NZ235265A

NZ235265A - Regulatory gene from 5' untranslated region of a plant chitinase gene, recombinant dna, vectors, hosts, transgenic plants (including some expressing chitinase), and method of repelling chitin-containing pathogens

Info

Publication number: NZ235265A
Application number: NZ235265A
Authority: NZ
Inventors: Frederick Meins; Shinshi Hideaki; Jean-Marc Neuhaus
Original assignee: Ciba Geigy Ag
Priority date: 1989-09-13
Filing date: 1990-09-11
Publication date: 1993-02-25
Also published as: IL95639A0; CA2025083A1; EP0418695A1; KR910006487A; HUT56140A; AU6245790A; AU645990B2; HU905883D0; IL95639A; ZA907264B; JPH03112488A

Description

<div class="application article clearfix" id="description"> 235265 Priority Date(s): . Xfc. fl^?l Completer..' IVPvftQ. Class: .OT-HOaIoi,,; ca«0v«\*i,sm9;,. W, Ui, qi IbS; .... ^9^.vW»;. to MS 92- DUi. . n 15 fEB 1993 Publication Dstu: P*0. Journal, Noi »••■•'■ .•^SSSSEXSsjj /?•' "'Is t V \\s <?i V^ec*-V Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION REGULATORY DNA SEQUENCE WE, CIBA-GEIGY AG, a Swiss corporation of Klybeckstrasse 141, 4002 Basle, SWITZERLAND hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page la) 235 - lo- Case GA/5-17740/= Regulatory DNA sequence The present invention relates to a novel, substantially pure, regulatory DNA sequence that is obtainable from the 5' region of a plant chitinase gene and that, in operable linkage with any desired expressible DNA, leads to greatly increased levels of expression in transformed plants. The present invention relates also to recombinant DNA molecules that contain the DNA sequence according to the invention in operable linkage with an expressible DNA, and to the vectors derived therefrom. The invention also includes host cells and/or host organisms, including transgenic plants, that contain the said recombinant DNA or the vectors derived therefrom. The present invention also includes a novel chitinase gene that is obtainable from Nicotiana tabacum L. c.v. Havana 425 plants and that contains in its 5* untranslated region the regulatory DNA sequence according to the invention. The present invention relates also to processes for the production of the DNA sequence according to the invention and of the recombinant DNA molecules and vectors containing that DNA sequence, and to the use thereof for the production of transgenic plants. One of the fundamental limiting factors in the production of transgenic plants having novel and useful properties is the level of expression of the inserted foreign genes, which in some cases is only low. As a result of this low expression of the foreign gene and the associated low protein concentrations in the plant, the desired effects often occur to only an unsatisfactory extent, if at all. In the intensively researched field of bacterial expression systems, various possible (followed by page 2)' 2 3 5 2 6 5 -2- methods have become available for increasing the level of transcription of a structural gene that has been cloned in. For example, a structural gene can be inserted behind a strong promoter of a replicon, which in many cases leads to an increase in the level of N gene expression. Furthermore, it is in principle possible to splice the entire operon unit of a gene, that is to say the promoter and also the operator and terminator, into a multi-copy plasmid. As a result of the increased gene expression, or in the second case as a result of the increased gene dosage, an increase in the protein concentration in the cell may possibly be achieved. By contrast, in the much less thoroughly researched field of plant systems, in which the mechanisms of regulation of transcription and translation are still largely unknown, the possible methods currently available for increasing the level of transcription are still very limited and, where they are available, can often be applied with only a poor level of success. Accordingly, it must be regarded a priority task of the present invention to provide a regulatory sequence that is capable of increasing the expression of an operably associated foreign gene or other useful DNA sequences, for example anti-sense DNA, in such a ; manner that the desired phenotypic effects, for example the development of resistance to certain pathogens or chemicals, become clearly visible and the plants in question are therefore effectively protected against those harmful influences. As a further field of application of the regulatory DNA sequence according to the ) invention, mention may here be made of the increase of the synthesis yields of transgenic plants provided for the production of useful and valuable compounds. Surprisingly, within the scope of this invention, it has now been possible to solve the task described above by the use of measures of which some are known. In detail, the present invention relates to a regulatory DNA sequence that is obtainable from the 5' untranslated region of a plant chitinase gene, preferably from the 5' untranslated region of a tobacco chitinase gene, and that, in operable linkage with suitable expression signals active in plant cells and with a structural gene or other expressible 235265 -3- DNA sequences, leads to a significant increase in the level of expression of the operably associated structural gene or other operably associated DNA sequences, for example an anti-sense DNA, in plant material. The present invention relates especially to a regulatory DNA sequence, preferably in substantially pure form, that is obtainable from the 5' untranslated region of a basic chitinase gene of Nicotiana tabacum L. cv. Havana 425 plants and that has essentially the following DNA sequence: 5' - TTGCATTTCACCAGTTTACTACTACATTAAA -3' . The invention relates also to all derivatives of that DNA sequence that are based on the mutation of one or more bases but that are still substantially homologous to the above sequence and still have the properties essential to the invention. Mutation is here to be understood as meaning the deletion or insertion of one or more bases and also, especially, the substitution of one or more bases. Within the scope of this invention, a DNA sequence is substantially homologous to a second DNA sequence if at least 60 %, preferably at least 80 % and very especially preferably at least 90 %, of the active sections of the DNA sequences are homologous to one another. The present invention also includes fragments or partial sequences that are obtainable from the DNA sequence described in greater detail above or from derivatives of that DNA sequence and that still have the specific properties of the starting sequence. Especially preferred is a partial sequence having the following DNA sequence: 5' -ACTACTACATTAAA-3' including all derivatives of that DNA sequence that are based on the mutation of one or more bases but that are still substantially homologous to the above sequence and still have 235 2 6 5 -4- the specific regulatory, expression-increasing properties of the starting sequence. The present invention relates further to.recombinant DNA molecules that contain a chimaeric genetic construction in which the regulatory DNA sequence according to the invention is operably linked to an expressible DNA and to further expression signals active in plant cells, such as promoter and termination sequences, and that, on transformation into a plant host, lead to a significant increase in the level of expression of the operably associated expressible DNA. Especially preferred is a recombinant DNA molecule that contains the above DNA sequence according to the invention in operable linkage with a 35S promoter from Cauliflower Mosaic Virus (CaMV) and with a chitinase or glucanase structural gene. The present invention also includes cloning, transformation and expression vectors that contain the said recombinant DNA molecule according to the invention, and to the host organisms transformed using the said vectors. The invention relates further to so-called shuttle vectors that contain the said recombinant DNA molecule according to the invention and are capable of stable replication both in E. coli and in A. tumefaciens. Of the host organisms, special preference is given to plant hosts selected from the group consisting of plant protoplasts, cells, callus, tissues, organs, zygotes, embryos, pollen and/or seeds and also, especially, whole, preferably fertile, plants that have been transformed using the said recombinant DNA. Whole plants can either be transformed directly as such using the recombinant DNA molecule according to the invention, or they can be obtained from previously transformed protoplasts, cells and/or tissues by regeneration. Very especially preferred are transgenic plants, especially transgenic fertile plants, having a chitinase and/or glucanase content that is significantly increased in comparison with the wild type. o 2 3 5 2 6 5 -5- The present invention also includes all propagation material of a transgenic plant, the said transgenic plant either having been formed by direct transformation using the recombinant DNA molecule according to the invention or being obtained from previously transformed protoplasts, cells, callus, tissues, organs, zygotes, embryos, pollen and/or seeds by regeneration, but without being limited thereto. Within the scope of this invention, propagation material is to be understood as being any plant material that can be propagated sexually or asexually and in vitro or in vivo, preference being given to protoplasts, cells, callus, tissue, organs, ovules, zygotes, embryos, pollen or seeds that are obtainable from a transgenic plant according to the invention. The invention relates also to the progeny of the said plants and to mutants and variants thereof, including those derived from plants obtained by somatic cell fusion, genetic modification or mutant selection. The present invention relates furthermore to processes (a) for the production of the regulatory DNA sequence according to the invention; (b) for the production of the recombinant DNA molecules according to the invention that contain the above DNA sequence according to the invention in operable linkage with a suitable promoter and with a structural gene; (c) for the production of cloning, transformation and/or expression vectors that contain the said recombinant DNA molecule according to the invention; (d) for the production of transformed host organisms, especially plant hosts selected from the group consisting of plant protoplasts, cells, callus, tissues, organs, zygotes, embryos, pollen and/or seeds and also, especially, whole, preferably fertile, plants; (e) for the production of propagation material from transformed plant material, but especially for the production of sexual and asexual progeny; and (f) for the protection of plants against chitin-containing pathogens, especially against pathogenic fungi and insects. The process for the production of the regulatory DNA sequence according to the invention essentially comprises (a) isolating the said sequence or a derivative thereof from the 5' untranslated region of 2 7 E 9 o vi «c. -6- a plant chitinase gene, preferably from the 5' untranslated region of a basic chitinase gene of tobacco, using known measures; or (b) synthesising the said sequence or a derivative thereof by means of chemical processes. In detail, process step (a) comprises the following steps, which are known per se: (a) extraction and purification of genomic DNA from tissues that are capable of expressing chitinase; (b) cleavage of the extracted and purified DNA preparations into fragments of a size suitable for subsequent insertion into a cloning vector; (c) cloning of the fragmented DNA in a cloning vector and creation of a gene library; (d) selection of clones that contain the chitinase gene or parts thereof by means of probe molecules; (e) isolation of those clones which exhibit a strong hybridisation signal with the probe molecule; (f) characterisation of the clones isolated in (e) by means of biochemical processes; and (g) identification and isolation of the regulatory DNA sequence responsible for the increase in expression. The process for the production of the recombinant DNA molecules according to the invention essentially comprises inserting one of the regulatory DNA sequences according to the invention directly in front of the start codon of a desired expressible DNA and linking that construction in operable manner to expression signals active in plant cells. Within the scope of the present invention, special preference is given to the production of a recombinant DNA molecule in which a structural gene that confers protection against pathogens, chemicals and adverse environmental factors on the transformed plant cells and the tissues developing therefrom, but especially on the plants, is associated with the regulatory DNA sequence according to the invention. The process for the production of an expression vector that contains a DNA expressible in plant cells in operable linkage with one of the regulatory DNA sequences according to the invention essentially comprises 35 2 -7- (a) inserting the said regulatory DNA sequence directly in front of the start codon of a desired expressible DNA; and (b) splicing that construct in operable manner into a known plant expression vector, which may contain a marker gene suitable for transformant selection, between expression signals active in plant cells. The process for the production of plant material selected from the group consisting of protoplasts, cells, callus, tissues, organs, seeds, embryos, pollen, ovules, zygotes, etc. essentially comprises transforming the said plant material by methods known per se using one of the recombinant DNA molecules described above. The process for the production of a transgenic plant having a protein content in the transformed cells and/or tissues that is increased in comparison with the wild type essentially comprises transforming the said plant by methods known per se using one of the recombinant DNA molecules described above. Especially preferred is a process for the production of a transgenic plant having a glucanase or chitinase content that is significantly increased in comparison with the wild type. The transformation of the plant material is preferably carried out by known methods selected from the group consisting of polyethylene glycol treatment, heat shock treatment and electroporation of plant protoplasts; microinjection into plant protoplasts, cells or embryos; bombardment of cells or tissues with microprojectiles; Agrobacterium-tae^ated transformation of plant protoplasts, cells or tissue or of whole plants; and CaMV-mediated transformation of plant protoplasts, cells or tissue or of whole plants. The process for the production of transgenic seeds essentially comprises sexually propagating the transgenic plants described in greater detail above and obtaining the seeds that develop, which still contain the inserted genetic material, by known methods. 235265 -8- The process for the production of transformed viable parts of transgenic plants essentially comprises obtaining from the transgenic plants described in greater detail above, by known methods, transformed parts of plants selected from the group consisting of protoplasts, cells, cell clones, cell agglomerates, callus and/or tissue cultures, seeds, pollen, ovules, zygotes and embryos, and selecting those parts which still contain the inserted genetic material. The process for the production of hybridisation and fusion products using the transgenic plant material described in greater detail above essentially comprises hybridising or fusing the said plant material with itself or with foreign material and selecting those hybridisation or fusion products which still contain the inserted genetic material and still have the properties essential to the invention. The process for the production of variants and mutants of the transgenic plants described in greater detail above essentially comprises mutating transgenic plants or viable parts thereof and also the sexual and asexual progeny thereof by known methods and then selecting those individuals which still contain the inserted genetic material and have the properties essential to the invention. The process for the production of transgenic parts of plants selected from the group consisting of blossoms, stems, fruits, leaves and roots essentially comprises isolating the said parts of plants and selecting those parts which still contain the inserted genetic material in the majority of their cells. The present invention also includes a process for protecting plants against chitin-containing pathogens, which process comprises transforming the said plants using a recombinant DNA molecule containing a chimaeric genetic construction in which the regulatory DNA sequence according to the invention is operably linked to a structural gene coding for chitinase and to further expression signals active in plant cells, and expressing the inserted chitinase gene in an amount sufficient to kill the pathogens or keep them under control. The present invention relates further to a process for controlling chitin-containing 2 3 5 2 6 5 -9- pathogens, which process comprises bringing the said pathogens into contact with a transgenic plant or parts of such a plant that have been transformed using a recombinant DNA molecule containing a chimaeric genetic construction in which the regulatory DNA sequence according to the invention is operably linked to a structural gene coding for chitinase and to further expression signals active in plant cells, so that the inserted chitinase gene is expressed in a transgenic plant or in parts of such a plant in an amount sufficient to kill the pathogens or keep them under control. The present invention relates also to the use of the regulatory DNA sequence according to the invention for increasing gene expression in plant material and for identifying homologous DNA sequences having the same function. Figures Before the present invention is described in detail below, the Figures will be explained briefly here. Figure 1 (Fig.l): shows a gene map of pSCH12, which shows the characteristic regions of that plasmid and their origin. (P35S-»CaMV 35S promoter, T35S-*CaMV termination sequence; TNOS—^termination sequence of the neomycin phosphotransferase gene; PNOS—^►promoter of the neomycin phosphotransferase gene; LB, RB—»left or right border sequence from the Ti-plasmid of Agrobacterium tumefaciens. The relevant part of the 5' region of the chimaeric gene construction coding for chitinase is shown in detail by means of the nucleotide sequence. Figure 2 (Fig.2): shows a restriction map of pCIB200. Figure 3 (Fig.3): is a general diagrammatic representation of the construction of plasmid pSCH12. Details are given in Example 8. Figure 4 (Fig.4): is a general diagrammatic representation of the construction of plasmid pSGL7. Details are given in Example 9. 2 3 5 2 6 5 -10- Figure 5 (Fig.5): shows in general manner the chitinase concentration in the leaves of transformedN. sylvestris SI plants (Km^/Km^) in comparison with controls (Km^/Km^). The leaves are numbered from bottom to top. Figure 6 (Fig.6): shows the nucleotide sequence of gene 48, a basic chitinase gene of N. tabacum. Definitions In the following description, a number of expressions are used that are customary in. recombinant DNA technology and in plant genetics. In order to ensure a clear and uniform understanding of the description and the claims and also of the scope to be accorded to the said expressions, the following definitions are listed. Plant material: Parts of plants that are viable in culture or that are viable as such, such as protoplasts, cells, callus, tissue, embryos, plant organs, buds, seeds, etc., and also whole plants. Plant cell: Structural and physiological unit of the plant, comprising a protoplast and a cell wall. Protoplast: "Naked" plant cell that has no cell wall and has been isolated from plant cells or plant tissue and has the potential to regenerate to a cell clone or a whole plant. Plant tissue: Group of plant cells organised in the form of a structural and functional unit Plant organ: Structural and functional unit comprising several tissues, for example root, stem, leaf or embryo. Phenotypic feature: Recognisable or at least detectable feature based on the expression of one or more genes. A phenotypic feature may, however, also be expressed through the suppression of the expression of a gene, for example when anti-sense DNA is used. Heterologous eenefs) or DNA: A DNA sequence that codes for a specific product or 2.35 2 -11- products or fulfils a biological function and that originates from a species other than that into which the said gene is to be inserted; the said DNA sequence is also referred to as a foreign gene or foreign DNA. Homologous gene(s) or DNA: A DNA sequence that codes for a specific product or products or fulfils a biological function and that originates from the same species as that into which the said gene is to be inserted. Synthetic gene(s') or DNA: A DNA sequence that codes for a specific product or products or fulfils a biological function and that is produced by synthetic means. Plant promoter: A control sequence for DNA expression that ensures the transcription of any desired homologous or heterologous DNA gene sequence in a plant, in so far as the said gene sequence is linked in operable manner to such a promoter. Termination sequence: DNA sequence at the end of a transcription unit that signals the end of the transcription process. Over-producing plant promoter (OPP"): Plant promoter that is capable, in a transgenic plant cell, of bringing about the expression of any operably linked functional gene sequence(s) to a degree (measured in the form of RNA or the amount of polypeptide) that is markedly higher than that observed in the natural state in host cells that have not been transformed with the said OPP. 375* untranslated region: DNA sections located downstream/upstream of the coding region which, although transcribed into mRNA, are not translated into a polypeptide. This region contains regulatory sequences, for example the ribosome binding site (5') or the polyadenylating signal (3'). DNA cloning vector: Cloning vehicle, for example a plasmid or a bacteriophage, containing all the signal sequences necessary for the cloning of an inserted DNA in a suitable host cell. 2 3 5 2 6 5 -12- DNA expression vector: Cloning vehicle, for example a plasmid or a bacteriophage, containing all the signal sequences necessary for the expression of an inserted DNA in a suitable host cell. DNA transfer vector: Transfer vehicle, for example a Ti-plasmid or a virus, that permits the insertion of genetic material into a suitable host cell. Mutants, variants of transgenic plants: Derivative of a transgenic plant that has been formed spontaneously or artificially using known process measures, for example UV treatment, treatment with mutagenic agents, etc., and that still has the features and properties of the starting plant that are essential to the invention. Substantially pure DNA sequence: A DNA sequence isolated in substantially pure form from a natural or non-natural source. Such a sequence may be present in a natural system, for example in bacteria, viruses or in plant or animal cells, or it may alternatively be made available in the form of synthetic DNA or of cDNA. Substantially pure DNA is generally isolated in the form of a vector that contains the said DNA as an insert. Substantially pure means that other DNA sequences are present in only negligible amounts and make up, for example, less than 5 %, preferably less than 1 % and very especially preferably less than 0.1%. Such sequences and the vectors containing those sequences are generally in aqueous solution, namely in a buffer solution or in one of the culture media customarily used. The present invention relates principally to a novel regulatory DNA sequence that is obtainable from the 5' untranslated region of a plant chitinase gene and that, in so far as it is operably linked to expressible DNA and to expression signals active in plant cells, leads to a significant increase in the level of expression of the operably associated expressible DNA on transformation into a plant host. The present invention relates especially to a novel, substantially pure, regulatory DNA sequence that is obtainable from the 5' untranslated region of a plant chitinase gene and that, in operable linkage with any desired structural gene, leads to a great increase in the levels of expression of the associated structural gene in transformed plant material. 