halves, ie the two fragment segments, are then gel isolated and combined with PrA.
The same fragments from K were each mixed with the complementary half of a non-mutant, ie, fragment segment, obtained by cutting in a similar manner. The prAK vector is then cut to isolate the large complementary fragment, this vector is mixed with the complementary halves of the two above, and a total of three DNA segments are ligated together to create a single point mutation. Modified prAK lasmids containing the mutations present in half of the fragments derived from the body or multiple mutant clones were generated. More specifically, the restriction enzyme endonuclease Xho
The sites for l were strategically limited to certain multiple mutated segments. It is clear that the use of the endonuclease Will.

Therefore, the multiple mutant plasmid was first treated with the restriction endonucleases N5il and 5stl, and the resulting 1430 base pair fragment was separated by gel isolation. Such fragments of 1430 bp each were then treated with restriction endonuclease X110 m and the resulting 3
30 bP (Nsil/Xhol) and 1100 b
Fragments of p(Xhol/5stl) were gel isolated for each multiple mutant. Then 330bp and 110
One non-mutant fragment of 0 bp and a larger N5i1/Sst l fragment of prAK of approximately 4 Kb were similarly obtained. More specifically, the thicker segment, the t), was mixed with the mutated 3301)p fragment and the 1100b p fragment of non-mutated prAK, and the resulting mixture was ligated to form p+-A. A series of hybrid mutant 'A Ilk grasmids were created. In a similar manner to this larger pl-8 segment busy and non-sudden r411rAK 300
1) I) 11 (1) with 7 ItI pieces and 1 mutant
0 bpHJi pieces and ligated these DNAs to create another series of hybrid 'JS: u bodies 1
Created a lasmid. The hybrid mutant V4 body obtained from the above strategy is summarized in Table C of F. is compared with plasmid prAK.

r Table C] Newly created 1) r A K λz 1 vector segment and source of (++00 bp) of Source mutation amino acid position prAKNsil/p48a14 prAKS
st+ <4 b) Glu-4Lys (101) Glu-kLys (116) B p26-3 prAK
Ala −= Thr (119)Cp48c5
prAK Glu-1Lys (116)D
prAK p48a14 Ar
g-His (217)EprAKp26-3het
-1ie(130)Gly -Asp (20+) F prAK p48c5
Ala −= Tolr (187) A second hybrid mutant cloning scheme involving one more round of methods similar to those applied for the cloning of Table 0 resulted from digestion with p rAK and N5il and 5stl. The large segment l- (approximately 4 Kb), 330 bρ obtained from one of the clones in Table B, Nsi I/
A series of mixed combination mutant plasmids were generated by three segment combinations consisting of the Xholl fragment and the 1100 bP Xho [1/Sst I fragment obtained from another clone in Table B. The hybrid mutant clone population obtained from this second experiment is shown in the table below.From this second design, two plasmids containing four amino acid changes compared to the parent pr'AK plasmid were generated. It turns out that it happened.

[Table D] New 1 year vector N5il/Kari υ■
/Created segment X1n11 fragment 5stl fragment prAK-J prAK Nsth p48a14 p
26-3/Sst I 4Kb Glu-4Lys (101) Glu →Lys (116) Met -11e (130) Gly →Asp (201) prAK-K p48a14 p48c5 Glu -+ Lys (101) Glu -Lys (116) Ala -p Thr (1B7) prAK-L 48c5 Glu -Lys (n6) Met -11e (130) Gly →Asp (201) prAK-M p48a14 Ala →Thr (119) Arg
-48is (217) [Continued] prAK-N prAK-0 p+-AK-P prAK-Q prAK-R prAK-3 pl・AK-T prAK-U prAK-53 prAK-68 348C5 48c5 48a14 48c5 ri48a14 99c62 99c62 48c5 Ala = ηlr (119) Ala −-Thr (187) 48a14 36a65 Glu −= Lys (116) Arg-IH
is (217) Ala-ηlr (119) η11-→Ile (122) Ala → Val (+25) 36a65 Glu -Lys (116) η1r→lie (+22> Ala → Val (125) 36a65 Glu-pLys (101) Glu → ys (n6) T hr -11e (122) Ala = Val (125) 95a86 Ala -Thr (n9) Tltr -p Ser (188) 95a86 Glu -Lys (116) Thr -+ Ser (lE18) 95a86 Glu - Lys (101) Glu -Lys (n6+ Thr -Ser (188) p107c25 Asn-1Tyr (105) Ph
e →lie (+84) Asn -Tyr (105) Shaishichi → Ile (130) Gly-4Asp (201) [Table 1] continued prAK-70n p26-3 p107c25 Ala -Thr
(119) Phe →lle (184) 1+rAK-39 II p99c62 p98cl ASI
I -Tyr (105) [Table E] A number of the hybrid mutants shown in Tables 1 and D were evaluated in the bollworm larva (T B W ) assay of Example A, especially the first mutation in Table B. woody from the body 1
Alternatively, the mutant clones in Table B were added to this for comparison of the effects of deletion or removal of two mutations. These toxicities, i.e., insecticidal activity 1 5:
Reference > 12) Equal amounts of 3M103al cells and 5G4044 cells that were not transformed with the plasmid were included as controls, and most, if not all, mutants were evaluated in both types of E. coli cells, and these It should be noted that the mutant results in the table with reference to both controls are shown as the average of the mutant results expressed from the two types of cells.

Furthermore, this invention demonstrates the possibility of general amino acid substitution at the amino acid position where an amino acid change occurs by initial random DNA mutation, thereby providing a wide variety of novel insecticidal active B, t, and endotoxin proteins. This possibility includes i & the so-called "codon-spin" that changes the selected codon involved in the first mutational change into a codon for another natural amino acid.
It is relatively easy to prove by experiment. These new mutants express endotoxin proteins that have insecticidal activity against bollworm larvae. In order to conduct such studies more efficiently, starting essentially from prAK as described in Example 1, additionally prAK-3,
producing other intermediate plasmids produced sequentially in the order of prAK-4, prAl(-5 and prAK-6;
Plasmid prAK-7 was arrived at to produce a series of unique plasmids of the prAK type. The plasmid pB8rll shown in Figure 2 contains a truncated endotoxin-encoding DNA that is slightly longer than the endotoxin encoded by prAK, and a truncated endotoxin structural gene DNA. This structural gene corresponds in all respects to plasmid prAK, except for a minor modification made to the parent plasmid in its ligation region to the downstream end, except that this structural gene contains Kpn 1 in the native gene, as shown in Figure 2. site is shown, whereas in prAK this site is deleted, resulting in pr
The end of the AK structural gene approximately corresponds to the position of this site. Plasmid prAK-7 is designed to express the same endotoxin amino acid sequence as plasmid prAK, but the modification of its DNA sequence is similar to that of prAK-3.
ru Not only contains one site (, Hind seal = Mst
ll and Bs5H11 sites, and the find site (which was originally present in prAK) is preferably removed. As shown in more detail in Example 1, pr
The order of producing prAK-7 from AK and the addition or deletion of sites at each step are as follows: prAK −+ prAK-3 (adds Nrul) p rAK-3p rAK-4 (adds old ndm) p
r A K-4--p r A K-5 (Mstn
E addition) prAK-5-prAK-6(Bsst(l+
added) prAK-6prAK-7 (original 1lin
dll deleted).

