ENHANCER-INCREASED GENE EXPRESSION IN PLANTS

Abstract: The invention provides enhancers for one or more gene promoters, which enhancers are nucleotide sequences rich in A and T bases, the total amount of A and T bases comprising more than 50 % of the nucleotide sequence. Particular sequences are identified from the pea plastocyanin promoter which are active as enhancers, as is a solely A/T nucleotide sequence, and methods of carrying out the invention are described.

Description: ENHANCER-INCREASED GENE EXPRESSION IN PLANTS This invention relates to increasing the expression of a gene, and in particular, to altering the activity of the promoter of the gene. Genetic manipulation depends on the introduction of chimaeric genes into plants and the expression of the introduced gene depends on the promoter. There are many reasons why it would be advantageous to have a method of improving the e ectiveness of the promoter in these genes. Different promoters work with different efficiencies in different tissues. Different promoters work with different efficiencies in the same tissue, some, such as the Cauliflower Mosaic Virus 35S promoter (35S CaMV) , are commonly considered to be a stronger promoter than the promoter from the nos gene. Promoters commonly consist of more that lOOObp and when shortened work less efficiently. However, long sections of DNA produce technical difficulties in recombinant DNA techniques. Therefore, there are many instances when improved expression may be required. In experiments involving antisense, the highest expression possible might be required to achieve a commercial result. It is, therefore, an advantage to have DNA constructs available which would enhance, under the appropriate conditions, the expression of a given gene. Sequences which activate transcription have been termed enhancers (Simpson et al . Nature (1986) 323, 551-554) and a sequence that is active as an enhancer has been obtained from the 35S promoter of CaMV (U.S. Patent No. 5,164,316) . The 35S promoter which contains this enhancer region is active in many plants and the promoter has been described as constitutive, acting in many tissues. However, while enhancer regions have been suggested for plant genes it has not been previously recognised that part of a plant promoter might have an enhancer activity in several different organs and in different species. For example, the -352 to -2 region of the pea RbcS gene was attached to the bacterial nos promoter and this gave strong light-induced expression in photosynthetic tissues. Similar experiments placing the element downstream of the coding sequence did not cause expression in tobacco (Fluhr et al , Science (1986) 232, 1106-1111) . The pea PetE gene was isolated by Last, D.I. and Gray, J.C. [Plant Molecular Biology (1989) 12, 655-666]. This gene encodes plastocyanin which is a lOkDa copper protein involved in photosynthetic electron transfer. Thus, expression of this gene is required in organs such as leaves and stems in cells which contain chloroplasts. Deletion studies with the promoter region of this gene suggested that the promoter was active in leaves, ste s and flowers, but not in roots, and that an element upstream from -784 to -992 repressed expression in leaves. Removal of this region produced a very 'strong' promoter (Pwee, K-H. and Gray, J.C. The Plant Journal (1993) 3 437-449) . The invention is based on the surprising finding that a gene expressed in green photosynthetic tissue of pea has an enhancer region that is active in other species and in other tissues, including non-photosynthetic tissues. It is an object of the present invention to provide a sequence of DNA which is active as an enhancer and causes an increase in expression of a promoter expressed in green tissues. It is a further object of the present invention to provide a sequence of DNA which is active as an enhancer and causes an increase in expression of any promoter which is expressed in one or more of the roots, tubers, stems, leaves, flowers or seeds of plants. It is also an object of the invention to provide a method of enhancing expression of genes in plants other than the plant from which the sequence was obtained. The present invention provides a method of increasing the expression of a gene promoter, the method providing an increased expression of one or more genes in one or more organs of a plant by enhancing the activity of a promoter of the one or more genes using an enhancer, the enhancer being a nucleotide sequence rich in A and T bases, the total amount of A and T bases comprising more than 50% of the nucleotide sequence. As used herein an increase in expression means that the expression of the gene when used with the enhancer of the invention is greater than would be seen without using the enhancer of the invention in expression of