2 3 5 2 6 5 -13- Within the scope of this invention, special preference is given to a DNA sequence that is present in the 5' untranslated region of a basic chitinase gene from tobacco and that can be obtained therefrom by techniques known per se. The present invention also includes a basic chitinase gene from tobacco that can act as starting material for obtaining the regulatory DNA sequence according to the invention and that has the following special structural features in its 5' untranslated region: (a) a transcription start within the CTACT sequence at positions 1967 to 1971; (b) a first possible start codon at position 1980,11 bp downstream of the transcription start site; (c) a TAAATA sequence ("TATA box") upstream of the transcription start site at positions -28 to -23 of the 5'-flanking region; (d) a CCA ATT sequence at position -114; (e) an imperfect inverted repeat (GCCGAATTCGAGC) comprising 6 bp at position -140; (f) a perfect repeat (ATGTCCAAAC) comprising 10 bp at positions -152 and -228; (g) an imperfect direct repeat (TTTTAACTAAATCTATGTCC) comprising 20 bp at positions -166 and -569; (h) an imperfect direct repeat (CAACTTTCAAAAATTATTTTTTAAA) comprising 25 bp at positions -191 and-217; (i) a palindrome (TAAAATATGAITCATGTTTTA) comprising 20 bp at position -289; (j) a perfect direct repeat (TAAGAGCCGCC) comprising 11 bp at positions -435 and -480; (k) an imperfect direct repeat (TAAAATACACGTCGA) comprising 15 bp at positions -514 and-644; (1) two AATAAA sequences at positions 52 and 120, downstream of the translation stop sequence TAA in the 3'-flanking region. Within the scope of this invention, special preference is given to a basic chitinase gene that is obtainable from Nicotiana tabacum L. c.v. Havana 425 plants and contains the DNA sequence shown in Figure 6. 2 3 5 2 -14- For the isolation of a suitable chitinase gene there are preferably used as starting material genomic or cDNA gene libraries that can be created by customary routine methods very well known to the person skilled in that field. The basic methods of creating genomic or cDNA gene libraries are described in detail, for example, in Maniatis et al (1982), while the transfer and application of those methods to plant systems are described, for example, in Mohnen (1979). Genomic DNA and cDNA can be obtained in various ways. Genomic DNA, for example, can, using known methods, be extracted from suitable cells and purified. For the production of cDNA, there is generally used as starting material mRNA, which can be isolated from selected cells or tissues, but especially from cells or tissues that are known to have high chitin concentrations. The isolated mRNA can then be used in a reverse transcription as the matrix for the production of a corresponding cDNA. Especially preferred as starting material for the production of cDNA are plant cells or tissue or other suitable plant material that has previously been stimulated by suitable measures to produce high chitinase levels. This can be achieved, for example, by inoculating cultivated cells or tissue or other suitable plant material onto a hormone-free medium and cultivating them for a period sufficient for the induction of high chitinase levels. Within the scope of this invention, special preference is given to a basal medium containing the salt and thiamine-HCl concentration proposed by Linsmaier and Skoog (1965) (LS medium). The methods of isolating poly(A+) RNA and of producing cDNA are known to the person skilled in the art and are described in detail below in the Examples. The extracted and purified DNA preparations are then cleaved into fragments for the subsequent cloning. The genomic DNA or cDNA to be cloned may be fragmented to a size suitable for insertion into a cloning vector either by mechanical shearing or, preferably, by cleavage with suitable restriction enzymes. Suitable cloning vectors which are already being used as a matter of routine for the creation of genomic and/or cDNA gene libraries include, for example, phage vectors, such as the X Charon phages, or bacterial vectors, such as the E. coli plasmid pBR322. Further suitable cloning vectors are known to the person skilled in the art. 235 2 65 -15- From the gene libraries created in that manner, suitable clones containing the chitinase gene or parts thereof can then be identified in a screening programme, for example with the aid of suitable oligonucleotide probes (probe molecule), and then isolated. Various methods are available for identifying suitable clones, for example differential colony hybridisation or plaque hybridisation. Immunological detection methods based on identification of the specific translation products may also be used. As probe molecule there may be used, for example, a DNA fragment that has already been isolated beforehand from the same chitinase gene or from a structurally related chitinase gene and that is capable of hybridisation with the corresponding section of sequence within the chitinase gene according to the invention. Provided that the amino acid sequence of the gene to be isolated or at least parts of that sequence are known, a corresponding DNA sequence can be drawn up on the basis of that sequence information. Since the genetic code is known to be degenerate, different codons can in the majority of cases be used for one and the same amino acid. As a result, apart from a few exceptional cases, a particular amino acid sequence can as a rule be coded for by a whole series of oligonucleotides that are similar to one another. However, care must be taken to ensure that only one member of that series of oligonucleotides actually corresponds with the corresponding sequence within the gene that is being sought. In order to limit from the outset the number of possible oligonucleotides, the rules on the use of codons laid down by Lathe R et al, (1985), which take account of the frequency with which a particular codon is actually used in eukaryotic cells, may, for example, be applied. On the basis of that information it is thus possible to draw up oligonucleotide molecules that can be used as probe molecules for the identification and isolation of suitable clones by hybridising the said probe molecules with genomic DNA or cDNA in one of the methods described above. In order to facilitate detection of the desired gene coding for chitinase, the above-described DNA probe molecule can be labelled with a suitable readily detectable group. Within the scope of this invention, a detectable group is to be understood as being any material having a particular readily identifiable physical or chemical property. Such 0> 2 3 5 2 6 5 -16- materials are already widely used especially in the field of immunoassays, and the majority of them may also be employed in the present Application. Special mention may be made at this point of enzymatically active groups, for example enzymes, enzyme substrates, coenzymes and enzyme inhibitors, and also of fluorescent and luminescent agents, chromophores and radioisotopes, for example •%, -^S, 32pt 125j an(j 14q The ready detectability of those labels is based on the one hand on their inherent physical properties (e.g. fluorescent labels, chromophores, radioisotopes) and on the other hand on their reaction and binding properties (e.g. enzymes, substrates, coenzymes, inhibitors). Also suitable as a probe molecule is a single-strand cDNA derived from a poly(A)+ RNA, which in turn is isolated from a tissue or a cell induced for the production of high chitinase levels. Especially preferred within the scope of this invention is the chitinase cDNA clone pCHN 50 which, although it contains the DNA sequence coding for the protein, does not contain the DNA sequence coding for the N-terminal signal peptide, and which is described in Shinshi et al (1987). General methods relating to hybridisation are described, for example, in Maniatis T et al (1982) and in Haymes BT et al (1985). Those clones within the above-described gene libraries which are capable of hybridisation with a probe molecule and which can be identified by means of one of the above-mentioned detection methods can then be analysed further in order to determine in detail the extent and nature of the sequence coding for chitinase. An alternative method of cloning chitinase genes is based on the creation of a gene library composed of expression vectors. In that method, analogously to the methods already described above, genomic DNA, but preferably cDNA, is first isolated from a cell or a tissue capable of expressing chitinase and is then spliced into a suitable expression vector. The gene libraries so created can then be screened using suitable measures, preferably using anti-chitinase antibodies, and those clones selected which contain the desired gene or at least part of that gene as an insert Using the methods described above it is thus possible to isolate a plant chitinase gene, but 0 1 C 0 \J J £ -17- especially a basic chitinase gene from tobacco, having in its 5' region a regulatory DNA sequence that, in operable linkage with any desired structural gene, leads to greatly increased levels of expression in transformed plant material. For further characterisation of that chitinase gene, the DNA sequences purified and isolated as described above are subjected to sequence analysis. The previously isolated chitinase gene is first cleaved into fragments by means of suitable restriction enzymes and then cloned in suitable cloning vectors, for example the M13 vectors mpl8 and mpl9. The sequencing is carried out in the 5'H3' direction, the dideoxynucleotide chain termination method according to Sanger [Sanger et al, 1977] or the method according to Maxam and Gilbert [Maxam and Gilbert, 1980] preferably being used. In order to avoid errors in sequencing, it is advantageous to sequence the two DNA strands in parallel. The analysis of the nucleotide sequence and of the corresponding amino acid sequence is advantageously computer assisted using suitable computer software. In a preferred form of the present invention, which is described in detail in the Examples below, a genomic gene library of chromosomal tobacco DNA is first created and screened by means of plaque hybridisation using a previously isolated cDNA clone (pCHN 50, described in Shinshi et al, 1987) as the probe molecule. After examination of a total of 5 x 10* plaques, 10 recombinants are obtained which are first purified and then partially characterised by means of Southern blot analysis. Two of those clones (XCHN14 and A.CHN17), which are derived from different areas within the genome, contain the complete chitinase gene. For further characterisation, clone A.CHN17 is selected since it produces a very strong hybridisation signal with the cDNA clones pCHN50 and pCHN48 (described in Example 4.4), which act as probe molecules. After digestion with the Hindm restriction enzyme a 5.4 kb DNA fragment is obtained which contains the complete chitinase gene and correlates with a fragment of the same size from tobacco DNA. Part of that fragment comprising 3850 bp and containing the entire coding sequence is then sequenced. The complete DNA sequence of the basic chitinase gene from tobacco is shown in Figure 6. The 5'- and 3'-flanking regions of the basic chitinase gene from tobacco show various areas which can be directly connected with regulatory functions analogously to other 2 3 5 2 -18- eukaiyotic genes. For example, the transcription start site of mRNA can be determined by means of SI nuclease mapping. A total of two bands are found, which leads to the supposition that several start sites exist on the chitinase gene. If the intensity of the bands in question is taken as a basis, the main transcription start site is identical with the A at position 1969 within the CTACT sequence. The first possible initiation signal is found at position 1980,11 bp downstream of the transcription start, within a base sequence TAAAATGAG, which, in line with other translation start sites of plant genes, is designated the consensus sequence. The 5'-flanking region is further characterised by the following base sequences: (a) a TAAATA sequence ("TATA box") upstream of the transcription start site at positions -28 to -23 of the 5'-flanking region; (b) a CCAATT sequence at position -114, similar to the CAAT box which is sometimes found in animal genes upstream of the TATA box; (c) an imperfect inverted repeat (GCCGAATTCGAGC) comprising 6 bp at position -140, which is similar to the regulatory heat shock consensus element of animal genes; (d) a perfect repeat (ATGTCCAAAC) comprising 10 bp at positions -152 and -228; (e) an imperfect direct repeat ( l 11 i AACTAAATCTATGTCC) comprising 20 bp at positions -166 and -569; (f) an imperfect direct repeat (CA AClllCA A A A ATTATlllllA AA) comprising 25 bp at positions -191 and-217; (g) a palindrome (TAAAATATGAlTCATG i in A') comprising 20 bp at position -289; (h) a perfect direct repeat (TAAGAGCCGCC) comprising 11 bp at positions -435 and -480; (i) an imperfect direct repeat (TAAAATACACGTCGA) comprising 15 bp at positions -514 and -644; The 3'-flanking region is characterised by two A ATA A A sequences at positions 52 and 120, downstream of the translation stop sequence TAA. The regulatory DNA sequence according to the invention can be obtained from the 5' region of the previously isolated chitinase gene by means of suitable methods that are very 2 3 5 2 6 5 -19- well known to the person skilled in the art. Especially preferred within the scope of this invention is the regulatory DNA sequence from the 5' region of a basic chitinase gene of Nicotiana tabacum. This section of sequence is a region rich in A/T that consists to the extent of at least 55 %, preferably at least 60 % and very especially preferably at least 70 % of A/T and contains at least 100, preferably at least 40 and very especially preferably at least 31 base pairs. In the case of the basic chitinase gene, isolated from a cloned line of parenchymatous pith tissue of Nicotiana tabacum L. c.v. Havana 425 plants [Eichholz et al, 1983], the said sequence extends from position -31 to position -1, the beginning of the translation start point. Using sequence analysis, the following nucleotide sequence can be determined for that section of sequence: 5'- TTGCATTTCACCAGTTTACTACTACATTAAA -3' . This regulatory DNA sequence, the base sequence of which is known, does not have to be newly isolated each time from a suitable chitinase gene but can of course be synthesised very easily at any time by chemical processes. Suitable processes for the synthesis of short DNA oligonucleotides, for example the phosphotriester or phosphite method, are known to the person skilled in the art. Today, the majority of oligonucleotide syntheses are mechanised and automated, so that short DNA fragments can be produced in a short period of time. By deletion, insertion or substitution of one or more base pairs, therefore, variants or mutants of the DNA sequence described in greater detail above can very easily be produced and checked for their suitability. In detail, the procedure may be that a gene containing one of the regulatory DNA sequences according to the invention is first identified and isolated. After splicing into a suitable cloning vector, that gene, especially the 5' regulatory sequence, can then be modified by means of known process measures. An especially suitable method of producing specific mutants is so-called oligonucleotide-mediated mutagenesis. In that method, short oligonucleotide fragments that, although substantially homologous to the o 235 2 -20- wild-type sequence, differ therefrom in individual nucleotides, are synthesised. The said differences may be insertions, deletions or a substitution of one or more nucleotides. These mutated fragments are then substituted for the homologous counterparts on the wild type gene by generally known methods. The finished construct can then be cloned in a suitable plant expression vector and transformed into a plant, as described in detail below. The present invention is therefore not limited to the base sequence described in greater detail above but also includes all mutants and/or variants of that DNA sequence that have been formed by deletion or insertion of one or more bases or, especially, by the substitution of one or more bases, and that still have the specific regulatory, expression-increasing properties of the starting sequence. The present invention also includes fragments or partial sequences that are obtainable from the DNA sequence described in greater detail above or from derivatives of that DNA sequence and that still have the specific regulatory, expression-increasing properties of the starting sequence. Especially preferred is a partial sequence having the following DNA sequence: 5'- ACTACTACATTAAA -3' The regulatory DNA sequence, or partial sequences thereof, that can be isolated in the manner described above can now be used to identify homologous DNA sequences having the same function, for example, by first creating genomic gene libraries and investigating them in the manner described above, using one of the regulatory DNA sequences according to the invention as the probe molecule, for the presence of homologous DNA sequences that are capable of hybridisation with that probe molecule. The present invention relates also to those processes for identifying homologous DNA sequences having the same function using one of the DNA sequences according to the invention. The present invention relates further to the construction of recombinant DNA molecules 2 3 5 2 -21 - that contain a chimaeric genetic construction in which the regulatory DNA sequence according to the invention is in operable linkage with a structural gene and also with further expression signals active in plant cells, such as promoter and termination sequences. It is often advantageous to incorporate a spacer sequence between the promoter sequence and the adjacent DNA sequence according to the invention, the length of the spacer sequence being so selected that the distance between the promoter and the regulatory DNA sequence according to the invention is the optimum distance for the expression of the associated structural gene. Especially preferred within the scope of this invention is a spacer sequence comprising from 1 bp to 100 bp, but preferably from 2 bp to 46 bp. When operably linked to a structural gene and also to further expression signals active in plant cells, such as promoter and termination sequences, the regulatory DNA sequence according to the invention leads to a significant increase in the level of expression, which may undergo an increase of up to 400 times or more. Accordingly, within the scope of the present invention it is now possible for the first time to improve significantly the phenotypic expression of particular features in plants, for example resistance to certain pathogens or chemical compounds, which hitherto could not be achieved or could be achieved to only an unsatisfactory extent because gene expression was too low and thus the protein concentration in the cell was low. This is especially important in cases where expression of a feature correlates directly with the protein concentration in the cell. Resistance to cytotoxins, for example, can be achieved by the transfer of a gene that codes for and expresses in the plant cell an enzyme that detoxifies the cytotoxin, for example type II neomycin phosphotransferase or type IV aminoglycoside phosphotransferase, which contribute towards detoxifying kanamycin, hygromycin and other aminoglycoside antibiotics, or a glutathione-S transferase, cytochrome P-450 or other