WO1992014826A1

WO1992014826A1 - Bacillus thuringiensis-promoter

Info

Publication number: WO1992014826A1
Application number: PCT/EP1992/000250
Authority: WO
Inventors: Martin Geiser
Original assignee: Ciba-Geigy Ag
Priority date: 1991-02-15
Filing date: 1992-02-05
Publication date: 1992-09-03
Also published as: AU1201492A

Abstract

The present invention relates to the sporulation-dependent promoter of the exoproteinase from B. thuringiensis (B.t), including its mutants, variants and homologues which are capable of functioning. It additionally relates to DNA fragments, transgenic cells and vectors which contain said promoter, and to the use of said promoter in regulatory sequences or for the regulation of the expression of homologous or, preferably, heterologous DNA.

Description

Bacillus thuringiensis-promoter

The present invention relates to the sporulation-dependent promoter of the exoproteinase from B . thuringiensis (B.t.), including its mutants, variants and homologues which are capable of functioning. It additionally relates to DNA fragments, transgenic cells and vectors which contain said promoter, and to the use of said promoter in regulatory sequences or for the regulation of the expression of homologous or, preferably, heterologous DNA.

The term promoter normally means a section of DNA at which initiation of transcription of coding sequences, which are normally located downstream therefrom, starts. It is the region to which RNA polymerase binds in order to start the synthesis of RNA from the so-called starting point of transcription. The particular nucleotide sequence of promoters of different genes and organisms may vary widely. It is decisive for the activity of the relevant promoter, for example whether and by what means a promoter can be regulated.

Continuous gene expression is called constitutive gene expression, and intermittent expression is called inducible or repressible. Responsible for inducible and repressible expression is the presence or absence of inducers and repressors, respectively, which interact with the promoters. Possible inducers and repressors are a wide variety of factors, for example physical factors such as heat and cold, inorganic factors such as metal ions, and organic compounds which are produced by the cell itself or come from outside. Of particular interest in this connection are the effects of limiting nutrient concentrations on the start of sporulation of microorganisms. If, for example, amino acids in the culture medium are used up, promoters are activated and are eventually responsible for the expression of completely different genes than previously and for the production of spores by the cells.

The promoters which control the expression of the protoxin genes of B. thuringiensis are sporulation-dependent and are, accordingly, active only during sporulation. Investigations by Rothnie and Geiser (1990) show that the individual protoxin genes are expressed to a different extent and that the relative level of expression in each case differs from strain to strain. The protoxins which, as a whole, are also called δ-endotoxin are of commercial interest because not only are they highly active but they also act very specifically on particular target insects, especially on various Lepidoptera, Coleoptera and Diptera larvae. Besides the high activity and specificity, a further advantage of the use of the protoxins of B.

thuringiensis is that they are harmless for humans and other mammals, as well as for birds, fish or insects, with the exception of the abovementioned target insects. The B.

thuringiensis δ-endotoxin thus represents an insecticide which is virtually ideally tailored by nature for important insect pests in agriculture, horticulture and forestry.

Because of the particular properties of δ-endotoxin, there is great interest in improving the productivity of natural B. thuringiensis strains. Attempts to increase the production of δ-endotoxin in other hosts, such as B. subtilis and B. megaterium, by the use of high copy number plasmids have hitherto failed. Shivakumar et al. (1989) were able to show that the entire process of sporulation is disrupted when additional copies of the protoxin genes are introduced into the cell.

Surprisingly, it has now been possible to solve this problem in a quite different way, by the choice of a suitable sporulation-dependent promoter. It has emerged that the promoter of the exoproteinase from B. thuringiensis represents a sequence which can be regulated and whose activity has its onset in the very early stage of sporulation. It is now possible, by combining this promoter with protoxin genes, to achieve onset of production of δ-endotoxin early and effectively, or in fact, which results in the required increase in productivity or in the expression of cryptic protoxin gene sequences (Dankocsik et al., 1990) in the case of transformed B. thuringiensis strains. It is, of course, also possible to couple, instead of a protoxin gene, any other homologous or heterologous DNA which is capable of expression to this promoter, for example a DNA which encodes a protease inhibitor (see EP 348,348) or a toxin which is produced by animals (see EP 374,753).

