AU641373B2 - Wound-induced DNA sequence from solanum tuberosum and its use - Google Patents

Abstract

Wound-induced DNA and part thereof, from Solanum tuberosum, use therof for the expression of gene products in higher plants in the case of wounding or pathogenic diseases, DNA-transmitting vectors containing them and plants or parts of plants containing them. The promotor part of this DNA sequence can be used in higher plants for the expression of gene products in the case of wounding and/or pathogenic diseases.

Description

P/00/011i8lM Regulation 3.2

AUSTRALIA

Patents Act 1990 ORIGINAL 6 1 7 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: 0CA0 ze1wel fr-- The following statement is a full description of this invention, including the best method of performing it known to me:- 2 The invention relates to wound-stimulated DNA from Solanum Tuberosum as well as parts thereof, the use of such for the production of gene products in higher plants using wound or pathogen attack, DNA-propagation vectors containing the same, and plants or plant parts containing the same.

It is known that the mechanical injury of plant tissue can cause morphological and physiological changes. Thus, the activity of various enzymes increases after the wounding, for example of phenylalanine-ammonialyase and peroxidases in potato tubers, extensin in the storage tissue of carrots, fatty acid synthetase in potato tubers and proteinaseinhibitors in tomato and potato leaves. For some of these enzymes, the increase in activity is associated with increased mRNA amounts. One of the best understood wound-induced genes is the prote.nase-inhibitor in tomatoes and potatoes; in wounded leaves, the mRNA for this gene is significantly increased.

The wound-induced development of genes in plants is of fundamental interest, because in this area the promoters that become active are capable of specifically activating the corresponding structure gene after wounding. Promoters of this kind in connection with other genes which, for example, code for resistance, or code substances that are effective as antibiotics, or promote the healing of wounding, are naturally of great economic interest, if they can be inserted into the genetic material of other plants.

It has now been found that relatively high concentration of induced mRNA are specifically present in wounded and/or microbially attacked potato plants.

-3 This mRNA can be isolated and characterized. It d..oes not appear in healthy, uninjured plants.

Accordingly, the invention has as an object a DNA-sequence from Solanum Tuberosum, which is stimulated through wounding and/or infection with pathogenic microbes, as well as the promoter part and the structural gene part. Said DNA-sequence corresponding essentially to: 1 AAATTTTATACATACATACAAACACAATCATTGATACATAACAA

TGATGTACATATATACAATTATCTAACCGATATATATATATATATATATATATATATATA

*105 TCACCTcTCTCCACTCTCTGCCCTCTCTTCATTCTCTCCCAATCTCGCTCACCACTCTAG 165 :225 TACCTrATTTATATAAATCGCCGTGATTTATACAAATCAGATGCTCCATAACAAACATAA :285 TTTGACCCATGGAGTGCATATAACAAAATTGTAGCTATAGAACCCTAATATGTTTTTCTT 345 AATCTTTGTTATTCCTAAAATTTACTCATAATAATACTCTTTATAAAAGCATAAGCTGGT 40~5 TTGGTTTAGGGTTAGAGTATTOI'CTAAAAATTCTAATTGAAATCAA.ATACATCTTATAGA 465 A TCCAAATTAGAATTGAACACGTCTTGTAGAGTCCCATAAATTTTTAATGTCTACAATGT .525 AATATCGTTAAAATATTTTAATATCTTGTTGAATTAATTTTTT:,TTTAGTAAAATAAT 585 ATGAGAATTAATTTT7TTTATTAATCTTCAC.~ACT.'7GATTTTTTTAAAATTTCA7GTAA **645 ATATATGGGCTAAGATGTGAGCCAACTGCTCAAACTCAAAAGTTAGTCGAGT77CAAT 705 GAAGTTAAAATTAAAAG-rATTGTTGTCATAACTCATATGTTGCAAGTTGCAACTC.TGTGT 0: a.

*765 ATAAGGTCAAAAAAGGTATGCTTGAAAGTTGAAACTTTAG ATATGACGATCATCTTCGTG 825 GGCCCTACCTAAAATAAAACGTCTCTTCATCATCCGAATATCACATCATCACGTAATCCC CGAGCACGTOGAA'rGGCGCGTAAATATrCATGrCGCCCTTTAAACCTAAATACACC7ACTA 945 TTACAATT ACTCACGCCZM CCAACC.C 1005 CTTTCTTCATTACCTACCATACCTTCTTCTCTCATCCTTCATAGCTAATAATATCATC TT 1065 CTGT-TTTACTGAACTGGCTAACTCTCAGTTAACTCTGGAGGAAACAACAAACAAAGGGG 1125 TGGTGCT :GCCCTATATGAAGCCTTGAGCTCACACGACGTCGTTCAGGTCCAGAAAcTCC 1185 TGGCCTCCGACCTCGAGTGGTGGTTCCATGGTCCTCCTTCTCATCAATTTTTGATGCAAA 3a mo I 1245 TACTCACCGGCACTG;CTAAATTCGATAACGCCrCTTTTCAATTCCTTCATAAGACCATTG L T G T A K F D NA S F Q F L H K T I D 1305 AC0TATTC:GGTTCCOTTGTTCTCDTCGAAGGC7 GTGACCCGACCCGATCTATTACTTGGG V F G S V V L V E G C 0 P T R S I T W V 1365 77,CACGCCJTGGACTCTTACGGATCZ;CGTAATTACCCAGGTTAGGGAGTATTTCAATACCT A W T V T D 0 V I T 0VR YFNTS 1425 CACTTACTGTC,\CCCG;TTTTGGGAAATCGC;ATATTTCCTCAATTACGACTCTGCATrGcC L T V T R F G K S D IS S IT T L H C P 1485 CATCTGTTTGGGAGAGTA'ZCTTACCAAATCGGGTCGGAAAATCTGTTCCGGGTCTTGTAT V W E S S L~ F N R V G K S V P G L V L TGGCTCTATAGAACGCCCCA TTGTGGCT GCGTTATATCTTGTGTCTAGTAGGATG L END 1605 TAAGATTAACGCGGCTGGTTTGG ,ATTCTGT-tGCTGrLTGTTTGGTTTGTTTTTATTTTTT 1665 AAGTTGGGATTTGTGTTCTr, TATTATTTGTTGTTG2 IATTGAGTTAAAGAAGGGGCCA 1725 ATGATAATTGGATATACCTTTCTACTCAATAGATTGTCTAATTATGTGTTGGTTTGCM- ~1785 AGCATATTGATTGCTGTTTACAAAATGTGTATATTTTTCAATTTGGTATT0CTTC IB145 T-TGTTTTCAACGAATGA CGATGGATACCGDGAAACAATTTCATT0AAAGGCAAATCTTA 1905 TTATTAATrGATGGAATCAGAATTTTTATTATGAGGTTTAAATAATTAAACATATAAAATA *1965 GTTAAATGAGGTTCGAAATCTATATAATGCATAAAAAAATAATTTTAACTATATATAAAA 2025 ATGTATAATTTTTTTCTGAGAATAGGAGGAACAATATATTTAAGAAGGAATCTTTGCTT a a.

a.2085 GCTTAGGATAGCTTCACATCATACTTTTCCCACTACATAGGTAATAGGAAGGCATATGCT a2145 CTATTCCTTcATAACTTACTCTTGGTATTTCTTATCCTAATCGCCAAAAAAAGAGAGrAT 2205 AATTTTTPATTTATAACACACTTTTTTATTTCCTATACAAGA.ATAAGTTGATTTTTCCATT 2265 TAAGTATAAACATCGAAACTTTTAGAGCGACTCTATAAGAGACTGCCCAATTTCATTGG 2325 ATTCTACAAGATATTTTCATTGATTCTTGGAGACTACTTAATC1T 2368 3b Further, the invention has as its object the use of this DNA-sequence for the expression of gene products in higher plants after wounding and/or pathogenic attack.

Furthermore, the invention has as its object DNA-propagation vectors into which DNA-sequences have been inserted, such sequences being defined above.

Finally, the invention has as a further object plants or plant materials which contain such a DNA-sequence.

The expression "transcriptionally active region" of a gene, of its promoter part and structural gene part, is to be understood to refer to those nucleotide-sequences which are unconditionally necessary in the promoter 9 o region of the gene for the activation of the structural gene part and in the structural gene part for the expression of an effective or active gene product.

The expression "structural gene" is to be understood to refer not only to the DNA-sequence from SSolanum Tuberosum in systems homologous to that herein described but also structural genes from other sources, thus from heterologous systems. An example would be the CAT-, NPT- and GUS-structural genes, which can be fused with the wound-stimulated promoter described herein, and can be used for the development' of the respective structural gene in potato or foreign plants.

I I 4 The propagation vectors can be provided by any suitable DNA-molecules which can be introduced into the final bacterium. Typically, these would be plasmids as are more fully described in the disclosure. The insertion techniques are known to the person skilled in the art.

The bacteria for the uptake and release of the propagation vectors can be constituted by bacteria of the genus Agrobacterium, in particular Agrobacterium tumefaciens and Agrobacterium rhizogenes.

EP-A 122,791 describes a DNA-propagation vector which contains T-DNA with a plant gene inserted therein. This plant gene consists of a plant promoter and a plant structural gene, wherein the plant promoter is joined to the 5'-end of the plant structural gene, and the plant structural gene is located behind the plant promoter in the transcription direction. EP-A 122,791 provides a detailed description of processes and steps for the insertion of genetic material in DNApropagation vectors, to which reference is now expressly made.

Research has shown that Erwinia-bacteria are latent in potato plants (Solanum tuberosum) and that they will preferentially attack the stem and the tubers of potatoes only after tissue has been wounded. This was induced in potato tubers of the species Datura, by slicing the tuber into discs and simultaneously incubating with Erwinia carotovora of the species atroseptica. As expected, these tubers exhibited a severely macerated surface after eighteen hours of incubation. The isolation of mRNA from the tissues lying under the maceration was not possible with standard methods. The reasons for this were a high contamination of the RNA with polysaccharides, poor precipitation and redissolvement of the RNA, a high level of RNA degradation, a small yield of RNA and substantial labor ard material expenditures. By variations and new combinations of existing methods, however, it was possible to develop a technique by which large amounts of non-degraded RNA cou.d be obtained with relatively small material and time expenditures, even from tissues containing polysaccharides. (Logemann et al, Anal. Bioch. 163, 16, 1987).