catabolically active enzymes known to detoxify triazines, sulfonylureas or other herbicides. Resistance to cytotoxins can also be mediated by a gene that expresses in a plant a particular form of a "target enzyme" (point of action of cytotoxin activity) that is resistant to the activity of the cytotoxin, for example a variant of hydroxyacetic acid synthase that is insensitive to the inhibitory activity of sulfonylureas, imidazolinones or other herbicides that interact with o 2^52 *■■■ \J *.J w -22- \ that specific metabolic step; or a variant of EPSP synthase that proves to be insensitive to the inhibitory activity of glyphosate. It may be advantageous to express those modified target enzymes in a form permitting their transport into the correct cellular compartment, for example in the above case into the chloroplasts. X J Moreover, in certain cases it may be advantageous to direct the gene products into the mitochondria, the vacuoles, the endoplasmic reticulum or into other cell regions, possibly even into the intercellular spaces (apoplasts). Resistance to certain classes of fungi can be achieved, for example, by the insertion of a gene that expresses chitinase in the plant tissues. A great many phytopathogenic fungi contain chitin as an integral part of their hypha and spore structures, for example Basidiomycetes (smut and rust fungi), Ascomycetes and Fungi imperfecti (including Alternaria and Bipolaris, Exerophilum turcicum, Colletotricum, Gleocercospora and Cercospora) . Chitinase is capable of inhibiting the mycelial growth of certain pathogens in vitro. A plant leaf or a root that expresses chitinase constitutively or in response to the penetration of a pathogen is protected from attack by a large number of different fungi. Depending on the particular situation, constitutive expression may be advantageous in comparison with inducible expression, which occurs in a great many plants as a normal reaction to attack by a pathogen, since the chitinase is present in a high concentration immediately without there first being a lag phase for new synthesis. Especially preferred within the scope of this invention is a basic chitinase gene that is ^ obtainable from tobacco, especially a basic chitinase gene that is obtainable from a cloned line of parenchymatous pith tissue of Nicotiana tabacum L. c.v. Havana 425 plants [Eichholz et al, 1983], and that contains the sequence shown in Figure 6. Resistance to insects can be conferred, for example, by a gene coding for a polypeptide that is toxic to the insects and/or their larvae, for example the crystalline protein of Bacillus thuringiensis. A second class of proteins mediating resistance to insects comprises the protease inhibitors. Protease inhibitors are a normal constituent of plant storage structures. It has been demonstrated that a Bowman-Birk protease inhibitor isolated from soybeans and purified inhibits the intestinal protease of Tenebrio larvae 235 2 6 5 -23- [Birk et al (1963)]. The gene that codes for the trypsin inhibitor from the cowpea is described in Hilder et al (1987). A gene that codes for a protease inhibitor can, in a suitable vector, be brought under the control of a plant promoter, especially of a constitutive promoter, for example the CaMV 35S promoter. The gene, for example the coding sequence of the Bowman-Birk protease inhibitor from the soybean, can be obtained by the cDNA cloning method. A further possible method of producing a protease inhibitor is synthetic manufacture, provided that the protease inhibitor contains less than 100 amino acids, for example the trypsin inhibitor of the lima bean. The coding sequence can be predicted by back translation of the amino acid sequence. In addition, there are incorporated at both ends restriction cleavage sites suitable for the vector desired in each particular case. The synthetic gene is produced by synthesis of overlapping oligonucleotide fragments of from 30 to 60 base pairs, by first subjecting those fragments to a kinase reaction and then linking them to one another [Maniatis et al (1982)] and finally cloning them in a suitable vector. By means of DNA sequencing it is then possible to identify a clone that has the insert in a correct orientation. For insertion into the protoplasts, isolated plasmid DNA can be used. The majority of insects have a cuticular skeleton in which chitin micelles in lamellar layers are embedded in a base substance. A further conceivable method of producing resistance or at least tolerance to pathogenic insects is, therefore, the use of a gene that codes for a chitinase enzyme and, after insertion into the plant, expresses it there. Especially preferred within the scope of this invention is a basic chitinase gene that is obtainable from tobacco, especially a basic chitinase gene that is obtainable from a cloned line of parenchymatous pith tissue of Nicotiana tabacum L. c. v. Havana 425 plants [Eichholz et al, 1983], and that contains the sequence shown in Figure 6. * A further enzyme which presumably plays a central role in the plant's defence mechanism against pathogens is |3-l,3-glucanase, which is therefore also preferred within the scope of this invention. In all the above cases it is to be expected that the protective effect for the plant will increase as expression of the inserted genes increases and as the protein (enzyme) concen- 235 2 -24- tration increases as a result. Surprisingly, within the scope of the present invention, it has now been possible to achieve the said increase in the level of expression of inserted foreign genes by operably linking a structural gene that, on expression in the plant, leads to a protective effect for the plant, to the regulatory DNA sequence according to the invention and, optionally, to further sections of sequence necessary for the expression and also for the regulation of transcription and translation, which sections of sequence may be of homologous and also of heterologous origin in relation to the target plant, and inserting the resulting chimaeric genetic construction into the target plant by methods known per se. Especially suitable for use in the process according to the invention, therefore, are all those genes which lead to a protective effect in the transformed plant cells and also in the tissues developing therefrom and especially in the plants, for example increased resistance to pathogens (for example to phytopathogenic fungi, bacteria, viruses, etc.); resistance to chemicals [for example to herbicides (e.g. triazines, sulfonylureas, imidazolinones, triazole pyrimidines, bialaphos, glyphosate, etc.), insecticides or other biocides]; resistance to adverse environmental factors (for example to heat, cold, wind, adverse soil conditions, moisture, dryness, etc.). The DNA sequence according to the invention can also be used in ideal manner to bring about a possible increase in the production of desirable and useful compounds in the plant cell as such or as part of a unit of higher organisation, for example a tissue, callus, organ, embryo or a whole plant. Genes that may be used within the scope of the present invention include, for example, those which lead to increased formation of reserve or stored substances in leaves, seeds, tubers, roots, stems, etc.. The desirable substances that can be produced by transgenic plants include, for example, proteins, starches, sugars, amino acids, vitamins, alkaloids, flavins, perfumes and colourings, fats, etc.. There may also be associated with the regulatory DNA sequence according to the invention structural genes that code for pharmaceutically acceptable active substances, for example certain alkaloids, steroids, hormones, immunomodulators and other physiologically active substances. 235 2 -25- The genes that can come into consideration within the scope of this invention therefore include known genes, but without being limited thereto, for example plant-specific genes, such as the zein gene from maize, the avenin gene from oats, the glutelin gene from rice, etc., mammal-specific genes, such as the insulin gene, the somatostatin gene, the interleucin genes, the t-PA gene, etc., or genes of microbial origin, such as the NPTII gene, etc. and synthetic genes, such as the insulin gene, etc.. Apart from naturally occurring structural genes that code for a useful and desirable property, within the scope of this invention it is also possible to use genes that have been modified previously in a specific manner using chemical or genetic engineering methods. Furthermore, the broad concept of the present invention also includes genes that are produced entirely by chemical synthesis. Genes or DNA sequences that may be used within the scope of the present invention are therefore both homologous and heterologous gene(s) or DNA and also synthetic gene(s) or DNA according to the definition given within the scope of the present invention. The insulin gene may be mentioned at this point as an example of a synthetic gene. Within the scope of the present invention it is also possible to use so-called anti-sense DNA which, in operable linkage with expression signals active in plant cells, leads to the production of an RNA molecule that is complementary to at least a part of an mRNA coded for by a sense DNA and is therefore capable of binding that mRNA. In this manner, the translation of a particular mRNA into the corresponding protein can be prevented or at least restricted, so that an instrument is thus available for effectively controlling the gene expression of selected genes in the plant. The DNA sequences that may be used within the scope of the present invention may be constructed exclusively from genomic DNA, from cDNA or from synthetic DNA. Another possibility is the construction of a hybrid DNA sequence comprising cDNA and also genomic DNA and/or synthetic DNA. In that case, the cDNA may originate from the same gene as the genomic DNA, or both 2 3 5 2 6 5 -26- the cDNA and the genomic DNA may originate from different genes. In any case, however, both the genomic DNA and/or the cDNA may each be produced individually from the same gene or from different genes. If the DNA sequence contains portions of more than one gene, these genes may originate from one and the same organism, from several organisms belonging to various strains or varieties of the same species or various species of the same genus, or from organisms belonging to more than one genus of the same or of a different taxonomic unit (kingdom). In order to ensure the expression of the said structural genes in the plant cell, it is advantageous for the coding gene sequences first to be linked in operable manner to expression sequences capable of functioning in plant cells. The hybrid gene constructions within the scope of the present invention therefore contain, in addition to the regulatory DNA sequence according to the invention, one or more structural gene(s) and expression signals which also include both promoter and terminator sequences and other regulatory sequences of the 3' and 5' untranslated regions. Any promoter and any terminator capable of bringing about an induction of the expression of a coding DNA sequence (structural gene) may be used as a constituent of the hybrid gene sequence. Especially suitable are expression signals originating from genes of plants or plant viruses. Examples of suitable promoters and terminators are those of the Cauliflower Mosaic Virus genes (CaMV) or homologous DNA sequences that still have the characteristic properties of the mentioned expression signals. Also suitable are bacterial expression signals, especially the expression signals of the nopaline synthase genes (nos) or the octopine synthase genes (ocs) from the Ti-plasmids of Agrobacterium tumefaciens. Within the scope of this invention, preference is given to the 35S and 19S expression signals of the CaMV genome or their homologues which can be isolated from the said genome using molecular biological methods, as described, for example, in Maniatis et al, (1982), and linked to the coding DNA sequence. Within the scope of this invention, homologues of the 35S and 19S expression signals are 2 35 2 -27- to be understood as being sequences that, despite slight sequence differences, are substantially homologous to the starting sequences and still fulfil the same function as those starting sequences. In accordance with the invention there may be used as starting material for the 35S transcription control sequences, for example, the Seal fragment of the CaMV strain "S", which includes the nucleotides 6808-7632 of the gene map [Frank G et al (1980)]. The 19S promoter and 5' untranslated region is located on a genome fragment between the PstI site (position 5386) and the HindHI site (position 5850) of the CaMV gene map [Hohn et al (1982)]. The corresponding terminator and 3' untranslated region is located on an EcoRV/BglH fragment between positions 7342 and 7643 of the CaMV genome. Also preferred within the scope of this invention are the expression signals of the CaMV strain CM 1841, the complete nucleotide sequence of which is described in Gardner RC etal (1981). A further effective representative of a plant promoter that may be used is an over-producing plant promoter. Provided that this type of promoter is operably linked to the gene sequence that codes for a desired gene product, it should be capable of mediating the expression of the said gene sequence. Over-producing plant promoters that may be used within the scope of the present invention include the promoter of the small subunit (ss) of ribulose-1,5 -bisphosphate carboxylase from soybeans and also the promoter of the chlorophyll-a/b-binding protein. These two promoters are known for the fact that they are induced by light in eukaryotic plant cells [see, for example, Genetic Engineering of Plants, An Agricultural Perspective, Cashmore A (1983)]. The present invention therefore relates also to recombinant DNA molecules that contain a chimaeric genetic construction in which the regulatory, expression-increasing DNA sequence according to the invention is operably linked to an expressible DNA and also to further expression signals active in plant cells so that, on transformation into a plant host, 2 3 5 2 -28- a significant increase in the level of expression of the operably associated expressible DNA is obtained. Especially preferred are recombinant DNA molecules that contain a chimaeric genetic construction in which the regulatory, expression-increasing DNA sequence according to the invention is operably associated with a structural gene that confers on the transformed plant cells and also on the tissues developing therefrom, and especially on the plants, a protective effect against pathogens, chemicals and also adverse (endaphic or atmospheric) environmental factors. Very especially preferred within the scope of this invention are recombinant DNA molecules that contain a chimaeric genetic construction in which the regulatory, expression-increasing DNA sequence according to the invention is operably associated with a structural gene that expresses chitinase or glucanase in plant cells. The present invention also includes recombinant DNA molecules that contain a chimaeric genetic construction in which the regulatory, expression-increasing DNA sequence according to the invention is operably associated with a structural gene that, on expression in the transformed plant cell as such or as part of a unit of higher organisation selected from the group consisting of a tissue, organ, callus, embryo and a whole plant, leads to an increase in the production of desirable and useful compounds. Further regulatory DNA sequences that may be used for the construction of chimaeric genes include, for example, sequences that are capable of regulating the transcription of an associated DNA sequence in plant tissues in the sense of induction or repression. There are, for example, individual plant genes that are known to be induced by various internal and external factors, such as plant hormones, heat shock, chemicals, pathogens, oxygen deficiency, light, etc.. As an example of gene regulation by a plant hormone, mention should here be made of abscisic acid (ABS), which is known to induce the excess of mRNAs that occurs during the late embryonal phase in cotton. A further example is gibberellic acid (GA3) which induces 2.35 2 -29- malate synthase transcripts in castor beans and isoenzymes of a-amylase in the aleurone layers of barley. The activity of glucanase and chitinase in bean leaves can be markedly increased by treatment with the stress hormone ethylene. In the case of chitinase, this induction effect is controlled via the promoter of the chitinase gene, and it was possible to demonstrate this by reporter gene tests using a promoter from the chitinase gene of beans (Phaseolus vulgaris). The regulation of heat-shock-sensitive protein genes of soybeans has been studied in detail. Treating the plants for several hours at a temperature of 40°C results in the de novo synthesis of so-called heat-shock proteins. A large number of those genes have since been isolated, and their regulation has been analysed in detail. The expression of those genes is controlled primarily at the transcription level. For example, if the promoter of the hps70 gene is fused with the neomycin phosphotransferase II (NPTII) gene, the chimaeric gene so formed can be induced by a heat shock (Spena et al, 1985). Another class of genes that are inducible in plants comprises the light-regulated genes, especially the nuclear-coded gene of the small subunit of ribuIose-l,5-bisphosphate carboxylase (RUBISCO). Morelli et al (1985) have shown that the 5'-flanking sequence of a RUBISCO gene from the pea is capable of transferring light-inducibility to a reporter gene, provided the latter is linked in chimaeric form to that sequence. It has also been possible to extend this observation to other light-induced genes, for example the chlorophyll-a/b-binding protein. The alcohol dehydrogenase genes (adh genes) of maize have been the subject of intensive research. The adhl-s gene from maize was isolated, and it was shown that a part of the 5'-flanking DNA is capable of inducing the expression of a chimaeric reporter gene (e.g. chloramphenicol acetyl transferase; CAT) when the temporarily transformed tissue was subjected to anaerobic conditions [Howard et al (1987)]. A further group of regulatable DNA sequences comprises chemically regulatable sequences that are present, for example, in the PR (pathogenesis-related) protein genes of tobacco and are inducible by means of chemical regulators. 2 3 5 2 -30- The regulatable DNA sequences mentioned by way of example above may be of both natural and synthetic origin, or they may comprise a mixture of natural and synthetic DNA sequences. The present invention therefore also includes chimaeric genetic constructions that contain, in addition to the regulatory DNA sequence according to the present invention in operable linkage with a structural gene, further regulatory sections of DNA sequence permitting, for example, specifically controlled induction or repression of gene expression. The various sections of sequence can be linked to one another by methods known per se to form a complete DNA sequence expressible in plant cells. Suitable methods include, for example, the in vivo recombination of DNA sequences having homologous sections and the in vitro linking of restriction fragments. As cloning vectors there are generally used plasmid or virus (bacteriophage) vectors having replication and control sequences originating from species that are compatible with the host cell. The cloning vector generally carries an origin of replication and, in addition, specific genes that lead to phenotypic selection features in the transformed host cell, especially to resistance to antibiotics or to specific herbicides. The transformed vectors can be selected on the basis of those phenotypic markers after transformation in a host cell. Selectable phenotypic markers that may be used within the scope of this invention include, for example, resistance to ampicillin, tetracycline, hygromycin, kanamycin, methotrexate, G418 and neomycin, but this list, which is given only by way of example, is not intended to limit the subject of the invention. Suitable host cells within the scope of this invention are prokaryotes, including bacterial hosts, for example A. tumefaciens, E. coli, S. typhimurium and Serratia marcescens, and also cyanobacteria. Eukaryotic hosts, such as yeasts, mycelium-forming fungi and plant cells, may also be used within the scope of this invention. 