The sites shown above are short 2-terminals that end with nucleotides that represent residues complementary to the restriction site residues that occur within prAK-7 upon cleavage with prAK-7 and two related restriction enzymes. The main DNA fragments are introduced approximately 40 base pairs apart so that they can be effectively generated using an automated DNA synthesizer. Such fragments are therefore easily prAK-
7 (see Example P), Example 1
As illustrated in column 2, prAK-7 within prAK-7
A plasmid can be provided that is capable of expressing an endotoxin protein that is identical to prAK endotoxin except for the amino acid changes encoded by the synthetic fragment substituted with the corresponding fragment of prAK.

Figures 3A and 3B show two short 2-lattice sequences ('P) consistent with the codon substitution l-latency described above to replace the former d-top/Mst■ portion in prAK-7.
The double-stranded fragment shown in Figure 3A, representing the heavy chain DNA fragment, was created by suitably choosing the XXX codons in accordance with the genetic code, so that the tip expressed in the prAK plasmid and the truncated B, t. . Amino acid position 1 of endotoxin
It is designed to combine any amino acid into 16 amino acids. Similarly, the double-stranded fragment shown in Figure 3B has an X
By suitably choosing the XX codon, prAK- Spe I /Nru + position in 7 (approximately 115 base pair fragment that can be created with an automatic DNA synthesizer), Nru1
/1ind1st million and Mst [1/Bs5)I 1
Other double-stranded fragments required for substitution at position 1 and double-stranded fragments designed to encode all amino acids at any mutation points found within these fragments are created by similar standard methods. It is possible to do so. Therefore, Nru+ and Bs5ll of prAK-7
■ At each mutation point between sites, changing or mutating the remaining 18 of the 19 natural amino acids is
This can be easily achieved by using plasmid prAK-7.

To cover all mutation points that favorably spin-engage related codons within a sequence involving 116 amino acids,
As described in Example 1, plasmid prA, i-7
was used to create plasmid prAK-8, which was then ultimately used to generate prAK-9.
Figure 5 shows all relevant restriction enzyme sites ultimately accumulated within prAK-9.
9 is a partially enlarged cross-sectional view. In addition, the Spe 1 site shown in Figure 5 is also prAK
This is a unique site already present in , prAK3, etc. It will also be appreciated that plasmids prAK7, prAK-8 and prAK-9 can be used to introduce multiple mutational changes between any pair of restriction enzyme sites and/or to encode at least one change in the DNA. By constructing B at two or more such positions, a large number of multiple mutation combinations encompassing the original mutation points discovered in this invention can be assembled to produce a variety of novel insecticidal activities. .

t, can produce endotoxin protein.

Also, as is obvious, plasmids prAK7, prAK-
Plasmids such as 8 and prAK, -9 are fully capable of changing any one or more codons present at any position between the pair of restriction enzyme sites provided on these plasmids. It is. If a plasmid such as prAK-4 or prAK-5 contains the desired changed positions, then preferably prAK is Those plasmids may be used after removing some native Hind1 sites or, if such plasmids are not suitable, introducing a pair of restriction enzyme sites used to modify prAK to create prAK-9. by modifying or limiting the right to
It will be appreciated that plasmids of the prAK type may be constructed containing any one or more such positions in the usual manner. Therefore, this invention also. A number of novel plasmids are provided that are useful in producing the mutations and combinations of mutations of this invention and ultimately contain any one or more restriction enzyme sites within prAK-9.

It will also be appreciated that DNA containing any one or more such restriction enzyme site pairs may be modified to contain the desired mutations, either before or after modification including one or more mutations of the present invention. It was excised from the prAK-type plasmid by cutting the corresponding restriction enzyme site found in another plasmid for B, t, endotoxin outside the mutated region, and the excised DNA segment was then cut into a similarly cut DNA fragment. Such other plasmids can be conveniently modified by insertion (ligation by standard means) into another B, t, endotoxin plasmid, or for the purpose of direct insertion into the mutations of this invention. possible.

To insert the mutations provided by this invention into the full length endotoxin coding sequence.

For convenience and availability, a plasmid into which the DNA structural gene of T. uhanensis δ-endotoxin is inserted is used. This plasmid pBT210
As shown in FIG. 4, as indicated by the dark bold line in FIG.
Also, by analogy with Figure 1, the sequence containing the B,t, liposome binding site obtained from B,t, Uhanensis is included together with the structural gene (indicated by the black square box in Figure 4). As shown in Figure 4, the 1 lasmid pBT210 contains the same E. coli promoter system as the prAK plasmid, the E. coli origin of replication (not shown) and the chloramphenicol resistance gene. Competent Escherichia coli expression vector. The arrows in Figure 4 indicate the reading directions of the B, t, and Uhanensis genes under the control of the Escherichia coli promoter and the chloramphenicol resistance gene. B.

t, the liposome binding sequence portion derived from E. coli and the E. coli expression promoter/operator sequence) are very similar to the operon of plasmid prAK, with only insignificant differences.

In particular, the 610 amino acids of the active part of B, t, endotoxin (Table A) and pBr310 extend at least approximately to the Kpn 1 site shown in Figure 4.
The DNA encoding the toxin region is plasmid prA
It is consistent with the corresponding DNA sequence of K. pBT210
B, t, downstream of the DNA region from the Kpn1 site of the structural gene to the end of the structural gene is B, t, Kurstaki HD-1, which was truncated to create prAK.
There are unknown but subtle differences compared to the corresponding parts of the gene. These differences are illustrated by restriction endonuclease mapping. As shown in Table A, plasmid pBT210 encodes the full-length endotoxin and the active portion (at least the prAK clone and B, t, Kurstaki HD- 1 to the amino acids produced up to the Kpn 1 site) are completely homologous,
and the balance of 10 toxin moieties has substantial homology. Finally, the liposome binding site of pBT210 is p
It is identical to that of rAK and has a liposome binding site (RB
The entire DNA extending from one degree before the origin Met (S) upstream through the m81 site and bound to a portion of the promoter is pBT210 and prAK.
) is the same except for two nucleotide changes after the m81 position (CC instead of GT in prAK)
, the three nucleotides (T T T ) found in prAK immediately after two such nucleotides are pBt2
10, these differences simply result from a different ligation strategy that joins the R83 portion from B,t to the Escherichia coli promoter portion via the B) m81 site. Does plasmid pB7210 have any one or more amino acid changes provided by this invention? can be used to produce full-length mutant B, t, δ-endotoxin proteins with For pBT210, the N5i1 site, two Xba1 sites, the Sst 1 site, and the first one appearing downstream (the grouped site is ρrAK) are shown in Figure 4.
(As shown above, the DNA for the two plasmids in this region is identical).

Examples] Example A 1 Trichobrusia ni (T, ni) test was carried out on second instar larvae. Throughout the test period, all larvae were kept in a climatic chamber under standard conditions such as temperature and humidity, and reared with standard artificial food. The test substance was given to the insects along with food, and groups of 20 insects were administered for each concentration of test substance.