A very essential aspect of the present invention is therefore the sporulation-dependent promoter of the exoproteinase from B. thuringiensis, including its mutants, variants and homologues which are capable of functioning. The promoter according to the invention can derive from the DNA from a subspecies of B. thuringiensis, preferably from one of the subspecies comprising kurstaki, berliner, alesti, sotto, tolworthi, dendrolimus, tenebrionis and israelensis, more preferably from the subspecies kurstaki HDI, or else be synthesised. Mutants, variants and homologues of the promoter which are capable of functioning are all natural, synthetic or partially synthetic DNA sequences which differ from the promoter sequence according to the invention by deletions and/or insertions and/or replacement of single or multiple nucleotides but retain its regulatory property.

The nucleotide sequence of a promoter according to the invention of this type is preferably located within the sequence SEQ ID NO:2, and a particularly preferred location is in the region from nucleotide 1 to nucleotide 709, in particular from nucleotide 300 to nucleotide 709 of the sequence SEQ ID NO:2.

The present invention also relates to a DNA fragment which contains the promoter according to the invention, preferably a DNA fragment which is selected with the aid of a suitable DNA probe from a mixture of restriction fragments of the complete DNA of B. thuringiensis. The fragment additionally comprises, if desired, the so-called prosequence and the so-called preprosequence (leader sequence) of the exoprotease.

Restriction fragments of the complete DNA are fragments which are obtained on cutting the complete DNA with the aid of a restriction endonuclease. Suitable according to the invention is a restriction endonuclease from the group comprising so-called six-cutters, such as XbaI, BglII, SpeI and HindIII, preferably XbaI, BglII and HindIII, more preferably HindIII.

A suitable DNA probe for the selection of restriction fragments is to be understood as meaning an oligonucleotide or a mixture of oligonucleotides with a DNA sequence which is homologous with that which is sought, or with one which is in the neighbourhood of that sought. "Homologous" is to be understood as meaning in this connection that homologous nucleotide sequences permit a base-pairing of at least 50 % under the usual hybridisation conditions. Usual hybridisation conditions have been adequately described (for example in Suggs et al., 1981).

Oligonucleotides which are particularly suitable as DNA probe within the scope of the present invention are those which are homologous with the coding sequence of the exoproteinase gene from B. thuringiensis, in particular the oligonucleotide HR5215 (SEQ ID NO:1). Another aspect of the present invention is a DNA fragment which comprises the promoter according to the invention. A DNA fragment is to be understood as meaning within the scope of the present invention a DNA sequence which is obtained by fragmentation of natural DNA, it being possible for the fragmentation to be carried out in various ways, for example by using restriction endonucleases.

The present invention also relates to a synthetic DNA sequence which is homologous with the promoter according to the invention because it is a synthetic equivalent of the promoter according to the invention and is suitable for the use according to the invention.

A synthetic DNA sequence is to be understood as meaning a sequence which is prepared by the usual means of nucleic acid synthesis (for example Applied Biosystems synthesiser, β-cyanoethyl phosphoramidite chemistry) or a natural DNA sequence which is modified by chemical means.

Of particular interest within the scope of the present invention is every recombinant DNA comprising the promoter according to the invention and, operably linked thereto, one or more directly consecutive coding homologous or heterologous sequences so that, when said DNA is used for the transformation of a cell, this sequence or these sequences is or are expressed during sporulation.

The coding sequences can be sequences according to the invention which can be transcribed into mRNA or else antisense RNA. Examples of mRNA are mRNAs which, during the course of translation, serve as template for the synthesis of proteins selected from the group comprising medically relevant proteins (expression systems for

heterologous proteins), β-galactosidase and protoxins, preferably β-galactosidase and protoxins, especially protoxins.