2 35 2 -31- The splicing of the hybrid gene construction according to the invention into a suitable cloning vector is carried out using standard methods, such as those described in Maniatis et al (1982). As a rule, the vector and the DNA sequence to be spliced in are first cleaved with suitable restriction enzymes. Suitable restriction enzymes are, for example, those that yield fragments having blunt ends, for example Smal, Hpal and EcoRV, or enzymes that form cohesive ends, for example EcoRI, SacI and BamHI. Both fragments having blunt ends and those having cohesive ends that are complementary to one another can be linked again using suitable DNA ligases to form a continuous uniform DNA molecule. Blunt ends can also be produced by treatment of DNA fragments that have projecting cohesive ends with the Klenow fragment of the E. coli DNA polymerase by filling the gaps with the corresponding complementary nucleotides. On the other hand, cohesive ends can also be produced by artificial means, for example by the addition of complementary homopolymeric tails to the ends of a desired DNA sequence and of the cleaved vector molecule using a terminal deoxynucleotidyl transferase, or by the addition of synthetic oligonucleotide sequences (linkers) that carry a restriction cleavage site, and subsequent cleavage with the appropriate enzyme. The cloning vector and the host cell transformed by that vector are generally used to increase the number of copies of the vector. With an increased number of copies it is possible to isolate the vector carrying the hybrid gene construction and prepare it, for example, for insertion of the chimaeric gene sequence into the plant cell. In a further process step, these plasmids are used to insert the structural genes coding for a desired gene product or non-coding DNA sequences having a regulatory function, for example anti-sense DNA, into the plant cell and, optionally, to integrate them into the plant genome. o 235 2 6 5 -32- The present invention therefore relates also to the production of recipient plant cells that contain the said structural gene or other desirable genes or gene fragments or other useful DNA sequences incorporated in their genome. .S The insertion of the chimaeric genetic construction is preferably carried out in plant protoplasts using known gene transfer processes. A number of very efficient processes have come into existence for introducing DNA into plant cells, which processes are based on the use of gene transfer vectors or on direct gene transfer processes. One possible method consists, for example, in bringing plant cells into contact with viruses or with Agrobacterium. This may be achieved by infecting sensitive plant cells or by co-cultivating protoplasts derived from plant cells. Within the scope of this invention, Cauliflower Mosaic Virus (CaMV) may also be used as a vector for the insertion of the chimaeric genetic construction according to the invention into the plant. The total viral DNA genome of CaMV is integrated into a bacterial parental plasmid to form a recombinant DNA molecule that can be propagated in bacteria. After cloning has been carried out, the recombinant plasmid is cleaved, using restriction enzymes, either randomly or at very specific, non-essential sites within the viral part of the recombinant plasmid, e.g. within the gene that codes for the transferability of the virus by aphids, and the hybrid gene construction is cloned into that cleavage site. A small oligonucleotide, a so-called linker, which has a single, specific restriction recognition site, may also be integrated. The recombinant plasmid so modified is cloned again and further modified by splicing the hybrid gene construction into a restriction site that occurs only once. The modified viral portion of the recombinant plasmid can then be cut out of the bacterial parental plasmid and used for the inoculation of the plant cells or of the whole plants. Another method of inserting the chimaeric gene construction into the cell makes use of the c 2 3 5 2 -33- infection of the plant cell with Agrobacterium tumefaciens and/or Agrobacterium rhizogenes, which has been transformed using the said gene construction. The transgenic plant cells are then cultured under suitable culture conditions known to the person skilled in the art, so that they form shoots and roots and whole plants are finally formed. A further possible method of transforming plant material comprises mixed infection using both Agrobacterium rhizogenes and transformed Agrobacterium tumefaciens, as described by Petit et al (1986) for the transformation of carrots. The mixing ratio must be such that the root colonies formed on the basis of the A. rhizogenes trans format ion also contain a high proportion of A. tumefaciens Ti-plasmids. This may be achieved, for example, by applying A. rhizogenes and A. tumefaciens together to the plant material in known manner in a mixing ratio of from 1:1 to 1:100, preferably in a mixing ratio of 1:10. The transgenic plants are then cultured under suitable culture conditions known to the person skilled in the art, so that they form shoots and roots and whole plants are finally formed. The two Agrobacterium species are advantageously not mixed until shortly before the actual inoculation of the plant material to be transformed. The chimaeric gene construction according to the invention can therefore be transferred "n into suitable plant cells by means of, for example, the Ti-plasmid of Agrobacterium ^ tumefaciens or the Ri-plasmid of Agrobacterium rhizogenes. The Ti-plasmid or Ri-plasmid is transferred to the plant in the course of infection by Agrobacterium and integrated in stable manner into the plant genome. -*\ Both Ti-plasmids and Ri-plasmids have two regions that are essential for the production of transformed cells. One of those regions, the transfer-DNA (T-DNA) region, is transferred to the plant and leads to the induction of tumours. The other region, the virulence-imparting (vir) region, is essential only for the formation of the tumours, not for their maintenance. The dimensions of the transfer-DNA region can be enlarged by incorporation of the chimaeric gene construction without the transferability being impaired. By removing the tumour-inducing genes and incorporating a selectable marker, the modified Ti- or Ri-plasmid can be used as a vector for the transfer of the gene construction according to the invention into a suitable plant cell. 2 3 5 26 5 -34- The vir region causes transfer of the T-DNA region of Agrobacterium to the genome of the plant cell irrespective of whether the T-DNA region and the vir region are present on the same vector or on different vectors within the same Agrobacterium cell. A vir region on a chromosome also induces the transfer of the T-DNA from a vector into a plant cell. Within the scope of this invention, therefore, there is preferably used a system for transferring a T-DNA region of Agrobacterium into plant cells, in which system the vir region and the T-DNA region are located on different vectors. Such a system is known as a "binary vector system", and the vector containing the T-DNA is accordingly designated a "binary vector". Any T-DNA-containing vector that can be transferred into plant cells and permits selection of the transformed cells is suitable for use within the scope of this invention. Especially preferred within the scope of this invention is a shuttle vector that contains the chimaeric genetic construction according to the invention cloned in between the left border sequence (LB) and the right border sequence (RB) and that is capable of stable replication both in E. coli and in A. tumefaciens. Using newly developed transformation techniques, it has also become possible to transform in vitro plant species that are not natural host plants for Agrobacterium. For example, monocotyledonous plants, especially the cereal species and various grasses, are not natural hosts for Agrobacterium. However, it has become increasingly evident that monocotyledons can also be transformed using Agrobacterium, so that, using new experimental formulations that are now becoming available, cereals and grass species are also amenable to transformation [Grimsley NH et al (1987)]. Preferred within the scope of this invention is so-called leaf disk transformation using Agrobacterium [Horsch et al (1985)]. Sterile leaf disks from a suitable target plant are incubated with Agrobacterium cells containing one of the chimaeric gene constructions according to the invention, and are then transferred into or onto a suitable nutrient 2 3 5 2 6 -35- medium. Especially suitable, and therefore preferred within the scope of this invention, are LS media that have been solidified by the addition of agar and enriched with one or more of the plant growth regulators customarily used, especially those selected from the group of the auxins consisting of a-naphthylacetic acid, picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lactic acid, indole-3-succinic acid, indole-3-acetic acid and p-chlorophenoxyacetic acid, and from the group of the cytokinins consisting of kinetin, 6-benzyladenine, 2-isopentenyladenine and zeatin. The preferred concentration of auxins and cytokinins is in the range of from 0.1 mg/1 to 10 mg/1. After incubation for several days, but preferably after incubation for 2 to 3 days at a temperature of from 20°C to 40°C, preferably from 23°C to 35°C and more especially at 25°C and in diffuse light, the leaf disks are transferred to a suitable medium for the purpose of shoot induction. Especially preferred within the scope of this invention is an LS medium that does not contain auxin but to which a selective substance has been added for the selection of the transformants. The cultures are kept in the light and are transferred to fresh medium at suitable intervals, but preferably at intervals of one week. Developing green shoots are cut out and cultured further in a medium that induces the shoots to form roots. Especially preferred within the scope of this invention is an LS medium that does not contain auxin or cytokinin but to which a selective substance has been added for the selection of the transformants. In addition to Agrobacterium-mediated transformation, within the scope of this invention it is possible to use direct transformation methods for the insertion of the gene constructions according to the invention into plant material. For example, the genetic material contained in a vector can be inserted directly into a plant cell, for example using purely physical procedures, for example by microinjection using finely drawn micropipettes [Neuhaus et al (1987)] or by bombarding the cells with microprojectiles that are coated with the transforming DNA ["Microprojectile Bombardment"; Wang Y-C etal (1988)]. Other possible methods for the direct transfer of genetic material into a plant cell comprise 235 2 -36- treatment of protoplasts using procedures that modify the plasma membrane, for example polyethylene glycol treatment, heat shock treatment or electroporation or a combination of those procedures [Shillito et al (1985)]. In the electroporation technique, plant protoplasts together with plasmids that contain the hybrid gene construction are subjected to electrical pulses of high field strength. This results in a reversible increase in the permeability of biomembranes and thus allows the insertion of the plasmids. Electroporated plant protoplasts renew their cell wall, divide and form callus tissue. Selection of the transformed plant cells can take place with the aid of the above-described phenotypic markers. A further method for the direct introduction of genetic material into plant cells which is based on purely chemical procedures and which enables the transformation to be carried out very efficiently and rapidly is described in Negrutiu I et al (1987). Also suitable for the transformation of plant material is direct gene transfer using co-transformation (Schocher RJ et al 1986). Co-transformation is a method that is based on the simultaneous taking up and integration of various DNA molecules (non-selectable and selectable genes) into the plant genome and that therefore allows the detection of cells that have been transformed with non-selectable genes. The list of possible transformation methods given above by way of example is not claimed to be complete and is not intended to limit the subject of the invention in any way. Those cell clones which contain the hybrid gene construction incorporated in their genome are selected using customary selection, screening and detection methods and are used for the regeneration of transgenic plants. The regeneration of protoplasts kept in culture to form whole plants is described, for example, in Potrykus I and Shillito RD (1986). o 2 35 2 -37- The regeneration processes differ from one plant species to another. In general, however, protoplasts in one of the known culture media are stimulated to divide and form cell walls. There are finally formed callus cultures which can be induced to form roots or shoots by treatment with specific active agents, for example auxins and cytokinins. The plantlets so obtained can then be transferred to soil and cultivated further in the same manner as normal seedlings. Efficient regeneration depends especially upon the medium, the genotype and the previous history of the culture. If these three variables are adequately controlled, the regeneration is completely reproducible and repeatable. The regenerated transgenic plants, which contain a structural gene, expressible in the plant cell, of the above-described hybrid gene construction as an integral component of the plant genome, can be propagated vegetatively, preferably in the form of sterile shoot cultures. The stable integration of an operative expressible gene into the plant genome of the regenerated transgenic plants is verified by reference to the mitotic stability of the integrated gene and on the basis of its behaviour as Mendelian characteristic during meiosis and using Southern blot analysis (Southern EM, 1975). The broad concept of this invention therefore also includes transgenic plant material, selected from the group consisting of protoplasts, cells, callus, tissues, organs, seeds, embryos, pollen, ovules, zygotes etc., including whole plants, but especially whole, fertile plants, that has been transformed by means of the processes described above and contains the recombinant DNA according to the invention in expressible form, and processes for the preparation of said transgenic plant material. The process for the production of transformed plant material that contains, in at least some of its cells, one of the recombinant DNA molecules according to the invention, essentially comprises: (a) first of all isolating the DNA sequence responsible for an overexpression from a -38- suitable source or synthesising it using known processes; (b) inserting the said DNA sequence in operable manner into the 5'-terminal end of any expressible DNA sequence; (c) cloning the finished construct in a plant expression vector under the control of expression signals active in plants; and {d) transforming the said expression vector into the plant material by means of known processes and expressing it therein. The broad concept of this invention therefore also includes transgenic plants, but especially transgenic fertile plants, that have been transformed by means of the above-described process according to the invention, and their asexual and/or sexual progeny that still have the novel and desirable property or properties resulting from the transformation of the parent plant. The expression "asexual and/or sexual progeny of transgenic plants", as defined within the scope of this invention, therefore also covers all mutants and variants that can be obtained by means of known processes, for example by cell fusion or mutant selection, and that still have the characteristic properties of the transformed starting plant, and to all hybridisation and fusion products obtained using the transformed plant material. Preferred within the scope of this invention are transgenic plants, especially fertile transgenic plants, the transformed cells and/or tissues of which exhibit an increased protein content in comparison with the wild type, and their sexual and asexual progeny. Especially preferred within the scope of this invention are transgenic plants, especially fertile transgenic plants, that have been transformed with a recombinant DNA molecule containing a chimaeric genetic construction in which the regulatory DNA sequence according to the invention is operably linked to a structural gene coding for chitinase and/or glucanase and to further expression signals active in plant cells, so that the chitinase and/or glucanase content in the plant is significantly increased in comparison with the wild type as a result of the greatly increased level of expression of the associated structural gene. o 235 2 6 5 -39- Very especially preferred are transgenic plants, especially fertile transgenic plants, that have a chitinase content of from 400 jxg/g fresh weight (FW) to 1200 jag/g fresh weight (FW). Also preferred are transgenic plants, especially fertile transgenic plants, that have a glucanase content of from 20 fig/g fresh weight (FW) to 130 fig/g fresh weight (FW). The invention relates also to the propagation material of transgenic plants. Within the scope of this invention, propagation material of transgenic plants is to be understood as being any plant material that can be propagated sexually or asexually and in vivo or in vitro. Propagation material that is to be regarded as especially preferred within the scope of this invention is especially protoplasts, cells, callus, tissue, organs, seeds, embryos, pollen, ovules and zygotes, as well as any other propagation material that can be obtained from transgenic plants. The invention relates also to parts of plants, for example blossoms, stems, fruits, leaves and roots, that originate from transgenic plants or their progeny that have been previously transformed by means of the process according to the invention and are therefore at least partially made up of transgenic cells. The process according to the invention is suitable for the transformation of all plants, especially those of the systematic groups Angiospermae and Gymnospermae. Of the Gymnospermae, the plants from the Coniferae class are of special interest. Of special interest among the Angiospermae are, in addition to deciduous trees and shrubs, plants of the families Solanaceae, Cruciferae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Alliaceae, Amaryllidaceae, Asparagaceae, Orchidaceae, Palmae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae, Malvaceae or Gramineae and of the order Leguminosae and, of these, especially the Papilionaceae family. Representatives of the Solanaceae, Cruciferae and Gramineae families are preferred. Target crops within the scope of the present invention also include, for example, those selected from the series: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, 2 3 5 2 -40- Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Gossypium, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus and Pisum. Owing to new developments in the field of in vitro culturing of plants, especially in the field of plant regeneration, it has now become possible, even with representatives of the Gramineae family, to regenerate whole plants starting from plant protoplasts. Examples of successful regeneration experiments with Gramineae are described, inter alia, in Yamada Y et al (1986) for rice protoplasts, in Rhodes et al (1988) and Shillito RD et al (1989) for maize protoplasts, and in Horn et al (1988) for Dactylis glomerata protoplasts. Within the scope of the present invention it is therefore also possible to use the following plants: Lolium, Zea, Triticum, Sorghum, Saccharum, Bromus, Oryzae, Avena, Hordeum, Secale and Setaria. Mature plants that have been raised from transformed plant cells are crossed with themselves to produce seed. Some of the seeds contain the genes that code for a useful and desirable property in a ratio that obeys exactly the established laws of heredity. These seeds can be used for the production of transgenic plants. Homozygotic lines can be obtained by repeated self-pollination and the production of inbred lines. These inbred lines can then be used in turn for the development of hybrids. In this process an inbred line that contains the said foreign gene is crossed with another inbred line for the purpose of production. After the general description of the present invention, for the purpose of better understanding reference will now be made to specific Examples which are incorporated into the description for illustrative purposes and are not of a limiting nature unless there is a specific indication to the contrary. 0 7 p; 0 ***S **'■ - 41 - Non-limiting Examples: General recombinant DNA techniques: Since many of the recombinant DNA techniques employed in this invention are a matter of routine for the person skilled in the art, it is better to give a short description of these generally used techniques here rather than to describe them every time they occur. Except where there is a specific indication to the contrary, all these procedures are described in the Maniatis et al (1982) reference. A. Cleaving with restriction endonucleases A reaction batch typically contains about 50 to 500 ng/ml of DNA in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA.. 2 to 5 units of restriction endonucleases are added for each |ig of DNA and the reaction batch is incubated for from one to three hours at the temperature recommended by the manufacturer. The reaction is terminated by heating at 65°C for 10 minutes or by extraction with phenol, followed by precipitation of the DNA with ethanol. This technique is also described on pages 104 to 106 of the Maniatis et al (1982) reference. ' 8. Treatment of the DNA with polymerase in order to produce blunt ends 50 to 500 |ig/ml of DNA fragments are added to a reaction batch in the buffer recommended by the manufacturer, New England Biolabs. The reaction batch contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction takes place over a period of 30 minutes at 15°C and is then terminated by heating at 65°C for 10 minutes. For fragments obtained by cleaving with restriction endonucleases that produce 5'-projecting ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of DNA polymerase is used. For fragments obtained by means of endonucleases that produce 3'-projecting ends, such as PstI and SacI, the T4-DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al (1982) reference. 