IE903825A1 - RNA having endonuclease and antisense activity, the preparation thereof and the use thereof - Google Patents

Abstract

Host cells can be transformed so that they express ribozyme RNA and antisense RNA, which are linked together in the loop of the ribozyme. The RNA molecules can, for example, be complementary to a particular viral RNA. Plants transformed with genes coding for such RNA display significantly improved defences against viruses.

Description

RNA having endonuclease and antisense activity, the preparation thereof and the use thereof RNA molecules can, under suitable conditions, catalyze reactions on other RNA molecules or autocatalytically cleave fragments from their own molecules without the participation of proteins. Thus an intron having 413 nucleotides is autocatalyically deleted from the 3' end of the 23S rRNA of Tetrahymena thermophila and converted into circular form. This takes place by a number of phosphoester-transfer reactions with the participation of guanosine cofactors (Cech, T.R., Nature 30, 578-583 (1983)). Depending on the RNA substrate or the reaction conditions chosen, the intron can function as specific ribonuclease, terminal transferase, phosphotransferase or acid phosphatase. In this connection an RNA molecule can carry out several reactions without being changed itself and, in this respect, behave like an enzyme. For this reason the term ribozyme has been coined for RNA molecules having these properties.

It has also been possible to show similar reactions without the participation of proteins for some viroid RNAs and satellite RNAs. Thus self-processing seems to be a reaction essential for the multiplication for avocado sunblotch viroid (ASBV) (Hutchins, C.J. et al. Nucleic Acids Res. 14, 3627-3640 (1986)), satellite RNA of tobacco ringspot virus (sTobRV) (Prody, G.A. et al., Science 231, 1577-1580 (1986)) and satellite RNA of lucerne transient streak virus (sLTSV) (Forster A.C. et al., Cell 49, 211-220 (1987)). During the replication of these RNAs, circular forms which, as templates, lead to the synthesis of RNAs with extensions are presumably formed. These transcripts are cut to the right genomic length by the autocatalytic endonucleolytic reactions.

The structures of the RNAs which the latter presumably assume for the reaction have been described as hammerheads (Forster A.C. et al., Cell 49., 211-220 (1987); Haseloff, J. et al., Nature 334. 585-591 (1988)).

The cleavage sites for these RNA enzymes are specific and have to have certain structural characteristics so that processing can take place.

It has now been found that host cells of any desired organism can be transformed using vectors which contain DNA coding for ribozymes coupled to antisense RNA, so that the said RNA is expressed.

It is known that antisense RNA inhibits gene expression in a number of prokaryotic and eukaryotic cells, inter alia also in plant cells (Green, P., et al., Ann. Res.

Biochem. 55, 569 (1986)). The mechanism of inhibition is still largely unclear. It is presumed that doublestranded RNA which interferes with the transport of the mRNA to the cytoplasm is formed in eukaryotic systems.

Rezaian, M. et al., Plant Mol. Biol. 11. 463 (1988) investigated, for example, the possibility of using antisense RNA as antiviral agent against cucumber mosaic virus (CMV) . However, the authors observed that the antiviral activity of the antisense RNA was unsatisfactory.

However, the coupling of the appropriate ribozyme RNA in the loop (see below for diagram) with an appropriate antisense RNA now results in a more effective resistance to viruses than Rezaian was able to show. Such a coupling of the RNA molecules thus effects an increased activity, which is directed against a substrate, in transformed organisms not only in relation to the activity as antiviral agent in plants but also in general.

The invention thus relates to: 1. A gene coding for a ribozyme RNA sequence in the loop coupled with a antisense RNA sequence. 2. Host cells which contain the gene defined under 1. 3. The use of the RNA encoded by the gene defined under 1. as an agent, which is directed against a substrate, in host cells.

The invention will be described in detail below, in particular in its preferred embodiments. The invention is furthermore defined by the contents of the claims.

The ribozyme/antisense RNA can be directed against substrates such as, for example, RNA coding for selectable marker genes (resistance to antibiotics) or RNA coding for any desired cell functions, such as, for example, dihydrofolate reductase, thymidine kinase, the ripening enzymes polygalacturonase, pectin esterase etc., proteins responsible for differentiation and development, or hormone receptors. In particular types of viruses which are harmful to plants can be combated advantageously by the system according to the invention. For this purpose the procedure described below is used, for example. A ribozyme/antisense RNA which is directed against other substrates can also be constructed in an analogous way.