On the basis of cleaned polyA+RNA from wounded tubers of the potato species granola, incubE' ed with Erwinia, a cDNA-bank was built up. Using diLferential colony hybridization techniques of 4000 cDNA-clones were hybridized with radioactively labelled RNA from nonwounded and Erwinia-wounded tubers. Two clones were identified, here named wunl and wun2, of which the complementary mRNA were induced by wounding of tubers of various tetraploidal potato species as well as the haploidal species AM 80/5793. Wunl-mRNA accumulated within thirty minutes after mechanical wounding of a tuber, and is thus involved in the primary processes induced by wounding, whereas wun2 was induced 3.5 hours after wounding. Twenty-four hours after wounding, both clones showed even higher concentration of m-RNA. If in addition to the mechanical wounding, the tuber is incubated with Erwinia carotovora of the species atroseptica, no alteration is found in the expresion pattern. Altogether, the expression studies with tubers reveal that wunl-mRNA and wun2-mRNA represent genes which are not Erwinia-specifically induced, but are stimulated by all processes which result in the destruction of the tuber tissue.

6 Wunl-mRNA accumulates in large quantities in wounded potato tubers (quantity-wise comparable with proteinase-inhibitor II-mRNA in non-wounded potato tubers, compare Sanchez-Serrano et al, Mol. Gen. Genet.

2032, 15 (1986)); whereas in non-wounded tubers, it is not detectable. It first appears thirty minutes after wounding with a maximum between the fourth and twentyfourth hour after injury. In smaller amounts, it was also detectable even after forty-eight hours.

The accumulation of wunl-mRNA and wun2-mRNA after wounding is not limited to the tuber, but also takes place in the leaves, stems and roots of various tetraploidal potato plants with comparable kinetics and intensity.

In contradistinction to wun2-mRNA, wunl-mRNA is induced in leaves also in the absence of wounding, when the leaves are sprayed with compatible phytophtora infestans spores. This result shows that wunl can be induced not only through mechanical wounding, but also through the presence of (fungoid) pathogens.

There were differences in the development of the wunl-gene, according to whether the tubers were wounded under aerobic conditions (tuber slitc yere sprayed with P-buffer and incubated for eighueen hours, then exposed to the air) or were wounded under anaerobic conditions (tuber slices were dipped in P-buffer and in this oxygen-starved condition incubated for eighteen hours). wunl-mRNA expression was significantly higher in aerobically wounded tubers than in the corresponding anaerobic situation.

For the use of promoters for the expression of genes in accordance with the invention, it is important 7 whether the detectable wunl-mRNA or wunl-mRNA arises through a new synthesis (transcriptional regulation), or is constitutively expressed and stabilizes only in the case of a wounding (post-transcriptional regulation).

"Run-off" transcription tests were able to demonstrate transcriptional regulation for both wuni and wun2, which means that the promoters of these genes are responsible for the new synthesis in case of an wounding.

Using known techniques, a series of wunlhomologous cDNA-clones were obtained from the tetraploidal potato "granola", from which some of them were sequenced. By cloning wunl-25A2 in M13mpl9-phages in both orientations and partial exonucleaseIII digestion, the nucleotide sequence was analysed in both directions according to the dideoxy method.

Figure 1 shows the relative sizes of the clones used for sequencing. The clone wunl-25A2 has been cloned in two orientations in the PstI-site, which, in respect to the position of the asymmetric XhoI section position are called 24A2*MP19 and 25A2MP19.

Using successive exonucleaseIII-digestions, it was possible to obtain deletion clones of different sizes, which 7ere available as clone 25A2*MP19delta 1-3 and 25A2MP19delta 1-4 for sequencing.

Figure 2 shows the nucleotide sequence of the cDNA-clone-wunl-25A2. Alternative open reading frames are underlined.

The size of the cDNA-clone-wunl-25A2 is 711 bp. At the 3-end is a poly-A tail of 14 bp (position 697-711). The largest open reading frames extends 8 over 105 aminoacids (position 121-438). Further, in position are two short open reading frames which code for three amino acids in position 23 and code for eight amino acids in position 95 (underlined). The last two mentioned open reading frames differ however from that which codes the 105 amino acids. By referring back to the genomic sequence data (Figure 6) the reading frame coding for 105 amino acids is related to the wunlprotein. The translational start point of the wunlprotein TTTTTGATGCAA fits only to a lower extent the consensus sequence for plant translation start TAAACAATGGCT, as reported by Joshi, Nuc. Acids Res. 6643, (1987).

Investigation of the genomic organization of the gene coding for wunl-mRNA in the haploid potato line AM80/5793 shows that this gene is available in only a limited number of copies. The hybridization of radioactively-marked wunl-cDNA against HindIII-, PstIand EcoRI-digested leaf-DNA leads, in the "Southern Blot" analysis, to the definition of one strong and one or two weak bands. The result of the "Southern Blot" analysis of wunl- and wun2-cDNA is illustrated in Figure 3.

For the haploid potato line AM80/5793, the "Southern Blot" analysis was able to identify only a few genes responsible for the development of wunl- and wun2mRNA. The likelihood of obtaining an active gene and no inactive pseudogene was particularly high here.

The presence of only one defined band in the "Southern Blot" of the haploid potato line points to a limited number of genes per genome. If this is considered along with high and wound-specific expression 9 of wunl-mRNA in the haploid line and transcriptional regulation, highly active genes become likely.

To build up a genomic bank, 10 micrograms of EcoRI-digested DNA from AM80/5793 was ligated with EcoRI-digested EMBL4-arms, and plated out on a C600bacteria-conteining medium. Approximately 500,000 plaques were obtained which, considered statistically, represent the genome of the potato. After the transfer of these plaques to nitrocellulose, plaque hybridization techniques with radioactively labelled wunl-cDNA permitted the identification and purification of the genomic clones wunl-22 and The isolation of recombinant EMBL4-DNA from wunl-22 and wunl-85, as well as the restriction mapping and hybridization thereof with radioactive wunl-cDNA gave the following organization.

The wunl-cDNA-equivalent fragment in has a size of 4 Kb. This exactly corresponds with the fragment size (hybridizing with wunl-cDNA) of EcoRIdigested DNA from the haploidal potato. On the basis of the asymmetric XhoI-site in the 5'-region of the cDNAclone, the orientation of the gene in the 4 Kb fragment could be established. According to this, a roughly 1 Kb sized promoter region lies upstream of the wunl-gene, whereas a rougly 2 Kb sized non-homologous portion is located 3' from the gene. The 8 Kb-large EcoRI fragment, also contained in wunl-85, could not be used for further analysis of the wunl-gene. On the basis of the total digestion of the potato DNA with EcoRI, it is likely that during ligation, two fragments that did not belong together were inserted into the EMBL4-vector, these fragments being not functional related with each other.

Figure 4 shows the arrangement of wunl in the genomic clone wunl-85. Located on the 4 Kb-large EcoRI fragment of the genomic clone wunl-85 are 1.0 Kb of wunl-promoter, 0.8 Kb of the wuni gene and 2.0 Kb of the 3'-end.

Sinice in the restriction pattern no differences emerged between wunl-85 and wunl-22, for further analysis the 4 Kb fragment of wunl-8F, was ligated intu EcoRI digested pUC8 (see Figure For the sequencing of the wunl promoter as well as the wunl gene, there followed a further recloning of the 4 Kb fragment in the EcoRI site of M13mplB. The determination of its orientation was carried out with control digestions using at the asymaetrically located XhoI-site. The clones 85*mpl8 and 85mpl8 represent both orientations of the fragment. The digestion of these plasmids with SphI and XbaI made it possible, with the help of exonucleaseIII, to successively digest the 3'end of the wunl-gene of the clone 85mp18, and the of the wunl-gene of the clone 85*mpl8.

Figure 5 shows the result of the deletion analysis of clone wunl-85 in schematic form.

The resulting deletion clone with different fragment sizes was used for the sequencing. Generally, it was possible to sequence the entire wunl-gene bidirectionally, and, in addition, to analyze about 400 bp of the 3'-end unidirectionally.

Figure 6 shows the nucleotide sequence of the wunl promoter and gene from wunl-85. The CAAT-box, TATA-box and Poly-A signal are indicated, and the transcriptional start- and stop site are marked with J 11 arrows. In order to identify the prerie transcriptional start site of the wunl-gene, the method of S1-nucJease-mapping was used. Figure 7 shows the determination of the size of the Sl-protected fragment of the wunl gene. The hybridization with wunl-mRNA of the region in pLSOOO lying 5' from the XhoI site leads to a 162-179 bp long DNA-RNA-hybrid which is protected from the singla-strand specific Sl-nuclease.

The actual transcriptional start site thus lies 162-197 bp 5' from the XhoI section location (A,C,G,T th'i sequence arrangement for the sizing the DNA fragment).

If one begins with the longest obtained fragment, then the transcription start begins 179 bp upFtream from the XhoI-position, i.e. with the sequence ACCATAC. This sequence agrees fully in the central region (CAT) with the concensus sequence or transcription start CTAATCA, discovered by Joshi et al (1987). From the position of the transcription start, further information is available (see Figure 6): Atpositian 33, as seen from the transcription start, a TATA-box C-ATATATT is found which agrees well with the concensus sequence TCACTATATATAG, as described by Joshi et al (1987).

The CAAT-box in the region between -60 and described by Benoist et al, Nuc. Acids Res. 8, 127-142 (1980), is found in position -58 (CAAACT) in the wunlpromoter.

The 5'-untranslated region of the wunl-gene has a size of 217 bp and, therefore, is relatively large.

The wunl-mRNA coded gene is 794 bp in size, which 12 very closely corresponds to the size of wunl-mRNA, on the basis of the "Northern-Blot" analysis.

97 bp of the 5'-untranslated region of the wunl gene are missing in the cDNA-clone, wunl-25A2.

Aside from the open reading frame already determined in the cDNA-clone, no others were found in the 5'-untranslated region of the genomic clone.

The wunl-gene contains no introns.

Figure 8 shows the arrangement and position of important regions in the wunl-gene. The wunl-promoter is marked in black, while the wunl-gene is hatched. The important recognition sequences are boxed. The mRNA is shown by a sinuous line, whereas the protein coding region is marked with Xes. The sizes of the individual regions are given in base pairs (bp).

Figure 9 gives the sequence comparison between the cDNA-clone wunl-25A2 and the genomic clone 2368 bp of the genomic clone wunl-5 were compared with 711 bp of the cDNA clone wunl-25A2. Homologous base pairs are marked by a vertical line. The missing nucleotides are shown by a period. The two arrows indicate the sequence of two 10-bp direct in the cDNAclone.