One embodiment comprises fusions of the exoprotease promoter according to the invention with DNA sequences which encode either a protoxin or a truncated version which corresponds to an activated toxin. Linkages with an appropriately constructed endotoxin DNA, for example with the kurhd 1 [cryΙA(b)] gene (Geiser et al., 1986) or with the cryΙA(c) gene (Adang et al., 1985) are preferred. For it to be possible for the endotoxin to be expressed as fusion protein, it must be in the same reading frame as the leader sequence. If the reading frames do not coincide, suitable linkers are if desired inserted by standard methods. Another embodiment comprises fusions with an exoprotease promoter fragment according to the invention which has been shortened by the leader sequence.

The linkage of the promoter fragment to a prepared coding gene is carried out by standard methods, for example by the PCR method of Yon and Fried (1989) in a plasmid, preferably pHP13 (obtainable from Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio, U.S.A., # 1E50).

The recombinant DNA according to the invention is suitable for the transformation of cells. The recombinant DNA can be introduced in a variety of ways into the cells to be transformed. Examples of conventional methods are direct methods such as the bombardment of the cells with microprojectiles which are loaded with the recombinant DNA, the treatment of the cells with polyethylene glycol, or electroporation, preferably electroporation, or else indirect methods using vectors. Transformation methods of these types are now familiar to those skilled in the art and described in detail in the specialist literature. Work is being done on further methods.

Also of great interest for the present invention are vectors and/or transgenic cells comprising a promoter according to the invention or a DNA fragment according to the invention or a synthetic DNA sequence according to the invention.

Suitable target cells within the scope of the present invention are cells which are able to activate the promoter according to the invention, preferably those cells which are able to form spores, particularly preferably Bacillus species, especially B.t. and its subspecies.

The present invention also relates to a 5'-flanking promoter region of a B. thuringiensis gene which is homologous with the exoproteinase, where said promoter fragment is sporulation-dependent so that the expression of DNA sequences which are functionally linked to the 3' end of said promoter fragment can be regulated in transformed cells.

Isolation of the sporulation-dependent promoter of the exoproteinase from B.

thuringiensis

The sporulation-dependent promoter according to the invention is obtained by identifying with a DNA probe the restriction fragments of the complete DNA of B. thuringiensis on which the promoter is located. The choice of the restriction endonuclease and of the probe sequence decides the size of the fragment on which the promoter which is fully capable of functioning is located. Particularly preferred in the present invention is the restriction endonuclease HindIII.

The ideal case for the choice of the probe is when the probe is directly homologous with the sequence which is sought. It can then be employed directly to find the correct fragment. However, only an indirect search is possible in the case of as yet unknown, specifically non-coding, sequences. The probe is then homologous with a sequence which is assumed to be in the neighbourhood of the sequence which is sought, so that it can be assumed that a restriction fragment which hybridises with this probe also comprises the sequence which is sought. It is advantageous to use in the present invention a probe which is homologous with the coding sequence, preferably with a portion of this sequence at the 5' end, whose expression is regulated by the promoter which is sought.

It is possible to use as probe all single-stranded DNAs and RNAs which are homologous with the DNA to be found. For example, it is possible to use mRNA in order to find an unknown coding sequence, or else a synthetically prepared oligonucleotide. The nucleotide sequence of the synthetically prepared oligonucleotide can be calculated with the aid of specific computer programs based on a known amino-acid sequence. One example of a program of this type is Backtranslate (GCG computer program package Backtranslate, Devereux et al., 1984). However, because the code is degenerate, not all nucleotides can be determined unambiguously in this way. In order nevertheless to obtain a probe capable of functioning it is therefore necessary to synthesise all possible oligonucleotides in order for there to be one in the mixture of these oligonucleotides which hybridises with the DNA sequence to be found. Particularly suitable within the scope of the present invention is the probe oligonucleotide HR5215 (SEQ ID NO:1).

The hybridisation is preferably carried out under the usual hybridisation conditions (Suggs et al., 1981).