The ribozyme moiety can be synthesized on the basis of the RNA sequence of the substrate to be inhibited. In the case of a virus as substrate, this RNA sequence is either the genome of RNA viruses or an RNA sequence which has been derived from the DNA sequence of a DNA virus. Any virus harmful to plants can therefore be used as basis. Preferred types of viruses are pathogenic viruses, in particular cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus, tobacco mosaic virus, potato virus X or Y, tomato ringspot virus, tomato aspermy virus or tobacco rattle virus. In particular the RNA sequences RNA1, RNA2 and/or RNA3 of cucumber mosaic virus are used as template, corresponding to the sequence from the publications by Rezaian, M. et al. Eur. J. Biochem. 150, 331 (1985), Eur. J. Biochem. 143, 277 (1989) and Gould, J. et al., Eur. J. Biochem. 126, 217, (1982) or in each case parts therefrom. At least 10 consecutive nucleotides, in particular 14 to 20 nucleotides, advant10 ageously selected from the middle of the RNA sequence of the appropriate virus, are preferred for the synthesis.

The ribozyme-encoding oligonucleotides are synthesized in such a way that the initial and terminal sequences which, in each case, consist of at least 5 nucleotides, advan15 tageously 7 to 10 nucleotides, are complementary to the RNA of the virus to be inhibited. The intermediate sequence consists in part of specific nucleotides which are predetermined for the functioning of the ribozyme, and in part of variable nucleotides.

Corresponding procedures are used for the preparation of the antisense RNA but the oligonucleotides are synthesized in such a way that they code for an RNA in the appropriate antisense orientation.

The ribozyme oligonucleotide is provided with an appro25 priate linker in the loop. Linkers of this kind have, for example, cleavage sites of EcoRI, Sail, BamHI, Hindlll, EcoRV, Smal, Xhol, Kpnl, preferably Xbal or Pstl. The synthesized ribozyme oligonucleotide is then coupled via these linkers to the oligonucleotide coding for the antisense RNA in the ribozyme loop.

The ribozyme/antisense RNA system hybridized with substrate RNA may be represented diagrammatically as follows: -NNNNNNNNNNNNNGUC NNNNNNNNNNNNNN-3 3-KKKKKKKKCA KKKKKKKK-5 substrate RNA C U A ®A CG Gy vv vv ribozyme where N are nucleotides of the substrate RNA, A, C, G or T, K are nucleotides complementary to N in the ribozyme, V are variable nucleotides in the ribozyme and VL are variable nucleotides in the loop of the ribozyme. The number of VL nucleotides can be 0-550. VL nucleotides are chosen such that a cleavage site into which the sequences can be inserted is produced at the DNA level.

The assembled oligonucleotides are cloned with the aid of the vectors pUC19, pUC18 or pBluescript (Stratagene, Heidelberg, Product Information) and sequenced.

The confirmed oligonucleotide is cloned into an intermediary vector having a plant promoter. Vectors of this type are, for example, the plasmids pPCV701 (Velten J. et al., EMBO J. £, 2723-2730 (1984)), pNCN (Fromm H. et al. PNAS £2, 5824-5826 (1985)) or pNOS (An, G. et al., EMBO J. 4., 277-276 (1985)). The vector pDH51 (Pietrzak, M. et al., NAR 14., 5857, (1986)) which has a 35S promoter is preferably used.

After subsequent transformation of E. coli, such as, for example, E. coli MC 1061, DH1, DK1, GM48 or XL-1, positive clones are identified by methods known per se (Maniatis et al., Lab. Manual), such as plasmid mini25 preparations and cleavage with an appropriate restriction enzyme. - 6 These positive clones are then subcloned into a binary plant vector. As plant vectors, pGV3850 (Zambrysk, P. et al., EMBO J. 2, 2143-2150 (1983)) or pOCA18 (Olszewski, N., NAR 16 . 10765-10782, (1988)) can be employed. Advan5 tageously pOCA18 is used.

The resulting binary plant vectors which contain a plant promoter with the attached DNA fragment for the ribozyme production in the T-DNA are used to transform plants. This can be carried out by techniques such as electro10 poration or microinjection.