2 3 5 2 -42- C. Agarose gel electrophoresis and purification of DNA fragments from gels Agarose gel electrophoresis is carried out in a horizontal apparatus, as described on pages 150 to 163 of the Maniatis et al. reference. The buffer used is the tris-borate buffer described therein. The DNA fragments are stained using 0.5 mg/ml of ethidium bromide which is either present in the gel or tank buffer during electrophoresis or is added after electrophoresis. The DNA is made visible by illumination with long-wave ultraviolet light. If the fragments are to be separated from the gel, an agarose is used that gels at low temperature and is obtainable from Sigma Chemical, St. Louis, Missouri. After the electrophoresis the desired fragment is cut out, placed in aplastics test tube, heated at 65'C for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This procedure is slightly different from that described by Maniatis et al (1982) on page 170. As an alternative, the DNA can be isolated from the agarose with the aid of the Geneclean kit (Bio 101 Inc., La Jolla, CA, USA). D. Addition of synthetic linker fragments to DNA ends If it is desired to add a new endonuclease cleavage site to the end of a DNA molecule, the molecule is optionally first treated with DNA-polymerase in order to produce blunt ends, as described in the above section. About 0.1 to 1.0 fig of this fragment is added to about 10 ng of phosphorylated linker DNA, obtained from New England Biolabs, in a volume of 20 to 30 jil with 2 nl of T4 DNA ligase from New England Biolabs, and 1 mM ATP in the buffer recommended by the manufacturer. After incubation overnight at 15°C, the reaction is terminated by heating at 65°C for 10 minutes. The reaction batch is diluted to about 100 pi in a buffer appropriate for the restriction endonuclease that cleaves the synthetic linker sequence. About 50 to 200 units of this endonuclease are added. The mixture is incubated for 2 to 6 hours at the appropriate temperature, then the fragment is subjected to agarose gel electrophoresis and purified as described above. The resulting fragment will then have ends with endings that were produced by cleaving with the restriction endonuclease. These ends are usually cohesive, so that the resulting fragment can then readily be linked to other fragments having the same cohesive ends. 235265 -43- E. Removal of 5'-terminal phosphates from DNA fragments During the plasmid cloning steps, the treatment of the vector plasmid with phosphatase reduces the recircularisation of the vector (discussed on page 13 of the Maniatis et al reference). After cleavage of the DNA with the correct restriction endonuclease, one unit of calf intestinal alkaline phosphatase obtained from Boehringer-Mannheim, Mannheim, is added. The DNA is incubated at 37°C for one hour and then extracted twice with phenol and precipitated with ethanol. F. Linking of the DNA fragments If fragments having complementary cofiesive ends are to be linked to one another, about 100 ng of each fragment are incubated in a reaction mixture of 20 to 40 p.1 containing about 0.2 unit of T4 DNA ligase from New England Biolabs in the buffer recommended by the manufacturer. Incubation is carried out for 1 to 20 hours at 15°C. If DNA fragments having blunt ends are to be linked, they are incubated as above except that the amount of T4 DNA ligase is increased to 2 to 4 units. G. Transformation of DNA into E. coli E. coli strain HB101 is used for most of the experiments. DNA is introduced into E. coli using the calcium chloride process, as described by Maniatis et al (1982), pages 250 and 251. H. Screening of E. coli for plasmids After transformation, the resulting colonies of E. coli are tested for the presence of the desired plasmid by means of a rapid plasmid isolation process. Two customary processes are described on pages 366 to 369 of the Maniatis et al (1982) reference. I. Large-scale isolation of plasmid DNA 235 2 6 5 -44- Processes for the isolation of plasmids from E. coli on a large scale are described on pages 88 to 94 of the Maniatis et al (1982) reference. J. Cloning in M13 phage vectors In the following description it is to be understood that the double-strand replicative form of the phage M13 derivatives is used for routine processes, such as cleaving with restriction endonuclease, linking etc.. Unless there is a specific indication to the contrary, enzymes can be obtained from Boehringer, Biolabs (BRL). They are used in accordance with the manufacturer's instructions unless otherwise indicated. K. Southern blot analysis The extracted DNA is first treated with restriction enzymes, then subjected to electrophoresis in a 0.8 % to 1 % agarose gel, transferred to a nitrocellulose membrane [Southern EM (1975)] and hybridised with the DNA to be detected which has previously been subjected to nick-translation (DNA-specific activities of 5 x 10^ to 10 x 10^ c.p.m./ng). In the present case a 3' AluI-PstI fragment of tobacco chitinase cDNA clone pCHN50 [Shinshi et al (1987)], which can be ^P-labelled using a 'Random Primer' labelling kit [Boehringer Mannheim], is used as hybridisation probe. The filters are washed three times for 1 hour each time with an aqueous solution of 0.03M sodium citrate and 0.3M sodium chloride at 65°C. The hybridised DNA is made visible by blackening an X-ray film over a period of 24 to 48 hours. L. Western blot analysis After SDS-polyacrylamide gel electrophoresis, the proteins are transferred electrophoretically to a nitrocellulose or nylon filter. This filter is then first pre-treated with a blocking agent (for example 5 % skim milk powder in PBS: in the following refened to as milk/PBS). The filter is then incubated for several hours with an antiserum that reacts with the compound to be detected (in the present case: rabbit anti-tobacco 235 2 6 5 -45- chitinase IgG). The filter pre-treated in this manner is washed several times with milk/PBS and then incubated with a commercially available secondary antibody that is coupled to an enzyme [for example peroxidase-coupled goat anti-rabbit antibody (BIORAD), 1:2000 diluted in milk/PBS]. The filter is again washed in PBS and then stained with chloronaphthol and hydrogen peroxide in accordance with the manufacturer's [BIORAD] instructions. Further details are given in Sambrook etal (1989). M. Northern blot analysis Total RNA can be prepared in accordance with the method described in Logemann et al (1987), but it is advantageous to introduce an additional precipitation step with 2M LiCl in order to eliminate any residues of contaminant DNA. The RNA (0.4 mg or 4 mg) is then fractionated by electrophoresis in a 1 % agarose gel containing 6.7 % formaldehyde [Maniatis et al (1982)]. The individual fractions are transferred to 'Zeta-Probe' membranes [BIORAD] and hybridised with a ^P-labelled PstI insert from the tobacco chitinase cDNA clone pCHN48. The size of the hybridising DNA can be estimated by reference to the standard run at the same time. N. Determination of enzyme activity The chitinase activity is determined radiometrically using ^H-labelled chitin [spec. activity 4.93 x 10^ dpm/mol of N-acetylglucosamine] as substrate [Boiler et al (1983)]. The a-mannosidase activity is determined in microtitre plates using p-nitrophenol as substrate [Boiler andKende (1979)]. The peroxidase activity is determined using guaiacol as substrate [Siegel and Galston (1967)]. For the purpose of illustrating the rather general description and for better understanding of the present invention, reference will now be made to specific Examples which are not of a limiting nature unless there is a specific indication to the contrary. The same applies also to all listings given by way of example in the above description. I. Production of cDNA and genomic gene libraries 2 35 2 65 -46- Example 1: Plant material Nicotiana tabacum L. c.v. Havana 425 plants are raised either in a greenhouse or starting from surface-sterilised seed. The surface-sterilisation of the seeds is carried out as follows: 20 to 30 seeds are put into a sieve having a pore size of about 200 ]xm and incubated in 10 ml of a 10 % commercially available bleach solution (NaOCl). The seeds are then rinsed repeatedly with sterile distilled water. In the following Examples there is preferably used a cloned line of parenchymatous pith tissue (N) which can be isolated from Nicotiana tabacum L. c.v. Havana 425 plants in accordance with Eichholz et al (1983). Example 2: Tissue culture The tobacco tissue is cultured on a basal medium that contains a salt and thiamine-HCl concentration according to Linsmeier and Skoog (1965) (LS) and that has been solidified by the addition of 10 g/1 of agar. As a further additive, this basic medium contains a pH indicator, for example 5 mg/1 of chlorophenol red. Other media that are based on this LS basic medium and that are used in the subsequent Examples contain as further additives, for example, kinetin (1.4 jlM) [cytokinin medium] or a-naphthylacetic acid (10.7 fiM) [auxin medium] or a mixture of kinetin and a-naphthylacetic acid [auxin/cytokinin medium (medium A)]. The selective media B and C do not contain a-naphthylacetic acid, but instead contain a selective substance by means of which the transformants can be selected from the large number of untransformed cells and tissue. The precise composition of these selective media is given in Section VII (media). Stock lines (275N) isolated from Nicotiana tabacum L. c.v. Havana 425 plants are subcultured at 21-day intervals on an auxin/cytokinin medium (10 ml), that is to say they are each time inoculated onto new medium (10 ml) and cultured at 25°C in the light. 235 2 -47- For the induction of high levels of chitinase in the cultured tissues, the tissues are inoculated from the auxin/cytokinin medium onto a hormone-free basal medium. Further details relating to the culturing of plant tissues and the induction of chitinase are described in Felix and Meins (1985). Example 3: Production of tobacco protoplasts 100 ml of a 2-day-old tobacco cell suspension according to Example 1 are mixed with an equal volume of a double-concentrated enzyme solution. The enzyme solution contains the following constituents: cellulase R.10 Onozuka^ 10.0 g/1 (Yakult Honsha, Tokyo, Japan) macerase (pectinase from Rhizopus sp., 2.5 g/1 Behring Diagnostics, La Jolla, CA) pectolyase Y-23^ 1.0 g/1 (Seishin Pharm. Co., Tokyo, Japan) The above enzyme solution is centrifuged and sterilised through a 0.2 iim filter. The batch consisting of the tobacco suspension culture and the enzyme solution is agitated carefully on a rotary shaker (40 ipm) for 5 hours at room temperature. At specific time intervals samples are taken from this batch and analysed microscopically. The enzymatic digestion is continued until about 80 % of the cells have changed into spherical protoplasts. The incubation vessels are then removed from the shaker. The cell/protoplast suspension is allowed to settle and the upper half of the medium, which does not contain cells or protoplasts, is removed by suction with a pipette and discarded. The remainder is transferred to 50 ml centrifuge test tubes and centrifuged for 10 minutes at 500 rpm in a clinical centrifuge (model HN-SII, IEC). The protoplast-containing pellets are resuspended in a rinse I solution [see Section VII] and then centrifuged again for 10 CaN03.4H20 MgS04.7H20 nh4h2po4 kno3 1.45 g/1 0.5 g/1 0.23 g/1 1.2 g/1 73.0 g/1 D-mannitol (pH 5.70) 235 2 65 -48- minutes at 1000 rpm. The band containing the protoplasts is located at the upper edge of the centrifuge test tube. The protoplast fraction is collected and then washed again in rinse I solution. The protoplasts are returned to a centrifuge test tube and stored in ice until required for further use. Example 4: Construction of a cDNA gene library A cDNA gene library is produced starting from poly(A)+ RNA, which can be obtained from a cloned line of parenchymatous pith tissue of Nicotiana tabacum L. c.v. Havana 425 plants (see Example 1). The tobacco tissue is first stimulated to produce high levels of chitinase in accordance with Example 2 by culturing the tissue on a hormone-free LS basal medium. 4.1. Isolation of total RNA The preparation of total RNA is effected substantially in accordance with the method described in Lagrimini LM et al (1987). Tobacco tissue deep-frozen in liquid nitrogen is first pounded coarsely in a mortar and then placed in a suitable homogenisation buffer [(1) 7.56M guanidine HC1,0.73M mercaptoethanol, 18.9 mM sodium acetate pH 5.0; or (2) 4 % (w/v) SDS, 0.1M tris-HCl pH 7.8 (1 volume) + 80 % phenol (v/v), 0.1 % (w/v) hydroxyquinoline, 0.1M tris-HCl pH 7.8 (1 volume); or (3) according to Lagrimini LM et al, (1987)] (2.5 ml of buffer per gram of tissue). After the addition of an equal volume of phenol, the batch is homogenised, for example in a Polytron homogeniser. A half volume of chloroform is then added and the emulsion is mixed carefully for about 15 minutes. The various phases are then separated from one another by centrifugation (10,400 g for 10 minutes); the aqueous phase is discarded. At this point it is possible, if desired, to add further extraction steps, for example in the form of an additional phenol/chloroform extraction or extraction twice with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1). The extraction is followed by the precipitation step. This step is effected by the addition 235 2 -49- of 0.3M sodium acetate and 2.5 parts by volume of ethanol. The precipitate is collected by centrifugation (10,400 g for about 15 minutes) and resuspended in 2 ml of sterile water. After the addition of lithium chloride in a final concentration of 3M, the entire batch is incubated overnight at 4°C. The precipitate is then again collected by centrifugation and the pellet formed is washed with ice-cooled ethanol. The pellet is then dried and resuspended in 500 fxl of sterile water. The concentration of total RNA in this preparation is determined by spectrophotometry. As an alternative to the process described above, the total RNA can also be isolated from callus tissue. In this case too, the above-described process steps are used, but the starting material used is callus tissue, cut into cubes (about 3 mm), which prior to the homogenisation step is first deep-frozen in liquid nitrogen and then pounded to a fine powder in a precooled mortar. 4.2. Isolation of polvadenylated RNA Poly(A)+ RNA is isolated by means of oligo-d(T) cellulose chromatography (Collaborative Research, Lexington, MA, USA) in accordance with methods known j>er se [see, for example, Mohnen (1979)]. 01igo-d(T) cellulose is first washed for 15 minutes in 20 volumes of 2.0M NaCl, then suspended in sterile water and packed into a column (1.5 cm in diameter and 20 cm in length). The column is washed with a buffer solution (440 mM NaCl, 0.9 mM EDTA, 9 mM tris-HCl, pH 7.5; or 0.5M NaCl, 10 mM tris-HCl, pH 7.5) until the eluate has a pH value of from 7.0 to 7.6. RNA solutions having an RNA content of from 0.6 mg to 6.0 mg of RNA in a volume of 4.0 ml are then adjusted to a final concentration of 1 mM EDTA and 10 mM piperazine-l,4-bis(2-ethanesuIfonic acid), pH 7.5. The RNA is denatured by heating for 5 minutes at 70°C and then cooling on ice. The entire solution is then adjusted to a value of 0.36M NaCl with 0.1 part by volume of 4M NaCl. The RNA solution is applied to the column and the non-polyadenylated RNA [poly(A)-RNA] is eluted with the above-mentioned buffer solution. The absorption of the eluates is determined by means of a spectrophotometer (Hitachi model 100-40, Hitachi, Tokyo, Japan) and a W+W recorder (Scientific Instruments, Basle, Switzerland) connected thereto. As soon as the absorption has reached the base line, the poly(A)+ RNA that is bound to the column is eluted with 235 2 -50- 1 mM EDTA, 10 mM tris-HCl pH 7.5. The eluates are introduced into a solution of 0.5 mM EDTA and 0.3M sodium acetate pH 5.0 and precipitated by the addition of 2.5 parts by volume of ethanol. The RNA is then collected by centrifugation at 83,000 x g (30 to 45 minutes), dried under nitrogen and resuspended in 1 mM EDTA, 10 mM tris, pH 7.5 or in water. 4.3. Construction and selection of cDNA clones Poly(A)+ RNA is separated into individual fractions by means of preparative ultracentrifugation (17 hours at 57,000 g) over a 5-25 % (w/v) saccharose gradient (1 mM EDTA; 10 mM tris-HCl, pH 7.5). The fractions that contain the information for chitinase can be identified after in vitro translation by anti-chitinase antibodies. These fractions are then combined and used for further working up. The procedure for the production of anti-chitinase antibodies is known to the person skilled in the art and can be carried out, for example, in accordance with the method described in Shinshi et al (1985) and Mohnen (1985). In that method, essentially emulsions containing purified chitinase preparations in complete Freund's adjuvant are injected into three-month-old female rabbits (New Zealand white rabbit). Further injections follow after one and two weeks and then at monthly intervals. Blood is taken 7 to 10 days after each injection, the serum samples being stored at -20°C. Immunoglobulin G is purified by means of affinity chromatography on a protein A-Sepharose CI 4B column (Pharmacia), then lyophilised and stored at -20°C. Synthesis of double-stranded DNA starting from the poly(A)+ RNA matrix, cloning of that double-stranded DNA in pBR322, differential colony hybridisation and plasmid isolation are carried out substantially in accordance with the instructions and description in Maniatis et al (1982). For the synthesis of about 0.8 \ig of double-stranded DNA, 3 (ig of poly(A)+ RNA previously isolated and enriched with chitinase mRNA are incubated with reverse transcriptase (Life Sciences, St. Petersburg, FL) and DNA polymerase I (New England Biolabs, Beverly, MA). The resulting cDNA is spliced into the PstI cleavage site of pBR322 by means of the homopolymeric dC-dG tailing which enables the cDNA and the 2 35 2 6 5 -51- vector DNA to be provided with complementary cohesive ends and which is described in detail in Maniatis et al (1982) [pages 217 - 219]. The Pstl-Iinearised vector pBR322 provided with oligo-dC ends is available commercially. The resulting recombinant plasmid is then used for the transformation of competent E.coli DH1 cells. The cloning in plasmids is described in detail in Maniatis et al (1982) on pages 242-246 and 391. The cDNA library constructed in this manner, which is in the form of bacterial colonies on agar plates, is then first screened by means of differential colony hybridisation using radioactively labelled cDNA. In the differential colony hybridisation, duplicates of the bacterial colonies are made on nitrocellulose filters by placing the filters on the agar plate and then carefully removing them again. The original agar plate is stored for later identification of positive colonies. Filters with adhering bacterial colonies are then placed on a nutrient medium and left there until the colonies have grown into clones approximately 2 mm in size. The filters are then treated with sodium hydroxide solution, which results in lysis of the bacterial cells and in denaturing and fixing of the bacterial DNA on the filter. After pH neutralisation, the filters are repeatedly washed, dried and finally "baked" at a temperature of 80°C in vacuo, so that the DNA is covalently bonded to the filters. The filters are hybridised twice one after another with radioactively labelled (non-cloned) cDNA probes. These cDNA probes are poly(A)+ RNA from tobacco tissue which has previously been induced to produce chitinase (incubation for 7 days on a basal medium without hormone additives) and poly(A)+ RNA from non-induced tobacco tissue (incubation for 7 days on auxin/cytokinin medium). Promising cDNA clones that react more strongly with the cDNA from induced tissue than they do with the control DNA from non-induced tissue, are then subjected to further analysis using the 'hybrid select' translation method in accordance with the method described in Mohnen et al (1985) and Mohnen (1985). The plasmid DNA is then denatured by boiling for 1 minute in 0.2M -0.3M NaOH, 3M NaCl and then cooled on ice. The hybridisation reaction is carried out on square BA/85 nitrocellulose filters (Schleicher and Schuell, Dassel, FRG) using 200 jig to o 23 5 2 65 -52- 250 Jig of total RNA per filter. The RNA hybridised on the filters is eluted, precipitated with ethanol and dissolved in 10 |il of water and analysed with the aid of in vitro translation (using a commercially available wheatgerm extract). The radioactively labelled products are precipitated with antibodies to the desired protein and analysed by SDS-! polyacrylamide gel electrophoresis. 