To demonstrate that the fragment identified by the probe and isolated by usual techniques in fact does contain the sequence which is sought, within the scope of the present invention the fragment is linked to a coding sequence which comprises no promoter, and it is checked whether gene expression takes place. Gene expression can be determined at two stages, once at the stage of transcription by measurement of mRNA or at the stage of translation by measurement of protein or enzyme activities. It is advantageous to link the fragment identified by the probe to a sequence which encodes a property which can be detected without extensive auxiliary means. Suitable for this purpose is, for example, the β-galactosidase gene whose expression can easily be detected, because the appropriate enzyme activity can be identified by a colour reaction.

The sporulation-dependent promoter of the present invention is suitable for linkage to coding sequences so that the expression of these coding sequences is controlled by the promoter according to the invention. In other words, the present invention likewise comprises the use of the promoter according to the invention or of a DNA fragment or of a synthetic DNA sequence which comprise said promoter in regulatory sequences for controlling transcription.

The promoter according to the invention is advantageously linked to the protoxin genes of B. thuringiensis so that expression thereof takes a more favourable course.

Examples

Deposits

The plasmid p_iW_iTh5 was deposited in compliance with the Budapest Treaty on February 7, 1991, at the Deutsche Sammlung von Mikroorganismen (DSM) under deposit number DSM 6345.

Media and buffer solutions

GYS medium (Yousten and Rogoff, 1969):

Glucose 1 g/l

Yeast extract 2 g/l

(NH₄)₂SO₄ 2 g/l

K₂HPO₄ 0,5 g/l

MgSO₄ · 7 H₂O 0,2 g/l

CaCl₂ · 2 H₂O 0,08 g/l

MnSO₄ · H₂O 0,05 g/l

The pH is adjusted to 7.3 before autoclaving. TE buffer

50 mM Tris pH 8 + 10 mM EDTA

PBS buffer

400 mM sucrose, 1 mM MgCl₂, 7 mM phosphate buffer pH 6

LB medium

Tryptone 10 g/l

Yeast extract 5 g/l

NaCl 5 g/l

General recombinant DNA techniques which are routine for the person skilled in the art are described in Sambrook et al. (1989).

Example 1: Isolation of the HindIII DNA fragment which encodes the

promoter and the N-terminal amino-acid sequence of the extracellular serine proteinase

It is possible to derive from the amino-acid sequence published by Stepanov et al. (1981) with the aid of the GCG computer program package Backtranslate (Devereux et al., 1984) a corresponding nucleotide sequence (see SEQ ID NO:1). The oligonucleotides corresponding to this sequence are synthesised, and the mixture is called oligonucleotide HR5215.

B. thuringiensis cells are cultured in LB medium at 30°C overnight and harvested as described for E. coli (Sambrook et al., 1989). The cells are resuspended in 25 % sucrose, 50 mM tris/HCl pH and 2 mM MgCl₂ and incubated with lysozyme (20 mg/ml) at 37°C for 90 minutes. The cells are then spun down and resuspended in 50 mM tris/HCl pH 8, 5 mM EDTA, 100 mM NaCl and 8 % sarkosyl. This solution is mixed twice with phenol and extracted twice with chloroform. The aqueous phase is precipitated with ethanol, and the sediment is taken up in water. The DNA-comprising solution is alternatively treated with 10 μg/ml RNAase A for 30 minutes.

The complete DNA of B. thuringiensis HD1-ETHZ 4449 (DSM 3667) is completely cut with HindIII, separated on an agarose gel and transferred to a nitrocellulose filter as described by Sambrook et al. (1989). The treatment with HindIII generates a 1.1 kb fragment which hybridises with the oligonucleotide HR5215. The chosen hybridisation temperature is 42°C. The filter is washed at 52°C (Suggs et al, 1981). The 1.1 kb fragment is then cloned into pUC8 (Sambrook et al., 1989). A positive clone identified by screening with the oligonucleotide HR5215 as probe is called pXI84.