Comparison analysis shows that the sequence of the genomic clone wunl-85 of the haploid potato AM80/5793 is up to 97% homologous with the sequence of the cDNA-clone wunl-25A2 of the tetraploidal potato "Granola". Altogether, 11 base pair changes were identified, of which five were located in the translated region. All five base pair changes in the translated 13 region resulted in no alteration of the amino acid sequence. The additionally sequenced 400 nucleotides downstream of the transcription stop exhibit no remarkable regions.

The gene codes for a protein of 12000 Dalton, which size can be approximately determined in hybridreleased-translation-experiments. Computer evaluation relative to the amino acid combination of the wunl protein indicates that a very hydrophilic proteii. The amino acid sequence is seen in Figures 2 and 6.

The size of the wunl-protein, its hydrophilic characteristics, the wound-inducibility in various tissues and the inducibility by pathogens, can be concluded on the basis of the fact that it belongs to the PR-proteins. The name PR- or "Pathogenesis- Related"-protein includes proteins of various plants which are inducible, inter alia, by pathogen attack, and in some cases show Chitinase-activity or Glucanaseactivity, i.e. the activity of enzymes which can destroy the cell walls of the fungus.

Using the agrobacterium-transformation system in transgenic tobacco plants, expression studies were possible on mRNA-level, since tobacco-mRNA crosshybridizes only very weakly with wunl, and moreover does not possess any wunl-homologous 5'-ends, as can be gathered from Sl-nuclease-mapping. Actually, a large quantity of approximately 800 bp-large wunl-mRNA was detected in transgenic tobacco plants in wounded leaves, which on the one hand shows an active wunl-promoter, and on the other indicates the use of the correct transcription start point.

14 A 4 Kb-fragment from pLS000, containing the wunl-promoter, the complete wunl-gene, and 2 Kb of the 3'-region of the gene (wunl-wunl), was cloned (pLS001) with its EcoRI-sites in the mobilization vector pMPK110, which is necessary for the Agrobacterium tumefaciens (At) transformation. After transfer to At3850km, the tobacco line Wisconsin 38 (W38) was transformed with this Agrobacterium on leaf discs. The small callus appearing some three weeks after Kanamycin selection was regenerated to the complete plant (LS1) through the use of a shoot-induction medium.

Aside from the capability of growing on Kanamycin, plants transformed with 3850Km have the capability to synthesize nopaline, a property which distinguishes them from untransformed plants (Zambryski et al, EMBO 7. 1 147-152 (1985)). Wunl-wunl transformed plants, here called LSl-plants (4 LS1-4; 6 LS1-6), were characterized by a high level of nopaline. By contrast, no nopaline was detectable in untransformed plants. Kanamycin-resistant and nopaline-positive LSlplants were transferred to the greenhouse and analyzed on the DNA- and RNA-planes.

Figure 10 shows the construction of wunl-wunlfusions and wunl-CAT-fusions which were utilized for expression studies on the basis of transient expression and stable transformations.

The creation of the plasmid pLS001: Beginning with the plasmid pLS000 (see Fiaure the 4 Kb-fragment which contains the wunl-promoter, the wunlgene and 2 Kb of the 3'-end was cloned (pLSO01) within the EcoRI-site of the vector pMPK110.

Creation of the plasmid pLSO11: The wunl-promoter, as well as 179 BP of the untranslated wunl-gene region, were fused (pLS010) at the XHOI site with the CAT-gene inclusive 3'-end.

Subcloning in the vector pMPK110 using PstI (pLS012 has the advantage that this plasmid can be used for transient studies in protoplasts and additionally can behave as a mobilization plasmid for the transfer of wunl-CAT-construction into the Agrobacterium 3850Km.

The "Southern Blot" analysis of EcoRI-digested DNA from transgenic LSl-tobacco plants and untransformed W38-tobacco plants resulted in a positive hybridization through the use of 32 P-radioactively labelled wunl-cDNA probe, both in the DNAs of untransformed plants and the DNAs of transformed plants. In both cases, a strong band and weak band were recognized. However, in the transgenic LS1-plants, there was additionally a 4 Kblarge band, of which the size exactly corresponded to the size of the EcoRI-insert from pLS000. By comparing different DNA amounts, there appeared a copy number of two to five of wunl-wunl in all tested LS1 plants. In some LS1 plants, there were found additional bands in high molecular regions, probably due to rearranged DNA or undigested DNA.

A functional analysis of the wunl-wunl-DNA of positive LS1 plants was possible on the RNA-level. Both in wounded as well as in non-wounded, untransformed tobacco plants, there was only a weak crosshybridization with a wunl-cDNA-probe. Additionally, testing was carried out on isolated RNA from untransformed tobacco plants against the 5'-region of the wunl-gene containing the 5'-untranslated region and the wunl-promoter (analogous to the fragment for determining transcription start for the wunl-gene) using Sl-analysis on homologous regions. This showed that no wunl-homologous sequences were present, either in nonwounded or in wounded untransformed tobacco leaves or stems.

Out of 30 nopaline-positive LS1 plants studied on the RNA-level, the "Northern Blot" experiment detected 5 plants with high levels of 800 bp-large mRNA hybridizing with wunl. An S1-analysis of the latter led to evidence of two equally large DNA fragments, coriesponding in size with fragments from wounded potato leaves. This was the proof to be wunl-homologous mRNA.

Several indicators supported the idea that wunlhomologous mRNA is expressed to counteract large-sc.±le TMV attack.

One goal of the invention is the construction and isolation of genes that have the ability to express fused proteins having the desired qualities. For this, it is necessary that the wunl-promoter be solely responsible for the expression of the 3'-fused structure gene, which is not always the case prcteinaseinhibitor II).

Transcriptional fusions, consisting of the wunl-promoter and 178 bp of the 5'-untranslated gene, were fused with various marker genes (wunl-CAT, wunl- NPT, wunl-GUS), proof thereof being possible through radioactive or fluorimetric methods. (GUS is the name for B-Glucuronidase (Jefferson et al, Proc. Nat. Acad.

Sci. 83, 8447-8451 (1987)).

The wunl-CAT construction was tested for transient expression in potato protoplasts. This method made it possible to analyze promoter activity within a 1 I 17 few '.ays. For this, highly purified plasmid-DNA containing the construction is transferred into the protoplasten by known methods, remeins there in stable condition and can be transcribed and translated. The requirements for expression is that the promoter is functional, and the inducing factors are present in protoplasts handled in this manner.

In these tests, the wunl-promoter, including 179 bp of the 5'-untransiated region, was transcriptionally fused with the Chloramphenicolacetyltransferase-gene (CAT). The construction of the wunl-CAT-fusion is shown in Figure The 35S-promoter of the pRT101-CAT plasmid was removed using HincII/XhoI, and instead of this, the Kb sized wunl-promoter together with 179 bp of the untranslated region was inserted using a filled-up EcoRI-site and Xhol. This chimeric gene wunl-CAT was able to be inserted into the PstI-site of the vector pMPK110 (pLS011).

Large amounts of endogenous wunl-mRNA were detected in the potato cell suspension used for transient expression experiments, as well as in the protoplasts derived therefrom. It was concluded that the liquid-cultivation of cells, and in particular the protoplasting step, produces a high stress in the cell, which is indicated by a large yield of wunl-mRNA, analogous to tae process in the potato tuber.

For the transient analysis of the wunl-CAT construction, 1 million protoplasts were isolated out of a suspension culture of potato stems of the variety 18 Datura (D12), these being then transformed using the CaCl 2 /PEG-method by adding 20 micrograms pLSO11-DNA.

The CAT-analysis of such protoplasts transformed with pLSO11-DNA showed high CAT-activity. A parallel test with a CAMV-35S-CAT-construction promoter from the cauliflower-mosaic virus (CAMV)) (pRT101-CAT) showed approximately the same CAT activity, whereas in a control test without the addition of DNA, no CAT-activity was detected. This means that, in a transient potato protoplast system, the activity of the wunl-promoter is comparable to that of the and thus must be classified as very high. Up to the present, the CAMV-promoter stands as the strongest known promoter in plants.

The plasmid pLSO11 (wunl-CAT), already used in the transient system, was also used further for the transformation of At 3850K. 3850Km::pLS0Ol Agrobacteria were used for the transformation of the tobacco species Samsum NN (SNN) using the method of leaf disc infection, and the resulting callus were regenerated to plants (LS2).

By the use of this special tobacco species that is resistant to tobacco mosaic virus (TMV), the (suspected) inducibility of wunl through TMV should be excluded. In LS2-plants, which were nopaline-positive and kanamycin resistant, tests were made on the DNAlevel and for the presence of CAT-activity. Pstldigested DNA from wunl-CAT transformed plants exhibited a 2.2 Kb sized fragment hybridized with CAT-cDNA. This exactly corresponded to the expected fragment size of wunl-CAT. No hybridizaticn could be detected in untransformed plants. By comparing DNA-amounts 2 to 4 I I 19 copies per haploid genome were found in all positive plants.

The functional analysis of LS2-plants continued with the measurement of CAT-activity in greenhouse plants and in plants of the Fl-generation grown in sterile culture. LS2-plants raised in the greenhouse showed in their non-wounded leaves a slightly increased CAT-activity, in comparison with the background activity of untransformed tobacco leaves. In wounded leaves an approximately 4-times higher CATactivity was noted by comparison with non-wounded leaves. However, if an excess of boiled-down potato tuber extract is added to the wounding condition, the increase is 6-times higher than in the non-wounded state. This effect was independent of the condition of the tuber prior to extraction (non-wounded or wounded tubers taken either fresh from the ground or from storage). Each extract had the same inducing effect, pointing to the permanent presence of an inductor in the extract.

The cause of the relatively low inducibility of wunl-CAT in transgenic tobacco plants is to be seen in the activity of non-wounded leaves. In order to eliminate possible causes determined by the greenhouse, the seeds of LS2-plants were harvested, and then germinated under sterile conditions. In young leaves of kanamycin-resistant plants, a very weak CAT-activity was measurable in the non-wounded condition, this being not much different from the CAT-activity of untransformed leaves. In wounded leaves, the CAT-activity rose at least by a factor of 40. The addition of boiled-down extracts from homogenized potato tubers to the wounding condition led to a 60-tines higher accumulation of CAT.

Further, it is to be noted that with increasing age of the plants, the activity of the wunlpromoter in the leaves became higher and higher, such that the strength equalled that of a transgenic CAMV- CAT-plant, There is a possibility that the senescence is also capable of inducing wunl.

With the help of the transient system, further tests were made to determine in which plant species the wunl-promoter is active, and correspondingly usable. It turned out that this promoter is not only active in potato protoplasts, tobacco protoplasts and parsley protoplasts, but also showed high expressior in rice protoplasts similar to the activity in its homologous system.