4.4. cDNA clone PCHN48 The cDNA gene library is screened in accordance with Maniatis et al (1982) using colony or plaque hybridisation. As DNA probe there is used the cDNA clone pCHN50 described Shinshi etal (1987) which contains the entire DNA sequence coding for the mature protein but lacks the complete N-terminal signal peptide sequence. In this manner, various clones of different lengths are obtained. The longest of these clones is selected and subjected to nucleotide sequence analysis. This clone, designated pCHN48, has an insert comprising 1.14 kb and having 7 adenosines at the poly(A) end, a single large reading frame (open reading frame) 987 nucleotides in length, corresponding to a polypeptide of 329 amino acids, and a nucleotide from the 5' untranslated region. The amino acid sequence derivable from the DNA sequence of the coding region tallies with the sequence for the first 20 N-terminal amino acids of known chitinases from tobacco. Example 5: Construction of a genomic gene library 5.1. Isolation of chromosomal tobacco DNA For the purpose of lysing the protoplasts, 100 ml of ice-cooled protoplast suspension are mixed with 400 ml of an ice-cooled TENP buffer (see Section VII). The nuclei are separated from this lysate by means of centrifugation for 10 minutes in an IEC clinical centrifuge at 2000 rpm. The pellet so obtainable is resuspended in 500 ml of ice-cooled TENP buffer and pelleted again as described above. Then 8 CsCl gradient test tubes are prepared; in each test tube a cell pellet comprising approximately 12.5 ml of protoplast suspension is added to 26 ml of tenfold concentrated TE buffer (see Section VII). For the purpose of lysing the cell nuclei, 5 ml of a 20 % (w/v) sodium lauryl sarcosine soludon and 32.2 g of CsCl and 2.89 ml of ethidium bromide solution (EtBr, 10 mg/ml) are added to the batch. The entire batch is stirred gently to dissolve the CsCl. 2 3 5 2 -53- The lysates so obtained are transferred to polyallomer test tubes (Beckman VTi50,39 ml test tubes) and centrifuged at 45,000 rpm and a temperature of 20°C for 16 hours in a VTi50 rotor (Beckman). Bands that are fluorescent in UV light are removed from the gradient by means of 3 ml syringes with 16 gauge needles and collected. Further working up of the DNA is effected by repetition of the above-described CsCl/EtBr equilibrium centrifugation. The fluorescent bands are again collected and adhering EtBr is removed in six successive extraction steps using isopropanol saturated with 20 x SSC (see Section VII) (equal volume). The DNA is precipitated from the gradient solution, which is purified in the manner described above, by adding in succession 2 volumes of distilled water, 0.1 volume of 3.0M sodium acetate, pH 5.4 and 2 volumes of ethanol. The filamentous DNA is wound up out of the solution using a Pasteur pipette, washed in 70 % ethanol and further extracted with ah equal volume of chloroform. The DNA is precipitated by the addition of 0.1 volume of 3M sodium acetate, pH 5.4 and 2.0 volumes of ethanol. The DNA filament is again wound up, washed in 70 % ethanol and dried in the air for 5 minutes. It is then dissolved in a total volume of 7 ml of TE buffer (see Section VII) and stored at 4°C until required for further use. 5.2. Construction of a genomic X gene library The previously isolated tobacco DNA is digested with the restriction enzyme Sau3A and separated for 20 hours by means of a 10 % - 40 % saccharose gradient in an SW41 rotor (Beckman) at 20,000 rpm. The fractions obtainable from that gradient are analysed by means of gel electrophoresis (0.5 % agarose gel in TBE buffer). Those fractions which contain fragments of the correct size are pooled. The DNA is precipitated using 1/10 volume of 3M sodium acetate (pH 4.8) and 2 volumes of ethanol, and ligated with phosphatase-treated X EMBL3 DNA digested with BamHI (Stratagen; La Jolla, CA). The linkage reaction is carried out in accordance with the manufacturer's instructions, there being used reaction batches of 5 jxl that contain 1 \ig of X-vector DNA and 0.1 |ig of the tobacco DNA to be incorporated. The incorporation of the DNA resulting from this linkage reaction into the heads of ^-phages is carried out using the Gigapack Plus kit by Stratagen in accordance with the manufacturer's instructions. The phage yield after infection of E. coli CES201 (Glover DM) is approximately 2 x 10^ phages per |ig of inserted DNA. n 235 2 65 -54- 5.3. Screening of the gene libraries and isolation of genomic clones The genomic gene library is screened in accordance with Maniatis et al (1982) using colony or plaque hybridisation. The cDNA clone pCHN50 described in Shinshi et al (1987) and the clone pCHN48 isolated in Section 4.4. are used as DNA probe. The recombinants obtainable in this manner are purified and partially characterised using Southern blot analysis. One of these clones, which exhibits a very strong hybridisation signal with the cDNA probe molecules pCHN50 and pCHN48, and according to Southern blot analysis contains the complete chitinase gene, is selected for further tests and is designated XCHN17. II. Characterisation of the genomic clone XCHN17 Example 6 DNA sequencing and sequence analysis After digestion with the restriction enzyme Hindlll there is obtained a 5.4 kb DNA fragment containing the complete chitinase gene and correlating with a fragment of the same size from tobacco DNA. A portion of this fragment, which comprises 3850 bp and contains the entire coding sequence, is then sequenced. Restriction fragments are cloned in suitable cloning vectors, for example M13mpl8 or M13mpl9 [Yanisch-Perron etal (1985)] and sequenced in both orientations by means of the dideoxynucleotide method ['Dideoxynucleotide chain-termination method'; Sanger et al (1977)]. The nucleotide and amino acid sequences determined are analysed further by computer using Genetics Computer Group software [Devereux et al (1984)]. The complete DNA sequence of the basic chitinase gene from tobacco, which is designated gene 48, is shown in Figure 6. A comparison of that sequence with the cDNA clones pCHN48 and pCHN50 makes it clear that within the coding region of gene 48 there are two intervening sequence sections (introns). The first of these introns comprises 274 bp and is located between the first and second nucleotide of codon 148 which codes for glycine. The second intron is 269 bp in length and is located between the second and third nucleotide of the codon 199 coding for 2 7i R 9 fi"" \J> >„> C -55- histidine. Both introns, in line with other known plant and animal introns, have consensus splicing sites which have a donor and an acceptor sequence (GTAAGTC and ACAG). Furthermore, both introns have the sequence CT(G/A)A(C/T), 33 nucleotides from the 3' border, having similarities with animal consensus sequences (PyTPuAPy). These consensus sequences participate in the formation of the lariat intermediate in excision. The nucleotide sequence of the exon of gene 48 is identical with the DNA sequence of the coding region of clone pCHN48. Example 7: S1 Mapping for the identification of the 3'- and 5'-flanking region 7.1. Primer extension 50 |xg of the previously isolated total RNA are lyophilised in a 500 fi.1 Eppendorf tube. The RNA is then resuspended in 30 (xl of a solution containing radioactively labelled probe molecules and is heated at 70°C for 10 minutes. The tube is slowly cooled to a temperature of 37°C in a water bath and incubated overnight. Without removing the tube from the 37°C water bath, the following solutions and reagents are added: reverse transcriptase buffer (tenfold) 2 jxl (500 mM tris-HCl, pH 7.5; 400 mM KCI; 30 mM MgCl2) bovine serum albumin 5 mg/ml dithiothreitol (100 mM) 5 |xl dNTPs (tenfold; 10 mM of each dNTP in H2O) 5 M-l H20 3 ill RNasin (80 units) 2 jil reverse transcriptase (400 units) 2 jil This reaction batch is incubated for 30 minutes at a temperature of 37°C. The reaction is stopped by the addition of 5 |xl of 3M sodium acetate (pH 5) and 150 (xl of absolute ethanol. The test tube is then left for 30 minutes at -20°C before the precipitate is obtained by centrifugation. This is followed by a washing (80 % ethanol) and drying step (air drying). The precipitate so obtained is resuspended in 10 p.1 to 20 jil of a dye-containing solution (90 % formamide, 0.05 % bromophenol blue, 0.05 % xylene 235265 -56- cyanol, 1 mM EDTA). The extension products are separated on a 6 % sequencing gel [Maniatis etal (1982)] and made visible with the aid of autoradiography. 7.2. SI mapping Analysis of the transcription start site of the mRNA is carried out by means of SI mapping which is based essentially on the description in Maniatis etal (1982). The genomic clone XCHN17, which contains the entire 5' untranslated region, is cleaved with Sau3A/PstI, and a fragment comprising 0.3 kb and containing a portion of the 5'-flanking region and a short section of the coding sequence, is subcloned in an M13 vector. The single-strand DNA obtained from this subcloning step is then used as a template for the subsequent primer extension. The resulting ^^P-labelled DNA sample is cleaved with SstI and Pstl. The extended DNA strand is then denatured and separated by means of polyacrylamide gel electrophoresis. The DNA sample is then incubated for 16 hours at a temperature of 52°C with 3 |o.g of poly(A)+ RNA and then digested at a temperature of 30°C with SI nuclease (about 400 units/ml). The optimum SI nuclease concentration can be determined in preliminary experiments. The digestion products are then separated on a 6 % sequencing gel. Poly(A)+ RNA is isolated in accordance with the above description from cultured tobacco tissue which has previously been induced to produce high levels of chitinase. The length of the digestion products is determined by means of size comparison markers. Suitable size comparison markers are obtained by performing sequencing reactions with single-strand DNA and digesting the reaction products with Pstl before electrophoresis. In this manner there are obtained fragments of different lengths having a dideoxy end, which fragments have the same 5'-ends as the protected fragments to be mapped. 7.3. Nucleotide sequence of the 3'- and 5'-flanking region of the chitinase gene Using S1 nuclease mapping it is possible to detect a total of two bands, which leads to the supposition that several start sites exist on the chitinase gene. If the intensity of the bands in question is taken as a basis, the main transcription start site is identical with the A at position 1969 within the CTACT sequence. The first possible initiation signal is found at 2 3 5 2 6 5 -57- position 1980,11 bp downstream of the transcription start, within a base sequence TAAAATGAG which, in line with other translation start sites of plant genes, is designated the consensus sequence. The 5'-flanking region is characterised by numerous base sequences which can be directly connected to regulatory functions analogously to other eukaryotic genes: (a) a TAAATA sequence (probable TATA box') upstream of the transcription start site at positions -28 to -23 of the 5'-flanking region; (b) a CCAATT sequence at position -114, similar to the CAAT box which is sometimes found in animal genes upstream of the TATA box; (c) an imperfect inverted repeat (GCCGAATTCGAGQ comprising 6 bp at position -140, which is similar to the regulatory heat shock consensus element of animal genes; (d) a perfect repeat (ATGTCCAAAC) comprising 10 bp at positions -152 and -228; (e) an imperfect direct repeat C l'l l l AACTAAATCTATGTCC) comprising 20 bp at positions -166 and -569; (f) an inperfect direct repeat (CAACl l i CAAAAATTATTl 11 l AAA) comprising 25 bp at positions -191 and -217; (g) a palindrome (TAAAATATGAITCATGTTTTA) comprising 20 bp at position -289; (h) a perfect direct repeat (TAAGAGCCGCC) comprising 11 bp at positions -435 and -480; (i) an imperfect direct repeat (T A A A ATACACGTCG A) comprising 15 bp at positions -514 and -644. The 3'-flanking region is characterised by two AATAAA sequences at positions 52 and 120, downstream of the translation stop sequence TAA. m. CONSTRUCTION OF CHIMAERIC GENES Example 8: Linking of the sequence from pCHN48 coding for chitinase to the regulatory 5'-sequence from gene 48 8.1. Construction of plasmid pGYl a 235 2 6 5 -58- ) The plasmid pGYl is derived from the plant expression vector pDH51 described in Pietrzak et al (1986). In plasmid pGYl, the Ncol cleavage site of the start plasmid pDH51 has been replaced by a Xhol cleavage site. v The plasmid pDH51 is cleaved with Ncol and the projecting ends are filled in using Klenow polymerase. The ends, which are then blunt, are then ligated with a Xhol linker (CCTCGAGG). 8.2. Construction of plasmid pCIB200 The construction of this binary vector is based on the plasmid pTJS75, a derivative of RK2 (An G et al, 1985), described in Schmidhauser and Helinski (1985), which has a wide range of hosts and has a tetracycline resistance gene. This plasmid is cleaved with the restriction enzyme Narl and then linked to the AccI fragment of pUC4K [Vierra and Messing (1982)], which carries the NptI gene. The plasmid pTJS75kan formed in this manner, which then also contains the NptI gene in addition to the tetracycline resistance gene, is then digested with the restriction enzyme Sail. At the same time, the plasmid pCIB7, which is described in Rothstein SJ et al (1987), is *\ cleaved with EcoRV and the resulting EcoRV fragment, which contains the left and right ^ T-DNA border sequence from the Ti-plasmid of Agrobacterium tumefaciens and a chimaeric Nos/NptH gene and the pUC polylinker region, is linked with Xhol linkers. The resulting construct is then digested with Xhol and cloned in the Sail cleavage site of the plasmid pTJS75kan. The resulting plasmid, the gene map of which is shown in Figure 2, is designated pCIB200. 8.3. Construction of plasmid pSCHIO Plasmid pSCHIO contains the sequence coding for chitinase, the 5'-transcribed non-coding sequence, 21 base pairs of the chitinase gene 48 located upstream of the transcription start site, and the 3'-transcribed, non-coding sequence, spliced into the BamHIZPstI cloning site of the CaMV 35S plant expression vector pGYl. The process steps necessary for the production of pSCHIO are shown in Figure 3 and are 235 2 -59- described in more detail below: (1) The genomic clone 1CHN17, which contains the chitinase gene 48, is cleaved with HindHI. The resulting 5.6 kb Hindin fragment is isolated and spliced into the Hindin cloning site of the plasmid pUC8, forming plasmid pCHN65. (2) Plasmid pCHN65 is cleaved with EcoRI and the resulting 2.5 kb fragment containing the 5'-end of the chitinase coding region is likewise cloned in pUC8. The resulting plasmid is designated pCHN68. (3) Plasmid pCHN68 is digested with Pstl and religated (linked to itself) with the loss of a 0.5 kb PstVEcoRI fragment. The resulting plasmid is designated pCHN74. (4) In order to clone the 5'-non-coding region of the transcript of gene 48 after a BamHI cleavage site, the plasmid pCHN74 is first digested with HphI, then provided with blunt ends by the action of T4 DNA polymerase and finally cleaved with Pstl. The Hphl/PstI fragment is isolated and spliced into the plasmid pUC8 digested with Hincll/Pstl [Vieira & Messing (1982)], forming pCHN87. After sequencing, it can be seen by reference to the DNA sequence that during the last process step a base of the 1/2 HincII cleavage site has been lost. (5) pCHN87 is digested with EcoRI and Hindin and spliced into the cloning vector pUC9 [Vieira & Messing (1982)]. The resulting plasmid is designated pCHN88. (6) The 1 kb Pstl fragment of the tobacco cDNA clone 48 (pCHN 48) is spliced into the Pstl cloning site of plasmid pUC8, forming pCHN78. (7) The Pstl insert of plasmid pCHN78 is freed by cleaving the plasmid with the restriction enzyme Pstl and spliced into the Pstl site of pCHN88, so that the complete DNA sequence coding for chitinase is re-established. The resulting plasmid is designated pCHN89. 2 3 5 ? W W iU* -does) The plasmid pCHN89 is then digested completely with BamHI and partially with Pstl. The resulting 1.5 kb fragment is cloned in the plant expression vector pGYl, which has been cleaved beforehand likewise with BamHI and Pstl, forming pSCHIO. As a result of this cloning step, the DNA sequence coding for chitinase is placed between the CaMV promoter and the CaMV termination sequence. 8.4. Construction of plasmid pSCH12 The plasmid pSCH12 is a so-called shuttle vector which, owing to its construction, is capable of stable replication both in E. coli and in Agrobacterium tumefaciens. For the construction of pSCH12, first the plasmid pSCHIO described above is cleaved with EcoRI and spliced into the binary vector pCIB 200. The desired plasmid pSCH12 is then formed. The binary Ti-plasmid vector pSCH12 contains 31 bp 5' from the ATG start codon, the coding sequence and the 145 bp 3'-non-coding region of the tobacco chitinase gene 48, spliced into the polylinker region between the CaMV 35S promoter and the terminator of plasmid pGYl. This vector also contains a Tn5 NPTII gene which is under the regulatory control of nopaline synthase expression signals and confers kanamycin resistance on the transformed cells. Example 9: Linking the sequence coding for b-1.3-glucanase to the regulatory 5'-sequence from gene 48 9.1. Construction of plasmid pSGL4 (a) The tobacco cDNA clone pGL36, which is described in Shinshi et al (1988), is cleaved with the restriction enzyme Pstl and cloned in the plasmid pUC8. The resulting plasmid is designated pGLN12. (b) A second cDNA clone from tobacco, pGL31 [Shinshi et al (1988)] is digested with AccI and Pstl, and a 0.85 kb fragment containing the 3'-portion of the sequence coding for glucanase is isolated. 2 35 2 -61- (c) pGLN12 from step (a) is digested with AccI and Pstl. A 0.5 kb fragment containing the 5'-portion of the sequence coding for glucanase is isolated. (d) The fragments from steps (b) and (c) are cloned together in the plasmid pUC8 cleaved beforehand with Pstl, the complete sequence coding for glucanase being re-established. The resulting plasmid is designated pGLN13. (e) In order to eliminate the 5'G-appendage of cDNA and at the same time to install a BamHI cleavage site on the 5' side of the sequence coding for glucanase, use is made of the fact that the 5' G-appendage forms, together with the first two bases of the cDNA, a Haelll cleavage site. pGLN12 is therefore cleaved with Haein and the 0.3 kb fragment is cloned in the Hindll site of pUC9, forming pGLN16. (f) pGLN16 is digested with Clal and Pstl, and the 0.25 kb fragment is replaced by the Clal/PstI fragment of plasmid pGLN13, so that the coding sequence of the glucanase is re-established. The resulting plasmid is designated pGLN17. (g) pGLN17 is digested with BamHI and Pstl. The BairiHI/Pstl fragment obtainable in this manner is spliced into the vector pGYl cleaved beforehand with BamHI and Pstl, the sequence coding for glucanase being placed between the 35 S promoter of CaMV and its termination sequence. The resulting plasmid is designated pSGL2. (h) The EcoRI fragment of pSGL2 is spliced into the binary vector pCIB200, forming pSGIA 9.2. Olieonucleotide-mediated mutagenesis for the production of pSGL7 In order to place the 5'-non-coding sequence of the chitinase gene immediately in front of the sequence coding for glucanase, oligonucleotide-mediated mutagenesis is used. First the EcoRI fragment of plasmid pSGL2 is cloned in the cloning vector M13mpl9, forming mSGLl. mSGLl is then cleaved with Xhol and religated, a portion of the insert being lost. mSGL5 is formed. 2 3 5 2 6 5 -62- Single-strand DNA produced by the growth of mSGLl on an E.coli dam" strain [z.B. E.coli GM1674] is hybridised with Xhol-cleaved mSGL5 DNA from a dam+ strain [z.B. E. coli HB101], forming double-strand DNA (duplex) interrupted by a single-strand gap. This is linked with a mutated oligonucleotide having the following base sequence: 5'TCATTTGGAGAGGACTACTACATTAAAATGGCTGCTATCA3'() by means of base pairing ('annealing'). A mutated clone that has lost both the BamHI and the Smal cleavage sites is identified and isolated. It is designated mSGL7. The Accl/Clal fragment from mSGL7 that contains the mutated sequence is then cloned in the Accl/Clal cleavage site of pSGL2, forming pSGL2/9. pSGL2/9 is digested with the restriction enzymes EcoRV and Pstl, and the EcoRV/PstI fragment that contains the mutated sequence is spliced into the EcoRV/PstI cleavage site of pSGL2, forming pSGL6. All sequences that were present in the mutagenesis are therefore replaced, with the exception of the short section of sequence between the EcoRV and Clal cleavage site. Finally, pSGL6 is digested with EcoRI, and the EcoRI fragment is cloned in the binary vector pCIB200. The resulting plasmid is designated pSGL7. This is a chimaeric construct consisting of: 1. a b-l,3-glucanase that is truncated at the 5'-end and begins with the start codon ATG at position +34; 2. the regulatory sequence from clone CHN17, which extends from position -14 to -1 (ATG=0) and is linked to the CaMV 35S promoter. 2 y S 9 -63- -14 -1 -11 i r11 i pSGL7 |P35S | chitinase 17 I pGL36 I pGL31 I T35S I +34 (ATG) AccI 1111 1294 As control there is used a corresponding construct without the regulatory sequence from the chitinase clone CHN17 designated pSGL5. pSGL5 I P35S J pGL36 J pGL31 1 J T35S | +34 (ATG) AccI 1111 1294 (Stop) Pstl A so-called empty vector (pCIB200) which does not contain a chitinase construct is used as a further control. o 235 2 6 5 -64- IV TRANSFORMATION AND PRODUCTION OF TRANSGENIC PLANTS Example 10.1: Transformation of Agrobacterium tumefaciens with binary vectors The binary vectors described above in Examples 8 and 9 are transformed into the Agrobacterium tumefaciens strain LB4404 using the following process. A. tumefaciens LBA 4404 contains a deleted Ti-plasmid that lacks the T-DNA region but still has an intact vir region [Hoekema etal (1983)]. Agrobacterium tumefaciens strain LB4404 is cultured at a temperature of 30°C in an overnight culture in 5 ml of MG/L medium [see Section VII]. 250 ml of MG/L medium are then added to these 5 ml overnight cultures and the whole batch is mixed thoroughly until an optical density of OD=0.6 (at 600 nm) has been obtained. The cells are then collected by means of centrifugation at 8000 x g and resuspended in 5 ml of MG/L medium. 200 |xl of this cell suspension are incubated with 0.2 |xg to 1 jig of binary plasmid DNA in MG/L medium, and after gentle mixing the batch is immediately deep-frozen in a dry ice/ethanol bath. After 5 minutes, the test tube is placed in a 37°C water bath and left there for 5 minutes. 2 ml of MG/L medium are then added. This suspension is then incubated for 2 to 3 hours in a 30°C water bath. The cells are then collected by means of centrifugation. The cells are resuspended in a small amount of MG/L medium and then plated out on selective media (MG/L plates with 100 ng/ml of gentamycin). The first colonies appear after 2 to 3 days at 30°C. Example 10.2: Transformation of Agrobacterium tumefaciens with binary vectors In an alternative embodiment, the binary vectors described above in Examples 8 and 9 are transferred by means of triparental mating [Rogers SG et al (1986)], using an E. coli helper strain that has a plasmid with a tra function, into Agrobacterium tumefaciens strain LBA4404. The E. coli helper strain used can be, for example, E. coli BHB1011 that contains the plasmid pRK2013 having the tra functions necessary for the transfer of the binary vectors. A. tumefaciens LBA4404 is cultured overnight at 28°C in LB medium with 20 mg/1 of rifampicin and 500 mg/1 of streptomycin. E. coli BHB 1011 and the E. coli strains having the binary vectors are cultured overnight at 37°C in LB medium with 25 mg/1 of o 235 2 -65- I kanamycin. 