Example 2: DNA sequence of the 1.1 kb HindIII fragment

Both strands of the DNA insert in pXI84 are sequenced by the method of Sanger et al. (1977) by using the universal and reverse primers which hybridise with the PUC8 vector and which permit sequencing of the cloned fragment. Novel oligonucleotides are identified from the resulting sequences and are chemically synthesised. It is possible with the aid of these oligonucleotides to sequence the complete 1.1 kb Hindiπ fragment (SEQ ID NO:2). An open reading frame starts at TTG (Position 710) within this sequence. The coding sequence for the amino-acid sequence which was described by Stepanov et al. (1981) and from which the oligonucleotide HR5215 is derived starts at position 1033. Nine nucleotides upstream of the TTG start codon is located the consensus sequence for the so-called Shine-Delgarno box (GGAGG), the ribosome binding site.

Example 3: Construction of a fusion protein between the N-terminal portion of the exoprotease and the E. coli β-galactosidase

The 1.1 kb Hindiπ fragment is treated with T4 polymerase in the presence of the 4 deoxynucleotide triphosphates (Sambrook et al., 1989) in order to fill in the 5 '-protruding ends. The fragment is then cut with BglII. The resulting 1000 bp fragment is isolated on an agarose gel (Sambrook et al., 1989).

The β-galactosidase gene is isolated from the plasmid p_iW_iTh5 from E. coli (DSM 6345). For this, first the plasmid is cut with SmaI and BglII, and the fragment which encodes the β-galactosidase is isolated on an agarose gel as described above.

The vector pHP13 is linearised, cut with BamHI and treated with alkaline phosphatase. Subsequently the two fragments described in the preceding paragraphs are linked to the vector pretreated in this way. E. coli cells are transformed with the ligation product. The clone which shows a positive result after restriction analysis is called pXI105. The pXI105 DNA is used in order to transform B. thuringiensis ΗD1 cryB (DSM 4574) by electoporation. For this, B. thuringiensis HDlcryB cells are cultivated to an optical density (OD₅₅₀) of 0.2 and pretreated by centrifugation and resuspension twice in 1/40 of the volume of an ice-cold PBS buffer. The electroporation of B. thuringiensis HDlcryB in the presence of pXI105 is carried out by a single discharge of a condenser with a voltage between 0.1 kV and 2.5 kV (electroporation apparatus: Gene Pulser Apparatus, BioRad, 1414 Harbor Way South, Richmond, CA 94804, U.S.A., # 165-2075) (EP 342633).

Successful transformation is demonstrated by cultivating the cells on plates which comprise X-Gal (5-bromo-4-chloro-3-indolyl β-galactoside), the chromogenic substrate for β-galactosidase, and GYS (sporulation) medium and by restriction analysis.

Example 4: Time course of the expression of the exoprotease gene

B. thuringiensis HDlcryB are raised in GYS. During sporulation, 100 ml aliquots are regularly removed and the cells are sedimented by centrifugation in a Sorval GS4 rotor at 6000 rpm and at 4°C for 10 minutes. The pellet is resuspended in 10 ml of TE buffer. The bacterial cells are lysed by forcing them through a so-called French press under 6000 psi directly into 10 ml of phenol. After careful mixing and a second extraction with phenol, the nucleic acids are precipitated with ethanol. The DNA can be removed directly from the tube with a small glass rod. The remaining DNA and RNA precipitate is centrifuged in a Sorval HB4 rotor at 10,000 rpm for 20 minutes and resuspended in TE buffer. The DNA is degraded by treatment twice with ribonuclease-free DN Aase (New England Biolabs) in a final concentration of 5 μg/ml at 37°C for 30 minutes. The RNA is then precipitated with ethanol and taken up in H₂O in a concentration of 1 μg/μl. The RNA is stored at -70°C.