Figure 11 shows the creation of wunl-NPTII constructs. Beginning from plasmid pLS001, the wunlpromoter is cloned including 179 bp of the untranslated region at EcoRI-XhoI-blunt of an NPT-II gene (Neomycinphosphotransferase-II). For improved handling in transient experiments, the wunl-NPT-II fusion was recloned in a high copy plasmid (pLS 020).

Using the wunl-NPTII construct, NPT-II activity was found in tobacco and in parsley protoplasts. Interestingly, wunl-dependent NPT-II activity was also detected in rice protoplast (Oryza sativa japonica c.v. Taipai), this activity being comparable in its intensity with the wunl-NPTII activity in potato protoplasts. Comparable transient expression of wunl-NPTII and NOS-NPTII (pGV1103) in rice protoplasts likewise showed similarly high values. In this connection, transient expression in potato protoplasts, similar to CAMV-35S-CAT were again indicated. Generally speaking, these studies indicate i\ T 21 that the wunl-promoter is capable, at least to some extent, of very high activity, both in homologous and in heterologous systems. Neither in parsley-protoplasts nor in tobacco-protoplasts nor in rice-protoplasts could there be found mRNA hybridized with wunl. Thus one must assume some factor stimulating the wunl-promoter, the factor being equally available in all tested plant species.

Generally speaking, all important DNA-regions are available in the 5'-region of wunl, in order to be able to express a subsequently fused gene in large quantity after wound-induction.

Additionally, the functionality of wunl-CAT and wunl-NPTII in diverse transient systems (potato, parsley, tobacco, rice) as well as in stable transformed tobacco, leads to the assumption of a similar molecular background.

Within the framework of the above invention, investigations further revealed that it can be advantageous to fuse the wunl-promoter of this invention with other promoters or enhancing elements.

An example is the fusing of the wunl-promoter with the known TR-promoter of the Agrobacterium tumefaciens TR-DNA.

The isolation of this TR-promoter from the Agrobacterium tumefaciens TR-DNA is known from the work of J. Velten and J. Schell "Selection-expression Vectors for use in Genetic Transformation of Higher Plants", published in Nucl. Acids Res., 13:6981-6997. From this literature reference, it is also known that this (j 22 promoter is responsible for the expression of Mpnnopinesynthase, and shows the following characteristics: It is bidirectionally active, i.e. if structural genes are fused to both sides, both can be read off in the same regulation manner and intensity. In this connection, reference should be made to the following: Langridge, W. H. Fitzgerald, Koncz, C., Schell, J and Szalay, A.A. (1989). "Dual promoter of Agrobact Tumefaciens Mannopine Synthase Genes is regulated by Plant Growth Hormones". Proc. Natl. Acad.

Sci. 86:3219-3223.

The orientation of the TR-promoter has been determined as 1' and 2'.

In transgenic plants, the TR-promoter can be induced by wounding. The fusion of the TR2' with the marker gene beta-qalactosidase (beta-gal) leads to a higher beta-gal-a,=tivity in wounded transgenic tobacco plants than in non-wounded plants.

The activity of the TR2'-promoter is primarily found in the leaf veins, thus in the vascular system of the leaf. For 2. and 3. reference is had to the following: Teeri, Lehvaslaiho, Franck, M., Uotila, Heino, Palva, Van Montagu, M. and Herrera-Estrella, L. (1989). "Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants". EMBO, 8:343-350.

The characteristics of the TR1'-promoter have not, up to now, been very intensively researched. However, it is krown that the TR1'-promoter is approximately 4-7 times weaker than the TR2'-promoter, although aside from this both of the promoters have similar characteristics.

*t r 23 Within the framework of researches aimed at a qualitative and quantitative optimization of the wunlpromoter, the TR-promoter was fused with the wunlpromoter, specifically in the orientation TR1'-wunl (Figure 12).

As evidence of the promoter activ.ty, the marker gene GUS (beta-Glucoronidase) was used (TR1'wunl-GUS). Using the Agrobacterium tumefaciens transformation system, this construction was transformed into tobacco and analyzed. The following characteristics distinguish this tobacco plant from wunl-GUS transgenic tobacco plants: 1. The tissue specificity had changed.

In wunl-GUS transgenic tobacco, the GUS-activity was found primarily in the epidermis of leaves and stems.

In TR1'-GUS transgenic tobacco, the GUS activity was primarily localized in the vascular system. There is thus the possibility, depending on the scientific requirements, to direct gene products to different locations of the tissue. As against this, the woundinducibility of TR1'-wunl-GUS transgenic tobacco plants is comparable with the wound-inducibility of wunl-GUS transgenic tobacco plants.

2. Generally, the activity of the TR1'-wunl-promoter in transgenjc tobacco leaves and tobacco stems is approximately 10 times higher than the activity of the wunl-promoter. Hence, the TR1'-wunl-promoter appears to be among the strongest promoters available for gene expression in plants.

3. While promoter activity in transgenic dicotyl plants currently presents little problem, there have been few 24 promoters up to now which are fully active in monocotyl plants (to which the commercially important grain plants belong). Transient development studies in rice protoplasts (these serve as substitute for studies in transgenic rice-plants, which are very difficult to obtain) show that the TR1'-wunl-promoter has roughly times higher activity than the wunl-promoter. This is all the more remarkable because even the which is so strong in dicotyl plants is here 30 times weaker than the TR1'-wunl-promoter.

Thus, the wunl-promoter, according to the invention, can, for example, be altered or optimized in the following characteristics, through fusion with the TR1'promoter: The specific activity of the wunl-promoter in the leaf or stem epidermis of transgenic tobacco can be targeted to the vascular sys'em through fusion with the TR1'-promoter; The capacity of the wunl-promoter for being induced through wounding is not changed through fusion; The TR1'-wunl-promoter is approximately 10 times stronger than the wunl-promoter alone, in transgenic tobacco leaves and stems; In transient experiments with a substitute for monocotyl plants (rice), the TR1'-wunl-promoter is roughly 30 times stronger than the wunl-promoter.

In accordance with the invention, for example, the expression of the wunl-promoter through fusion of the same with an enhancing element of the promoter can be increased. Thus, for example, the

I

fusion of the enhancing element of CamV-35S-promoter at the 5'-end of the wunl-promoter leads to an increased activity of the wunl-promoter in transgenic tobacco and potatoes.

Figure 12 shows the organization of the clones wunl-GUS, TR1'-GUS and TR1'-wunlGUS.

Figure 13 shows the Northern-Blot-analysis of wounded and non-wounded transgenic tobacco leaves. Nonwounded (NW) and wounded leaves of the transgenic tobacco plants wunl-GUS, TR1'-wunl-GUS Nr.5 and Nr.ll and 35S-GUS were used for the isolation of RNA. In each case, 50 micrograms of the total RNA was separated gelelectrophoreticaljy using a 1.2% formaldehyde gel, then transferred to a nylon membrane, then hybridized with a radioactively labelled GUS-DNA probe, and visualized on an autoradiogram. shows a 2-day old exposure, (B) shows a 6-hour old exposure.

The examples below serve to clarify the invention: Examples 1-8 Materials Media Culture media for bacteria: The media used for the growth of bacteria were established on the basis of data from Maniatis et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbour Laboratory, Cold Spring, Harbour, New York (1982): Plant Media: The media utilized were derived from the media given by Murashige and Skoog, Physiol. Plan. 15 473-497 (1962),

(MS).

3MS MS 3% Sucrose 3MSC MS 3% Sucrose, 500 micrograms/mi Claforan MSC1O MS 2% Sucrose. 500 micrograms/mi Ciarofan, 0.2 micrograms/mI. NAA, 1 microgram/mi BAP 100 micrograms/mi Kanamycinsuiphate MS 2% Sucrose, 500 micrograms/mi Ciaforan, 100 micrograms/mi Kanamycinsulphate MS 2% Sucrose, IuJ0 micrograms/mi Kanamycin-suiphate For solid media, 8g/1 of Bacto-Agar was added.

Strains and Vectors E. coli-strain: BM4H 71-18 C 600 GJ2 i (lac-proAB), thi, supE; F' (iaciq. proA+B+) (Messing et al., Proc. Nat. Acad. Sci.74, 3642-2646 (1977)) CR34 (Maniatis et al. i982) AB1157 (R64drdil) (pGCT28) (Van Haute et al., EMBO J.

2, 411-418, (1983)) C58CIpGV3850KM (Zambryski et al., EMBO J. 1. 147- 152, (1983)) pUC8 (Vieira and Messing, Genes 19, 259-268 (1982)) pMPKiiO (EckP3 et al., Moi. Gen. Genet. 205, 14- 22 (1986)) Agrobacteria-strains; Piasmids pRTl01-cat (Prols et al., Plant Cell Reports, in print (1988)) pGV1103 (Hain et al., Mol.

Gen. Genet. 199, 166-168 (1985)) Phages EMBL4 (Frischauf et al., J.Mol. Biol. 170, 827-842 (1983)) M13mpl8 (Yanisch-Perron et Gene 33, 103-119, (1985)) M13mpl9 (Yanisch-Perron et al., (1985)) Plants Solanum tuberosum AM 80/5793 (haploid) Berolina (tetraploid) Datura (D12) Granola Nicotinia tabacum Wisconsin 38 (W38) Samsum NN (SNN) Oryza sativa japonica c.v. Taipai All molecular biological standard methods, such as restriction analysis, plasmid isolation, minipreparation of plasmid-DNA, transformation of bacteria and so on, were carried out as described by Maniatis et al., (1982) unless otherwise indicated.

Wounded Plant Tissue Potato tuber material was cut into slices 3 mm thick, and incubated in a phosphate solution phosphate buffer, pH 7.0, with chloramphenicol micrograms/ml)) for 18 hours at 28°C in the absence of light. Leaf material, stem material and root material was cut into small pieces and incubated under the same *t 28 conditions. After the completion of incubation, the material was either directly processed or stored at Non-Wounded Plant Tissue Leaf material, stem material, root material and tuber material was processed immediately after removal from the living plant, or frozen in liquid nitrogen and stored at Aerobic Wounding of Potato Tubers Treatment as described under "wounding", in w.ich during the incubation the tuber slices were only lightly we.ted with the phosphate buffer. After the incubation, the potato surfaces were brown.

Anaerobic Wounding of Potato Tubers Treatment as described under "wounding", in which during the incubation the potato slices were fully submerged in the phosphate buffer. After the incubation, the tuber surfaces were bright yellow-white.