that gene. Advantageously the enhancer is either obtained from a plant gene or is synthetically produced. The enhancer may be a homologue of the plant gene or of the synthetically produced sequence. Advantageously the enhancer is obtained from a gene expressed in pea. More advantageously, the gene is expressed in the green photosynthetic tissues of pea, in particular, the leaves of the pea plant. Preferably the enhanced expression of the gene to be incorporated into one or more organs of the plant is in a plant which is different from the plant from which the enhancer was obtained. The difference may be a difference in plant type, i.e. family, or another plant of the same plant family. The present invention provides an enhancer for a gene promoter, which enhancer is a nucleotide sequence rich in A and T bases, the total amount of A and T bases comprising more than 50% of the nucleotide sequence. The enhancer sequence may suitably be an isolated and/or purified sequence. Preferably the sequence comprises at least 20% A bases and at least 20% T bases. Preferably the sequence comprises at least 25%, and more preferably at least 30%, and even more preferably at least 35% of A and T bases respectively. One of the A or T bases may even be present as 40, 45% or 50%, or more, of the sequence. The sequence may comprise solely A and T bases. Advantageously the enhancer is the isolated and/or purified sequence, SEQ. IDl, of Figure 1 of the drawings hereof or a similar sequence thereto. More advantageously the enhancer is an isolated and/or purified sub-sequence of SEQ. IDl, or a similar sequence thereto, which sub-sequence is active as an enhancer. Preferably the sub-sequence of the enhancer is a 31bp region of SEQ. IDl described and known herein as SEQ. ID2, or any similar sequence thereto. The enhancer may alternatively preferably be any one or more of the following base pair sequences: -444 to -389, -388 to -284, -283 to -179, -444 to - 284, -388 to -179 or any similar sequence having the required enhancer activity. Avantageously the enhancer is the isolated and/or purified sequence, SEQ. ID3 , of Figure 8 of the drawings hereof, or a similar sequence thereto. A similar sequence may also be known as a homologue. As used herein the term homologue means a nucleic acid which has a nucleotide sequence which is identical, or similar, to another nucleotide sequence. The similarity must be sufficient to enable the nucleotide sequence to act as an enhancer according to the invention. More advantageously the enhancer is a purified sub¬ sequence of SEQ.ID 3, or a similar sequence thereto, which sub¬ sequence is active as an enhancer. Preferably the gene promoter is a gene promoter in plants. Preferably the sequence or sub-sequence of the enhancer causes increased expression of the gene in green or non-green tissues of plants, and in particular in the roots, tubers, seeds, stems, flowers or leaves of such plants. Advantageously, the enhancer increases expression of a gene in plants other than the plant from which the enhancer is obtained. Advantageously, the enhancer may comprise a plurality of enhancers. The enhancer may suitably operate both in normal or reverse orientation. Suitably the enhancer may also be operable attached to either the promoter or terminator of the gene to be expressed. The present invention also provides a chimaeric gene comprising an enhancer according to the invention, a gene promoter, a coding or non-coding sequence and a terminator sequence. As used herein the term chimaeric gene means a recombinant DNA molecule containing sequences from more than one organism. The chimaeric gene may comprise more than one of the enhancer and more than one promoter. The enhancer may be in normal or reverse orientation when contained in the chimaeric gene. The chimaeric gene may contain a reporter sequence or any other sequence which confers an identifiable character to a transformed plant. The present invention, moreover, provides a transformed plant, which may have been transformed by the method of the invention, having an increased expression of one or more genes in the transformed plant by virtue of the use of one or more enhancers according to the present invention. The transformed plant may be a dicotyledonous species, such as potato, tobacco, cotton, lettuce, melon, squash, cucumber, pea, rape, soyabean, sugar beet or sunflower, or a monocotyledonous species, such as wheat, barley, rye, rice or maize. Suitable alternative transformation systems for such crops will be known to the skilled reader and need not be elucidated here. The present invention also provides propagules of a plant transformed using an enhancer according to the present invention. The present invention also provides a cell which harbours a gene having increased expression as a result of the method or