It is demonstrated by the so-called Northern blot method and the subsequent hybridisation with the oligonucleotides which have been prepared in accordance with the known sequence of the exoprotease gene on pXI84 that the gene for the extracellular serine proteinase is expressed only during a short period from 3 to 6 hours after the start of sporulation. No RNA is detectable after 6 hours (Northern blot analysis of B. thuringiensis RNA). This expression pattern differs from that of the regulation of the δ-endotoxin promoter of B. thuringiensis HD1-4449. In that case RNA is present up to the end of sporulation (Rothnie and Geiser, 1990). Example 5: Time course of the expression of the β-galactosidase gene under the control of the exoprotease-promoter

B. thuringiensis HD1cryB (pXI105) are raised in GYS. At various times during growth aliquots are taken from the culture and tested for β-galactosidase activity (Miller, 1972, Experiments 48 and 49). The lacZ activity is sporulation-dependent, and the activity is induced 4.5 hours after the start of sporulation.

Example 6: β-Galactosidase activity under the control of various promoters lacZ Promoter: The reading frame of the β-galactosidase gene is restored by inserting a sequence, which is suitable in terms of length, into the XbaI cleavage site of the vector P_iW_iTh5, so that the β-galactosidase activity depends on the lacZ promoter. The

XbaI/BamHI fragment with the lacZ gene is, after the ends have been made blunt with the Klenow fragment of DNA polymerase I, subcloned in the vector pHP13.

One correct clone is called pXIlacZ and transformed into B . thuringiensis. B.

thuringiensis(pXIlacZ) cells are plated onto GYS plates which comprise X-Gal. Light blue or colourless cells are to be seen at the end of sporulation.

E. coliipXllacZ) cells become dark blue on LB plates in the presence of IPTG (isopropyl thio-β-D-galactoside) and X-Gal.

Exoprotease promoter: B. thuringiensis(pXI105) cells become dark blue when grown on GYS plates which comprise X-Gal.

B. thuringiensis expresses β-galactosidase with the exoprotease promoter, not with the lacZ promoter. References:

Adang, M.J., Staver, M.J., Rocheleau, T.A., Leighton, J., Barker, R.F., Thompson, D.V.,

Gene 36:289-300 (1985)

Dankocsik, C, Donovan, W.P., Jany, C.S., Molecular Microbiol. 4:2087-2094 (1990) Devereux, J., Haeberli, P., Smithies, O., Nucleic Acids Res. 12:387-395 (1984)

Geiser, M., Schweitzer, S., Grimm, C, Gene 48:109-118 (1986)

Miller, J.H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold

Spring Harbor (1972)

Rothnie, H., Geiser, M., in: Baker, R., Dunn, P. (Eds.), New Directions in Biological

Control: Alternatives for Suppressing Agricultural Pests and Diseases, Alan R. Liss,

New York, 599-607 (1990)

Sambrook, J., Fritsch, E.F., Maniatis, T., Molecular cloning, a laboratory manual, Cold

Spring Harbor Laboratory, Cold Spring Harbor (1989)

Sanger, F., Nicklen, S., Coulson, A.R., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977) Shivakumar, A.G., Vanags, R.I., Wilcox, D.R., Katz, L., Vary, P.S., Fox, J.L., Gene

79:21-31 (1989)

Stepanov, V.M. Chestukhina, G.G., Rudenskaya, G.N., Epremyan, A.S., Osterman, A.L.,

Khodova, O.M., Belyanova, L.P., Biochem. Biophys. Res. Comm. 100:1680-1687

(1981)

Suggs, S.V., Hirose, T., Miyake, T., Kawashima, E.H., Johnson, M.J., Itakura, K.,

Wallace, R.B., in: Brown, D.D. (Ed.), Developmental biology using purified genes,

Academic Press, New York, 683-693 (1981)

Yon, J., Fried, M., Nucl. Acids Res. 17:4895 (1989)

Yousten, A.A., Rogoff, M.H., J. Bacteriol. 100:1229-1236 (1969)