After the treatment, the insects were observed for 7 days and after that period the insects' LD
,. was tested. L D so can be calculated from the dose of substance required to cause death in 50% of the test subjects. f
&Final results are shown in the activity ratio below.

Using the above method, the interpretation of the results is such that all activity ratio values higher than the standard indicate higher activity than the standard! I
I-Ds made of leather. can provide the absolute value of Furthermore, a substance with an activity ratio of 1, for example 400, has an activity four times higher than that of a parrot with an activity ratio of 100.

In the above test, the activity ratio was not 100, and tl was plasmid pr.
3T301, this plasmid contains the full-length wild-type δ-endotoxin sequence and two Escherichia coli promoters, each individually linked to pBT.
It is the same as that contained in pBT210, except that it contains the same as that contained in pBT210.

The controls used in the above tests were (a) an Escherichia coli strain that does not contain the CAG629-δ-endotoxin-producing plasmid and does not itself have insecticidal activity [CA Gross, Department of Bacteriology (1) ep31- jment of Bac
terioloi(y), University of Wisc
onsin).

(b) SAN 415 - Commercially available.

Insecticide "available under the trade name JAVELIN".

21) Bollworm larva test (T B W test) TBW
The assay was basically performed using the method reported by Dalmage et al.
Invertebr, Path, ), 18 volumes, 197
1 year), pp. 240-245], prepare 3 1 oz clean plastic soufflé kelp for each sample and add 2nd instar TBW larvae (4 to 5 days old,
Average ff1i1.6gm>1 animal of each was added with the sample. The samples were prepared as described by Dalmage [USDA Techni-c
al Bulletin), No. 1428, pp. 1-5 (1
(976) was mixed with 1.15 ml of feed (containing nutritional powder, vitamin powder, agar, and other ingredients). After allowing it to cool for half an hour, it was distributed evenly among three bait pots. TBWI fish were added to each pot (using a size 00 camel hairbrush) and the lids were tightened. These samples were incubated at 27°C.
The size of the number of groups used for the TBW toxicity assay evaluation corresponding to the 0 weight range placed in an incubator at 50% relative humidity for 4-5 days is shown in the table below.

Group Size Weight Range Average Weight 1.0
1.3 1.5 2.7 2.0 5.5 2.5 11.7 3.0 17.3 3.5 30.8 4.0 50.1 4.5 76.6 5.0 119. 9 6.0 +, 19.8 ~ 1° 9 1.6 ~ 3.1 2.8 ~ 6.3 5.8 ~ 12.3 12.0 ~ 22.2 19.6 ~ 34,2 32. 8 ~52.4 51.1 ~94.3 84.8 ~114.7 11.3.5 ~134.3 >140.0 The "group size" above is directly equivalent to the toxicity score reported here. , thus measuring the larval weight after each test and assigning an equal toxicity score to the group corresponding to the weight range in which it falls.

Hereinafter, unless otherwise specified, all dead larvae were treated as one group size, and the toxicity score was 0. Toxicity scores from all replicates were averaged and the results obtained are reported here. As a general rule, all results are based on at least 30 repetitions.

After the TBW fit into each size range, I opened the tip. These TBWs were weighed until one TBW fell into one of the weight ranges. Sample chips containing a given weight range of TBW were then labeled with the corresponding group size number. These chips were arranged on a shelf in the laboratory in ascending order of number. The weighed sample TBW and the remaining samples were then scored by visually comparing them, and the group numbers were entered in the scoring sheet. Dead larvae were recorded on a scale of 10 and given a score of 0.

Dead larvae that appeared bright pink or liquefied were suspected to have died from causes unrelated to δ-entodoxin. These larvae were scored with a ``1'' and were not included in the results.

p r A K medium W 1 ml standard sample size 3
When divided into individual groups, growth retardation occurred in the middle of the toxicity rating range. This assay was therefore useful in distinguishing between clones with increased toxicity from those with equal or reduced toxicity than their parent non-mutants. The assay produced a dose response that was dependent on the amount of prAK culture added to the assay. Therefore, an increase in the degree of toxicity to TBW was observed as more p r A K-containing bacterial pellets were added to the sample. prA
Dose-response curves for K were useful in assessing the degree to which a clone was more virulent than its parent non-mutant.

The table below contains prAK! This figure shows the dose-response of I bacteria.

Static culture rAK-1 without supplements Toxicity score m1 0.25 Based on the above evaluation, compared to the standard prAK (and the non-mutant plasmid from that pond), its All cultures were assayed using lml culture to determine whether the sample range was more or less active.

'rThe nutritional powder used for the BW test is Duram! I
Mixed at TJl ratio: soybean fine powder 1103.4g, wheat germ 429.4g, wetulin salt mixed ¥292.4g,
Sucrose 164.2g, methyl rosebenzoate 24.6g
g, sorbic acid 14.8 g.

The vitamin powder used for the TBW assay was mixed at the following gram density ratio: 12.0 g of calcium bantothenate;
Nicotinamide 6, Og, Riboflavin 3.0g, Leaf 1
i! 3.0g, thiamine hydrochloride 1.5g, pyridoxine hydrochloride 1.5g, biotin 0.12g, vitamin B+z6.
0g.

The following three "l" BW test screenings (first stage).

(secondary and dilution series) were carried out at each stage of the evaluation and are cited for the purpose of explanation in this specification.

Primary assay: Take 1 ml aliquots from two separate clone cultures incubated for 18 hours and divide into 5 Q ml circles #
Mixed with TBW diet in centrifuge tubes and divided equally into 3 tips (ITBW per tip).

These samples were combined with wild-type p rAK or pBT21O
Transformed cells and controls of untransformed cells prepared in a similar manner were compared.

Second-order Assay Samples that showed increased toxicity to TBW in the second-order assay were screened in the second-order assay. ! YT/
Inoculated into Amp Pros (1 colony per culture), 1
ml sample 101! I prefer the elbow! It was evaluated together with 1ζI. Clones with increased toxicity against TBW were designated as “
The third evaluation was conducted under the title "Possible Top Variants - 'S Body". Samples that similarly reproduced the "superior" phenotype in the 3rd [1] were confirmed as "position-mutated gastric corpus," and a dilution series assay was performed to more accurately measure the improved activity compared to the wild-type parent. carried out.

One-piece series test: for non-mutant assemblies [10
Mutants confirmed to have the 1-phenotype and lyophilized polycystophores containing either the mutant or wild-type toxin (obtained from prAK or pB 7210 clones grown for 18 hours). Screening was performed by dilution series T B W assay (as dry weight cells), IJ! (cells were pelleted before drying), and samples were diluted in three stages (167/1 g, 67 μg, 33 μg).
In g), the final agricultural yield was adjusted to 15 rnl. Proteins obtained from these mutants were conjugated to 5DS-PA.
As a general rule, 75 μg dry weight cells per run should be used for GE and Western (immunoplotting) analysis.
The results of the dilution series assay essentially confirmed the previous assay results and are either not reported herein or presented as average values in the tables herein reporting the biological results.

Example P (General Procedures) In the numbered examples below and other portions of the specification, the following general or standard laboratory procedures are used in the cited portions or where appropriate, unless otherwise specified. there was.