Development of wunl-mRNA in Pathogen-Attacked Potato Leaves Leaves of the species "Datura" were cut away at the leaf stem and placed in water along with the stem. Then the leaves were sprayed with a suspension containing phytophtora-spores. At different times, the complete RNA of a given leaf could be isolated and investigated using the "Northern-Slotblot-Analysis".

Three different materials were used: Hater, containing the spores of a Datura-compatible phytophtora infestans species Pil; Water, with the spores of a Datura-incompatible phytophtora infestans species P14; Water, without addition of spores (control) Two hours after the treatment of the leaves with water and spores of the incompatible phytophtora species, densitometric evaluation of the strength of the 29 wunl-hybridization indicated a 4-times increase in the amount of wunl-mRNA. From the fourth to the eighth hour, the amount remained approximately the same, whereupon it fell gradually up to the 30th hour back to the 1.5 to 2-times value. The same development ratios were found for wunl-mRNA when using water without spores (control).

In the analogous trial using water a:-d compatible phytophtora spores, a 4-times increase in wanl-mRNA was seen 2 hours after the attack, this factor only slightly reducing up to the 10th hour. From the 16th hour at the latest, the factor increased to about 6-times the control value, and then constantly rose to reach a factor of 9 after 30 hours.

Wounding with the addition of potato extract Potato tubers were homogenized at 4 0 C without the addition of buffers, and then centrifuged for minutes at 10,000 rpm in an SS34-rotor.

The remaining material was cooked for minutes and then again centrifuged. The clear supernatant was added in the ratio of 1:10 to 20 mM phosphate buffer, pH 7.0, and the tissues to be tested were then incubated under the usual conditions.

Example 1 RNA-Isolation The isolation of RNA from various organs of the potato and the tobacco plant were carried out as described by Logemann et al. in Anal. Biochem., 163:16- (1987).

The selective concentration of PolyA+RNA was done using mAP-papers (Werner et al., Analytical Biochem. 141, 329=336 (1984)).

"Northern-Blot" Analysis The RNA was separated electrophoretically on a 1.5% formaldehyde-agarose-gel (Lehrach et al., Biochemistry 16, 4743, (1977)). As described by Willmitzer et al., EMBO J. 1. 139-146, (1982), the RNA was next transferred to nitrocellulose, fixed and hybridized with 32 p*radioactively marked cDNA, grown and exposed.

DNA-Isolation Nuclear DNA was obtained according to the method of Bedbrook, PMB Newsletter II, 24 (1981) from potato leaves and used for the cloning procedure in Lambda Phages EMBL4. DNA from the transformed tissues was isolated and purified using Triton X-100, SDS, and proteinase K (Wassenegger, Dissertation, Koln (1988)).

"Southern-Blot"-Analysis DNA was electrophoretically separated on 0.8 to 1.2% Agarose gel, transferred to nitrocellulose and fixed (Southern, J. Mol. Biol. 98, 503-517 (1975)), and hybridized and washed as described by Willmitzer et al (1982).

"Run-Off" Experiments A method which relatively accurately distinguishes between the two regulation types (transcriptional and translational regulation) is the "run-off" method. In this method, cell nuclei from wounded and non-wounded tubers are isolated, and newly synthesized mRNA therefrom is radioactively pulselabelled and hybridized with single strand DNA of the individual clones. In cell nuclei from non-wounded clones, no wunl-mRNA was produced. By contrast, in cell nuclei from wounded tubers, this material was produced in high quantity.

The isolation of cell nuclei from potato tubers, as well as the subsequent in-vitro transcription, were carried out in accordance with Willmitzer et al., Nucl. Acids Res. 9, 19 (1981). The radioactively labelled RNA-transcripts obtained by this method were hybridized with bidirectional M13 cDNAclones.

Protein Isolation and Splitting The isolation of proteins fron potato tubers as well as the 1-dimensional and 2-dimensional gelelectrophoretical separation thereof, has been described by Mayer et al., Plant Cell Reports 6, 77-81, (1987).

Example 2 "Hybrid-released-translation"-Experiments The 800 bp fragment of the cDNA-clone wunl- 25A2 and the 700 bp fragment of the cDNA-clone wun2- 29C12 were cloned in M13mpl8 using the PstI sites. The orientation of the insertions in the M13-clones were determined by complementation analyses (Messing, in: Genetic Engineering of Plants, (1983), Plenum Press, New York, 1983). The identification of the M13-clone containing the coding strand resulted from positive hybridization with radioactively labelled polyA+RNA from wounded potato tubers.

The isolation of single strand DNA from M13 phages (they contain the coding strand), the fixation of this DNA on nitrocellulose and the hybridization with potato tuber RNA, as well as the wash and release of selected RNA, has been described by Maniatis et al (1982). The selected RNA as vell as the polyA+RNA isolated from the potato tubers were translated in a rabbit-reticulocyte-lysate (Pelham and Jackson, Eur. J,.

Biochem. 67, 247 (1976)) in the presence of Methionin. The 1-dimensional and 2-dimensional electrophoretic separation on a polyacrylamide-gel was carried out according to Mayer et al (1987).

Example 3 Establishment and Screening of a genomic bank Isolation of o'enomic DNA:

I

32 According to the methods of Bedbrook (1981), isolated leaf DNA of the haploid line AM 80/5793 was used as genomic potato DNA.

This DNA was fully digested with EcoRI.

Isolation of the phage-DNA: EMBL4-DNA was cut into three fragments with EcoRI, wherein after gel-electrophoretic separation and subsequent fragment isolation, the two vector arms of the middle fragment could be separated. In addition, commercially available purified EMBL4-arms were also used (Amersham).

Ligation and Loading: After subsequent ligation of the EMBL4-arms with the EcoRI-digested genomic DNA, the ligated, highmolecular DNA was loaded in vitro into phage heads.(Hohn and Murray, Proc. Nat. Acad. Sci. USA, 74, 3259-63, (1977); Hohn, Meth. Enzym. 68, 299-309 (1979)). The loading material derived from a "Lambda in vitro packaging kit" of the company Amersham. The genomic bank was plated out with a concentration 25.000 plaques per plate (25X25cm).

Plaque hybridization: The detection of cDNA-homologous Lambda-clones resulted from a plaque hybridization according to Benton and Davies, Science 196, 180 (1977) using a radioactively labelled cDNA. Positively hybridising plaques were isolated and again tested.

DNA-Preparation of Recombinant Phages: ml of a C600-lysed bacterial culture was mixed with chloroform in order to obtain, after subsequent centrifugation, a bacteria-free supernatant.

The sedimentation of the phages from the supernatant was 1 Sj 33 carried out by a 4-hour centrifugation at 10,000 rpm.

The phage sediment was removed in 500 microlitres phagebuffer (10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 and treated with DNase and RNase. After the extraction of the DNA through repeated phenolysation, the phage DNA was EtOHprecipitated, washed with 70% EtOH and removed in TE.

Example 4 Creation of deletion mutants after exonuclease-IIItreatment The fragment to be investigated was cloned in both orientations in M13mpl9, in accordance with Henikoff et al., Gene 28, 351-359 (1984). Since the exonuclease III digests 5' overhanging ends, while the 3' overhanging ends remain, 20 microgram of the DNA to be analyzed was digested by two restriction enzymes, creating 5'-end and a 3'-end. Because the 5'-end was located towards the fragment, an exonuclease-digestion of the fragment could take place. Using repeated stopping of this reaction, it was possible to isolate 200 bp smaller fragments, of which the sticky ends were changed into ligatable blunt ends through a subsequent Sl treatment. Finally, there followed the transformation of this DNA in BMH 7118-cells, and then the single strand DNA isolation.

i 34 Sequencing The sequencing of single strand DNA was carried out according to the chain interruption method of Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463, (1977). The separation of the reaction materials was done on 6% and 8% sequence gel (Ansorge amd de Mayer, J.

Chromatogr. 202, 45-53 (1980); Ansorge and Barker, J.

Biochem. Biophys. Meth. 9, '3 (1984)).

Cloning and sequencing of wunl-25A2 The approximately 711 bp-sized insert of the clone wunl-25A2 was cloned with its PstI-site in the M13mpl9-vector. The orientation of the insert was analysed using the asymmetric XhoI-site. The clones 25A2-4-mpl9 and 25A-5-mpl9, differing in the orientation of the insert, were digested with KpnI/XbaI, in order to make possible a bidirectional exonuclease III digestion of the fragment. Deletion clones of different sizes could be obtained by successive stopping of the exonuclease reaction. These deletion clones served as purified single strand DNA for sequencing in accordance with the method of Sanger et al (1977).

Example Sl-Analysis The principle of Sl-nuclease mapping for the determination of transcription starting sites is based on the concept that, through a hybridization of single strand DNA from the 5'-region of the wunl-gene with wunl-mRNA, only those regions are protected against destruction by the single strand-specific nuclease S1, which on the basis of its homology, can form a double strand. The size of the protected DNA fragment can be determined on a sequencing gel, and thus the transcription start can be traced back.

The determination of the transcription start point was carried out according to Berk and Sharp, Proc.

Natl. Acad. Sci. USA 75, 1274 (1987). For this, the 1.2 kb fragment was isolated from the plasmid pLS000 with EcoRI and XhoI, and dephosphorylised with phosphatase.

Finally, there followed a radioactive labeling of the through the combination of polynucleotidekinase and y- 32 P-ATP. After denaturing the DNAfragments, they were hybridized with 50 micrograms of whole RNA (hybridization buffer: 80% formamide, 0.4 mM NaC1, 40 mM PIPES pH 6.4 and 1 mM EDTA). This condition favours RNA-DNA-hybrids in comparison to DNA-DNA hybrids (Casey and Davidson, Nucl. Acids Res. 4, 1539-1552 (1977)). The hybridization temperature started near 80 0 C and was lowered overnight to 40 0 C. A subsequent Sl-nuclease digestion (120 U/ml) removed unpaired strands. It was expected that the radioactively labelled XhoI site located at the 5'-untranslated region of the wunl-gene would be protected due to its homology to mRNA. Finally, the Sl-protected DNA strand was electrophoretically separated on a sequencing gel, so that the sequence of a known DNA-fragment would serve as a size marker.

Example 6 Transient Expression in Potato Protoplasts Quantities of 20 micrograms of highly purified (CsCl-gradient) plasmid-DNA was transformed in potato protoplasts treated with PEG/CaC1 2 (Lipphardt, Dissertation, K61n, 1988). The protoplasts were isolated from a potato stem suspension culture of the species Datura.

Transient Expression in Rice Protoplasts micrograms of highly purified (CsClgradient) plasmid DNA was transformed in accordance with the modified method by L6rz et al., Mol. Gen., Genet.