Sequence listing

SEQ ID NO: 1

TYPE OF SEQUENCE: Nucleotide with corresponding peptide

LENGTH OF SEQUENCE: 27 Bases/13 amino acids

TGG ACN CCN AAY GAY CCN TAY TTY AAY

Trp Thr Pro Asn Asp Pro Tyr Phe Asn Asn Gln Tyr Gly

SEQ ID NO:2

TYPE OF SEQUENCE: Nucleotide

LENGTH OF SEQUENCE: 1100 Base pairs

AAGCTTTAGT TTCAATGACT GGTACTTTTC TAGATACGTT TATTGTATGT 50

ACAATTACAG GACTAGTATT AATTACTACA GGTGCTTGGA GATCTGGTAA 100

AACTGGCGTT GAAGCTACAA CACTTGCATT CCAATCTGTA TTTGGTACTG 150

CTGGTAGTAT GATTCTCGGT ATCGCAATTA TTTTATTTGC CTACTCTACT 200

ATTTTAGGCT GGTCGTATTA TGGAGAAAAA TGTGTAGCTT ATTTATTCGG 250

AGAAAGTGCA GTTAAATATT ATAAAGCAAT CTTTATTGTT ATGATTGCAA 300

TTGGTGCTAA TTTAAAGCTA GGAATCGTAT GGACATTTGC TGATATTGCA 350

AATGGACTTA TGGCAATTCC AAACTTGATT GGTCTAATTG GATTAAGTAG 400

TATTGTTGTT GCTGAAACGA ATCGCTTTTT ACAAGCAGAG AAATTGAAAG 450

AAAATAATAA AAAACAGGCA AGTTAATCTA TGAAAAAAGG AATGACTCAT 500

ACATGACTGA GCGTTCCTTT TTTCATCCCC TCTTTTACTT AATTACTATC 550

ATTAAAAATA TATTTATATC AATATTTACT CCTTTTTATT CCTTCAAAAG 600

TTTTTCACAT AAATGTCATA AATCGTATGG TTTAACTATA TAGTTGAAAA 650

GGAATGCGAC ATTAAGGTGT CACTGAAAAA CTCATCCAAG AAAAGGGAGG 700

AAAAATCTTT TGAAAAACAA AATCATCGTT TTCCTATCTG TTTTGTCATT 750

TATTATTGGT GGTTTCTTCT TTAACACGAA TACTTCAAGC GCTGAAACAT 800

CATCTACTGA TTACGTTCCT AACCAATTAA TCGTTAAGTT CAAACAAAAT 850

GCATCTTTAA GTAATGTGCA ATCTTTTCAT AAATCTGTCG GAGCTAATGT 900

CTTATCTAAA GATGATAAGT TAGGTTTTGA AGTCGTACAA TTTTCAAAAG 950

GTACTGTAAA AGAAAAAATA AAGAGCTATA AAAATAATCC AGATGTGGAA 1000

TATGCAGAAC CGAATTATTA CGTTCACGCC TTTTGGACTC CAAACGACCC 1050

ATATTTTAAT AATCAATACG GGTTACAAAA GATTCAAGCT CCACAAGCTT 1100

Claims

WHAT IS CLAIMED IS:

1. A sporulation-dependent promoter of the exoproteinase from B. thuringiensis (B.t.), including its mutants, variants and homologues which are capable of functioning.

2. A promoter according to claim 1, which derives from the DNA of a subspecies of B. thuringiensis.

3. A promoter according to claim 2, wherein the subspecies is selected from one of the subspecies comprising kurstaki, berliner, alesti, sotto, tolworthi, dendrolimus, tenebrionis and israelensis.

