NZ236945A - Hybrid gene having self-splicing autocatalytic activity - Google Patents

Description

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NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION EXPRESSION OF A MULTIGENE RNA HAVING SELF-SPLICING ACTIVITY We, HOECHST AKTIENGESELLSHAFT, a Corporation organized under the laws of the Federal Republic of Germany, of D-6230 Frankfurt am Main 80, Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement - 1 - (Followed by 1A) -I A- 2 3 6 9 4 p.j i -H&EeHST—AKT-rENGESEHjGeiiftFT Dr-rSP/gm HOE 90/F 034 •Seocription Expression of a multigene RNA having self-splicing activity RNA molecules can, under suitable conditions, catalyze reactions on other RNA molecules or autocatalytically cleave off fragments from their own molecules without the participation of proteins. For example, an intron having 413 nucleotides is deleted autocatalytically at the 10 3' end of the 23S rRNA of Tetrahymena thermophila and transformed into a circular form. This takes place by means of a number of phosphoester transfer reactions with guanosine cofactors participating (Cech, T.R., Nature 30. 578-583 (1983)). Depending on the RNA substrate or the 15 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 a few conversions without being changed itself and has the characteristics 20 of an enzyme in this respect. For this reason, the term ribozyme has been introduced for RNA molecules having these properties.

It was possible to show similar reactions without participation of proteins also for some viroid RNAs and 25 satellite RNAs. For example 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 30 streak virus (sLTSV) (Forster A.C. et al., Cell 49., 211- 220 (1987)), self-processing seems to be a reaction essential for multiplication. During the replication of these RNAs circular forms are presumably formed which, as templates, lead to the synthesis of RNAs which are 35 overlong. These transcripts are cut to the right genome 23 6 9 4 length by the self-catalyzed endonucleolytic reactions.

The structures of the RNAs, which these presumably take on for the reaction, have been described as hammerheads (Forster A.C. et al., Cell 4J., 211-220 (1987)); Haseloff, J. et al., Nature 334, 585-591 (1988)).

The cleavage sites for these RNA enzymes are specific and must have certain structural prerequisites in order to allow processing.

Surprisingly, it has now been found that host cells of any desired organisms can be transformed using vectors which contain DNA coding for ribozyme RNA linked to functional genes, so that said RNA is expressed and subsequently spliced.

The invention thus relates to: 1. A hybrid gene comprising one or more copies of a gene sequence coding for ribozyme RNA and of one or various functional genes, the gene sequences being linked via a spacer which, on the RNA level, represents a substrate for the ribozyme. 2. Host cells containing the gene characterized in 1.

In the following, the invention is described in detail, in particular in its preferred embodiments. Furthermore, the invention is defined by the contents of the claims.

A functional gene is a DNA section in the genome, which codes for a polypeptide. The polypeptide can, on its own, be a functional protein, or function as sub-unit of an enzyme complex.

The gene according to the invention is constructed in such a way that, starting from a promoter, several genes, for example entire synthetic pathways for the production n 236 9 of e.g. amino acids, such as glycine, nucleotides, secondary metabolites such as antibiotics, cofactors of enzymes, hormones, such as thyroid hormone, can be expressed in plants or microorganisms. The intention of 5 this is, on the one hand, to produce foreign substances in appropriate plants or microorganisms and, on the other hand, to increase the yield in plants or microorganisms which naturally synthesize these substances. Genes coding for appropriate polypeptides can be employed according to 10 the invention by using control sequences.

It is furthermore possible to employ several copies of a gene for a particular functional protein, e.g. in order to increase the yield of a protein such as insulin or transaminase.

Furthermore, the expression of one or more selection markers using the system according to the invention is possible. For this purpose, for example, genes for the following proteins can be employed: yS-lactamase, ^-galactosidase, phosphinotricine acetyl transferase, 20 chloramphenicol acetyl transferase or thymidine kinase.

The acetyl transferase gene for the herbicide phosphinotricine and the Cat gene from the transposon Tn9 for the chloramphenicol acetyl transferase are preferably used.

The acetyl transferase gene from Streptomyces viridochro-25 mogenes (Wohleben, W. et al., Gene 7J0, 25-37 (1988)) can be assembled from synthetically prepared oligonucleotides, there also being the possibility of modifying it for the expression in plants. The gene gives resistance to phosphinotricine to transgenic plants which express 30 the product constitutively. The Cat gene effects resistance to chloramphenicol. Like the acetyl transferase gene, the Cat gene acetylates its product. The Cat gene is derived from Tn9 (Alton, N.K. et al., Nature 15. 282, 864-866, 1979) but is also commercially 35 available. fif) V , , ^ "o' '•o' ■< The genes are linked by ribozyme substrate sequences, so-called spacers, and are released by a ribozyme structure domain of the same molecule. In this way, at least 2 and up to about 25 or more gene sequences can be 5 expressed in one organism. The release can also take place via a separately expressed ribozyme molecule.