1 ml of each of those cultures is centrifuged off at 8000 x g, washed in 1 ml of sterile water or 10 mM MgS04, again centrifuged off and resuspended in 100 ill of water or of a MgSC>4 solution. A plate containing an LB solid medium is divided into four sectors. Drops of the three bacterial cultures are applied one over another in these sectors in such a manner that in three of the four sectors the three possible combinations of two cultures are mixed. These act as controls. In the fourth sector, however, all three cultures are mixed together. After the drops have dried, the plates are incubated overnight at 28°C. Then a sample is taken from each sector and suspended in water or MgSCty solution. Dilutions of those suspensions are prepared and plated out on LB plates containing 20 mg/1 of rifampicin, 500 mg/1 of streptomycin and 25 mg/1 of kanamycin and incubated for two to three days at about 28°C. Colonies that grow at a high dilution of the triparental hybrid [nothing is able to grow at a similar dilution of the control hybrids] are freed of parent bacteria which may still be present by repeated plating-out of individual colonies. Example 11: Leaf disk transformation of N. svlvestris and N. tabacum The leaf disk transformation is carried out substantially in accordance with the method described in Horsch et al, (1985). A. tumefaciens LBA 4404 (pSCH12; pSGL7) is cultured overnight at a temperature of 28°C in a glutamate salt medium enriched with 20 mg/1 of rifampicin, 500 mg/1 of streptomycin and 25 mg/1 of kanamycin and adjusted to a pH value of 5.6. In this overnight culture, which contains approximately 3.3 x 10^ cells, sterile leaf disks (5 mm to 10 mm diameter) of N. sylvestris or N. tabacum c.v. Havana 425 are incubated for 5 minutes. The disks are then removed from the culture and dabbed dry on sterile paper towels before being transferred to 100 mm diameter Petri dishes containing a nutrient culture. This nutrient culture consists of a basal medium (30 ml), according to Linsmeier and Skoog (1965), solidified with 1 % agar (DEFCO) and containing as further additives a pH indicator (chlorophenol red; 5 mg/1) and the plant growth substances kinetin (0.3 mg/1) and a-naphthylacetic acid (2 mg/1). This agar medium (medium A) is covered with a layer of from 1 ml to 2 ml of a 2-week-old suspension culture of S275N cells derived from pith tissue of N. tabacum c.v. Havana 425 (Eichholz et al, 1983) and covered with a filter 2 35 2 -66- paper (No.l Whatman filter paper). The leaf disks pretreated in accordance with the above description are then placed on the filter paper. After 48 hours, for the purpose of shoot induction the explantates axe placed on a selective medium having the same composition but also containing 350 mg/1 of cefotaxim and 100 mg/1 of kanamycin (medium B) and are incubated at 25°C and in diffuse light (80 to 100 ^Einstein). Co-cultivated control tissue is inoculated onto the same medium without kanamycin. The explantates are transferred to fresh medium B at weekly intervals. 4 to 8 weeks after the co-cultivation, the green shoots developing from the explantates are harvested and transferred onto 25 ml of medium C (solid medium containing 0.6 % Phytagar) in 50 ml containers. The entire tissue is cultured at a temperature of 24°C to 28°C with a light intensity of 80 to 100 (lEinstein. The shoots form roots after 1 to 2 weeks. V. ANALYSIS OF TRANSGENIC PLANTS Example 12.1: Selection of the transformants The plantlets that are obtained from the various transformation processes can be tested for transformation by two different methods. 1) The vector-specific kanamycin resistance marker serves as a criterion for successful transformation. Leaf explantates (about 5 mm in diameter) of previously transformed plantlets are subcultured on an auxin/cytokinin medium (medium A) containing 50 mg/1 of kanamycin. Explantates of the same plant cultured on an auxin/cytokinin medium (medium A) without selective markers are used as control. The leaf explantates are incubated for a period of 21 days in accordance with the description in Meins & Lutz (1979). The plantlets that grow both on the kanamycin medium and on the control medium can be assumed to be kanamycin-resistant. 2) The second test for transformation has the aim of detecting the overexpression of chitinase and p-l,3-glucanase. An overexpression can be detected by measuring the chitinase or glucanase content in the leaf tissue using "rocket" immunoelectrophoresis 2 3 5 2 6 -67- [Laurell and McKay (1981)] using purified anti-tobacco chitinase or glucanase IgG antibodies [Shinshi et al, (1987)]. Leaf tissue is first homogenised in 2 to 5 volumes of an extraction buffer (0.1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 200 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol, pH 8.0). The soluble protein fraction is obtained by centrifugation (15 min. at 10,000 x g) at 6°C. The content of chitinase and (5-1,3-glucanase is determined by means of "rocket" immunoelectrophoresis using anti-tobacco chitinase or (3-1,3-glucanase IgG. Samples containing chitinase or p-l,3-glucanase are separated by means of a 1 % (w/v) "low mr" agarose gel (BioRad) that contains anti-tobacco chitinase or 0-1,3-glucanase IgG in a final concentration of 10 jxg/ml, at a temperature of 6°C and a maximum of 200 V and 15 mA for 12-16 hours. The dried gels are stained with Coomassie blue and the content of chitinase or (3-1,3-glucanase is determined. As standards there are used authentic enzyme samples isolated directly from Havana 425 tobacco tissue. (3) The chitinase activity is determined radiometrically using %-labelled chitin [spec, activity 4.93 x 10^ dpm/mol of N-acetylglucosamine] as substrate [Boiler etal (1983)]. Example 12.2: Extraction of the intercellular fluid (ICF) The extraction of intercellular fluid from plant tissue can be carried out in accordance with the method described in Parent and Asselin (1984). In that method leaves of regenerated, transgenic plants are first collected and cut into pieces of 4 to 5 cm^ which are then infiltrated with a large excess of cold (about 4°C) buffer, in vacuo for 30 seconds each, with gentle shaking. The said buffer advantageously has the following composition: tris-HCl [pH 7.8] with 0.5M saccharose MgCl2 CaCl2 25.0 mM 10.0 mM 10.0 mM 2 1 k 9 J -J c -68- phenylmethylsulfonyl fluoride (PMSF) 2-mercaptoethanol 0.5 mM 5.0 mM Alternatively, it is also possible to use a 50 mM citrate buffer (pH 5.5). The operation can also be carried out at room temperature. On removal of the vacuum, the buffer penetrates the leaves. The pieces of leaf are then carefully dried and transferred to a 20 ml syringe. The syringe is suspended in a centrifugation test tube and centrifuged for 10 minutes at low speeds (about 1000 x g) and at low temperature (4°C). Example 12.3: Extraction of the intracellular protein fraction The pieces of leaf treated in accordance with Section 13.2 are then homogenised in the same buffer. The coarse particles are separated off by centrifugation. Example 13: Development of transgenic, homozygotic SI plants The plantlets of the SO generation that contain kanamycin-resistant tissue and have an increased level of chitinase or glucanase are transferred to soil and after self-pollination are left to form seed in a greenhouse. The seeds are surface-sterilised and caused to germinate, without secondary contamination, in Petri dishes containing 30 ml of medium D (medium A without kinetin and a-naphthylacetic acid with the addition of 400 mg/1 of kanamycin). The kanamycin-resistant seedlings are raised to maturity in a greenhouse (SI generation) and likewise caused to form seed. The seeds of various plants within the SI generation are tested for kanamycin resistance in the manner described above. If all of 100 tested seeds of an SI plant have the feature of kanamycin resistance, then it can be assumed that the plant is homozygotic in respect of that antibiotic resistance marker. The homozygotic Kmr/Kmr seeds from transformed Nicotiana sylvestris plants (SSC2.3) are then used for the biological tests. The controls used are (a) plants that are regenerated from non-transformed, untreated leaf disks and (b) plants that are regenerated starting from leaf disks that have been transformed beforehand with the vector pCIB200 (SCIB2). 2,3 *5 2 -69- ) Example 14: Overexpression is inherited further as a monogenetic Mendelian characteristic Seeds of the S1 generation of transformed plants and of control plants are caused to germinate. The leaf tissues are tested for kanamycin resistance, chitinase or glucanase content ("rocket" immunoelectrophoresis) and for chitinase or glucanase activity. The chitinase activity is determined with the aid of a radiometric assay using tritiated chitinase in accordance with the description in Boiler etal (1983). The glucanase activity is determined on the basis of the rate of production of non-reduced sugars that are formed with reduced laminarin as substrate [Felix G and Meins FJr (1985)]. VI Results (A) Chitinase transformation Chitinase content, chitinase activity (protein level) The results given in Table 1 and Figure 5 show that (1) Leaf tissue from SI SSC2.3 plants has a chitinase content that is up to 400 times y higher than that of the vector-transformed controls (SCIB2). (2) There is absolute correlation between the overexpression of chitinase and the occurrence of the kanamycin resistance marker (Kmr). (3) The features of kanamycin resistance and chitinase overexpression segregate in a > manner such as is to be expected for a monogenic Mendelian characteristic. (4) The chitinase activity and the content of chitinase antigen in the transformed plants correlate clearly with one another. This is a clear indication that the inserted chitinase gene is transcribed and translated into an enzymologically active protein. It is known that the basic isoenzyme of chitinase is induced to a considerable extent in leaves treated with the stress hormone ethylene. Table 2 shows a comparison of the distribution of the chitinase protein in homozygotic SSC2.3 plants and control plants with and without ethylene induction. The results show that the chitinase content in all the tissues and organs tested has increased dramatically in comparison with the controls. Although o 2 ? 5 2 -70- j y the chitinase gene is under the control of a constitutive promoter, an increased chitinase level can nevertheless be determined in some cases after ethylene induction. Despite the greatly increased chitinase levels, the transformed transgenic plants exhibit normal growth and development behaviour. (5) Chitinases from leaf extracts of control plants and transformed plants are fractionated by means of an SDS-PAGE, transferred to nitrocellulose paper and stained using anti-tobacco chitinase IgG as the primary antibody. Purified tobacco chitinase gives as reference two bands, which correspond to chitinase A (approximately 34 kD) and chitinase B (approximately 32 kD) [Shinshi et al (1987)]. The blots of those gels in which equal amounts of chitinase antigen are loaded onto each lane give a single band of approximately 32 kD in the extracts of the non-transformed N. sylvestris plants and a single band of approximately 34 kD in the extracts of the SSC 2.3 transformants. Blots resulting from greatly overloaded gels also exhibit an additional, weak band at approximately 32 kD, which corresponds to the N. sylvestris chitinase, in the case of the SSC transformants. Mixing experiments show that the chitinase in the extracts of the SSC 2.3 plants has approximately the same molecular size as the tobacco chitinase A, the expression product of gene 48, which is used for the transformation. These results therefore confirm that the tobacco chitinase gene which is chimaeric according to the invention is expressed in N. sylvestris plants and the gene product is correctly processed to a polypeptide having the size of the mature chitinase gene. Determination of RNA content (RNA level) The RNA content is determined by Northern blot analysis. Total RNA is isolated from the inferior leaves of three homozygotic control plants having a low chitinase level [15 ' 3.0 ng/g FW] and of three homozygotic chitinase transformants having a high chitinase level [827 ' 66 jig/g FW] and analysed. As the probe there is used the Pstl insert of the chitinase cDNA clone pCHN48, which hybridises both with chitinase sequences from tobacco and with chitinase sequences from N. sylvestris. The total RNA from control plants yields a band of approximately 1.2-1.3 kb. By 2 3 5 2 6 5 -71- contrast, comparable amounts of RNA from SSC2.3 plants yield two hybridisation bands, the intensity of which is at least 10 times greater than that of the controls. As regards their size, the two bands correspond to the 1.198 kb and 1.41 kb transcripts which can be expected on transcription of the chimaeric tobacco chitinase gene comprising CaMV 35S transcription start, the DNA sequence coding for tobacco chitinase, and the CaMV terminator. The two bands are of approximately equal intensity, which leads to the conclusion that the tobacco gene is transcribed with approximately the same effectiveness irrespective of the terminator used. Location of the expression products in the correct compartments As a rule, basic chitinases are located in the intracellular compartment. In order to permit location of the chitinase coded for by gene 48 in the transgenic N. sylvestris plants, the chitinase content of intact tissue is compared with that of the intercellular washing fluid (IWF), which is obtainable by vacuum infiltration of leaves using buffers having a high salt concentration. It is assumed that, under these conditions, an IWF is obtained that contains principally the proteins found in the interstitial fluid and the proteins associated loosely with the cell wall. There is used as control a-mannosidase, which is a marker for the central vacuole [Boiler and Kende (1979)], and peroxidase, which is to be found both in the central vacuole and in the extracellular compartment. Table 5 shows the percentage of the above-mentioned enzymes in the IWF fraction of leaf tissue from non-transformed and SSC 2.3 leaves. Less than 1 % of the a-mannosidase marker enzyme is found in the IWF fraction, which shows that, when the described process for producing the IWF is used, no appreciable amounts of enzymes located in the vacuole are extracted. By contrast, approximately 30 % of the peroxidase activity is found in the IWF fraction. The concentration of chitinase antigen in the leaves of the SSC 2.3 plants is 20 times greater than that in the control plants. In both cases, however, the chitinase concentration in the IWF is less than 2 %. These results show that the chitinase is coiTectly located in the cellular compartment of the N. sylvestris leaves even at very high chitinase levels, as are produced by the chimaeric construct according to the invention. -72- (B) 3-1,3-Glucanase transformation The results of two transformations (SSG7.1 and SSG7.2) conducted independently are shown in Table 3. The transformed plants have in their leaf tissue (S-l,3-glucanase concentrations which are up to 100 times greater than those of the controls. The control plants have been transformed using the control plasmid pSGL5 (see Section 9.2), which contains a genetic construction in which the sequence according to the invention from the 5' untranslated region of the chitinase gene is not linked to the glucanase structural gene. \ 235265 -73- iQt Tables Table 1: Chitinase activity [nKat/g] of pSCHIO transformed N. sylvestris plants [Kmr/Kmr; Kms/Kmr] in comparison with controls [Kms/Kms], Selfed P-1,3-Glucanase Chitinase Chitinase SO plants [jxg/g antigen] [nKat/g] Kms/Kms 18.7 ±0.2 (3) 45.7 ±1.64 22.4 ±2.58 Kms/Kmr 15.8 ±2.17(8) 553.0 ±78.4 149.0 ±17.3 Kmr/Kmr 19.9 ±2.33(3) 750.0 ±22.5 312.0 ±46.0 \ J ) 2 3 5 2 -74- Table 2: Chitinase concentration [|xg/g fresh weight (FW)] in various tissues of pSCHIO transformed N. sylvestris plants in comparison with controls, with [4 days at 20 ppm] and without ethylene treatment. Transformants Tissue Chitinase [|xg/g FW] -ethylene +ethylene Vector (SIB2) superior leaves inferior leaves 10.4 53.2 intact 10.0 42.8 epidermis 90.8 104.0 lower epidermis removed 9.8 38.8 stems 8.4 14.0 roots 58.8 148.0 Chitinase (SSC2.3) superior leaves inferior leaves 452.0 704.0 intact 520.0 760.0 epidermis 756.0 828.0 lower epidermis removed 652.0 1116.0 - stems 161.0 1200.0 roots 1200.0 1200.0 0 r c 9 \J -75- Table 3: p-l,3-Glucanase concentration [|xg/g fresh weight (FW)] in the leaf tissue of transformedN. sylvestris SI plants in comparison with controls (SYL3) Plants Superior leaves Inferior leaves Glucanase Chitinase Glucanase Chitinase [jig/gFW] [M-g/gFW] SSG7.1-1 128 11.4 74.1 54.6 SSG7.1-4 93.9 21.9 90.3 51.0 SSG7.2-23 93.9 15.0 5.4 46.5 SSG7.2-24 100.8 13.5 0.0 41.7 SYL3 1.2 6.3 3.0 15.6 235 2 -76- Table 4: P-1,3-Glucanase concentration in N. sylvestris Km^ plants of the SI generation in comparison with controls transformed using an empty vector (pCIB200). Plants Chitinase concentration in the superior leaves [|Xg/g FW] SCIB (control) 1.5 ± 1.5* (4)# SSG7.1 77.0 ±6.4 (20) SSG7.2 63.4 ±1.0 (20) ♦mean value ' SE; ^number of plants Table 5; Distribution of chitinase between the extracellular and the intracellular compartment in the leaves of control plants and transformed//, sylvestris plants % in the IWFa Plant Peroxidase a-Mannosidase Chitinase untransf. 28.2 ± 4.8b 0.65 ±0.13 1.48 ±0.54 SSC 2.3 30.5 ±6.2 0.89 ±0.16 1.78 ±0.65 a Percentage of the total enzyme content in the IWF present in the leaf tissue, based on the determination of chitinase antigen and of peroxidase andmannosidase activity. ° Mean value ± SEM of 4 independent tests. 235 2 6 5 -77- VII. MEDIA AND BUFFER SOLUTIONS Medium A nh4n03 1650 mg/1 kno3 1900 mg/1 CaCl2.2H20 440 mg/1 MgS04.7H20 370 mg/1 kh2po4 170 mg/1 Na2EDTA 37.3 mg/1 FeS04.7H20 27.8 mg/1 h3bo3 6.2 mg/1 MnS04.4H20 22.3 mg/1 ZnS04.7H20 8.6 mg/1 ki 0.83 mg/1 Na2Mo04.2H20 0.25 mg/1 CuS04.5H20 0.025 mg/1 C0CI2.6H2O 0.025 mg/1 saccharose 30.0 g/1 thiamine.HCl 0.400 mg/1 myo-inositol 100.0 mg/1 kinetin 0.3 mg/1 a-naphthylacetic acid 2.0 mg/1 chlorophenolled 5.0 mg/1 agar 10.0 g/1 Medium B same composition as medium A but without oc-naphthylacetic acid and with the following additional constituents: cefotaxim kanamycin 500 mg/1 75 mg/1 -78- Medium C same composition as medium B but without kinetin MG/L medium for Aerobacterium mannitol-glutamate medium (Holsters et al 1978) 50% W5 salt solution pH 5.6-6.0 Rinse I solution saccharose 154.0 g/1 MES 0.59 g/1 kno3 250.0 mg/1 nh4no3 25.0 mg/1 NaH2P04.H20 15.0 mg/1 CaCl2.2H20 90.0 mg/1 MgS04.7H20 25.0 mg/1 (NH4)2S04 13.4 mg/1 -79- TENP buffer tris-HCl (pH 8.0) 100 mM EDTA 10 mM NP-40 (Sigma Chem.) 1 % (v/v) TBE buffer tris-borate 89 mM EDTA 2mM TE buffer tris-HCl (pH 8.0) 10 mM EDTA 1 mM SSC NaCl sodium citrate (pH 7.0) 1.54 mM 0.154 mM 235 2 6 5 -80- Vin BIBLIOGRAPHY An G et al, EMBO J.. 4: 277-284 (1985). Birk Y et al, Biochim. Biophvs. Acta, 67: 326-328 (1963). Boiler et al, Planta, 157; 22-31 (1983) . Boiler T and Kende H, Plant Physiol., 63: 1123-1132 (197 9) Cashmore A, Genetic Engineering of Plants, an Agricultural Perspective. Plenum, New York 1983, pp. 29-38. Devereux et al, Nucl. Acids Res., 12: 387-395 (1984) . Eichholz R et al, Planta, 158: 410-415 (1983). Facciotti and Pilet, Plant Science Letters, 15: 1-7 (197 9). Felix G and Meins FJr, Planta, 164: 423-428 (1985) . Frank G et al. Cell, 21: 285-294 (1980). Gardner RC et al, Nucl. Acids Res., 9: 2871-2888 (1981). Garfinkel and Nester, J. Bact., 144: 732-743 (1980). Glover DM, DNA cloning, volume 1: a practical approach; DM Glover ed./ IRL Press# Oxford and Washington DC/ p. 33 (198) GrimsleyNH et al, Nature, 325: 177-179 (1987) . Haymes BT et al, Nucleic Acid Hybridisation a Practical Approach, IRL Press, Oxford, England (1985). Hilder et al. Nature, 330: 160-163 (1987). Hoekema et al, Nature, 303: 179-180 (1983) . Hohn T et al, in: "Molecular Biology of Plant Tumors", Academic Press, New York, pp. 549-560 (1982). Horn et al. Plant Cell Reports, 7:469-472 (1988). Horsch et al, Science, 227: 1229 (1985). Howard et al, Planta, 170: 535 (1987). Lagrimini LM et al, Proc. Natl. Acad. Sci., USA 84: 7542, (1987). Laurell and McKay, Methods Enzvmology, 73: 339-361 (1981) . Lathe R et al, J. Mol. Bio.. 183: 1-12 (1985) . Linsmeier and Skoog, Physiol. Plant., 18: 101-127 (1965). Logemann J et al, Analvt. Biochem., 163: 16-20 (1987) 2 X £ 9 \,f -j -81- Maniatis et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982. Maxam and Gilbert, ' Sequencing end-labelled DNA with base-specific chemical cleavage', in: Methods in Enzymology 65: 499-560, Academic Press, New York, London, (1980) . Meins & Lutz. Differentiation, 15: 1-6 (1979). Mohnen, "Regulation of Glucanohvdrolases in Nicotiana tabacum on the messenger RNA level", Dissertation University of Illinois at Urbana-Champaign, 1985. Mohnen et al, EMBO J., 4: 1631-1635 (1985). Morelli et al, Nature, 315: 200 (1985) . Murashige and Skoog, Physiol. Plant., 15: 473-497 (1962). Negrutiu I et al, Plant Mol. Biol., 8: 363-373 (1987). Neuhaus et al, Theor. AppI. Genet., 74: 30 -36 (1987). Parent JG and Asselin A, Can J. Bot., 62: 564-569 (1984). Petit et al, Mol. Gen. Genet., 202: 388 (1986). Pietrzak et al, Nucl. Acids Res.. 14: 5857-5868 (1986). Potrykus I and Shillito RD, Methods in Enzvmologv, Vol 118. Plant Molecular Biology, eds.A and HWeissbach, Academic Press, Orlando, 1986. Rhodes et al, Biotechnology, 6: 56-60 (1988) . Rogers SG et al, Methods in Enzvroology, 118: 630-633 (1986) Rothstein SJ et al, Gene. 53: 153-161 (1987) . Sambrook et al, Molecular Cloning, A Laboratory Manual, Second Edition (1989). Sanger et al, Proc. Natl. Acad. Sci., USA 74: 5463-5467 (1977) . Schmidhauser and Helinski, J. Bacteriol., 164: 446-455 (1985) . Schocher RJ et al. Bio/Technology, 4: 1093-1096(1986). Shillito RD et al, Biotechnology, 7: 581-587 (1989). Shinshi et al, Proc. Natl. Acad. Sci.. USA 84: 89-93 (1987). Shinshi et al, Planta. 164: 423-428 (1985). o 235 2 6 5 -82- Shinshi et al, Proc. Natl. Acad. Sci., USA 85: 5541-5545 (1988) . Shillito et al, Bio Technology, 3: 1099-1103 (1985); Siegel BZ and Galston AW, Plant Physiol, 42: 221-226 (1967) Southern EM, J. Mol. Biol. 98:503-517 (1975). Spena et al, EMBO J., 4: 2736 (1985). Vierra and Messing, Gene, 19: 259-268 (1982). Wang Y-C et al, Plant Mol. Biol., 11: 433-439 (1988). Yaroada Y et al, Plant Cell Rep# 5:85-88 (1986) . Yanisch-Perron et al, Gene, 33: 103-119 (1985). </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 235265 -83 WHAT WE CLAIM IS:-