199, 178-182 (1985). The concentration of polyethylene glycol and osmoticum were altered.

Example 7 DNA-Transfer in Agrobacteria Conjugation: DNA cloned in E. coli was transferred using the helper strains GJ23 into the Agrobacteria species 38 50km, according to the method described by Van Haute et al. (1983).

DNA-analysis of Agrobacteria: The monitoring of the DNA transfer into the Agrobacterium was carried out through the isolation of Agrobacteria DNA according to the method described by Murray and Thompson, Nuc. Acid Res. 8, 4321-4325 (1980).

Restriction digestion of the DNA, the transfer to nitrocellulose and the hybridization with the corresponding radioactive probe, point out a successful DNA-transfer into the Agrobacteria.

Example 8 Transformation of Tobacco Growth of Agrobacteria: The AgrDbacteria 3850 Km necessary for infection, were grown in selective antibiotic media (Zambryski et al., 1983), sedimented through centrifugation and washed i a YEB-medium without antibiotics. After another sedimentation and resuspension in YEB-medium, the bacteria can be used for infection.

Leaf Disc-Infection: Sterile leaves of the tobacco lines SNN and W38 were used for the leaf disc-infection. Leaf pieces about 1 cm in size were sterilized by a 37 incubation in 0.1% HgC1 2 0.1% SDS, and then washed three times in sterile water. There followed the submersion in an Agrobacterium suspension as described above, and the subsequent transfer to 3MS-medium. After a two-day incubation with 16 hours under light and at about 25*C to 27°C, the leaf pieces were washed several times in liquid 3MS-medium, and transferred to 3MSCmedium. After 4-6 weeks, shoots which emerged were isolated and incubated on a 2MSC-medium. As further evidence of a successful transformation, the nopaline test was carried out.

Analysis of the Transformed Plants Evidence of nopaline-synthase activity: The detection of nopaline-synthase activity in transformed plant leaves was carried out according to Otten and Schilperoort, Biochem. Biophys. Acta 527, 497 (1978).

Proof of NPT-II-Activity: The NPT-II-activity in transformed plants was established in accordance with Reiss et al., Gene 217-223 (1984) and Schreier et al., EMBO 4, 25-32 (1985).

Proof of CAT-Activity: Chloramphenicol-acetyl-transferase (CAT) activity was determined according to the method of Velten and Schell, Nuc. Acids, Res. 13, 6981-6997 (1985) and Herrera-Estrella et al., EMBO J. 2, 987 (1983).

Example 9 Optimization of the wunl-promoter by fusion with the TRpromoter (TR1'wunl) For the media used, reference may be had to Examples 1-8.

2.1.4. Species and Vectors Agrobacterial species: GV3101pmp90RK (Koncz, C. and Schell, J. "The promoter of TL-DNA gene

V

4 38 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector", (1986), Mol. Gen. Gent. 204:383-396).

Plasmids: pPCV720 (thus far unpublished Plasmid by Dr.

Koncz, MPI f. Zachtungsforschung, K61n) pRT99-GUS (T6pfer, Schell, J.

and SteinbiB, H.H. "Versatile cloning vectors for transient gene expression and direct gene transfer in plant cells (1988) wunl-GUS (Siebertz, Logemann, J., Willmitzer, Schell, J. "Cisanalysis of the wound-inducible promoter wuni in transgenic tobacco plants anr histochemical localisation of its expression".

The Plant Cell, in press (1989) TR1'-wunl-GUS (Logemann et al., in press) TRI'-GUS (Logemann et al., in press) Plants: Nicotiana bacum Wisconsin 38 (W38) Methods All molecular biological standard methods, such as, for example, Restriction Analysis, Plasmid Isolation, Mini-preparation of Plasmid-DNA, Transformation of Bacteria and so on, were carried out as described by Maniatis et al, (1982), unless otherwise indicated.

Wounded and non-wounded Plant Tissue was obtained as described in Examples 1-8.

Reference to Examples 1-8 can also be had in connection with the RNA-isolation, the "Northern Blot" analysis, the DNA-isolation, the transient expression in rice protoplasts, the "Southern Blot" analysis and the DNA-analysis of Agrobacteria.

39 DNA-Transfer into Aqrobacteria The DNA cloned in E. coli was transferred into the Agrobacteria species GV3101pmp90RK, in accordance with the method described by Van Houte, Joos, H., Maes, Warren, Van Montagu, Schell, J.

"Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for reversed genetics of Ti-plasmids of Agrobacterium tumefaciens", EMBO 2:411-418.

Transformation of tobacco Culture of Agrobacteria The Agrobacteria GV3101pmp90RK, containing the desired plasmid, were grown in a selective antibiotic medium (Hygromycin), sedimented by centrifugation and washed in YEB-medium (Maniatis et al, 1982) without antibiotics. After further sedimentation and removal in YEB-medium, the bacteria could be used for infection.

Leaf Disc-Infection This was carried out as described in Example 8, however without the use of the nopaline test.

AnAvysis of the transformed plants Proof of GUS-activity A: The fluorometric analysis of beta-glucoronidaseactivity (GUS-activity) was carried out in accordance with the methods of Jefferson, Kavanagh, T.A., Bevan, M.W. "Gus-Fusions: 8-Glucoronidase as a sensitive and versatile gene fusion marker in higher plants", EMBO J. 6:3901-3907 (1987).

B: Blue staining of tissue in relation to GUSactivity could be obtained using X-Gluc solution. X- Gluc solution. 1-2mM X-Gluc (5-Bromo-4-chloro-3indolyl-glucuronide), and dimethyl formamide were dissolved in 50 mM phosphate-buffer at pH Wounded Tissues: Thin tissue slices were incubated as described under "Wounded Plant Tissues" and then stained overnight at 37*C in X-Gluc solution. Finally, the chlorophyll was removed by the addition of 100% methanol at Non-wounded Tissues: Thin tissue slices were incubated in X-Gluc solution which additionally contained the translational inhibitor Cycloheximide (1.8 mM). The incubation period and the methanol treatmern are the same as with the wounded tissue.

X-Gluc staining of Pollen: Pollen was incubated overnight at 28"C in sucrose, leading to the development of pollen tubes.

Finally, a doubly concentrated 30% X-Gluc solution was added and the pollen were incubated overnight at 28"C.

Alternatively, the pollen grains are incubated directly in X-Gluc, without developing pollen tubes.

Results Oranization of TR1'-wunl-Gus. TR1'-GUS and wunl-GUS (Figure 12) wunl.-GUS: 1022 bp of the wunl-promoter and 178 bp of the wuni 5'-untranslated region were transcriptionally fused with the beta-Glucuronidase-Gene (GUS). The polyadenylation signal (pA) of the nopaline-synthase-gene was used ('NOS) as a termination sequence.

TR1'-GUS: The TR1'-promoter was fused transcriptionally with the GUS-gene, and terminated by the pA-signal of the NOS-gene.

TR1'-wunl-GUS: Behind the 1'-promoter of the TR-promoter the pA-signal of the octopine-synthase-gene (OCS) was cloned in order to suppress a transcription through the wunlpromoter. Behind the OCS pA-signal the wunl-promoter (1022 bp) is located, including (in homology to the wunl-GUS construction) 179 bp of the untranslated region of the wunl-gene which are transcriptionally 41 fused with the GUS-gene. The pA-signal of the 35-S gene serves as the termination sequence for the GUS-gene.

This construction (TRI'-wunl-GUS) is present in the binary vector pPCV720, which can replicate in both E.

coli and in Agrobacteria (with the help of a helper plasmid).

RNA-Analysis of TR1'-wunl-GUS transqenic tobacco RNA from wounded and non-wounded leaves of various TR1'-wunl-GUS transgenic tobacco plants, as well as from wunl-GUS and 35S-GUS transgenic tobacco leaves, was isolated and hybridized with radioactively labelled

GUS-DNA.

As is shown in the Northern-Blot in Figure 13, 1. The GUS-mRNA in transgenic TR1'-wunl-GUS-leaves is exactly as large as in wunl-GUS transgenic leaves.

It appears that in both constructions, the same transcription sta-'t was used.

2. The amount of RNA in wounded leaves, both in TR1'wunl-GUS-plants and in wunl-GUS-plants, is higher than in non-wounded leaves. The woundinducibility already known for wui i-GUS was also true for TR1'-wunl-GUS.

3. From an RNA quantity comparison, it was realized that somewhat more RNA was to be found in wounded TR1'-wunl-transgenic leaves than in transgenic leaves. Since the 35S-promoter is one of the strongest promoters in plants, the TR1'wunl-promoter is estimated of similar strength.

Analysis of the GUS-activity in transqenic tobacco A quantitative analysis of GUS-activity of transgenic TR1'-wunl-GUS plants shows that the TR1'wunl-promoter in transgenic leaves is inducible by wounding at a factor of 2 times up to 13 times (on average about six times). This observation is similar to the wound inducibility of the wunl-promoter in transgenic leaves (Logemann Lipphardt L6rz, H.,

M

42 Hauser, Willmitzer, L. and Schell J. sequen:es from the wunl-gene are responsible for gene activation by wounding in transgenic plants" The Plant Cell 1:151-158 (1989A)). Analogously to the trangenic wunl-GUS tobacco plants, the wound inducibility in the higher, younger leaves is the highest, while, in the lower, older leaves it is the smallest. The reason for this difference in inducibility is the increasing activity, from top to bottom, of the TR1'l-wunl-promoter in non-wounded tissue. In old leaves of old plants, the activity of the TR1'-wunl-promoter in non-wounded condition is already so high that wounding cannot induce any further activity (analogous to wunl-GUS plants).

A relative comparison of promoter-activities in transgenic tobacco leaves and stems shows that the wunl-promoter is roughly 10 times stronger than the TR1'-promoter. The TR1'-wunl-GUS promoter, on the other hand, is 10 times stronger than the wunl-promoter.

Consequently, the TR1'-wunl-promoter is roughly 100 times stronger than the TR1'-promoter.

Localization of GUS-activity in transaenic tobacco Leaf, Stem: The GUS-activity in the tissue slices from wounded and non-wounded leaves and stems of wunl-GUS and TR1'-wunl-GUS transgenic tobacco was localized by the use of the substrate X-Gluc.

wunl-GUS: As can be seen from the blue colored regions, the activity of the wunl-promoter is limited primarily to the epidermis (including trichome), and to a lesser extent the vascular system of wounded leaves In non-wounded leaves on the other hand, only a small wunl-promoter-activity can be seen in the epidermis, and no activity in the vascular system. The same results were obtained also with stem cross-sections.