Example P-1 (Storage and Propagation of Bacterial and Phage Strains) Enoerichia coli (E, Co11) strains SG 4.044 and JMI03 were used primarily as 1n in all plasmid constructions. Enerihia coli (E.

Co11) strain JM103 and phages MI8 and MPI9 were obtained from New England Bioraps. These bacterial strains were grown in YT medium (5g/a yeast extract, IOg/Q bactodributon, 5g/a yeast extract, 5g/a yeast extract, IOg/a
C0,). The medium contains 50 mg/ml for growth of cells containing PrΔK or derivatives of this plasmid.
Add f2 ampinoline and 20 mg/m for cells containing PBT2IO or derivatives of this plasmid.
Q of chloramphenicol was added.

Example P-2 (Proliferation and isolation of phage DNA) Production of M13-derived recombinant phage stock and isolation of phage DNA were carried out using previously reported methods [Mesong (1983), Men's in Enzyme Now (Methods Enzymol, Month 01 Volume 20
- This was done using page 78.

Example P-3 (Preparation of synthetic oligonucleotides) Synthetic oligonucleotides were manufactured by Applied Pion Stems (Foster City, Calif.) 380
A-Manufactured by automatic synthesis using a DNA synthesizer.

The purification steps after completion of the cycle are as follows.

Synthetic oligonucleotides were deprotected by adding an equal volume of ammonium hydroxide and incubating overnight at 55°C. After removing ammonium hydroxide by rapid centrifugation and continuous rotation of resuspension in distilled water, urea/polyacrylamide gel electrophoresis (urea/P A, G E
) the oligonucleotide was further purified. To visualize the bands, the gel was placed on a two-layer chromatography plate covered with Saran™ wrap and the oligonucleotides were irradiated with short wavelength ultraviolet light. After cutting the oligonucleotide-containing portion of the gel with a laser blade, the oligonucleotide was extracted from the gel with 0.5M ammonium acetate,
Elution was performed by stirring overnight at 37° C. with 1 mM EDTA (pH 8,0). - After night elution, the gel fragments were pelleted at 6000 rtPM in a JA20 rotor and the supernatant containing the eluted oligonucleotides was taken into a separate tube. Oligonucleotides were concentrated by ethanol precipitation and quantified by absorption at 0D260. 200 pmoles of oligonucleotides were kinased in a 40 μQ reaction mixture of 2 units of T4 polynucleotide kinase and 0.05 mM ATP.

Example P-4 (DNA of Escherichia coli (E, Co11) cells)
A) Escherichia coli (E, Co11) eligible for DNA conversion
) JMI O3 or 5G4044 were prepared according to previously reported general methods and described in Niechen, Chuck, and Huss (1972), Proceedings of the National Academy of Sciences (eroc, Natl. , Acad.

Sci, USA), Vol. 69, pp. 2110-2114]. For site-directed oligonucleotide mutagenesis experiments,
Hetero double-stranded recombinant phage (M13) DNA (60
ng) was added to 0.2 ml of qualified cells and kept at 0°C for 15 minutes. The cells were then kept at 42°C for 2 minutes, and 30 μg of this mixture was added to 3 m YT broth containing 0.7% Bacto agar.
0.21 of a fresh overnight culture of Q (held at 42°C to prevent solidification) and JM103 or 5G4044. Immediately transfer the mixture to YT agar plates (YT broth n.
5% bacto agar) and place the plate (inverted) for 3
Incubate overnight at 7°C.

B) Mutagenesis experiments or other prAK described herein
Alternatively, plasmid DNA (lOOng) obtained in a ligation experiment using pBT210 vector was added to competent cells (JM1
03 or 5G4044) was added and kept at 0°C for 30 minutes. The cells were then kept at 42°C for 2 minutes,
Transferred to water bath. 2μm2-200μg amount to 50μg/
YT containing mQ ampicillin (for prAK-based clones) and YT containing 20 μg/mQ chloramphenicol (for pBT210-based clones) and incubated overnight at 37°C.

Example P-5 (Restriction Enzyme Digestion) All restriction enzymes were purchased from Vetesta Research Lapus (Gethersburg, MD) or Niney England Biolaps. Incubation conditions were according to the manufacturer's instructions.

Example P-6 (Ligation of DNA fragment) Δ) DNA ligation reaction (20μQ) contains 6 (laM Tris-HCQ(1) 117.5), lomM MgC
(!t, 1mM dithiothreital, 50mM-ATP
, 2 OnM DNA ends and 20 units of T4 DNA ligase (Ninny England Biolapse) were included. Incubation was carried out at 14°C for 4 hours.

B) Ligation of three 7-ragments was performed with the fragments present in a 20 μQ reaction volume in a molar ratio of 1:1, each at a concentration of 0.04 pmol.

Incubation was for 4 hours at 16°C if only sticky ends were ligated (eg, Xholl internal site), or overnight if there were blunt ends (eg, FnuD2 site).

New England Biolapse (NEB) T4 ligase was used at a concentration of IO units (NEB standards)/μQ reaction volume.

Example P-7 (DNA sequencing) DNA of the delta endotoxin gene and its derivatives
A-sequencing was performed by the reading method of Heidetzker et al. [Heidetzker, Messing, and Gronenborn (1980), Gene, Vol. 10, pp. 68-73].

Example 1 (vector prAK-3, prAK-4, prA-5
, production of prAK-6 and prAK-7) Step a) p
Production of rAK-3! 11g plasmid pB8rII (illustrated in FIG. 2 and described above) and the well-known M13 cloning vectors MP+8 and MP! The DNA reproduction form obtained from 9 was treated with restriction enzyme B) m[l! (8 units) and Kpnl (10 units).
M MgCQv, lOOμg/mQ Time digested in 100mM buffered saline containing bovine serum albumin for 60 minutes at 37°C. The desired fragments from the resulting DNA mixture were purified by running the total mixture on a 1% preparative agarose gel and separating by size.

Bands were visualized by staining with ethidium bromide and irradiating with long wavelength ultraviolet light. Gel fragment I containing the target DNA was cut, and the DNA was eluted by electrophoresis in a dialysis tube.