enhancer hereof. In order that the invention may be easily understood and readily carried into effect, reference will now be made, by way of example, to the diagrammatic drawings hereof, in which: Figure 1 shows the specific activity of GUS in bar chart form of SEQ. IDl behaving as an enhancer in the leaves of transgenic tobacco plants when fused in normal and reverse orientation to a minimal PetE promoter, GUS reporter and nos terminato , Figure 2 shows the specific activity of GUS in bar chart form of SEQ. ID2 behaving as an enhancer in the leaves of transgenic tobacco plants in single and multiple copies when fused to a minimal PetE promoter, GUS reporter and nos terminator, Figure 3 shows the specific activity of GUS in bar chart form of SEQ. IDl behaving as an enhancer in the roots of transgenic tobacco plants when fused in normal and reverse orientation upstream or downstream of a minimal 35S CaMV(-90) promoter, GUS reporter and nos terminator, Figure 4 shows the specific activity of GUS in bar chart form of sub-sequences of SEQ. IDl in normal orientation behaving as enhancers in the leaves of transgenic tobacco plants when fused to a minimal PetE promoter, GUS reporter and nos terminator, Figure 5 shows the specific activity of GUS in bar chart form of SEQ. IDl behaving as an enhancer in the micro-tubers of transgenic potato plants when fused in normal and reverse orientation to a minimal or full length patatin promoter, GUS reporter and nos terminator, and Figure 6 shows the coding sequence of the pea plastocyanin promoter (-444 to -179) sequence, known also herein as SEQ. IDl, Figure 7 shows the coding sequence for the sub-sequence of the pea pastocyanin promoter of Figure 6, this sub-sequence being known as SEQ. ID2 , Figure 8 shows the nucleotide sequence active as an enhancer and known herein as SEQ.ID3, Figure 9 shows the specific activity of GUS in bar chart form of SEQ.ID3 behaving as an enhancer in the leaves of transgenic tobacco plants when fused to a minimal PetE promoter, GUS reporter and nos terminator, Figure 10 shows the construct pATC, and Figure 11 shows the constructs pATC 21040, pATC 25040, pATC 26040, pATC 27040, pATC 28040 and pATC 29040. Example 1 The sequence known herein as SEQ. IDl (see Figure 6) was isolated from the leaves of pea in the manner described by Last, D.I. and Gray, J.C. [Plant Molecular Biology (1989) 12, 655-666]. This sequence was joined either in the normal or the reverse orientation to the -175 to +4 section of the PetE promoter fused to a GUS reporter coding sequence and nos terminator as shown in Figure 1. The resulting chimaeric gene in the Agrobacterium tumefaciens vector pBIN19 (Jefferson, R.A. et al . , EMBO J, 6, 3901-3907) was used to transform tobacco plants ( Nicotiana tobacum cv . Samsun ) . Figure 1 shows in graphical form the results for four different constructs. For each construct several independent transformed lines were analysed. Table 1 shows the actual values of specific activity of GUS obtained for each line. As expected, the PetE promoter is only expressed in the leaves but not in the roots. Nevertheless, the activity figures indicate the surprising result that the -179 to -444 upstream region of this promoter will enhance expression in either orientation, i.e. normal or reverse orientation. The methods for the production of the plants are detailed below but, as would be recognised by one skilled in the art, other methods for the production and assay of these or other plants would be equally suitable. Transgenic plants Recombinant fusion constructs containing the enhancer and PetE promoter linked to a GUS reporter and a nos terminator were mobilised into Agrobacterium tumefaciens LBA4404 (Ooms, G; Hooykaas, P.J.J; Van Veen R.J.M; Van Beelen, P; Regensburg, T.J.G; Schilpoort R.A. (1982a) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T region. Plasmid 7, 15-29) using electroporation according to Shen, W.J. and Forde, B.J. [Nucleic acid research (1989) 17, 8385] and the transformed Agrobacterium cells were used to infect tobacco leaf discs according to Horsch, R.B., Fry, J.E. , Hoffmanr, N.L., Eichholtz , D. , Rogers, S.G. and Fraley, R.T. 1985 [A simple and general method for transferring genes into plants. Science, 22, 1229-1231]. Individual kanamycm-resistant regenerated shoots were dissected away from callusing leaf discs and rooted in media without growth regulators. Rooted transgenic plants were maintained in tissue culture on media with lOOμg ml -1 kanamycin and 200μg ml -1 carbenicillin, and subcultured every 7-8 weeks. Material used in GUS assays was harvested from young, healthy, expanded leaves (25-35mm long) , fairly close to the shoot apex. Roots were washed extensively in distilled water before use. Fluorometric GUS assay GUS enzyme assays were performed essentially according to Jefferson et al . 