The appropriately transcribed RNA is essentially composed so that the ribozymes are preferably at the 3' or 5' end of the RNA molecule. A sequence of 40-50 nucleotides, 10 which is entirely or in a partial region comprising at least 10, preferably 15-35, nucleotides complementary to the sequence of the ribozyme, is inserted as spacer. The ribozyme sequence of the RNA can, in this way, associate itself with the spacer and cut the latter immediately 15 downstream of a defined sequence. A GUC triplet is preferably used as ribozyme cleavage site. The number of linked genes can, in each case, be multiplied by introducing further spacers, it being possible for the sequence of the latter to be the same or different from 20 the first spacer introduced. If it is different, a further ribozyme structure domain matching this sequence is created in the same RNA molecule.

The spacer and ribozyme sequences necessary for this RNA can be prepared synthetically. The linkage to the genes 25 is carried out by means of suitable linkers which have been synthesized on.

Spacers and ribozyme are synthesized to be analogous to ribozyme structures in nature (Uhlenbeck, O.C., Nature 328. 596-600, (1987). In this connection, these 30 sequences can mimic naturally occurring ribozymes (Forster A.C. et al., Cell 4j}., 211-220, (1987)) or be constructed in such a way that the essential structures of the ribozyme are present, but that other sequences are chosen for nonessential parts. In the basic construction, 35 the procedure by Haseloff, J. et al., Nature 334. 585-591, 1988 can essentially be carried out. A GUC sequence 23 6 9 around which sequences which are complementary to a described ribozyme sequence are located in the direction of 5' and 3' is incorporated into the spacer part of the RNA.

Spacer and ribozyme on the RNA level can diagrammatically be described as follows: -NNNNNNNNNNNNNGUC NNNNNNNNNNNNNN-3 — Subatnt-RNA 3-KKKKKKKKCA KKKKKKKK-5 A C A UG G * ®A C G Gy yV v > Ribozym VV where N are nucleotides of the substrate RNA, A, C, G or U K are nucleotides complementary to N in the ribozyme V are variable nucleotides A, C, G or U in the ribozyme and VL are variable nucleotides A, C, G or U in the loop of the ribozyme.

The number of nucleotides of VL can be 0-550.

The gene according to the invention is cloned into an intermediary vector having a plant promoter. Vectors of this type are for example the plasmids pNCN (Fromm M. et al., PNAS 82, 5824-5826 (1985)) or pNOS (An G. et al. EMBO J. 4., 277-276 (1985)), or preferably pDH51 (Pietrzak, M. et al. NAR 14, 5857-5861, (1986)).

After subsequent transformation of E. coli, such as e.g. E. coli MC 1061, DH1, DK1, GM48 or XL-1, positive clones fTj) y uL. \J. are identified by methods known per se (Maniatis et al., Lab. Manual), such as plasmid minipreparation and cleavage using an appropriate restriction enzyme. These positive clones are then subcloned in a binary plant 5 vector. pGV3850 (Zambrysky, P. et al., EMBO J. 2, 2143-2150 (1983)) or pOCA18 (Olszewski, N., NAR 16, 10765-10782, (1988)) can be employed as plant vectors. Advantageously pOCA18 is used.

The binary plant vectors obtained which contain in the 10 T-DNA a plant promoter with the attached DNA fragment which is constructed as specified above are used to transform plants. This can be carried out by techniques such as electroporation or microinjection.

Preferably, the cocultivation of protoplasts or the 15 transformation of pieces of leaf by agrobacteria is used.

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)), in Agrobacterium 20 tumefaciens such as A 282 via triparental mating using a Ti plasmid. Direct transformation and triparental mating were carried out as described in "Plant Molecular Biology Manual" (Kluwer Academic Publishers, Dardrecht (1988)).

Basically all plants can be transformed using the binary 25 plant vectors according to the invention and carrying constructed DNA. Dicotyledonous plants, in particular useful plants, which produce or store for example starch, carbohydrates, proteins or fats in usable amounts in their organs, or produce fruit and vegetables, or provide 30 spices, fibers and technically usable products or medica ments, dyes or waxes, as well as fodder plants are preferred. Examples which may be mentioned are tomato, strawberry, avocado, and plants which carry tropical fruit, e.g. papaya, mango, but also pear, apple, 35 nectarine, apricot or peach. Furthermore, as examples of plants to be transformed all types of cereal, rape, 23 6 9 potatoes, soya bean, cotton, corn, sugar beet or sunflower may be listed.