B) mtl I /Kpn l fragment from pB8r[l, mpl8 and mpl 9B) mHIKp
60 mM l.Lis.HC4 (pH 7, 5), l OmM MgCf22 to the nl-cleaved and gel-purified vector.
．． 1mM dithiothreitol, 50mM-ATP, 20
14°C in a total volume of 20 liters containing μM-DNA ends.
ligation by incubation with D obtained
Example P- except that the NA and YT plates contained isopropylthiogalactonodide (lPTG) and 5-bromo-4-chloro-3-indolyl-D-galactonodide (X-gal) (both handcrafted from Nogma Chemical Company). 4
Similarly qualified Escherichia coli (EColi) J M
103 was transformed. Recombinant phage (pB8
I) containing the NA insert cleavage from rll) produces clear plaques under these conditions, while M2S and MI9 produce blue plaques, so clear plaques were added to Escherichia coli (lEColi) JM103 (2 m
&), incubated overnight at 37°C, and meshed single-stranded DNA from the phage using Methods Enzymol.
) (1983) Vol. 101, p. 2078. These target clones containing the pL38r11 Yamaki large single-stranded DNA inserter h were confirmed using agarose gel electrophoresis based on their slower migration compared to mpl8 or mpl9. Sequencing of endotoxin DNA-containing portions in these clones using the chain termination method confirmed the presence of endodoquinone DNA, indicating that mpl8 had acquired an antisense strand and mpl9 had acquired a sense strand. It showed that (these) were (taken). Sequence 5'GTCCTT CTA ATCGCG AAA T
A 26-base andysense oligonucleotide containing GG CTT GG 3° was prepared by automatic 1) solid-phase synthesis in a NA synthesizer using T4 polynucleotide kinase and ATP. Maniades et al., Molecular Cloning
ngXI 982) A Laboratory Manual (
The enzyme was kinased as described in the AL Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Recombinant phage mp19.20 containing 0.4 μg of endotoxin antisense strand
mM1 Lis-I-IC (!(pi(7,5), 7 m
M-MgCQt, ImM dithiothreicool,
A total volume of 60 μQ of the mixture containing 50 mM sodium chloride and lOng of 26 base antisense oligonucleotide was heated at 68°C for 15 minutes, then at 37°C for 10 minutes and brought to 0°C. The resulting mutagenized oligoannealed mp19 recombinant DNA-containing mixture was diluted with 0.2mM each
dATP.

dcTI), dTTP and dGTP,
lmM-ATP.

4 units of DNA polymerase Eklenow Fragment (New England Bioraps), 0.5 μg of Escherichia coli (E, Co11) single-stranded binding protein (Pharmacia, Bispower Taway, Ninny Jersey)
and 20 units of T4DNA ligase (New England Bioraps). 1 of the resulting mixture
Incubate for 4 hours at 4°C to polymerize and ligate;
The resulting circular double-stranded heteroduplex DNA was transformed into Escherichia coli (EColi) J MI O3 and plated as described in Example P-1. Twenty individual plaques were then selected and single in DNA isolated from the phage using the method described by Maniatis et al. (supra). The phage DNA from each of the 20 plaques was collected in 50mM) Lys HCl! (pl-18, 0'), 50mM-KCL
Total volume containing 10 mM MgC (h, I Ong of the above antisense 26 base oligonucleotide and 0.2 μg of another circular single-stranded phage I) NA molecules 20
Heat in a bottle at 68°C for 15 minutes and heat to 37°C for 10 minutes. Restriction endonuclease Nr in each product mixture
ul (8 units) was added and incubation at 37°C continued for 1 hour. The mixture was electrophoresed on a 0.8% agarose gel. Approximately 10% of the sample contains DNA that migrates as linear DNA, thereby providing the desired Nru1
The site clone is Escherichia coli (E, Co11
) J M l 03 to ensure purification.

Bawl-11 site and X ba in positive clones
The DNA between one site is excised by simultaneous cleavage with an endonuclease for both sites (after double stranding with ml 3 annealing, oligo sequencing and polymerization as described above for the Nrur oligo), and two identical A large fragment (approximately 5004 base pairs, B of prAK) obtained after simultaneous complete digestion of prAK with restriction enzymes and gel purified.
(about 749 bp between the HI site and the second Xba1 site) and ligation (Example P-6A) L (both of which are carried out by conventional methods) to form plasmid prAK-3.

Step b) Preparation of prΔ−4 Following essentially the entire manufacturing method similar to step a) above, step a
) The single-stranded phage DNA obtained in Table A is nucleotide position number 327 and 35 of the nucleotide sequence in Table A! A sequence designed to introduce a Hind [site between 5 CCACTCTCTAAAGCTTTCTGC
GTAA3° was annealed to a 25 base synthetic antisense nucleotide. Desired Nrul and llin
Positive clones with both dIll sites showed approximately 6.6% cleavage as assessed by nuclease 11indlll.
was identified with a frequency of 50 from prAK, similar to step a).
B) m1 with a new site in the 04 base pair dog fragment
By ligating the 11/Xbal fragment, p
It was used to produce rAK-4.

Step c) Preparation of prAK-5 Following essentially the entire manufacturing method analogous to step a) above, step a)
The single-stranded phage DNA obtained in
ATT8.

was annealed to a 26 base synthetic antisense nucleotide having the following: desired Nrul, HindlI [and Ms
Positive clones with a total of three L11 sites were identified with a frequency of approximately 91% by cleavage evaluation with the restriction enzyme Mst■ and were used for the production of prAK-5.

Step d) Preparation of prAK-6 Following essentially the entire manufacturing method analogous to step a) above, step C
) was converted to single-stranded phage DNA and the sequence 5' GCGGTTGTAAGCGCGCTGTTCA was designed to introduce a Bs5H[1 site between nucleotide positions 406 and 431 of the nucleotide sequence in Table A.
TGTCj.

was annealed to a 26 base synthetic antisense nucleotide having the following: Positive clones with a total of four sites ultimately desired for prAK-7 were identified with a frequency of approximately 12.5% by cleavage evaluation with the enzyme Bs514
AK Step c) Preparation of prA-7 Following essentially the entire manufacturing method similar to step a), the positive-heavy chain Furno DNA obtained in step d) was converted to the nucleotide position of the nucleotide sequence in Table A. Sequence 5 designed to remove the flindl11 site between numbers 1682 and 1707 CCTAAATCTTCCGGA to TAC8G
CTGTA3 was annealed to a 26 base synthetic antisense nucleotide (T).
Recombinant phages I) due to deletion of I) were identified by restriction endonuclease screening with a frequency of approximately 28%. rI from the mutant strain
Two pieces separated by i g111) NA was used in the production of prAK-7.

Development of a prAK-based plasmid in order to completely offset the supply of a plasmid (prAK-9) with a unique and unique restriction site suitable for performing codon spin experiments at a mutation point between two Xba1 sites. was used in the production of 6.

Plasmid pr as shown in steps f) and g) below
The following steps can be followed to produce AK-8 and prAK-9 therefrom.

Step r) Preparation of prAK-8 Following essentially the entire manufacturing method analogous to step a) above, step e
) The single-stranded phage DNA obtained in Table 1 is a sequence designed to introduce a Ba1l restriction site at approximately nucleotide position number 534 of the nucleotide sequence 5' TCCCCCACCTTTTGGCCAAACAC.
It was annealed to a 27 base synthetic antisense oligomer bearing TGAAAC3°. Desired Nrul, Hind
l■, MsL[, Bs5l- (a positive clone lacking ll1ndllT (prA
K-8) was identified by the method of step a) above.

Step g) Preparation of prAK-9 Following essentially the entire manufacturing process analogous to step a) above, step r
The single-stranded Furno DNA obtained in
TCATTATA3 (f) was annealed to a 30 salt J tongue synthetic antisense oligomer. Desired Nrul, l1
indllL MsLII, 13ss II II,
All six Bal I and Mlul restriction sites
A positive clone lacking tlindII, which was removed during formation of 1LprAK-7, was identified by the method of step a) above and named plasmid prAK-9.

