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  • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
  • C12N2310/121—Hammerhead

Definitions

• the present invention is directed generally to synthetic ribozymes and their mutants and derivatives capable of catalysing cleavage of virus specified mRNA and, more particulary, DNA virus specified mRNA.
• the present invention further contemplates the use of these ribozymes in the generation of transgenic cells and, in particular, transgenic plant cells and plants capable of inhibiting infection by the viruses.
• Viruses are the causative agents of a large number of serious and potentially serious diseases in humans, animals and plant. Plant viruses in particular have the potential to destroy or reduce crop yield and to otherwise have a deleterious effect on agricultural and horticultural industries to economically significant levels.
• viruses particularly important viruses in this regard are the DNA viruses, including the Caulimo- and the Geminiviruses and two unclassified viruses, the subterranean Clover Stunt Virus (SCSV, Shu and helms, Virology, 1986, 167, 38-49) and Coconut Foliar Decay Virus (CFDV, Rohde et al, Virology, 1990, 176, 648-51) .
• the geminiviruses are a group of small viruses which infect plant cells. They contain a single stand of circular DNA referred to as "virion- sense” or "positive-sense" DNA of less than about 2900 base pairs.
• the viral coat protein Upon infection of a host cell by a geminivirus, the viral coat protein is removed and a double stranded replicative form of DNA is synthesised comprising the virion-sense strand and a "complementary-sense" strand. Transcription occurs from both the virion-sense strand and from the "complementary sense” strand, giving rise to (+) and (-) sense RNA transcripts respectively.
• geminiviruses examples include Maize Streak Virus (MSV) , Bean Golden Mosaic Virus (BGMV) , African Cassava Mosaic Virus (ACMV) , Chloris Striate Mosaic Virus (CSMV) , Tomato Yellow Leaf Curl Virus (TYLCV) , Tomato Golden Mosaic Virus (TGMV) , Beet Curly Top Virus (BCTV) and Tomato Leaf Curl Virus (TLCV) amongst many others (see also the viruses mentioned in table 1 below) . These viruses cause economically important diseases in cultivated plants including leaf curling and other leaf distortion.
• MSV Maize Streak Virus
• BGMV Bean Golden Mosaic Virus
• ACMV African Cassava Mosaic Virus
• CSMV Chloris Striate Mosaic Virus
• TYLCV Tomato Yellow Leaf Curl Virus
• TGMV Tomato Golden Mosaic Virus
• BCTV Beet Curly Top Virus
• TLCV Tomato Leaf
• TLCV in particular causes severe crop damage in northern parts of Australia and infected plants show symptoms similar to those caused by TYLCV in the Middle East and Europe. These tomato viruses are transmitted by the white-fly Bemisia tabaci and constitute a major disease problem world ⁇ wide.
• TLCV and TYLCV are used interchangeably and reference herein to Israeli and Sardinian isolates of TLCV should be taken as TYLCV isolates.
• TLCV refers to the Australian isolate.
• Geminiviruses replicate in the nucleus of the infected cell and are thought to employ a rolling- circle mechanism similar to one used by the single- stranded DNA containing coliphages (e.g. QX174) and certain Staphylococcus aureus and Bacillus subtilis plas ids.
• the other known plant viruses including other plant DNA viruses, replicate via RNA intermediates.
• Geminivirus particles accumulate in the nuclei of infected cells where DNA replication and virus assembly probably take place (Davies et al., J. Cell. Sci (Suppl) (1987) 7 : 95-107). Their putative replicative forms are double-stranded covalently closed circular DNA Ca. 2,7 Kb in chromatine-like structures and are likely to be the transcriptionally active forms of the virus (Abouzid et al., Mol. Gen. Genet. (1988) 212 : 252-258) .
• viruses which infect dicotyledonous hosts, have bipartite genomes and are transmitted by white-fly vectors Bemisia tabaci (Genn.) (ACMV, African Cassava Mosaic Virus (Stanley and Gay, Nature (1983) 301 : 260) ; TGMV, Tomato Golden Mosaic Virus (Hamilton et al, EMBO J.
• Bemisia tabaci Genn.
• ACMV African Cassava Mosaic Virus
• TGMV Tomato Golden Mosaic Virus
• CMV Cucumber Mosaic Virus
• antisense technology has been applied to tobacco plants expressing an antisense DNA sequence of virally encoded AL1 gene of TGMV transcriptionally fused to a drug resistance gene (Day, A. G. et al Proc. Nad. Acad. Sci. USA 88:6721-6725, 1991).