1987, EMBO J, 6, 3901-3907. Extracts were made from l0-40mg of plant tissue in 500μl of GUS lysis buffer (50mM NaPi pH 7.0, 10 MM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, lOmM 2-mercaptoethanol) and 5-50μl of extract was used in each assay containing lmM 4-methyl umbelliferyl glucuronide. Fluorescence was measured using an LS50 fluorescence spectrophotometer (Perkin Elmer, Connecticut, USA) . Protein was determined using the Bradford (1976) microassay procedure. Table l Specific activity of GUS from leaf and root extracts of tissue- cultured tobacco plants transformed with construct containing SEQ. IDl in both orientations upstream of PetE minimal -175/+4 promoter, GUS reporter, nos terminator. Specific Activity of GUS (pmole MU/min/μg protein) Construct 40 PetE -175/+4 promoter, GUS reporter, nos terminator as a control Plant No. Leaves Roots 14 4.6 18 3.3 6 5.3 11 4.6 15 6.1 21 2.4 Mean±SEM 4.30±0.54 Construct 38 SEQ.IDl in normal orientation upstream of PetE -175/+4 promoter, GUS reporter, nos terminator Plant No. Leaves Roots 3 147.5 4 21.3 6 57.4 7 56.9 8 41.7 11 37.1 14 79.7 19 26.9 25 34.5 31 97.2 Mean±SEM 60.0±12.26 Table 1 (continued) Specific Activity of GUS (pmole MU/min/μg protein) Construct 39 SEQ.IDl in reverse orientation upstream of PetE -175/+4 promoter, GUS reporter, nos terminator Plant No. Leaves Roots 2 60.6 5 43.8 8 55.4 14 160.5 16 24.2 18 85.2 23 78.7 24 16.7 Mean: :SEM 65.6±15.96 MU = 4 methyl umbelliferone Example 2 In a further experiment, instead of using the whole of SEQ. IDl, a 31bp region from -289 to -259 of SEQ. IDl was used. The sequence was modified to give SEQ. ID2 described in Figure 7. In construct 110, one copy of the sequence SEQ. ID2 was attached to the upstream of PetE -175/+4 promoter, which promoter is attached to a GUS reporter coding sequence and nos terminator as shown in Figure 2. In construct 108, 3 copies of SEQ. ID2 were attached to the -175 to +4 PetE promoter. The results obtained after transformation and regeneration of tobacco plants are shown for the population in Figure 2 and indicate that more than one copy of the SEQ. ID2 region increases the expression of the promoter. Table 2 shows the actual values of specific activity of GUS obtained for each line. The same control as in Example 1 was used and as shown in Figure 2. TABLE 2 Specific activity of GUS from leaf extracts of transgenic tobacco plants transformed with constructs containing a single copy of 31 bp fragment or 3 copies of the 31 bp fragment from the pea plastocyanin promoter that shows high affinity for protein binding. Specific Activity of GUS (pmole MU/min/μg protein) construct no 31 bp oligonucleotide in normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos promoter Plant No. Leaves 5 61 13 99 3 4 41 4 3 20 5 3 35 6 25 30 7 10 85 7.35 9 4.76 10 4.58 Mean±SEM 8.34±6.54 construct 108 3 copies of 31 bp oligonucleotide in normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos terminator Plant No. Leaves 1 18. .29 2 40, .24 3 14. .80 4 23. .67 5 14, .73 6 12. .12 7 28. .46 8 12. .77 9 21. .83 10 10, .88 11 19, .42 12 54, .13 Mean±SEM 22, .61±12 37 Example 3 To establish whether the enhancer region SEQ. IDl had a similar effect on other promoters SEQ. IDl waε joined to the "minimal" 35S CaMV (-90) promoter in normal and reverse orientation, and also joined in normal and reverse orientation to the terminator region of the chimaeric gene prepared with the minimal CaMV promot Tobacco was transformed and assayed as deεcribed in Example 1. Figure 3 showε the mean values for the populationε obtained with theεe constructs. Table 3 presents the values for each line both for leaves (shown in Figure 3) and for roots. The region identified as an enhancer for the PetE gene acts as an enhancer for the heterologous CaMV promoter and is active in both orientations, whether it is present upstream or downstream of the promoter. The enhancer is active with a promoter which s expressed m roots and leaves and, therefore, is not tissue specific. TABLE 3 Specific activities of GUS from root and leaf extracts of transgenic tobacco plants transformed with constructs containing minimal 35S CaMV (-90) promoter, GUS reporter, nos terminator and the enhancer sequence. Specific Activity of GUS (pmole MU/min/μg protein) Construct 33 35S CaMV (-90) promoter, GUS reporter, and nos terminator as a control Plant No. Roots Leaves 4 0.07 0.30 39 0.89 0.42 48 3.21 0.35 50 0.95 0.27 51 0.98 0.39 52 1.99 0.37 53 3.44 0.33 67 5.90 0.21 77 1.25 0.47 86 2.30 0.34 Mean±SEM 2.09±0.54 0.34±0.02 Construct 34 SEQ.IDl in normal orientation upstream of 35S CaMV (-90) promoter, GUS reporter and nos terminator Plant No. ROOtS Leaves 8 2.08 0.61 11 11.34 0.51 17 13.45 0.45 19 12.67 0.32 21 14.69 0.43 22 23.11 0.60 55 13.45 0.43 72 13.75 0.47 86 4.91 0.54 Mean±SEM 12.16±1.99 0.48±0.03 Table 3 (continued) Specific Activity of GUS (pmole MU/min/μg protein) Construct 35 SEQ.IDl in reverse orientation upstream of 358 CaMV (-90) promoter, GUS reporter and nos terminator Plant No. Roots Leaves 7 17. 