As is clear, Ba1l and Mlul of prAK-9
Suitable cassette DNA for substitutions between the mutated amino acid positions !8/I, 187, +88 and 194, and -4' to induce possible codon and amino acid changes. can be encrypted.

In a similar manner, the cassette l-DNA suitable for substitution between the Mlul and the second can be encrypted.

FIG.
FIG. 2 is a diagram of the unique restriction sites that can be introduced between the two original Xba[ sites.

At various stages in Example 2, the underlining of the andysense oligonucleotides shown on the left and right indicates changes or changes to be introduced.

Example 2 Codon Spin Test Δ) Using a DNA synthesizer programmed to randomly insert all of the nucleotides A, T, C and G into each X position of each clay shown in Figure 3A, Example P
Single-stranded oligonucleotides having the sequences of each of the two strands shown in Figures 3A and 3B were prepared as described in Figure 3-3.

The above single strands (corresponding to a family of identical oligonucleotides except for the manufactured by the method. 68 pmoles of sense and antisense 6 strands for each codon spin
It was bonded and annealed in bulk by heating for 10 minutes at 0.degree. C., then 10 minutes at 37.degree. C., cooling to room temperature and placing on water. Matches the DNA shown in Figure 3Δ,
The resulting mass of double-stranded oligomers contains a stop signal at position XXX (amino acid position ll6) and each set is made possible by the genetic code containing multiple but varying numbers of codons for each of the 20 natural amino acids and the stop signal. It was determined that it contained a mixture. Two pmoles of these double-stranded oligomers or cassettes are then phosphorylated using T4 polynucleotide kinase from New England Piolabs and! 00 mmol NaCl, 10 mmol Tris 1-I CI (pH 7,5), 10 mmol M
gCI! , obtained by gel separation (less than 10 times molar The resulting mixture was ligated according to Example P-6(A). Escherichia coli JM1 under the conditions of Example P-4(B) using the resulting ligation mixture
03 was transformed. Some (200) of the resulting transformants were individually subjected to the TBW and T.2 assays (without knowing in advance which codons of XXX are in which particular clones), and all the various different mutants were tested. It was well shown to produce a pesticidally active protein product with an activity at least nearly identical to that of the regulated cleavage endotoxin, and was found to induce activity levels also exhibited by the sharp up-mutant (5x control). Prior to this, random cells from the transformation were selected, plated, and colonies were grown as in Example pt.
Plasmid DNA for DNA sequence analysis was isolated by cloning the [1/Xba fragment into sequencing vectors mp18 and mp19 cut with similar enzymes. The DNA in each region of the 11 above-mentioned position -116 mutations and the identified up mutants is
It was sequenced to determine the amino acid encoded at position 116. It was found that one of the two up mutants was a mutant of the original species (116-Lys1ΔA
(encrypted by A). Other up mutants are CO
It turned out to be 116-Arg encrypted by T. One of the other clones is the natural l l 6
-Glu (encrypted by GAA). Seven of the other mild variants are 116-11e
(ATT), 116Cys (TGC), l l 6-L
eu (CTC), n6Asp (GAT), l I
6-Asn(AAC), 116-11e(A
TT) and r16-Gly (GGA). All characteristics except new 116-11e (ATT) are 2
demonstrated by two clones. The final clone is 116
- Encrypted stop (TGA).

B) For amino acid position -119, the method of part A) of this Example 2 was repeated using the DNA shown in Figure 3B. Furthermore, random evaluation of the large number of resulting clones exhibited activity levels in the TBW and T.2 assays that were well indicative of all mutant clones in the pool being active. (The % of inactive molecules is approximately equal to the frequency of stop codons UAA, UAG and UGA expected from mechanical random insertion of nucleotides at position XXX). Seven randomly selected individual clones were
Although both of them were shown to have an activity level at least equivalent to that of the truncated native sequence endotoxin produced by prAK, there was no Niru-up mutant at group 44.11 of our alpha. Ta. Sequencing of seven individual clones shows that they are all 514 and specific, as shown below. l f 9 -
L, eu(CTA), I19-Tyr(T
AC), I I 9-A5p (GAT), I I 9
-1ris(C,A.

'1゛), l l 9-Pro (CCA), l I 9
-11e (AT'1゛) and l I 9-9er (T
CT).

Example 3 (Bacillus ti, Lingenos entoginno 1 restriction endonuclease + 3)m of Tris IICI (pl + 7.9), 6 mmol of M g C!
18g) was cut into the same pieces. A larger fragment of approximately 7180 base pairs was gel purified for use as a hector. Then A la-”Thr (119th), Met-Ile (130th) and cry-Δ5
18 g of plasmid prAK26-3 containing the p (position 201) mutation was also simultaneously injected with 6 mmol of Tris-HCI (
pH 7,9), 6 mmol MgCl, and 100μ
Cleavage was performed using the restriction endonucleases Ban Hl (8 units) and Sst I (8 units) in 100 mmol sodium chloride buffer containing g/ml bovine serum albumin. A smaller fragment of approximately 1428 base pairs containing the mutation and the BtRI3S portion was gel purified and the 1-3 self fragment obtained with 25 self, 0,
0.02 pmol of the 7180 base pair Hector fragment and 60 mmol of TrisHC1 (
Pt! 7, 9), 10 mmol MgClt,
Combined with 20Q ligation solvent containing 1 mmol nodiothreitol, 50 MATP, and 20 units of T4DNA ligase, and the resulting mixture was incubated for 4 hours at 14 °C.
Incubated with The resulting plasmid designated pBt2B-3 was mixed with 5 μl of the above ligation mixture into 0.2 ml.
of E. coli and transformed into E. coli JM103 by first pulsating at 0° C. for 30 min and then at 42° C. for 2 min before returning to water. Various (2 μm, 20 It I and 180
Immediately spread the resulting filtrate mixture on YT agar plates with 20 gg/ml chloramphenicol and then
The plates were incubated overnight at 37°C. Only transformed cells that are resistant to chloramphenicol can grow on these plates. Flora J, phenicol-resistant colonies were grown in liquid medium overnight at 37°C and plasmid I)
NA was prepared for restriction digestion with IΣcort I reagent and I)N, which actually determines the presence of partial mutations, for 11 determinations. Plasmid p l-3T26-3 *fT'
4-infected transformants were evaluated by 'r13W and T.2 assays.

Another full-length endotokin strain shown below was produced in a manner similar to Example 3 above and evaluated in the 'I' 13 W and T.2 assays. The first table below is
Another full-length female 51+ manufactured! enodotokin-producing plasmids/cell lines and approximate results of their evaluation in the T13W assay and the product of Example 3 above, Bacillus thuringenos uhanensus natural endotoxin produced by Escherichia coli JMI03 and untransformed JMI03 cells. Approximate results of similar assays for controls are shown.