Accordingly, the wunl-promoter activity is to be regarded as epidermis-specific in leaves and stems.

In TR1'-wunl-GUS plants, an intensive blue coloring in the vascular system of wounded plant slices is recognized. In other regions (epidermis, parenchyme), the coloring is weak. Non-wounded leaf slices show a low level of blue coloring in the vascular system, and no color in the epidermis. The same results were found with stem cross sections.

This appears to suggest, in connection with TR1'-wunl-GUS, a vascular-specific promoter in leaves and stems.

Leaf cross-sections of TR1'-GUS plants were used as control. Because of the weak promoter activity of the TR1'-promoter (10 times weaker than the wunlpromoter), blue staining was not found in either wounded or non-wounded leaf cross sections.

Anthers: An X-Gluc staining of anther cross-sections as well as of pollen shows the following characteristics, which apply both for wunl-GUS and for TR1'-wunl-GUS: A blue staining (and GUS-activity) was detected in the stomium (the natural perforation location of the anther in order to release pollen) and in the pollen. In separated ungerminated pollen and germinated pollen, there was found a blue staining in the pollen grain and in the pollen tube.

Transient Expression with Rice-protoplasts The constructions TR1'-wunl-GUS, wunl-GUS, TRI'-GUS and 35S-GUS were used for studies of transient development with rice protoplasts of the variety Oryza sativa japonica c.v. Taipai. As shown in Table 1, the activity of TRI'-GUS is not significantly different from the control (GUS-activity of rice-protoplasts), which were transformed without DNA). wunl-GUS and show a 2 times to 3 times higher activity than the 44 control. By contrast, TR1'-wunl-GUS DNA exhibits on average a 57-times higher activity than the control, and therefore is a highly active promoter in rice protoplasts.

Table 1 Construction GUS-Activity Relative nmol MU/mq/min GUS-Activity TR-GUS 31 1.2 wun-GUS 59 2.3 TR-wun-GUS 1416 56.7 66 2.6 Control 25 Legend for Table 1: The given GUS-activity was based on 5 independent tests.

The results of Table 1 are seen through the staining of the protoplasts with X-Gluc. Only protoplasts transformed with TR1'-wunl-GUS show an intensive blue staining.

Claims (8)

1. A DNA-sequence from Solanum tuberosun, which is stimulated through wounding and/or infection with pathogenic microbes, having a nucleotide sequence corresponding essentially to: 9 o S. 9 9 9S *9 .9 9 .9 99 0 9*99 9 .1 105 165 225 285 345 405 465 525 585 645 705 765 825 885 945 1005 1065 1125 1185 TGATGTACATATATACAATTATCTAACCGATATATATATATATATATATATATATATATA TCACCTCTCTCCACTCTCTGCCCTCTCTTCATTCTCTCCCAATCTCGCTCACCACTCTAG TACCTGATTTATATAAATCGCCGTGATTTATACAAATCAGATGCTCCATACAAACATAA TTTGACCCATGGAGTGCATATAACAAAATTGTAGCTATAGAACGCTAATATGTTTTTCTT AATCTTTGTTATTCCTAAAkATTTACTCATAATAATACTCTTTATAAAAGCATAAGCTGGT TTGGTTTAGGGTTAGAGTATTCrCTAAAAATTCTAATTGAAATCAAATACATCTTrTAGA ATCCAAATTAGAATTGAACACGTCTTGTAGAGTCCCATAAATTTTTAATGTCTACAATGT"* AA'rATCGTTAAAATATTTTAATATCTTGTTGAAATATAATTTTTTATTTAGTAAAATAAT AT0AGAATTAATTTTTTTTATTAATCTTCACAkACTA7GATTTTTTTAAAATTTCATGTAA ATATATGGGCTAAGA7TGTGAGCCAACT0GrCAAC7CAAAAGTTAGTCGAGTTTG;.AT GAAGTTAAkAATTAAAAGTATTGTTGTCATA-ACTCATATGTTGCAAGTTGCAACTGTGTGT ATAAGGTCAAAAAAGGTATGCTTGAAAGTTGAAACT TTAC'ATATGACGATCATCTT.CGTG GGCCCTACCTAAAATAAAAcGTCTCTTCATCATcCCAATATCA CAT CATCACGTAATCcC CGAGCACGTGGAATGGCGCGTAAATATCATGTCGCCCTTTA.AACCTAAATACACCTACTA TTACAATT CTCACTCU CCCTAAC CTTTCTTcATTACCTACCATACCTTCTTCTCTCATCCTTCATAGCTAATAATATCAT' 'TT CTGTTTTTACTAACTGCTAACTCTCAGTTAACTCTGGAGGAAACAACt*AACAAAGGGG TGGTGCTCGCCCTATATGAAGCCTTGAGCTcACACGACGTCGTTCAGGTCCAGAAACTCC TGGCCTCCGACCTCGAGTGGTGGTTCCATGGTCCTCCTTCTCATCAAT'TTTGATGcA-AA 1 *99S 9* 49 S. 46 M Q1 1245 T ACTCACCGQCACTGCTAAATTCGATAACGCCTCTTTTCAATTCCTTCATAAGACCATTG L T G T A K F D N A S FQ F L H K T I D 1305 ACGTATTCGGTTCCGTTGTTCTCGTCGAAGGCTGTGACWCGACCCGATCTATTACTTGG V F G S V V L V E G C P T R S IT W V 1365 TTCACGCCTGGACTGTiTACGGATGGGGTAATTACCCAGGTTAGGGAGTATTTCAAITACCT HMAW T V T C G V T T Q V R E Y F N T S 1425 CACTTACTGTCACCCO TTTTGGGAAATCGGATATTTCCTCAATTACGACTCTGC kTTGCC L T V T R F G K S D01S S IT T L H CFP 1485 CATCTGTTTGGGAGAGTAGCT'.ACCAAATCGGGTCGGAAAATCTGTTCCGGGTCTTGTAT .S V W E S S L P N R VG K S V P G LV L 0*~.1545 TGGCTCTATAAGAAACGACCCGATTTGTGGCTGGCGTTATATCTTGTGTCTAGTAGGATG A L END 1605 TAAGATTAACGCGGC-TGGTTTGGGATTCTGTTGCTGCTGTTTGGTTTGTTTTTATTTTTT 1665 AAGTTGGGATTTGTGTTCTGTTTATTATTTGTTGTTGTGATTGAGTTAAAGAAGGGGCCA 1725 ATGATAATTGGATATACCTTTCTACTCAATAATTGTCTAATTATGTGTTGGTTTC~ :~~:1785 AGCATATTGATTGGCTGTTTACAAAATGTGTATM'TrTTTCAATTTGGTATTGCTTC :1845 T-1GTTTTCAACGAATGACGATGGATACCGTGGAAACAAT'TTCATTGAAAGGCAAATC'rTA *1905 TTATTAATGAT0GGATCAGAATTTTTATTATGAGTTTAAATAATTAPAACATATAAAATA 1965 GTTAAATGAGGTTCGAAATCTATATAATGCATAAAAAAATAATTTT;', ~CTATATATAAAA 2025 ATGTATAATTTTTT0TCTAGAATAGGAGGAACAATATATTTAAGAAGGAATCTTTGCTT *2085 GCTTAGGATAGCTTCACATCATACTTTTCCCACTACATAGGTAATAGGAAGGCATATGCT .2145 CTATTCCTTCATAACTTACTCTTGGTATTTCTTATCCTAATCGCCAAAAAAAGAGAGTAT 2205 MATTTTTATTTATAACACACTTTTTTATTTCCTATACAAGATLAGTT0ATTTTTCCATT 2265 TAAGTATXAACATCGAAACTTTTAGAGCGACTCTATAAGAGACTGCCCAATTTCATTGGG 2325 ATTCTACAAGATATTTTCATTGATTCTTGGAGACTACTTAATCT 2368 47

2. The promoter portion of the DNA-sequence in accordance with claim 1, or its transcriptionally active region.

3. The promoter portion of the DNA-sequence according to claim 1, or its transcriptionally active region, fused with the Mannopine-Synthase-Promoter.

4. The structural gene portion of the DNA-sequence according to claim 1, or its transcriptionally active region. The use of the DNA-sequence according to claim 1, or the promotor portions according to claim 2 or claim 3, ;for the development of gene products in higher plants using wounding and/or pathogen attack.

6. The use claimed in claim 5, characterized in that, the promoter portion of the DNA-sequence according to claim 2 or claim 3, or their transcriptionally active regions, is/are fused with a structural gene of a different origin. S' 7. A DNA-transfer vector with an inserted DNA-sequence according to claim 1, 2, 3 or 4. l" 8. A DNA-transfer vector according to claim 7, characterized in that, it contains the promoter portion or its transcriptionally active regions in association with a structure gene of a different origin.

9. A plant or plant material containing a DNA-sequence according to claim 1, 2, 3 or 4. 0: ^I -48 ABSTRACT WOUMiD-STINULATED DNA-SEOUENCE MROM SOLANM TUBEROSUI4 AND ITS USE The invention relates to wound-stimulated DNA from Solaium tub~rosum as well as parts thereof, their use for the development of gene products in higher plants usincr wounding or pathogen attack, DNA-transf er vectors containing these, and plants or plant portions containing these. The promoter of this DNA-sequence can be used for the expression of gene products in higher plants using wound and/or pathogen attack. 0 to 1/13 98 Xh 01 i25A2aMP19 98 1 25A2 MP19 794 Pal IyA 794 a 0* a o a, a a a a. a a a. a a. a a a a a a. *a a a a a a. ac a. a a a a., B. a a a, a aaa*a a 25 A 24 mpiga 3 ZS A2 IP 19A FIG. I. 2/13 121 ATGCAAAACCACCGCAC-GCTrAAA7CGTAACGCCTCTTTTCAATCCTTCArAAG M Q I L T G T A X F D N A S F 0 F X. H X 181 ACAT~CTTCGTCTGTTG-TAGTGCCCACGTT~ T I D v F G S V V L V E G C D PT R S 1 241 ACTG=CACCCTGGACTTTACAATGOCTAATTACCCAGOTAGGGArTAT-.C T W V H AW T V T V G V I T Q VR E Y F 301 A~ACCCTCCMCGTTGAATGAATC- ATAG~CG N T S L T VT R rG KS D I S S IT T L *361 C.TGCACG.TGAG.GTAC.A ~.AATTTCG- H C P S V W E S S L P N EV G K S V P 0 :.421 CTGA-- G.CAAGACACCTT7=GGTTTTGGCA, o L V I. A L Xnd ~481 AGOATQTAGATTACGCGTTTTGG,\TTCTGTTGCATTTGGTTTGATTTGrT ,**541 GTTTTTTTTTAA'T0COATTTGTGTCTGTTTATTATTTGTTGTTGTGATTGA-TT 601 AAG,\GGCCAATGATAATTOGTATACCTTCTCTCATOATTGTCTAATTATGT 000661 ATTG: G AA=gCATATTGATTOOCGTTTA )~AAAAAAA FIG. 2 -3 IIPUIH I'SI **eQ e .8*O* 09* S. S S S S. S 4/13 L rm Ri Rl I kb I ,wurl I S A S *9 S A AS SO S A S AO A. A A* 9S A A. A A 0 A 0 5* A S A S A S 90 9. A. S A* S* S 5 'Rl RArrm EM BLk Rl (27 kb) it RI PLS 000 ECVRI (6.7 kb). 4- FIG. 4 5/13 Xho I 2.2 as mpla 0.8 Xho I 3' MrC.C.-C.AG.AAAT h 6e 9 9 9 .9 9 9 9 9 9. 99 9 9 99 9* 9 9. 9 a a 9