• transgenic plant lines expressing the antisense transcript showed reduced symptom development, it is possible that the transgenic plants were infertile which would prevent large scale production of virus resistant plants.
• the inventors sought to overcome the disadvantages of the antisense approach to generating plant DNA virus- resistant plants by employing the strategy of ribozymes.
• Ribozymes are synthetic RNA molecules which possess highly specific endoribonuclease activity.
• they comprise a hybridising region which is complementary, in nucleotide sequence to at least part of a target RNA and a catalytic region which is adapted to cleave the target RNA.
• Ribozymes are well described by Haseloff J. and Gerlach L. Nature 334:586-591, 1988 and in International Patent Application No. WO89/05852.
• the inventors have successfully developed ribozymes capable of targeting plant DNA viruses thereby providing an economically viable and efficient means of controlling such viruses.
• one aspect of the present invention contemplates a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target RNA transcribed from a DNA molecule associated with a DNA virus, wherein said catalytic region is capable of cleaving said target mRNA sequence thereby substantially reducing replication, infection and/or assembly of said DNA virus.
• the present invention is directed to a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target mRNA sequence transcribed from a virion-sense DNA strand or a complementary-sense DNA strand of a plant single stranded DNA virus, wherein said catalytic region is capable of cleaving said target mRNA sequence thereby substantially reducing replication, infection and/or assembly of said DNA virus.
• reference to a plant single stranded DNA virus includes reference to its double stranded replicative form.
• the plant single stranded DNA virus is a geminivirus and the target mRNA encodes a protein essential for replication.
• essential is meant a protein specified by the viral genome (or a complementary form thereof) and required or substantially required for a particular function such as replication, infection and/or viral assembly. A decrease in the level of the essential protein results in a reduction in the level of that function. The decrease in the level of the particular function.
• the level of essential protein is 20 % less, more preferably at least 30 % less, even more preferably at least 45 % less, still more preferably at least 55 % less and still even more preferably 65-7 5% less and yet more preferably more than 85-90 % less relative to the level of essential protein (or level of particular function) in the absence of the ribozyme.
• the level of essential protein can be tested in vitro within the limits of biological variation in vivo.
• Such a test for. example, can be by analysis of mRNA degradation products in vitro.
• a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target mRNA sequence transcribed from a virion-sense DNA molecule of a geminivirus or a DNA molecule complementary to said virion-sense DNA molecule, said mRNA encoding a protein essential for replication of said geminivirus in a plant cell wherein said catalytic region is capable of cleaving said target mRNA sequence thereby substantially reducing replication of said geminivirus.
• the target mRNA sequence is present in low abundance within the cell.
• An example of such a mRNA molecule is the transcript of the Cl gene or its equivalent in geminiviruses, such as in TLCV or TYLCV and its mutants, derivatives and variants.
• the single stranded DNA of TLCV contains six open reading frames (ORFs) , two on the virion-sense strand and four on the complementary-sense strand of the replicative form DNA produced in leaf curl infected plants.
• ORFs open reading frames
• Analysis of the viral mRNAs in infected plant cells reveal that the mRNA encoding the Cl gene, which is essential for replication, occurs at lower abundance than the other mRNA transcripts specified by the TLCV genome.
• Equivalent forms of the Cl gene include the genes LI, ALl and ACl and reference herein to the "Cl gene" is taken as including equivalents thereof.
• targets include the C2, C3 and C4 transcripts which play roles in transactivation of virion-sense gene expression and spread of the virus, expression of the disease phenotype, and development of the disease respectively.
• the present invention is directed to a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target mRNA sequence encoding the Cl gene of geminivirus and in particular TLCV and wherein said catalytic region is capable of cleaving said target mRNA sequence thereby substantialy reducing replication of said geminivirus (e, g, TLCV) .
• the ribozymes of the present invention can be defined as nucleic acid compounds having endoribonuclease activity and being capable of inhibiting the replication, infection or assembly of a DNA virus, said compound comprising at least one unit, "R", having the general formula :
• - X and X' each represent, independently, a nucleotide sequence having at least 4 bases and being complementary to at least a part of either the viral genome, or the (-)sense RNA transcript of a DNA virus ;
• Y represents the catalytic region of a ribozyme.
• the target RNA is preferably the (-)sense RNA transcript, i.e. the mRNA originating from the transcription of the complementary sense strand of the double-stranded replicative form of the DNA virus genome, particularly a geminivirus.
• the sequences X and X* are therefore complementary to at least a part of the (-)sense RNA transcript (which is equivalent to being complementary to at least a part of the single stranded genomic DNA) .
• hybridising arms are sufficiently long to allow both stable hybridisation of the ribozyme to the substrate RNA, and efficient cleavage of the substrate.