Table 1 S Table F Example 4 1 Another full-length nighty6-body B. thuringiensis endotogycin] Restricting plasmid pfJ'l'210 (lμg) endotoxin Il)m III (8 units) and Ss mouth (
8 units) and the resulting 7180 bp large fragment was gel isolated. In a series of isolation tests, restriction endonucleases r3ata III (8 units) and 5st1 (position 81Ti) were used to isolate each (1 μg) of plasmid prA1 (, prAK-F, p26-3, p36
a65 and p95a86 were simultaneously digested and each resulting 1428 bp fragment consisting of a portion of the endotoxin coding sequence was gel isolated. restriction endonuclease Fnu
D2 (6 mmol NaCl, 6 mmol Tris H
Each amount of these 1428 bp fragments was digested separately using C1 (9h7,4), 6 mmol MgClt, 6 mmol 2-mercaptoethanol, 4 units in 100 μg/ml bovine serum albumin). Restriction endonuclease Fnu D2 (also known as Acc2) produces a variety of 428 bpB) mLl r/S
Cut each of the slo fragments once, 640b
pB) m H[/Fnu D2 fragment and 788b9Fnu D2/5stl fragment. Different fragments from each test side were separated into agarose
Purified by gel chromatography.

Thus, a series of different 640 bp I3) m H1/
Fr+uD2 fragment and a series of different 788b
The pFnu D2/5stl fragment was obtained.

By selecting one fragment from each of these two series and ligating it with the large 718 Obp fragment obtained from pBT210, a large number of new plasmids harboring different full-length mutant B. thuringiensis endotoxin genes were generated. Obtained. By the method of Example P-4(A), as shown in Table 6 below,
The resulting new plasmid was used to transform Essoerychia coli JMI03 and the resulting cells were evaluated for production of B. thuringiensis endotoxin by the TBW assay of Example A.

” The approximate results obtained with the bipedal assay are also shown in Section 6 below.
Reported in table. Various transformants containing plasmids such as those described in Tables F and G were evaluated in T. ni. and the results are shown in Table 1I below.

Table 6 -E -F 10 days prAK p26-3 pru-B prAK Su I + table BTA BTC BT66 pBT107c25 BTP BTS pB↑67 BT106 standard pBT301 control 5AN415 control C AG629 201-A So 30Jle Example 5 Encoding mutant endotoxin sequence Using DNA that transforms
Transformed cells were grown in fermentation beds under known and specified conditions. Immediately at the end point of cell growth and harvest,
The entire fermentation layer was brought to a temperature of about 70-80°C. This temperature was held for 10 minutes before cooling, which is sufficient to inactivate the recombinant microorganism without affecting the biological activity of the endotoxin protein.

The contents of the fermentation layer were evaporated under pressure, reducing the content to one-third of the original amount!
The raw filter concentrate was spray dried using a heated countercurrent air flow at a population temperature of 140-1609 C1 and an outlet temperature of 20-50° C. at an insertion pressure of about 2000 psi. The resulting powder was mixed with a carrier such as defatted soybean to obtain a wet concentrate. The preferred ratio described above depends, for example, on the desired strength of the final product, but may be, for example, an agent to carrier ratio of 6040 parts (by weight). For spores or entodoquinone protein, the resulting concentrate is between 0.4 and 10%
, preferably 0.8 to 8% of active ingredients. For spray applications, wet powders are suitably diluted with water, as is known in the art.

Person A CCA GAA T'TCAcT? M' CCG Cr
A TAT GGA ACT ATOGGA AA?
GCA GIJPro GLu Phe Thr Ph
e Pro Leu? yr Gly Thr Mat
GLy Asn Ala^1aILe Pro Se
r Sat Gin Il* Thr Gin Els
Pro Leu Thr Lys Sat ThrA
A'l' CTT GGCTCT GGA ACT
TIC'! 'GTCGTT AM GGA
CCA GGA TT'gekif ACA Asn Leu GLy Ser Gly Thr S
er Val Val Lys Gly Pro Gl
y Phs ThrGLY S+IIr Leu
Trp Pro Leu s@r ALa
I'rOSat Pro Engineering 1 @ 65y @ L
Iys cys Pn+lI TM Tnr pro Ph@
Asn phe sir Asn GAY
5tlr Sir Vak PH@Tnr 11 (a) f month (++mll l i!l: Samurai (l+)
11 S li+・1,)ζ(Sl(c) is X hr+
i! l jI''/((1) is the X ha I portion (+7
19 ('rhr), 130 (lie) and! , 8
8 (S er) including Norano, the combination is pl'
3T2 G-3, p[('f'-106, pBT-6
8, pl(T-C, p+'3'F-67 and pB
T-70 (some of these are seen in Table 6) consisting of one or more "-" as found in Table 6. In particular, regarding the end-cut enodotkin, the individual increase and combination U-change in p36a65 are also preferable ↓1!
It is.

The 25 amino acids (Table A) that are hypothesized to form the pretoquinone or butoquinone moiety of entotoginone and are stripped away by proteases in the intestine to form the active or more active enotoquinone (Table A). 1111 to 25), the mutation at amino acid position 4 discovered and tolerated by this invention is of interest. Therefore, the mutations allowed at position 1 are suitable for achieving substantial (at least 670%) homology with or to the 25 amino acid endotoquinone sequence, and especially at position 4. Under severe conditions, prior to the mutation of
Equivalent codes for the corresponding amino acids as discussed above in connection with the 116 amino acid sequence region encoded by DNA that hybridizes with A and related to those endokines encoded for the above region. Show that it is appropriate for endotokino.

The mutation revealed by our study at amino acid position 217, listed in Table 13 above, and evaluated as further found throughout this text, indicates that all naturally encoded amino acids are shown in Table A. It has a sequence equivalent to that of the 116 amino acid standard sequence, indicating that it can exist at this position in active endoquinones extending from the end of the standard sequence of 116 amino acids to and including amino acid position 217. Therefore, the condition in which the amino acid at position 2+7 in the above sequence or its equivalent is any naturally encoded amino acid except Arg:
be within the scope of this invention. However, position 21
Since the directed mutation at 7 produces less interesting results, it is of interest in combination with the other mutations described here, and furthermore, position 2+7 suggests that Arg is the naturally occurring endotoxin at this position. It shows that things can change.

[Brief explanation of the drawing]

FIG. 1 is used at various stages related to this invention. Fl, t, truncated B, t derived from Kurstaki 11[)・1, L encoded with endotoxin
This is a map of hybrid plasmids p r A K and p 'AK-3, which contain INA and have insecticidal activity against Lepidoptera. 1 lasmid pB 8 r l which contains DNA encoding the endotoxin protein and is also derived from B, t Kurstaki HD-1 and has insecticidal activity against Lepidoptera. Figures 3a and 3b show maps of B, t, endotoxin D, which were synthesized to carry out the so-called codon...
Two representative dual 111 DNA strands useful for conveniently introducing desired mutations into the NA coding sequence are shown. 41:21 shows a map of plasmid pB7210 containing DNA encoding the full length natural endotoxin from B. t. Uhanensis. Figure 5 is an abbreviated diagram of plasmid n'Δ1ri-9, together with an enlarged view of its partial fragment. Engraving of the drawing (no changes to the content) Figure 1 Special Feature 1.'1 Applicant: Zand・Akdiengesellschaft agent FuFI'l! 1:1 Susamaya Haka 1 person'11
sr 1 Engraving of the drawing (no change to the content) Fig. 2 Engraving of the drawing (no change to the content) Fig. 4 Sr co R1 Engraving of the drawing (no change to the content) Fig. 3A Fig. 1 Oligo No. 3 Bl at G pupil; J 9 points] 5'Hind III Mstl1 6'+J4 58 so