1. A regulatory DNA sequence in substantially pure form that can be obtained from the 5' untranslated region of a plant basic chitinase gene and has an A/T content of at least 55% and that, in so far as it is operably linked to expressible DNA and to expression signals active in plant cells, leads to an increase in the level of expression of the operably associated expressible DNA on transformation into a plant host that leads to concentrations of the expression product that are up to 100 times greater than those obtained without the regulatory DNA sequence, including all derivatives of the said DNA sequence that are still substantially homologous thereto and still have the specific regulatory, expression-increasing propenies thereof.

2. A regulatory DNA sequence according to claim 1, wherein this section of sequence is a region rich in A/T that consists to the extent of at least 70 % of A/T.

3. A regulatory DNA sequence according to claim 1 that is obtained from the 5* untranslated region of a basic chitinase gene of Nicotiana tabacum L. c.v. Havana 425 plants and that has essentially the following DNA sequence: 5'- TTGCATTTCACCAGTTTACTACTACATTAAA -3' including all derivatives of that DNA sequence that are still substantially homologous to the above sequence and still have the specific regulatory, expression-increasing properties of the starting sequence. in substantially pure form

4. Fragments or partial sequences/that are can be obtained from one of the regulatory DNA sequences or from derivatives of those DNA sequences according to any one of claims 1 to 3 and that still have the specific regulatoiv, expression-increasing properties of the starting sequence.

5. A partial sequence according to claim 4 that has the following DNA sequence: 5'-ACT ACTACATTAAA-3' including all derivatives of that DN'A sequence that are still substantially hom^&^us O 235265 -84- the above sequence and still have the specific regulatory, expression-increasing properties of the starting sequence.

6. A basic chitinase gene obtained from Nicotiana tabacum L. c.v. Havana 425 plants that has the following special structural features in its 5' untranslated region: (a) a transcription start within the CTACT sequence at positions 1967 to 1971; (b) a first possible start codon at position 1980, 11 bp downstream of the transcription start site; (c) a TAAATA sequence ("TATA box") upstream of the transcription start site at positions -28 to -23 of the 5'-flanking region; (d) a CCA ATT sequence at position -114; (e) an imperfect inverted repeat (GCCGAATTCGAGC) comprising 6 bp at position -140; (f) a perfect repeat (ATGTCCAAAC) comprising 10 bp at positions -152 and -228; (g) an imperfect direct repeat (TTTTAACTAAATCTATGTCC) comprising 20 bp at positions -166 and -569; (h) an imperfect direct repeat (CAACTTTCAAAAATTATTTTTTAAA) comprising 25 bp at positions -191 and -217; (i) a palindrome (TAAAATATGAITCATGTTTTA) comprising 20 bp at position -289; (j) a perfect direct repeat (TAAGAGCCGCC) comprising 11 bp at positions -435 and -480; (k) an imperfect direct repeat (TAAAATACACGTCGA) comprising 15 bp at positions -514 and-644; (1) two AATAAA sequences at positions 52 and 120, downstream of the translation stop sequence TAA in the 3'-flanking region.

7. A basic chitinase gene obtained from Nicotiana tabacum L. c.v. Havana 425 plants that has the nucleotide sequence shown in Figure 6.

8. A recombinant DNA molecule that is a chimaeric genetic construction in which a regulatory DNA sequence according to any one of claims 1 to 5 is operably linked to an expressible DNA and to further expression signals active in plant cells so that, on transformation into a plant host, a significant increase in the level of expression of the operably associated expressible DNA is obtained.

9. A recombinant DNA molecule according to claim 8, wherein a spacer s 235265 -85- inserted between the promoter sequence and the adjacent regulatory DNA sequence, the length of the spacer sequence being so selected that the distance between the promoter and the regulatory DNA sequence is the optimum distance for the expression of the associated structural gene.

10. A recombinant DNA molecule according to claim 9, wherein the spacer sequence comprises from 1 bp to 100 bp.

11. A recombinant DNA molecule according to claim 8, wherein the expression signals originate from genes of plants or plant viruses.

12. A recombinant DNA molecule according to claim 8, wherein the expression signals are bacterial expression signals.

13. A recombinant DNA molecule according to claim 11, wherein the expression signals are promoter and/or termination signals of Cauliflower Mosaic Virus genes (CaMV).

14. A recombinant DNA molecule according to claim 12, wherein the expression signals are the expression signals of the nopaline synthase genes (nos) and/or the octopine synthase genes (ocs) from the Ti-plasmid of Agrobacterium tumefaciens.

15. A recombinant DNA molecule according to claim 8 that contains additional regulatory DNA sequences that are capable of regulating the transcription of an associated DNA sequence in plant tissues in the sense of induction or repression.

16. A recombinant DNA molecule according to claim 15, wherein the said regulatory DNA sequences are induced by various internal and/or external factors selected from the group consisting of plant hormones, heat shock, chemicals, pathogens, oxygen deficiency and light.

17. A recombinant DNA molecule according to claim 8, wherein the expressible DNA associated with the regulatory DNA sequence is a structural gene that confers on the transformed plant cells and also on the tissues developing therefrom, and especially on. the. plants, a protective effect against pathogens, chemicals and adverse environmentali|a<?tors. £''i> ✓ To % 235265 -86-

18. A recombinant DNA molecule according to claim 17, wherein the said structural gene is a gene that expresses chitinase in plant cells.

19. A recombinant DNA molecule according to claim 17, wherein the said structural gene is a gene that expresses glucanase in plant cells.

20. A recombinant DNA molecule according to claim 17, wherein the said structural gene is a gene that expresses a protease inhibitor in plant cells.

21. A recombinant DNA molecule according to claim 17, wherein the said structural gene is a herbicide resistance gene.

22. A recombinant DNA molecule according to claim 17, wherein the said structural gene is a gene that expresses a 5-endotoxin from Bacillus thuringiensis in plant cells.

23. A recombinant DNA molecule according to claim 8, wherein the structural genes associated with the regulatory DNA sequence, on expression in the transformed plant cell as such or as part of a unit of higher organisation selected from the group consisting of a tissue, organ, callus, embryo and a whole plant, lead to an increase in the production of desirable and useful compounds.

24. A recombinant DNA molecule according to claim 8, wherein the said expressible DNA sequence is anti-sense DNA.

25. A recombinant DNA molecule according to claim 8 that contains in addition a DNA sequence that codes for a selectable phenotypic marker.

26. A recombinant DNA molecule according to claim 25, wherein the said selectable phenotypic marker is resistance to antibiotics selected from the group consisting of ampicillin, tetracycline, hygromycin, kanamycin, methotrexate, G418 and neomycin.

27. A recombinant DNA molecule according to claim 8 that contains in addition an origin of replication permitting replication in microorganisms.

28. A recombinant DNA molecule according to claim 27, wherein the said ori0n?o^ ** replication is capable of functioning in E. coli, in Agrobacterium or in both. / , I ' Hi 235265 -87-

29. A cloning vector containing a recombinant DNA molecule according to any one of claims 8 to 28.

30. A transformation vector containing a recombinant DNA molecule according to any one of claims 8 to 28.

31. A shuttle vector containing a recombinant DNA molecule according to claim 28 that is capable of stable replication both in E. coli and in A. tumefaciens.

32. A shuttle vector according to claim 31 obtainable from the Ti-plasmid of A. tumefaciens, wherein the chimaeric genetic construction is cloned in between the left border sequence (LB) and the right border sequence (RB).

33. A host organism containing a recombinant DNA molecule according to any one of claims 8 to 28.

34. A host organism containing a vector according to any one of claims 39 to 32.

35. A host organism according to claim 33 or 34, wherein the said host organism is a bacterium.

36. A host organism according to claim 33 or 34, wherein the said host organism is plant material selected from the group consisting of protoplasts, cells, callus, tissues, organs, seeds, embryos, pollen, ovules, zygotes, etc..

37. A transgenic plant, including the sexual and asexual progeny thereof, containing a recombinant DNA molecule according to any one of claims 8 to 28.

38. A transgenic plant, including the sexual and asexual progeny thereof, comprising a recombinant DNA molecule according to claim 17, having a protein content in the transformed cells and/or tissues that is up to 100 times greater than that obtained without the regulatory DNA sequence according to claim 1. ,:^TaFX

39. A transgenic plant, including the sexual and asexual progeny thereof, composing a *\ recombinant DNA molecule according to claim 18, having a chitinase content in th\>A ^® | 0 Til * /<?« ■?/> V & "'t'jr) ,/y 235265 -88- transformed cells and/or tissues that is up to 100 times greater than that obtained without the regulatory DNA sequence according to claim 1.

40. A transgenic plant, including the sexual and asexual progeny thereof, comprising a recombinant DNA molecule according to claim 18, having a glucanase content in the transformed cells and/or tissues that is up to 100 times greater than that obtained without the regulatory DNA sequence according to claim 1.

41. A transgenic plant regenerated from transformed plant material according to claim 36.

42. A transgenic plant according to any one of claims 37 to 41, wherein the transgenic plant is a fertile plant.

43. Propagation material of a transgenic plant according to any one of claims 37 to 42.

44. Parts of a transgenic plant according to any one of claims 37 to 41.

45. A process for the preparation of the regulatory, expression-increasing DNA sequence characterised in claim 1, which comprises isolating the said sequence from the 5' untranslated region of a basic chitinase gene or synthesising it using known measures.

46. A process according to claim 45 wherein the said regulatory DNA sequence is essentially the following nucleotide sequence: 5'- TTGCATTTCACCAGTTTACTACTACATTAAA -3' including all derivatives of that DNA sequence that are based on the mutation of one or more bases but that are still substantially homologous to the above sequence and still have the specific regulatory, expression-increasing properties of the starting sequence.

47. A process according to claim 45, which comprises the following process measures: (a) extraction and purification of genomic DNA from tissues that are capable of expressing chitinase; (b) cleavage of the extracted and purified DNA preparations into fragment i size 235265 -89- suitable for subsequent insertion into a cloning vector; (c) cloning of the fragmented DNA in a cloning vector and creation of a gene library; (d) selection of clones that contain the chitinase gene or parts thereof by means of probe molecules; (e) isolation of those clones which exhibit a strong hybridisation signal with the probe molecule; (f) characterisation of the clones isolated in (e) by means of biochemical processes; and (g) identification and isolation of the regulatory DNA sequence responsible for the increase in expression.

48. A process according to claim 47, wherein expression vectors are used for the creation of a genomic gene library.

49. A process according to claim 47, wherein a known chitinase clone or parts thereof is/are used as the probe molecule.

50. A process according to claim 45, wherein the said regulatory DNA sequence and the derivatives having the same function derived therefrom are synthesised by means of chemical processes.

51. A process for the production of a recombinant DNA molecule according to claim 8, which comprises inserting a regulatory DNA sequence according to claim 1 directly in front of the start codon of a desired structural gene and linking that construction in operable manner to expression signals active in plant cells.

52. A process according to claim 51, wherein a spacer sequence is inserted between the promoter and the regulatory DNA sequence.

53. A process according to claim 51, wherein the said structural gene is a chitinase or glucanase gene.

54. A process according to claim 51, wherein the said structural gene is a gene coding for a protease inhibitor.

55. A process according to claim 51, wherein the said structural gene is a he?b,jcicfeA ? ^ resistance gene. -90-

56. A process according to claim 51, wherein the said structural gene is a d-endotoxin gene from Bacillus thuringiensis.

57. A process for the production of an expression vector containing a DNA expressible in plant cells in operable linkage with a regulatory DNA sequence according to claim 1, which process comprises (a) inserting the said regulatory DNA sequence directly in front of the start codon of a desired expressible DNA; and (b) splicing that construct in operable manner into a known plant expression vector, which may contain a marker gene suitable for transformant selection, between expression signals active in plant cells.

58. A process according to claim 57, wherein the construct is spliced into the polylinker region of the plant expression vector, which region is located between the promoter and the terminator.

59. A process for the production of a transgenic plant according to claim 38, which process comprises transforming the said plant by known methods using a recombinant DNA molecule according to any one of claims 8 to 28.

60. A process for the production of a transgenic plant according to claim 39 which process comprises transforming the said plant using a recombinant DNA molecule according to claim 18 and expressing the inserted chitinase gene.

61. A process for the production of a transgenic plant according to claim 40, which process comprises transforming the said plant using a recombinant DNA molecule according to claim 19 and expressing the inserted glucanase gene.

62. A process for protecting plants against chitin-containing pathogens, which comprises transforming the said plants using a recombinant DNA molecule according to claim 18 and expressing the inserted chitinase gene in an amount sufficient to kill the pathogens or keej^them under control. .... ^ ^ f

63. A process for protecting plants against chitin-containing pathogens, wniah i. to. % J 1 i ' \ ii OUkW»J n 5 -91 comprises transforming the said plants using a recombinant DNA molecule according to claim 19 and expressing the inserted glucanase gene in an amount sufficient to kill the pathogens or keep them under control.

64. A process for controlling chitin-containing pathogens, which comprises bringing the said pathogens into contact with a transgenic plant or parts of that plant that have been transformed using a recombinant DNA molecule according to claim 18 or 19 and that express the inserted chitinase or glucanase gene in an amount sufficient to kill the pathogens or keep them under control.

65. A process for the production of a basic chitinase gene according to claim 6, which comprises the following process measures: (1) extraction and purification of genomic DNA from tissues that are capable of expressing chitinase; (2) cleavage of the extracted and purified DNA preparations into fragments of a size suitable for subsequent insertion into a cloning vector; (3) cloning of the fragmented DNA in a cloning vector and creation of a gene library; (4) selection of clones that contain the chitinase gene or parts thereof by means of probe molecules; (5) isolation of those clones which exhibit a strong hybridisation signal with the probe molecule; (6) characterisation of the clones isolated in (5) by means of biochemical processes.

66. A process according to claim 65, wherein expression vectors are used for the creation of a genomic gene library.

67. A process according to claim 65 or 66, wherein the genomic gene library is screened by means of plaque hybridisation or differential colony hybridisation using a previously isolated cDNA clone as the probe molecule or using immunological detection methods based on identification of the specific translation products.

68. A process according to claim 67, wherein the said cDNA clone is pCHN48 or pCHN50 or functional equivalents thereof.

69. A process for the identification of novel regulatory DNA sequences^which ; \> © 1 ffl C. rn !; f A V?, l>??n o .C O t;•- ()«) -92- comprises using a regulatory DNA sequence or partial sequences thereof according to any one of claims 1 to 5 to identify homologous DNA sequences having the same function by first creating genomic or cDNA gene libraries and investigating them, using the said regulatory DNA sequence as the probe molecule, for the presence of homologous DNA sequences that are capable of hybridisation with that probe molecule.

70. The use of the regulatory DNA sequence characterised in claim 1 for increasing gene expression in plant material.

71. The use of the regulatory DNA sequence characterised in claim 1 for identifying homologous DNA sequences having the same function, which comprises creating genomic gene libraries and investigating them, using the said regulatory DNA sequence as the probe molecule, for the presence of homologous DNA sequences that are capable of hybridisation with that probe molecule. ciba-geigy ag BY THEIR ATTORNEYS BALDWiN, SON & CAREY ) </div>