The cocultivation of protoplasts or the transformation of leaf pieces using agrobacteria is preferably employed. For this purpose the plant vector construct is transferred by transformation with purified DNA or, mediated by a helper strain such as E. coli SM10 (Simon R. et al., Biotechnology 1, 784-791 (1983)), into Agrobakterium tumefaciens, such as A282, having a Ti plasmid via a triparental mating. Direct transformation and triparental mating were carried out as described in Plant Molecular Biology Manual (Kluwer Academic Publishers, Dordrecht (1988)) .

In principle, all plants can be transformed by the binary plant vectors carrying the DNA constructed according to the invention. Dicotyledonous plants, in particular useful plants, which produce or store starch, other carbohydrates, proteins or fats in usable amounts in their organs or which produce fruit and vegetables or supply spices, fibers and technically usable products or medicaments, dyes or waxes, and also fodder plants.

Examples which may be mentioned are tomato, strawberry, avocado, and plants which carry tropical fruit, for example papaya, mango, but also pear, apple, nectarine, apricot or peach. Furthermore, examples which may be listed as plants to be transformed, are all types of cereal, rape, turnip rape, potatoes, soybean, cotton, corn, sugarbeet or sunflowers. The transformed cells are - Ί selected for with the aid of a selection medium, are cultured to a callus and regenerated to the plant on an appropriate medium (Shain et al., Theor. appl. Genet. 72, 770-770 (1986); Masson, J. et al., Plant Science 53, 1675 176 (1987), Zhan et al., Plant Mol. Biol. 11. 551-559 (1988); McGranaham et al., Bio/Technology 6, 800-804 (1988) Novrate et al., Bio/Technology T., 154-159 (1989).

The resulting plant is changed by the transformation in such a way that, with the aid of the constructed oligo10 nucleotides, the corresponding RNA is expressed in the cells, it being possible that ribozyme RNA together with antisense RNA becomes active against virus RNA.

The examples which follow are intended to illustrate the invention further.

Examples Percentages relate to the weight if not stated otherwise. 1. Synthesis of the DNA for the expression of RNA The synthesis of the nucleotide for the ribozyme was carried out on the basis of the CMV RNA1 sequence, position 3248 to 3264, (Rezaian M. et al., Eur. J.

Biochem 150, 331-339 (1985)) provided with an Xbal linker on the 5' end and a Pstl linker on the 3' end. The constant regions are evident from the attached diagram for the ribozyme: DNA for the expression of a ribozyme with an Sail site in the variable part of the loop: ’-ctagaggtagctcctgatgagtcgtcgacgacgaaacaaccttctgca-3’ tccatcgaggactactcagcagctgctgctttgttggaag or: - 8 5'-CTAGATTTCAAGGGTAGCTCCTGATGAGTCGTC 3' TAAAGTTCCCATGGAGGACTACTCAGGAG GACGACGAAACAACCTTGTAGGATGTCTGCA-3' CTGCTGCTTTGTTGGAACATCCTACAG - 5' The synthesis of the nucleotide for the antisense RNA was carried out on the basis of the CMV RNA4 sequence (Gould I. et al., Eur. J. Biochem. 126. 217-26 (1982)) from position 2179 to 2193 with an Xbal linker on the 5' end and with a Pstl linker on the 3' end, which were filled in order to create a Sail site for cloning.

DNA for the production of an antisense RNA: 5’-CTAGATGGTCTCCTTATGGAGAACCTGTGGAAAACCACAGCTGCA-3 ’ TACCAGAGGAATACCTCTTGGACACCTTTTGGTGTCG or: ’-TCGACATGGTCTCCTTATGGAGAACCTGTGGAAAACCACAGCG-3’ RG ΠGTACCAGAGGAATACCTCTTGGACACCTTTTGGTGTCGCAGCT-5’ 2. Cloning in pBluescript SK+ and sequencing The plasmid pBluescript SK+ (Stratagene, Product Informa15 tion) was cut open with the particular enzymes and, after treatment with calf intestinal phosphatase (CIP), was ligated with a five-fold excess of the double-stranded, phosphorylated oligonucleotides. After induction with isopropylgalactoside (IPTG) it was possible to identify positive clones in the strain XL Blue (Stratagene) as white colonies on plates containing 5-bromo-4-chloro-3indolyl 0-D-galactoside (X-gal). of the colonies were isolated in each case and sequenced by the dideoxy method (Boehringer, Sequencing kit), in order to determine the orientation of the inserted oligonucleotide. ,ε 903825 Fig. 1: pBluescript SK clones containing the desired oligonucleotide DNA after digestion with Xba/Pst. 3. Cloning in pDH51 The plasmid pDH51 is reproducibly described in the publication Pietrzak, M. et al., NAR 14. 5857-5868, 1986.