99.. 99 9 9 99 9. .9 9. 9 9 999 *99* .4 9. .4 99,999 9 Xthoi to 08 if I 3F .5 MpI8a -t I 7j IP 8I 41 8smp18a 1 3 FIG* U- 6/13 S AAArrTTATAACACA7TAcuAACAACA??."ATACAAACAA 45 TGATG7ACAATAACAATTAAC=AATAT7kAAAAAAATATATATATATh 105 TCCTT-CCCCGCTTTCTCCCCAC-CTACCCA 165 CC-AACATArCAGAACAAACATATACAAACACAATTTCATAGACACA7GGAATTAA 225 TACC7CATTTATATAAATCGCCGGATFMACAATCtCATGC-CCATMCAAACATAA 285 TTGCCTAT TTAAAATTAC.TkAG"ATTTTTT 345 AACTTGT-?ATC=?AAATTTACTCAThAA7 %C.CTAAAACTAC= 405 TT=TGOTG=TCCAAATTATAACAAAC A~. 465 ATCATAATGAA=.T.AG=CAAATTATTTCAG 525 AAAC AATTTATT--GrrAAAATTTATA;AATA 585 ATCAG"-.TAA7'7?T?1ATTAATC. CACAACTATCATTTTFFAAAAT'TTCAG7TAA 64.5 ATATAGGC-.AAATTGGAGCCAAC G"-,CAACCAAG *Ax7CGACTTTAAT 705 GAAGTAA-AAGT7CTGT.ATCTCAT770CAAGTGCAACT rGG 765 ATAAGCAAA)ACG7ATGC77TGAAGrrGAAAe77AGATITGACaATCATCTiCGrG 825 GGCCCACCA7AATAAAC07C.CCATCACAATACCACACACGAATCCC 885 COACACTGGAAGGCGCTAATACTGCCCCCTAC7A.AAL7ACACCAC7A 945 TTCACCTAAATTC ACCC77-CCAACTCCOrCCCCCCA:,CACC= 1005 c~C:TCCATACCACCAACCTC7-TCCATCCTCATAGCAATAATATCATCTT ***1065 C7GTTTTAC-.AACGGCAACCAGAACTGGAGAAACAACAACAAAGGWC S112S TGG7,GC=CGCCC7XA70AAGCC?7GCAC7CACLNGACGTC=CAG7CCAGAACCC .a1185 TGGCC.CCACC7C3AGGGTGGTCCAGGC=CCTCT CTCAcATT?7G.TGCAAA 1245 TACT CACCCAC-..AAATCATACCCC-CT-TCAN-.=CAAAGACCATG L. T G T A K F 0 N4 A S F 0 F L~ H K T 1 0 ***1305 ACGACCC-7TC7G7C--GAAGGCTTGACGCCCC--ATC.-ArTAr-,TGr. V 7 G S V V L V 0 C 0 P R S Z7 ~T V ***1365 TTCACCCC'CGAc -G-7ACATGCG7AAT7ACCCAGG-TAGCCAG7AT77CAATACO7 FI1G. 6 H A W4 T V T 0 G V I T 0 V A Z Y F N T S 1425 CAC77ACT G7CACCC07T77CG0AAA7CG:%7AT?CCCAATACAC-C-CCATTCCC 0 e L TV T R F C K S 0 ZS St I7,7 L H C 1485 CATCGTTGAGGAGCACCATCGCGAA7TC-7GC,CGTC-7G7,AT oo* o S v w4 a S S L P N R V G K S V G L V L *1545 TG0C-c-7CAGAACACCCAT70CCCCGC77ATAC.-G-77C.AGAGATG A L END3 1665 AATGGT.T- -CG-AT C- GCTGGTAGAGGC

177.5 AT0\AAAGCAATACC77CACAAGAG 77AATG7iC--GCM7 1845 M ,TTCAGACCAOAACCGACATCTGAG;AACT 1905 TTTATAGATAAT-TTACG'TAAATACkAAAT 1965 GTrATCAGC-TCATCW7TAAC ATAATCAMMT-T-AAA7T~k .085 GC.G;TC-TAACTU;iCCCAAAGAAGkrGAAC =OS AATT77T-7 kCCC7TT!T-=A, TA "265 TAGTMACAC-TAAC;C.-TAAA-Cr;A--TG Z325 ATcACAAGATAT7 7C 0A77C7C0AGA AC. ^CT :368 3 7/13 -wQ 0O 8 S S. o SO 8 8 0 55 S S V 55 S S S 5 5 *SS5 SOS S* 0 5 55 88 *88 5558 0* S 55 *-179 S162 FIG. 7 Oe 5 5 C@ 0 See C 063 69 Promoter *0*5 55 0605 *5 5 6 5* 5 5 a gS CC SC 55 55 C. C S SC 55 C 555 9 55 55000 S S S S C 5 a a S S Ce so.. so wun1 IF -Ge n EcoRi NcaI 1022 L 31 rn~mRN A EUOVW99MProtein ATG TAA 217 318 259 179 aCAT P vunl-CAT 1- jrd-NPT I 1198 1 iga NPTIJ 1- 'olyA, PoIyA -1 H i Figure 8 9/13 54 TTTATACGCACCCGTACCCAGACAAA 96 AAAGGGGTCCCCAACAC- TCGTCCACCT 147 I WAGCGCTGG CTCCCCTATATGAAGCC-*rAGC)CACC0ACG- C-. 149 TCAGG7CCAGA. ACTCCTG3C 7CCGACC7C0ACGGTCGTTCCA7.T. 197 51 TCAGG7CCG A CACTGGCzCCCGACC-CGAG'rGGTGGT-1CCATGG.C 198 C CCZCC7ATTTACA.TCACC^c. CrA-- 247 Ic j. jillTGTGAATC CAC II lii C7 I I I IS 248 GAACC-CTTAT'CTCTA *C.,TAG- TCG.C 297 151 GAT ACoCCTC TTC7T .TAAAAl-C.A I ;ACG-TATTCCGGTC 200 298 CGO GTTCTCC7CGAAGG3CTG7GACCCGACCCGATCTATTAC TGGTC 347 201 CO TGTTCTC07TTGAGGTTTGACCCACCCATCTATTAC-,TGGGTTC 250 348 ACOCCTCGAC'O TTACOGATOGGGTAATTACCCAGGTTAGGSGAGTATTTC 397 11111111II1I1I1Hil III 1111111111111 liii 1 251 ACOCCTGGAC-.O7T-ACAC:ATGGGTAATTACCCAGGTTAGGGAGTATTTC 300 *398 AATACCTCAC TACTGTCACCCG-TTTTGGGcAAATCGGATATTTCCTCA.AT 447 ::301 AAtACCTCAC.!TACTGi .TCACCCGTTTTGGGAAATCGGATATTTCCT.CAAT 350 00 44 .AG~~GATCCTGlTCf-AATGTACATCG 9 00351 TACGACTCTG TTGCCCATCTGTTTGGGAGAGTAGCTTACCrAATCGGG 400 .:too* 498 TCGGAA ATC=GTTCCGGGTC--TGTATTGGC rCTATAAGAAACGACCCGA 547 I I I I I I I I I I I I I I I I I I I I I I I 1 1 1 1 I I I 401 rCGGA kATC-TGTTCCGGrTCTGTATTGGCr--TATAAGACGACCCGA 450 548 TTGCG- Cr--TATC TGCAGATGTAAGATTAACGCG 597 TTTGT GC-McTTGTATC OGTGTCTAGTAGGATG-7AAGATAACGCG 499 :::598 GCTGGTTTGGGATTC1'GTTCCTGC" GTTTGGTTTGTTTTTATT 640 oW500 GCTGGTTTGGGATTCTOTGC'-ATTTGGTTTGATTI'GGTTTTTTTATT 549 *641 TTTTAAGTTGGGATTTGTGTTC STTTATT TTTGTTGTTGTGATTGAGT 690 550 TTAGTGATTTGTGTTrCTGTTTA'I 'ATTTG'rTGTTGATTGAGT 59 0.*0691 TAAGAACGGGCCATOATATTGGATATACCTTCTACTCAATA ATTG 740 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 7 1 1 *600 TAAAAAOGGGCCAATGATAATTGGATATACCTTTCTACTCAATAGATTG 649 741 TCTAATTATGrOTTGGTTTOCAATAAGC7.TATTGATTGGCTOTTTAC 790 650 1C I ATA AI ATTOGTTTGCAkATAA kAgCATATTGATTGGCTOTTTAA 699 791 AAATTTATATTTTTCAATTTGGTATTCTTTrTTTCAACAATG 700 AAAAAAkAkh11X 711 FIG. 9 10/13 to as a OLSOOO Nit 16",kbi N..d Owl 1 pot -pRTIOK.AT PLSOID 4* 4 4. .4 4 *.44 0444 4 4. .4 4.. 4. 44 PW.l 9 38SOKM Wunl-Wunl Pal Zhal III !A PLS 011 9 38509m Wuni- CAT Nmwl FIG 11/13 RI Prm I r- pLSOCI M' 3 m 1 of Lmaml Ma3mI l8 wlw Al Pt~~H MPI Pa 3.' 1" FIG. 1 a a a. a a a a a a. a. C. *a a. a a a. a a a a a a a a a a a a a wuni-GUS TRf -GUS IZFwEI1- GUS FI~AI U~ETh oUS F- GUS TRI f-wun 1-GUS lkb Pig, 1 2 13/13 z z .a NW W NW Bto FIG. 13 0 SS