Claims (25)

[Claims] (1) Contains a DNA encoding an endotoxin protein that has toxic activity against insects, and the DNA has a 116 amino acid sequence starting from position m-1 and spanning position m-116 in Table A. contain portions encoding amino acid sequences that exhibit substantial homology, apply that position number to homologous sequences without regard to any deletions or additions therein, and at the indicated amino acid reference position ( a) Any natural amino acid other than Asn at position m-5 (b) Any natural amino acid other than Gln at position m-6 (c) Any natural amino acid other than Glu at position m-12 (d) Any natural amino acid other than Asn at position m-16 (e) Any natural amino acid other than Glu at position m-27 (f) Any natural amino acid other than Ala at position m-30 (g) m- Any natural amino acid other than Thr at position 33 (h) Any natural amino acid other than Asr at position m-34 (i) Any natural amino acid other than Ala at position m-36 (j) Any natural amino acid other than Ala at position m-41 Any natural amino acid other than Met at position (k) Any natural amino acid other than Phe at position m-95 (l) Any natural amino acid other than Ala at position m-98 (m) Any natural amino acid other than Ala at position m-99 any one or more natural amino acids other than Thr; (n) any natural amino acid other than Asn at position m-105; and (o) any natural amino acid other than Gly at position m-112. A structural gene containing modified DNA to encode amino acids. (2) The amino acid sequence encoded by a portion of the structural gene DNA has at least 70 homologs to the 116 amino acid sequences starting from position m-1 and spanning position m-116 shown in Table A. % of claim 1
Structural genes described in section. (3) At the sequence reference positions shown, the DNA contains (a) Lys at position m-5, (b) Lys at position m-6, (c) Lys at position m-12, and (d) Lys at position m-16. Tyr at position (e) Lys or Arg at position m-27 (f) Thr at position m-30 (g) Ile at position m-33 (h) Tyr at position m-34 (i) m- Val at position 36 (j) Ile at position m-41 (k) Ile at position m-95 (l) Thr at position m-98 (m) Ser at position m-99 (n) m- Claims 1 or 2 modified to encode any one or more amino acids consisting of Lys at position 105 and Asp at position (o)m-112. structural genes. (4) The structural gene according to claim 3, wherein the DNA is modified by incorporating a change in one or both of its amino acid reference positions m-27 and m-30. (5) A DNA portion that does not have any of the modifications described in any one of claims 1 to 4 is hybridized under strict hybridization conditions, and the starting point is position n-1 in Table A. 5. The structural gene according to claim 1, which is a 348 nucleotide oligomer having a nucleotide sequence spanning position n-348. (6) Before the modification described in any one of claims 1 to 4, the DNA contains 116 amino acids starting from position m-1 in Table A and spanning position m-116. The structural gene according to claim 5, which encodes the following. (7) The DNA encoding endotoxin is shown in Table A
starting from amino acid position 1 and starting at amino acid position 205
6. The structural gene according to claim 5, which comprises DNA encoding an amino acid sequence spanning. (8) DNA encoding an endotoxin protein having toxic activity against insects, which DNA is
starting from amino acid position 1 and starting at amino acid position 205
A structural gene that has a portion encoding an amino acid sequence showing substantial amino acid homology with a 205-amino acid sequence spanning the 205-amino acid sequence, and in which the amino acid at position 4 is any amino acid other than Asn. (9) The structural gene according to claim 8, wherein the amino acid at position 4 is Tyr. (10) An expression vector containing the structural gene according to any one of claims 1 to 9, which can operate under the control of DNA to cause expression of the structural gene in a bacterial host. (11) The expression vector according to claim 10, which is capable of causing expression of the gene in Escherichia coli by operating the gene under the control of DNA. (12) By activating genes under the control of DNA, Bacillus
11. The expression vector according to claim 10, which is capable of causing expression of the gene in S. thuringiensis. (13) An endotoxin protein having toxic activity against insects, the protein having substantial homology with a 116 amino acid sequence starting from position m-1 and spanning position m-116 in Table A. At the indicated amino acid reference position, (a) m Any natural amino acid other than Asn at position -5 (b) Any natural amino acid other than Gln at position m-6 (c) Any natural amino acid other than Glu at position m-12 (d) Any natural amino acid other than Glu at position m-16 Any natural amino acid other than Asn at position (e) Any natural amino acid other than Glu at position m-27 (f) Any natural amino acid other than Ala at position m-30 (g) Any natural amino acid other than Ala at position m-33 any natural amino acid other than Thr (h) any natural amino acid other than Asn at position m-34 (i) any natural amino acid other than Ala at position m-36 (j) Met at position m-41 (k) Any natural amino acid other than Phe at position m-95 (l) Any natural amino acid other than Ala at position m-98 (m) Any natural amino acid other than Thr at position m-99 Any natural amino acid (n) Any natural amino acid other than Asn at position m-105 (o) Any one or more amino acids consisting of any natural amino acid other than Gly at position m-112. An endotoxin protein containing a sequence part. (14) The endotoxin according to claim 13, wherein the amino acid sequence portion has at least 70% homology with a 116 amino acid sequence starting from position m-1 and spanning m-116 in Table A. protein. (15) At the sequence reference positions shown (a) Lys at position m-5 (b) Lys at position m-6 (c) Lys at position m-12 (d) Tyr at position m-16 (e) Lys or Arg at position m-27 (f) Thr at position m-30 (g) Ile at position m-33 (h) Tyr at position m-34 (i) Position at m-36 Val (j) Ile at m-41 position (k) Ile at m-95 position (l) Thr at m-98 position (m) Ser at m-99 position (n) m-105 position 15. The endotoxin protein according to claim 13, wherein the endotoxin protein encodes one or more amino acids consisting of Lys at position (o) and Asp at position (o)m-112. (16) The endotoxin protein according to claim 15, wherein one or both of the amino acids at reference positions m-27 and m-30 are changed. (17) An endotoxin protein produced from the gene according to claim 8 or 9. (18) An endotoxin protein obtained by transforming or transfecting cells with the expression vector according to any one of claims 8 to 11, and culturing the obtained cells to produce the desired endotoxin. production method. (19) The method according to claim 18, wherein the cells to be transformed or transfected are plant cells. (20) The method according to claim 18, wherein the cell to be transformed or transfected is a cell. (21) A plant containing cells containing the structural gene according to any one of claims 1 to 9. (22) A bacterial cell containing the expression vector according to any one of claims 10 to 12. (23) Having a sequence spanning from nucleotide position n-1 to nucleotide position n-348 in Table A or a mutational change thereof, HindIII, MstII, BsshII, Ba
DN having one or more remotely located restriction enzyme sites selected from the group consisting of l I and Mlu I
A, and these sites do not change the amino acid sequence encoded by those DNA sequences. (24) An insecticidal composition comprising both an insecticidal effective amount of the protein produced from the DNA according to any one of claims 1 to 9 and an agriculturally acceptable carrier. (25) An insecticidal composition comprising both an insecticidal effective amount of the protein according to any one of claims 13 to 17 and an agriculturally acceptable carrier.