11 1. 40 14 16.70 1.11 22 14.24 0.91 36 13.25 0, 59 41 18.20 1, 00 68 16.90 1.11 77 7, 23 1, 24 78 37, 61 0, 75 83 34.62 0, 96 85 14, 87 1 05 94 7 93 1 12 Mean±SEM 18 06±2 90 1.01±0.06 Construct 36 SEQ. IDl in normal orientation downstream of 35S CaMV (-90) promoter, GUS reporter and nos terminator Plant No. Roots Leaves 5 3.34 0. ,36 11 2.46 0. ,63 14 1.49 0. ,51 16 1.73 0. .31 21 3.28 0. .26 34 7.61 0. .29 36 4.29 0. .38 43 2.61 0, .37 49 7.67 0. .36 Mean±SEM 3.83±0.77 0. .38±0.03 Table 3 (continued) Specific Activity of GUS (pmole MU/min/μg protein) Construct 37 SEQ.IDl in reverse orientation downstream of 35S CaMV (-90) promoter, GUS reporter and nos terminator Plant No. Roots Leaves 4 4 67 0 37 9 3 67 0 29 13 4 32 0 32 17 2 19 0 61 20 2 98 0 18 21 9 34 0 27 46 2 25 0 42 48 3 45 0 35 49 3 55 0 33 73 3 98 0 37 MeanrSEM 4 04±0.64 0 35±0.03 Example 4 To identify whether regions of the SEQ. IDl other than SEQ. ID2 contained enhancer-like activity, five further chimaeric genes were constructed as depicted in Figure 4. These constructs used regions of SEQ. IDl both upstream and downstream of SEQ. ID2. Transgenic tobacco plants were obtained as described in Example 1. Analysis of the transgenic plants containing these constructs shows that all the sub- regions of SEQ. IDl selected contain enhancer-like activity and Table 4 indicates that the activity demonstrated by SEQ. IDl is not wholly caused by SEQ. ID2. Table 4 Specific activities of GUS in leaf extracts of transgenic tobaccco plants transformed with constructs containing fragments of SEQ. IDl. Specific Activity of GUS (pmole MU/min/μg protein) Construct 74 -444 to -389 fragment from the pea plastocyanin promoter in normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos terminator Plant No. Leaves 1 56. .89 2 9. .91 3 87. .11 4 11. .38 5 13. .96 6 20. .79 7 17. .24 8 12. .76 9 38, .98 10 43, .00 Mean SEM 31, .40±8.00 Construct 83 -388 to -284 fragment from the pea plastocyanin promoter in normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos terminator Plant No. Leaves 1 45 55 2 16 71 3 22 32 4 21 66 5 22 49 6 17 72 7 16 86 8 11 33 9 10 81 MeanrSEM 20 .60±3 43 Table 4 (continued) Specific Activity of GUS (pmole MU/min/μg protein) Construct 90 -283 to -179 fragment from the pea plastocyanin promoter in normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos terminator Plant No. Leaves 1 2.19 2 16.70 3 11.65 4 15.61 5 8.95 6 17.77 7 32.41 MeanrSEM 15.04±3.53 Construct 105 -444 to -284 fragment from the pea plastocyanin promoter in normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos terminator Plant No. Leaves 1 42, 81 2 30 29 3 28 63 4 31 82 5 24 68 6 34 67 7 33 54 22 95 9 25 61 10 44 81 11 37 89 Mean±SEM 32.51±2.15 Table 4 (continued) Specific Activity of GUS (pmole MU/min/μg protein) Construct 111 -388 to -179 fragment from the pea plastocyanin promoter in the normal orientation upstream of PetE -175/+4 promoter, GUS reporter and nos terminator Plant No. Leaves 1 11.43 2 36.72 3 51.10 4 21.45 5 32.22 6 17.61 7 8.75 Mean±SEM 25.61±5.73 Example 5 To establish whether the enhancer region was active in other species the -179 to -444 region was joined to the -330 to -t-l region of the patatin promoter PS20 described by Mignery, G.A. ; Pikaard, CS. and Park, W.D. 1988 [Molecular characterisation of the patatin multigene family of potato. Gene, 62, 27-44]. The chimaeric gene produced was transferred into potato by transformation. Plant Material Potato shoot cultures were maintained in vitro on Murashige and Skoog (MS) medium in Magenta GA-7 containers at 22°C (16 hours/8 hours light/dark) . These were nυdally sub¬ cultured every 3 weeks. In vitro shoots of 2-3 inches (5-7.5cm) height were potted in 2.5 inches (6.4cm) pots of Levingtons Fl compost. They were weaned in a propagator for one week in a growth room at 18°C (16 hours/8 hours light/dark) . The propagator was removed and the plants repotted at 3 weeks into 5 inch (12.7cm) pots. At 5-7 weeks the plants were used for transformation. Agrobacterium tumefaciens Liquid overnight cultures of suitable strains, e.g. LBA4404, C58#3, were grown at 28°C to an ODg 00 (Pharmacia LKB ULTRASPEC II) of 0.8 in L-broth (see below) . Cocultivation The youngest four most expanded leaves were taken and surface sterilised in 10% commercial bleach (Domestos RTM) for 15 minutes. Leaves were rinsed thoroughly with sterile water and then cut into discs with a 7mm cork borer. The diεcs were mixed with the Agrobacterium for 1-5 minutes, blotted dry on filter paper (Whatman No. 1) and then placed on callusing medium (see below) in 