4. A promoter according to claim 1, which is located within the sequence SEQ ID NO:2 of the following structure

AAGCTTTAGT TTCAATGACT GGTACTTTTC TAGATACGTT TATTGTATGT 50

ACAATTACAG GACTAGTATT AATTACTACA GGTGCTTGGA GATCTGGTAA 100

AACTGGCGTT GAAGCTACAA CACTTGCATT CCAATCTGTA TTTGGTACTG 150

CTGGTAGTAT GATTCTCGGT ATCGCAATTA TTTTATTTGC CTACTCTACT 200

ATTTTAGGCT GGTCGTATTA TGGAGAAAAA TGTGTAGCTT ATTTATTCGG 250

AGAAAGTGCA GTTAAATATT ATAAAGCAAT CTTTATTGTT ATGATTGCAA 300

TTGGTGCTAA TTTAAAGCTA GGAATCGTAT GGACATTTGC TGATATTGCA 350

AATGGACTTA TGGCAATTCC AAACTTGATT GGTCTAATTG GATTAAGTAG 400

TATTGTTGTT GCTGAAACGA ATCGCTTTTT ACAAGCAGAG AAATTGAAAG 450

AAAATAATAA AAAACAGGCA AGTTAATCTA TGAAAAAAGG AATGACTCAT 500

ACATGACTGA GCGTTCCTTT TTTCATCCCC TCTTTTACTT AATTACTATC 550

ATTAAAAATA TATTTATATC AATATTTACT CCTTTTTATT CCTTCAAAAG 600

TTTTTCACAT AAATGTCATA AATCGTATGG TTTAACTATA TAGTTGAAAA 650

GGAATGCGAC ATTAAGGTGT CACTGAAAAA CTCATCCAAG AAAAGGGAGG 700

AAAAATCTTT TGAAAAACAA AATCATCGTT TTCCTATCTG TTTTGTCATT 750

TATTATTGGT GGTTTCTTCT TTAACACGAA TACTTCAAGC GCTGAAACAT 800

CATCTACTGA TTACGTTCCT AACCAATTAA TCGTTAAGTT CAAACAAAAT 850

GCATCTTTAA GTAATGTGCA ATCTTTTCAT AAATCTGTCG GAGCTAATGT 900

CTTATCTAAA GATGATAAGT TAGGTTTTGA AGTCGTACAA TTTTCAAAAG 950

GTACTGTAAA AGAAAAAATA AAGAGCTATA AAAATAATCC AGATGTGGAA 1000

TATGCAGAAC CGAATTATTA CGTTCACGCC TTTTGGACTC CAAACGACCC 1050

ATATTTTAAT AATCAATACG GGTTACAAAA GATTCAAGCT CCACAAGCTT 1100

5. A promoter according to claim 4, which is located within the nucleotide sequence in the region from nucleotide 1 to nucleotide 709.

6. A promoter according to claim 5, which is located within the nucleotide sequence in the region from nucleotide 300 to nucleotide 709.

7. A DNA fragment which comprises the promoter according to claim 1.

8. A probe for finding a promoter according to claim 1, which comprises a DNA which is homologous with the coding sequence of the exoproteinase gene from B. thuringiensis.

9. A DNA fragment according to claim 7, which comprises the promoter according to claim 4.

10. A synthetic DNA sequence which is homologous with a promoter according to claim 1 or is a functional equivalent of this promoter.

11. A recombinant DNA which is capable of expression during sporulation and comprises a promoter according to claim 1 and, operably linked thereto, one or more, directly consecutive coding homologous or heterologous sequences.

12. A 5'-flanking promoter region of a B. thuringiensis gene which is homologous with the exoproteinase, wherein said promoter fragment is sporulation-dependent, for the controlled expression in transformed cells of DNA sequences which are functionally linked to the 3' end of said promoter fragment.

13. A vector comprising a promoter according to claim 1.

14. A vector comprising a DNA fragment according to claim 7.

15. A vector comprising a synthetic DNA sequence according to claim 10.

16. A transgenic cell capable of sporulation and comprising a promoter according to claim 1.

17. A transgenic cell capable of sporulation and comprising a DNA fragment according to claim 7.

18. A transgenic cell capable of sporulation and comprising a synthetic DNA sequence according to claim 9.

19. The use of a promoter according to claim 1 in regulatory sequences for controlling transcription.

20. The use of a DNA fragment according to claim 7 in regulatory sequences for controlling transcription.

21. The use of a synthetic DNA sequence according to claim 10 in regulatory sequences for controlling transcription.