The transformed cells are selected with the aid of a selection medium, grown to give a callus and regenerated 5 to the plant on an appropriate medium (Shain et al., Theor. appl. Genet. 22/ 770-770 (1986); Masson, J. et al., Plant Science 53., 167-176 (1987); Zhan et al., Plant Mol. Biol. 11, 551-559 (1988); McGranaham et al., Bio/Technology 6., 800-804 (1988); Novrate et al., 10 Bio/Technology 2f 154-159 (1989)).

The resulting plant is altered by the transformation insofar as the RNA which is expressed with the aid of the constructed oligonucleotides is cleaved open in the cells at GUC cleavage sites by the ribozyme activity in order 15 to release the genes.

It is also possible to use the described system in bacteria, cell cultures, yeasts or other eukaryotic organisms.

The examples which follow illustrate the invention in 20 more detail. fN «- Examples 1) DNA structures used a) Acetyl transferase gene having Sail linkers 9 IB 27 36 45 ' GTC GAC ATG TCT CCG GAG AGG AGA CCA GTT GAG ATT AGG CCA GCT 3' G TAC AGA GGC CTC TCC TCT GGT CAA CTC TAA TCC GGT CGA 54 63 72 81 90 ACA GCA GCT GAT ATG GCC GCG GTT TGT GAT ATC GTT AAC CAT TAC TGT CGT CGA CTA TAC CGG CGC CAA ACA CTA TAG CAA TTG GTA ATG 99 108 117 126 135 ATT GAG ACG TCT ACA GTG AAC TTT AGG ACA GAG CCA CAA ACA CCA TAA CTC TGC AGA TGT CAC TTG AAA TCC TGT CTC GGT GTT TGT GGT 144 153 162 171 180 CAA GAG TGG ATT GAT GAT CTA GAG AGG TTG CAA GAT AGA TAC CCT GTT CTC ACC TAA CTA CTA GAT CTC TCC AAC GTT CTA TCT ATG GGA 189 198 207 216 225 TGG TTG GTT GCT GAG GTT GAG GGT GTT GTG GCT GGT ATT GCT TAC ACC AAC CAA CGA CTC CAA CTC CCA CAA CAC CGA CCA TAA CGA ATG 234 243 252 261 270 GCT GGG CCC TGG AAG GCT AGG AAC GCT TAC GAT TGG ACA GTT GAG CGA CCC GGG ACC TTC CGA TCC TTG CGA ATG CTA ACC TGT CAA CTC 279 2BB 297 306 315 AGT ACT GTT TAC GTG TCA CAT AGG CAT CAA AGG TTG GGC CTA GGA TCA TGA CAA ATG CAC AGT GTA TCC GTA GTT TCC AAC CCG GAT CCT 324 333 342 351 360 TCC ACA TTG TAC ACA CAT TTG CTT AAG TCT ATG GAG GCG CAA GGT AGG TGT AAC ATG TGT GTA AAC GAA TTC AGA TAC CTC CGC GTT CCA 369 378 387 396 405 TTT AAG TCT GTG GTT GCT GTT ATA GGC CTT CCA AAC GAT CCA TCT AAA TTC AGA CAC CAA CGA CAA TAT CCG GAA GGT TTG CTA GGT AGA 414 423 432 441 450 GTT AGG TTG CAT GAG GCT TTG GGA TAC ACA GCC CGG GGT ACA TTG CAA TCC AAC GTA CTC CGA AAC CCT ATG TGT CGG GCC CCA TGT AAC 459 468 477 486 495 CGC GCA GCT GGA TAC AAG CAT GGT GGA TGG CAT GAT GTT GGT TTT GCG CGT CGA CCT ATG TTC GTA CCA CCT ACC GTA CTA CAA CCA AAA 504 513 522 531 540 TGG CAA AGG GAT TTT GAG TTG CCA GCT CCT CCA AGG CCA GTT AGG ACC GTT TCC CTA AAA CTC AAC GGT CGA GGA GGT TCC GGT CAA TCC 54 9 553 CCA GTT ACC CAG ATC TGA G• 3' GGT CAA TGG GTC TAG ACT CAG CTG 5' b) Spacer having Sail and Hindlll linkers 9 18 27 36 ' TCG ACT TAC GGC TAA AAT GGT CAG TAT CCC CCA AAG GCG GCC GC 3' ( 3' GA ATG CCG ATT TTA CCA GTC ATA GGG GGT TTC CGC CGG CGT t&A * 23 5 9 c) Cat gene from Tn9 according to Alton et al. Nature 282, 864-866 (1979) d) Ribozyme structure domain having Hindlll and PstI linkers 9 IB 27 36 45 AGC TGC GGC CGC TTA CGG CTA AAA TGG TCA GTA TCC CCC AAA GGG CG CCG GCG AAT GCC GAT TTT ACC AGT CAT AGG GGG -TTT CCC 54 63 72 81 90 GTA CCC CTT TCG GGC ATA CTC TGA TGA GTC CGT GAG GAC GAA ACC CAT GGG GAA AGC CCG TAT GAG ACT ACT CAG GCA CTC CTG CTT TGG 99 108 ATT TTA GCC GTA ACT GCA 3' TAA AAT CGG CAT TG ' The oligonucleotides under a), b) and d) were synthesized by means of a DNA synthesizer by the phosphoramidite method. 2) Cloning the fragments The DNA specified under la)-d) was ligated in equal molar amounts and incorporated into the Sall/PstI sites of the vector pDH51. Positive clones were identified by hybridization with all 4 radioactively labeled DNA sections used. 3) Cloning into pOCAl8 The plasmid pOCAl8 is reproducibly described in Olszewski, N. et al. NAR 16., 10765-10782 (1988).