90mm triple vented petri dishes, lower epidermis down. The 90mm triple vented petri dishes were sealed with tape, cut to allow gas exchange and then incubated at 22°C (16 hours/8 hours light/dark) . The discs were transferred to callusing medium plus 500μg ml- 1 of claforan and 30μg ml -1 kanamycin after 48 hours. This removes bacteria and selects for transformed cells. Regeneration of Transformed Shoots After 1 week, the discs were transferred to shooting medium containing the same antibiotics. L-broth lOg l -1 bactotryptone 5g l -1 yeast extract 5g l -1 sodium chloride lg l" 1 glucose Callusing medium MS with 3% sucrose 0.5mg 1 2,4-D 2.5mg 1 BAP Shooting medium MS with 3% sucrose 2.5mg 1 " BAP l.Omg l -1 GA 3 Further transfers were made onto the same medium until shoots could be excised (usually about 4 weeks) . Shoots with calli were transferred to MS medium with claforan (500μg/ml) in well ventilated containers, e.g. Magenta. Transformants were maintained, after several passages with cefotaxime to remove bacteria, on MS medium. They were removed from in vitro , weaned and grown to maturity as described above for plant material. The process yields transformed potato plants at a frequency of up to 30% of the discs cocultivated. Microtubers produced in the presence of Ancymidol (180μg/ml) from these plants were assayed for GUS activity as described in Example 1. Figure 5 shows the results and indicates that the enhancer sequence in either orientation can increase the activity of the patatin promoter and Table 5 shows that the gene was expressed in micro-tubers but not in leaves. TABLE 5 Specific activity of GUS from micro-tubers induced on transgenic potato plants containing SEQ. IDl upstream of a -330 to +1 patatin class 1 promoter compared with a minimal patatin promoter and a full length PS20 patatin promoter. Specific GUS activity (pmole MU/min/μg protein) Construct p250 2562 bp patatin promoter, GUS reporter and nos terminator Plant NO. Micro-tubers Leaves 1 51. ,38 2 6. .17 3 32. .50 4 49. .28 5 15. .96 6 10. .36 Mean±, SEM 27. ,55±8 06 Construct 116 330/+1 bp minimal patatin promoter, GUS reporter and nos terminator Plant No. Micro-tubers Leaves 1 3. ,85 2 14. .01 3 10. .15 4 8, .45 5 9. .03 6 8, .79 7 8, .81 Mean±SEM 9. .Ol±l. .12 Table 5 (continued) Specific Activity of GUS (pmole MU/min/μg protein) Construct 112 -444 to -179 fragment from the pea plastocyanin promoter in normal orientation upstream of minimal patatin -330/+1 promoter, GUS reporter and nos terminator Plant No. Micro- ubers Leaves 1 18. 76 2 15.28 3 8. 79 4 14.66 5 11, 29 6 84, 08 1 16, 28 8 61, 15 9 12, 77 10 10 14 11 9 49 Mean±SEM 23.88±7.48 Construct 114 -444 to -179 fragment of the pea plastocyanin promoter in reverse orientation upstream of minimal patatin -330/+1 promoter, GUS reporter and nos terminator Plant No. Micro-tubers Leaves 1 18. .36 2 40. ,82 3 40. .49 4 11. .81 5 98. .54 6 59. .89 7 11. .48 8 15, .26 9 7 , .50 Mean±SEM 33, .69110 01 Example 6 The sequence known herein as SEQ.ID3 (see Figure 8) was designed by coin flipping and constructed from two complementary oligonucleotides (5' AAT TAT AAT ATA ATT TTA ATT TAA AA3 ' ) and (5 ' AAT TTT TTA AAT TAA AAT TAT ATT AT 3 ' ) containing EcoRI overhangs at the 5 ' ends to allow multi erisation without any intervening G/C bp. Oligonucleotides were annealed, phosphorylated and concatamers were inserted in the EcoRI site of pIC19H (Marsh et al , 1984). Sequencing identified three plasmids containing inserts of 4 , 2 and 1 copies of the oligonucleotide, respectively. Inserts were excised as Hindlll-Sal l fragments and inserted in pKHd7 (Pwee, K-H and Gray, J.C. (1993) The Plant Journal) , known herein as pJSΞ22, to give pJΞS139, pJSS140 and pJSS141 containing 2,1 and 4 copies respectively. The resulting chimaeric gene in the Agrobacterium tumefaciens vector pBIN19 was used to transform tobacco plants (Nicotiana tobacum cv. Samsum) according to the methods described above. Figure 9 showε in graphical form the results for five different constructs. For each construct several independent transformed lines were analysed. Table 6 shows the actual values of specific activity of GUS obtained for each line. As expected, the PetE promoter is expressed in the leaves but also indicate the surprising result that multiples of ΞEQ.ID3 will enhance expression in a dose related manner. Table 6 Specific GUS activity from leaf extracts of transgenic tobacco plants transformed with a construct containing single copy of 26 mer oligonucleotide at EcoRI site upstream of petE minimal -175/+4 promoter, GUS reporter and nos terminator. Specific GUS activity (pmol MU/min/μo; protein) Plant No. No. 26 mer 1-26 mer 2-26 mer 3-26 mer 1 6.14 20.73 18.87 2 4.01 12.60 52.40 3 5.83 6.39 27.38 4 2.68 2.56 7.59 6 2.21 42.95 7 7.57 8 5.57 9 10.08 10 4.82 Mean±SEM 