• X and X 1 will have a length of at least 4 nucleotides each, and preferably at least 6.
• the upper limit of the lengths of the hybridising arms is generally determined by the length of the targetted substrate and can be as long as 800 or 1000 bases or more. Preferred lengths are between 6 and 300 bases, for example 10 to 150.
• the complementarity of the hybridising arm to the substrate or to a part of the substrate normally extends along the entire length of the hybridisisng arm.
• the catalytic region of the ribozyme, "Y” is derived from any type of suitable ribozyme, for example "hammerhead” or "hairpin". These ribozymes are described in patent applications EP-A-321201 and EP- A-360257 respectively. Particularly preferred are “hammerhead” catalytic regions (see for example, figure 7) , and which will be described in detail hereafter.
• the ribozymes may be either “monoribozymes” possessing one single "R” unit of the formula [X-Y- X'], and having only one catalytic region.
• the ribozymes may be "polyribozymes" comprising a plurality of "R” units and having the general formula : R-*-R 2 R restroom, wherein n > 1, and may have a value as high as 50 or 100. In practice, a value for "n" of about 5 to 10 is frequent.
• the polyribozymes are thus constituted by a head-to-tail series of ribozymes, the monoribozyme "R” or [X-Y-X'] unit, being the unit motif. In other words, it is a series of catalytic regions connected together by hybridising arms.
• the aggregate of the X and X' motifs contributed by all the "R" units of a polyribozyme will be designated collectively as the “complementary” or “hybridising” sequence.
• the polyribozyme normally acts as a "uni-molecule" against a single transcript, i.e. the cleavage sites of each of the catalytic regions are located on the same transcript.
• the precise structure of the polyribozyme is in fact primarily determined by the nature of the substrate it is designed to cleave. The number of catalytic regions, their position in the complementary sequence and the distance between two consecutive catalytic regions are dependent to a large degree on the position of the "three-base target" sites in the substrate.
• the "hammerhead” ribozymes cleave the substrate immediately downstream from a "target" site XXX, preferably XUX, in which X represents one of the 4 bases A, C, G, U and U represents uracil.
• X represents one of the 4 bases A, C, G, U and U represents uracil.
• One particularly preferred target sequence is X°UY in which X° is any ribonucleotide, U is uracil and Y represents adenine, cytosine or uracil.
• X°UY forms part of a base pair flanking region and Y is not base paired.
• Preferred triplets defining X+OUY include, but are not limited to GUC, GUA, GUU, UUC and CUC.
• Target sites are possible , but less efficient, for example CAC, UAC and AAC.
• Perriman et al (Gene, 1992, 113:157) have studied extended target sites for hammerhead ribozymes. It has been found that GUC, UUC, CUC, GUA and GUU targets show equivalent rates of studies. For GUG, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. There are some targets with nucleotides other than U in the center position which show significant, discernable cleavage.
• a preferred target sequence is AGUC.
• each catalytic region of the polyribozyme must be situated at a site in the complementary sequence which corresponds to a XUX site of the transcript. For example, if one XUX site is situated at position 412 of the C, gene and another at position 711, a catalytic region is inserted at the position corresponding to 412 in the complementary sequence, and another at 711.
• the X and X' hybridising sequences which flank the catalytic domain in each "R" unit are chosen so as to be complementary to the bases surrounding the XUX site targeted in the substrate.
• the motif XUX is a motif which occurs very frequently in the RNA sequences. For example, on average there is a GUC motif every 64 bases in a sequence having a random and equal distribution of bases. This signifies that the substrate usually contains a plurality of XUX cleavage sites.
• the catalytic regions of the polyribozymes are situated at positions of the complementary sequence which correspond to the XUX sites.
• the polyribozyme variant of the invention may thus contain a very variable number of catalytic regions.
• the polyribozyme may contain from about 2 to about 6 catalytic regions, when the target sequence is GUC.
• the choice of the sites selected may be made by respecting the following criteria :
• the distance between the 2 XUX sites targeted and, consequently, between 2 catalytic regions in the polyribozyme must be long enough to enable the hybridizing arms of the polyribozyme situated between the corresponding catalytic regions to hybridize with the substrate in a stable manner and to prevent the catalytic regions hybridizing with themselves.
• a distance of at least 8 bases, and preferably at least 14 bases, for example about 20 bases is particularly advantageous.
• this criterion must only be taken into consideration when the substrate contains XUX sites very close together. Otherwise, if the XUX sites of the substrate are separated from each other by more than 8 to 20 bases, this selection criterion is not important.