The plasmid pBluescript SK+ containing the oligonucleotide was digested with Xbal and Pstl in order to be able to isolate the oligonucleotide DNA after the fragment fractionation in a 1% low melting agarose gel.

The isolated oligonucleotide was incorporated into the Xba/Pst site of pDH51 in a five-fold excess. It was possible to detect positive clones, after the transformation of E. coli MC 1061 cells and transfer of the .5 colonies onto nitrocellulose filters, by hybridization with 32P-labeled oligonucleotide DNA and subsequent washing of the filters in SSC (1. lx SSC; 2. 0.1 x SSC; 3. 0.1 x SSC for 30 min each time) at 65*C.

A DNA sequence for the synthesis of the antisense oligo20 nucleotide DNA was via the newly created Sail cleavage site cloned into a vector generated in this way. - 10 After the transformation of E. coli MC 1061 cells, the colonies were streaked onto nitrocellulose filters and hybridized overnight with 32P-labeled oligonucleotide DNA which was isolated from an appropriate pBluescript SK+. The filters were washed as mentioned above.

Positive clones were identified by autoradiography. 4. Cloning in pOCA18 The plasmid pOCA18 is reproducibly described in publica10 tion Olszewski, N. et al., NAR 10765-10782, 1988. The about 1.0 kb long EcoRI fragment from pDH51 with the oligonucleotide after the 35S promoter was isolated on a CsCl gradient, and the 0.8 kb fragment resulting from EcoRI digestion was incorporated into the EcoRI cleavage site of pOCA18.

The positive clones were identified by means of a DNA preparation and a subsequent EcoRI step.

Fig. 3: Lane 1+2: Lane 3: Fig. 4: Lane 1 + 3: Lane 2+4: Clones cut with EcoRI after the CsCl purification λ DNA cut with EcoRI/HindiII DNA cut with EcoRI, from the ligase reaction of vector pOCA18 with the 0.8 kb fragment As above, but uncut . Transformation of agrobacteria The vector pOCA18 containing the 35S promoter/oligonucleotide insert was transferred into the agrobacterial strain A282 (Pharmacia, Freiburg, FR Germany or ATCC 37349 USA). This was carried out by a triparental mating with the aid of the E. coli strain SH10 (Simon, R. et al., Bio/Technology 1., 784-791, 1983). For this purpose equal amounts of the bacteria were placed together on a filter overnight, incubated at 28*C, and the filters were rinsed with 2 ml of 10 mM MgSO4 and aliquots thereof were placed on yeast extract containing tetracycline and rifampicin (YEB: 1% yeast extract, 1% peptone, 0.5% NaCI) in order to select for positive clones. The subsequent hybridization of the colonies, which had been transferred onto nitrocellulose membranes, with radiolabeled oligo5 nucleotide DNA again demonstrated the presence of positive clones.

Fig. 5: Transformed agrobacteria were streaked onto filters, incubated at 28eC overnight and hybridized with radiolabeled oligonucleotide DNA. 6. Transformation of tobacco The agrobacteria were grown in YEB medium containing tetracycline and rifampicin. 20 ml of the bacteria were spun down, washed once in YEB medium and, suspended in ml of 10 mM MgSO*, placed in a Petri dish. The plant material used was Nicotiana tabacum Wisconsin 38. The plants had been cultivated for 4 weeks under sterile conditions on 2MS medium (Murashige, T. et al., Physiol. Plant 15, 473-497 (1962)) at 25eC with 16 h of light per day. A 1 cm2 leaf piece was cut off these plants, damaged with sterile emery paper and immersed in the overnight fe903825 - 13 bacterial culture for 30 sec.