An Nosl/Hindlll fragment with a length of 2.4 kbp was isolated from the described vector pDH51 with the inserted construction and, after filling in the ends, cloned into a pOCA18 vector which had been cut using BamHI and filled in. Positive clones were detected by hybridization with 3ZP-labeled DNA. r* • ■ 4) Transformation of agrobacteria The pOCA18 vector with the described 35S promoter/insert was transferred into the agrobacteria strain A 282 (Pharmacia Freiburg, FR Germany, or ATCC 37349, USA). This was carried out by triparental mating with the aid of the E. coli strain SM10 (Simon, R. et al. Bio/Technology 1, 784-791, 1983)). For this purpose, equal amounts of the bacteria were applied together onto a filter overnight, rinsing with 2 ml of 10 mM MgS04 was carried out and aliquots thereof were applied to YEB plates containing tetracycline and rifampicin (YEB: 1 % yeast extract, 1 % peptone, 0.5 % NaCl). It was possible to detect positive agrobacteria by hybridization.

) Transformation of tobacco The agrobacteria were grown in YEB medium (1 % yeast extract, 1 % peptone, 0.5 % NaCl) containing tetracycline and rifampicin. 20 ml of the bacteria were spun down, washed once in YEB medium and suspended in 20 ml of 10 mM MgS04 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 25°C with 16 hours of light per day. A 1 cm2 leaf piece was cut off from these plants, wounded using sterile emery paper and immersed in the bacteria culture for 30 sec. The leaf pieces were maintained on MS medium, as described above for 2MS, at 25°C for 2 days and were then washed with liquid 2MS medium. The leaf pieces were then transferred onto MSC 10 plates (as MS containing 1.5 % agar) containing 100 pg/ml of kanamycin. After 56 weeks, it was possible to replant regenerated plants into larger vessels where they formed roots after 2 3 weeks. 236 94 5 6) Detection of transformation DNA was isolated from transformed tobacco plants with an age of about 8 weeks using standard methods (Maniatis et al., Lab. Journal), transferred to nitrocellulose mem-5 branes and hybridized with 32P-labeled insert DNA.

It was possible to demonstrate an incorporation of the desired sequences in the DNA of the plant. 7) Detection of the expression of the RNA RNA was isolated from the abovementioned tobacco plants 10 from a second leaf sample, was transferred from a formaldehyde gel to nitrocellulose and hybridized as above. It was possible to detect several bands which showed the expected sizes. 8) Detection of in vitro function of the ribozyme RNA The multifunctional RNA was produced from the pBluescript SK+ clones containing the inserted entire oligo using T3 or T7 polymerase in a reaction mixture (Stratagene, Product Information for SK+) and was then isolated. Hybridization of this RNA with individual 20 components showed that the RNA was cleaved open. 9) Detection of in vivo activity of the genes Transformed plants showed growth on 2MS medium containing phosphinotricine. In a spray experiment, the plants likewise proved to be resistant. An experiment in order 25 to acetylate chloramphenicol showed that the plants express an active enzyme. \ 2 3 T, 1) 4 T)

Claims (6)

WHAT WE CLAIM IS:

1. A hybrid gene comprising one or more copies of a gene sequence coding for ribozyme RNA and of one or more functional genes, the gene sequences being linked in each case via a spacer which, on the RNA level, represents a substrate for the ribozyme.

2. A hybrid gene as claimed in claim 1, wherein the functional gene codes for phosphinotricin acetyl transferase and/or for chloramphenicol acetyl transferase.

3. . A plant, plant cell, part of a plant, or a seed of a plant containing a gene as claimed in claim 1 or 2.

4. A microorganism or a plant, plant cell, and a part or a seed of the plant, containing a gene as claimed in claim 1 or 2.

5. A hybrid gene according to claim 1 substantially as herein described or exemplified.

6. A host cell according to claim 3 substantially as herein described or exemplified. HOECHST AKTiENGESELLSCHAFT