4.3010.54 3.9110.69 16.4615.39 35.68111.70 Example 7 To further establish that SEQ IDl was active with other promoters the pea metallothionein promoter, known herein as PsMT A , (Marta Evans, I., et al . , FEBS 262 (1) 29-32) was obtained. The region -806 to -1 of that promoter was ligated to the GUS coding region which contained an intron (Vancanneyt, G. et al . , (1990) Mol. Gen. Genet 220 245-250) and a nos terminator, which resulted in construct pKS 21040 (see Figure 10) . SEQ ID 1 was joined at the 5' end to this chimaeric gene in either the normal or reverse orientation. The enhancer- pro oter-GUS-nos terminator fusions were moved into the binary vector pATC (a pBIN19 derivative with modified restriction sites) , shown in Figure 10. This resulted in the constructs pATC 25040 and pATC 26040 respectively (see Figure 11) which were used to transform Nicotiana tabacum cv Heavy Western tobacco with Agrobacterium tumefaciens R1000 to produce hairy roots. Hairy root transformation: Agrobacterium tumefaciens R1000 (McAfee, B et al., Plant Cell Tissue and Organ Culture (1993) 34, 53-62) was transformed by electroporation (Shen, W.J. and Forde, B.J. Nucleic acid research (1989) 17, 8385) and the resulting bacteria used to transform Nicotiana tabacum by the leaf disc method (Horsch, R.B; Fry, E.J; Hoffman, N.L; Eichholtz, D; Rogers, S.G; Fraley, R.T. Science (1985) 22, 1229-1231) . Roots which were resistant to kanamycin were dissected away from discs and transferred to tissue culture media which contained lOOμg/ml kanamycin and 500μg/ml claforan. Roots were maintained in this media and subcultured every three weeks. The roots were then transferred to media with lOOμg/ml kanamycin and 200μg/ml claforan. After a further 7 days the roots were transferred to media without the claforan and subcultured every week. Example 8 Monomer, dimer and tetramers of the synthetic oligomer (SEQ. ID 3) were synthesised to contain EcoRI overhangs so that they could be ligated into the EcoRI site of pIC19H (Marsh et al (1984) Gene 32, 481-485) . The Xhol- Sail fragment was the excised from the resulting plasmidε and ligated into the corresponding site in pKS 21040. The synthetic oligomer promoter-GUS-nos terminator fusions were moved into the binary vector pATC. The resulting constructε pATC27040, pATC28040 and pATC29040 (see Figure ll) were used to transform Nicotiana tabacum cv Heavy Western to produce hairy roots. REFERENCES Bradford, M.M. , (1976) A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein di-binding. Analytical Biochemistry 72, 248. Fluhr, R. , Kuhlmeier, C. , Nagy, F. and Chua, N.H. (1986) Organ specific and light-induced expression of plant genes. Science 232, 1106-1111. Horsch, R.B. , Fry, J.E. , Hoffmann, N.L., Eichholtz, D. , Rogers, S.G. and Fraley, R.T. (1985) A simple and general method for transferring genes into plants. Science, 22, 1229-1231. Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987) [GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. European Molecular Biology Organisation J. 6, 3901-3907] Last, D.I. and Gray, J.C. (1989) Plastocyanin is encoded by a single-copy gene in the pea haploid. Plant Molecular Biology 12, 655-666. Marsh, J.L. , Erfle, M. , Wykes, E.J. (1984) The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32, 481-485. Marta Evans, I., Gatehouse, L.N., Gatehouse, J.A. , Robinson, N.J. and Croy, R.R.D. (1990) A gene from pea (Pisum sativum L) with homology to metallothionein genes. Federation of European Biochemical Sciences, 262 (1) 29-32 McAfee, B. , White, E., Pelcher, L. and Lapp, M. (1993) Root induction in pine ( Pinuε ) and larch (Larix) spp. using Agrobacterium tumefaciens. Plant Cell Tissue and Organ Culture 34, 53-62. Mignery, G.A.; Pikaard, CS. and Park, W.D. (1988) Molecular characterisation of the patatin multigene family of potato. Gene, 62, 27-44. Ooms, G; Hooykaas, P.J.J; Van Veen R.J.M; Van Beelen, P; Regensburg, T.J.G; Schilpoort R.A. (1982a) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T region. Plasmid 7, 15-29. Pwee, K-H. and Gray, J.C. (1993) The pea plaεtocyanin promoter directs cell specific but not full light regulated expression in transgenic tobacco plants. The Plant Journal 3, 437-449 Shen, W.J. and Forde, B.J. (1989) Efficient transformation of Agrobacterium spp. by high voltage. Nucleic acid research 17, 8385. Simpson, J., Schell, J. , Van Montagu, M. and Herrera-Estrella, L. (1986) Light-inducible and tissue-specific pea lhcp gene expression involves an upstream element combining enhancer- and silencer-like properties. Nature 323, 551-554. Vancanneyt, G. , Schmidt, R. , O'Connor-Sanchez, A., Willmitzer, L. and Rochsa-Ξoεa, M. (1990) Construction of an intron- containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium- mediated plant transformation. Mol. Gen. Genet. 220, 245-250.