The leaf pieces were, as described above for 2 MS, maintained on MS medium for 2 days at 25 °C and then washed with liquid 2MS medium. Then these leaf pieces were placed on MSC 10 plates (MS medium containing 1.5% agar) containing kanamycin. After 5-6 weeks, it was possible to replant regenerated plants into larger vessels where they formed roots after 2-3 weeks.

The expression of the desired RNA in the transgenic 10 plants was detected by transferring the total RNA (10 Mg), which had been fractionated in a gel (1% agarose gel containing 2.2 M formaldehyde), onto nitrocellulose filters and subsequent hybridization with radioactive oligonucleotide DNA.

Fig. 6: Lane 1: 10 μς of total RNA of the control plant were, after fractionation in a gel, - 14 transferred onto nitrocellulose. The hybridization was carried out with radiolabeled oligonucleotide DNA.

Lane 2-24: As above, but 10 /*g of total RNA of the transgenic plants were applied in each case. 7. Detection of the transformation DNA was isolated from about 8-week old transformed tobacco plants by standard methods (Maniatis et al., Lab.

Journal) and cut with EcoRI, and 10 μς of DNA were transferred onto nitrocellulose membranes in each case and hybridized with 32P-labeled oligonucleotide DNA. The incorporation of the desired sequence in the DNA of the transgenic plant was detectable by hybridization with the characteristic EcoRI DNA.

Fig. 7: Lane 1-9: 10 ^g of total DNA of transgenic plants were, in each case, hybridized with radiolabeled oligonucleotide DNA on nitrocellulose.

Lane 10: As above, but total DNA of the control plant. - 15 8. Detection of the expression of the RNA RNA was isolated from a second leaf sample from the abovementioned tobacco plants, transferred onto nitrocellulose from a formaldehyde gel and hybridized as above. It was possible to detect bands which showed the expected sizes. 9. Detection of the in vitro activity of the multifunctional RNA RNA was produced from the pBluescript SK+ clones contain10 ing the inserted entire oligo by addition of T3 or T7 polymerase in a reaction mixture (Stratagene, Product Information for SK+) and then isolated. Hybridization of this RNA with 32P-labeled CMV RNA showed a cleavage of the virus RNA.

. Infection with CMV strain Q About 8-week old tobacco plants were infected, with the aid of carborundum, with the CMV strain Q (Rezalan, M. et al., Eur. J. Biochem. 150, 331 (1985); 143, 277 (1984); Gould, J. et al., Eur. J. Biochem. 126, 217 (1982)).

Control plants showed distinct symptoms after 12-15 days.

It was possible to detect virus particles in these plants. The transgenic plants proved to be resistant to the virus to varying degrees. - 16 Table: Percentage of infected plants with distinct symptoms after the time stated in each case (10 plants of a clone were infected together and evaluated, in each case) 12 d 20 d Wild type W 38 80 80 10 Transgenic CLONE NUMBERS: plants 3 40 50 7 60 60 10 40 60 15 13 30 50 18 60 60 19 30 40 20 30 30 21 20 10 20 22 50 50

Claims (11)

1. Patent claims:

1. A gene coding for a ribozyme RNA sequence in the loop coupled to a antisense RNA sequence.

2. The gene as claimed in claim 1, with the encoded RNA being complementary to a virus RNA.

3. An RNA encoded by a gene as claimed in claim 1 or 2.

4. A host cell containing a gene as claimed in one of claims 1 or 2.

5. A host cell containing an RNA as claimed in claim 3. 6 . Plants, plant cells and parts or seeds of the plants, containing the gene as claimed in claim 1 or 2. 7. Plants, plant cells and parts or seeds of the plants, containing the RNA as claimed in claim 3.

6. 8. The use of the RNA encoded by the gene as claimed in claim 1 as agent directed against the substrate, in host cells.

7. 9. The use of the RNA encoded by the gene as claimed in claim 2 as antiviral agent.

8. 10. A gene according to claim 1, substantially as hereinbefore described and exemplified.

9. 11. An RNA according to claim 3, substantially as hereinbefore described and exemplified.

10. 12. A host cell according to claim 4 or 5, substantially as hereinbefore described and exemplified.

11. 13. Plants, plant cells and parts or seeds of the plants according to claim 6 or 7, substantially as hereinbefore described and exemplified. -1814. Use according to claim 8 or 9, substantially described.