Claims: CLAIMS 1. An enhancer for a gene promoter, which enhancer is a nucleotide sequence rich in A and T bases, the total amount of A and T bases comprising more than 50% of the nucleotide sequence. 2. An enhancer according to Claim 1, wherein the enhancer is an isolated and/or purified sequence. 3. An enhancer according to Claim 1 or 2, wherein said nucleotide sequence comprises at leaεt 20% A bases and at least 20% T bases. 4. An enhancer according to Claim 3 , wherein the sequence comprises at least 25% of A and T bases respectively. 5. An enhancer according to Claim 4, wherein the sequence comprises at least 30% of A and T bases respectively. 6. An enhancer according to Claim 5, wherein the sequence comprises least 35% of A and T bases respectively. 7. An enhancer according to any one of Claims 1 to 6, wherein one of the A or T bases is present as 40, 45% or 50%, or more, of the sequence. 8. An enhancer according to any one of Claims 1 to 7 , wherein the sequence may comprise solely A and T bases. 9. An enhancer according to Claim 1, wherein the enhancer is the isolated and/or purified sequence, SEQ. IDl, of Figure 1 of the drawings hereof or a similar sequence thereto. 10. An enhancer according to Claim 9, wherein the enhancer is an isolated and/or purified εub-sequence of SEQ. IDl, or a similar sequence thereto, which sub-sequence is active as an enhancer. 11. An enhancer according to Claim 10, wherein the sub¬ sequence of the enhancer is a 31bp region of SEQ. IDl described and known herein as SEQ. ID2. 12. An enhancer accirding to Claim 10, wherein the enhancer is alternatively any one or more of the following base pair sequences: -444 to -389, -388 to -284, -283 to -179, -444 to -284, -388 to -179, or any similar sequence having the required enhancer activity. 13. An enhancer according to Claim 1, wherein the enhancer is the isolated and/or purified sequence, SEQ. ID3 , of Figure 8 of the drawings hereof, or a similar sequence thereto. 14. An enhancer according to Claim 13, wherein the enhancer is a purified sub-sequence of SEQ.ID 3, or a similar sequence thereto, which sub-sequence is active aε an enhancer. 15. A method of increasing the expression of a gene promoter, the method providing an increased expression of one or more genes in one or more organs of a plant by enhancing the activity of a promoter of the one or more genes using an enhancer, the enhancer being a nucleotide sequence rich in A and T bases, the total amount of A and T bases comprising more than 50% of the nucleotide sequence. 16. A method of increasing the expression of a gene promoter according to Claim 15, wherein the enhancer is obtained from a plant gene or is synthetically produced. 17. A method of increasing the expression of a gene promoter according to Claim 16, wherein the enhancer is a homologue of the plant gene or the synthetically produced sequence. 18. A method of increasing the expression of a gene promoter according to any one of Claims 15 to 17, wherein the gene promoter is a gene promoter in plantε. 19. A method of increasing the expression of a gene promoter according to any one of Claims 15 to 18, wherein the sequence or sub-sequence of the enhancer causes increased expresεion of the gene in green or non-green tissues of plants, and in particular in the roots, tubers, seeds, stems, flowers or leaves of such plantε. 20. A method of increasing the expression of a gene promoter according to any one of Claims 15 to 19, wherein the enhancer comprises a plurality of enhancers. 21. A method of increasing the expression of a gene promoter according to any one of Claims 15 to 20, wherein the enhancer operates both in normal or reverse orientation. 22. A method of increasing the expression of a gene promoter according to any one of Claims 15 to 21, wherein the enhancer s attached to either the promoter or terminator of the gene to be expresεed. 23. A method of increasing the expresεion of a gene promoter according to any one of Claimε 15 to 23, wherein the enhancer is any one of Claims 1 to 14. 24. A chimaeric gene comprising an enhancer according to any one of Claims 1 to 14 , a gene promoter, a coding or non- coding sequence nd a terminator sequence. 25. A chimaeric gene according to Claim 24, said chimaeric gene comprising more than one of said enhancer and more than one promoter. 26. A plant having an increased expression of one or more genes ins by virtue of the use of one or more enhancers according to any one of Claims 1 to 14. 27. A plant according to Claim 26, wherein the transformed plant may be a dicotyledonous species, such as potato, tobacco, cotton, lettuce, melon, εquash, cucumber, pea, rape, soyabean, sugar beet or sunflower, or a monocotyledonous species, such aε wheat, barley, rye, rice or maize. 28. Propagules of a plant tranεformed using an enhancer according to any one of Claims 1 to 14. 29. A cell which harbours a gene having increased expression as a result of the method of any one of Claims 15 to 23 or the enhancer of any one of Claims 1 to 14 hereof. 30. A chimaeric gene substantially as hereinabove described with reference to any one of Figures 1 to 5 and 11 hereof. 31. A nucleotide sequence substantially as hereinabove described with reference to any one of Figures 6, 7 or 8 hereof m use as an enhancer.

Inventors: GRAY JOHN CLINTON, SANDHU JAGDEEP SINGH, WEBSTER CARL INNES, GRAY, JOHN, CLINTON, SANDHU, JAGDEEP, SINGH, WEBSTER, CARL, INNES
Applicants: CAMBRIDGE ADVANCED TECH, GRAY JOHN CLINTON, SANDHU JAGDEEP SINGH, WEBSTER CARL INNES, ADVANCED TECHNOLOGIES (CAMBRIDGE) LIMITED, GRAY, JOHN, CLINTON, SANDHU, JAGDEEP, SINGH, WEBSTER, CARL, INNES
Application year: 1996
Main classification: C12N15
IPC codes: A01H5, C07K14, C12N5, C12N15