• the XUX sites targeted are preferably situated in a part of the substrate which does not have significant secondary structure. This facilitates the access of the polyribozyme to the substrate and increases its efficacy. Three-dimensional folding analyses may be carried out.
• the XUX sites targeted may form part of the regions of homology conserved between different strains or variants or mutants of one and the same virus, or between different related viruses. This aspect of the invention is considered in detail later.
• XUX sites targeted is the absence of homology with endogenous genes of the plant to be transformed. In fact, although they are rare, some viruses possess sequences which find a homology in the genome of plants. It is thus important to avoid XUX sites situated within such a sequence.
• the hybridising X* and X motifs within any one "R" unit are often complementary to adjacent, contiguous partions of the substrate.
• the ribozyme can be thought of as “symetric", and the catalytic domain acts at a unique site determined by the complementarity of its two flanking arms.
• a preferred example of this symetric type of ribozyme is the "purely complementary" polyribozyme in which the 3• hybridising arm of a given "R" unit and the 5' hybridising arm of the adjacent "R” unit are complementary to sequences which are contiguous in the (-)sense RNA transcript or in the DNA genome.
• the aggregate of the hybridising arms gives a sequence of uninterrupted complementarity to the target sequence.
• This type of polyribozyme may be considered as a complementary sequence having, inserted at distinct sites in that sequence, a plurality of ribozyme catalytic regions.
• the complementary sequence may hybridise with the entire length of the target transcript.
• it may hybridise with only a fragment of the targeted transcript.
• the fragment in question must be long enough to allow the inclusion of at least 2 catalytic regions in the corresponding sequence of the polyribozyme.
• the length of the complementary sequence, not counting the catalytic regions may vary from about 40 to 2000 bases. Lengths of 400 to 1000 is particularly preferred.
• the ribozyme may be, or may possess at least one "R" unit which is "asy etrical".
• R the X and X 1 hybridising arms within a given "R” unit are complementary to two sequences which are non ⁇ contiguous in the substrate.
• the catalytic domain of the "asymetric" "R” unit can act at two distinct XUX sites in the substrate, one being the XUX site flanked by the sequence complementary to its 5 1 arm, and the other being the XUX site flanked by the sequence complementary to its 3' arm.
• This type of ribozyme may be specifically constructed, or may be the result of a spontaneous re-arrangement during synthesis.
• the locations of the sequence to which, on the one hand, the 3' flanking arm hybridises, and the sequence to which, on the other hand, the 5 1 flanking arm hybridises, may be separated by hundreds of bases or by as little as 10 or 20 bases.
• This type of structure is advantageous since it allows a significant "economy” in catalytic regions : for example five "asymetric" "R" units can achieve the same result as 10 symetric units.
• the 3* hybridising arm (X') of a given "R” unit and the 5' hybridising arm of the adjacent "R” unit are complementary to non-contiguous portions of the genome or of the (-)sense RNA transcript.
• This type of polyribozyme will be referred to hereinafter as a "short arm polyribozyme”.
• the structure of these polyribozymes is basically a sequence complementary to the target sequence in which ribozyme catalytic regions are embedded and which has undergone deletions in the complementary sequence.
• the distance between two catalytic regions in the polyribozyme is shorter than the distance between the two corresponding XUX sites in the target sequence.
• the distance between ribozymes can be reduced significantly with respect to the distance between the corresponding target sites in the substrate, for example, if two adjacent targeted XUX sites in the substrate are separated by a distance of 1000 nucleotides, the distance between the catalytic regions of the "short arm ribozyme" may be as little as 30 or 40 nucleotides. Reductions of over 90 % of the length of the hybridising sequence may therefore be made.
• the minimum length between two adjacent catalytic regions is normally about 8 nucleotides and preferably at least 20.
• the present inventors have shown that such shortened hybridising sequences do not, surprisingly, prevent the polyribozyme from efficiently cleaving the substrate and can, in fact, in many cases improve the efficiency of the inactivation.
• the reduction in the length of the complementary sequence avoids problems associated with the formation of secondary structure leading to the inactivation of the ribozyme.
• more catalytic regions may be inserted targeting more cleavage sites in the substrate.
• the use of the short arm polyribozyme means that efficient polyribozymes may be constructed even if only parts of the sequence of the substrate are known. It is sufficient to know the 10 or 15 bases surrounding an XUX target site.
• Another important advantage is the possibility to use, as the shortened hybridising arms, exclusively those sequences which are conserved in different viruses, for example viruses of the same type. In this way, different viruses may be inactivated by the same polyribozyme even if the sequences between the conserved regions are totally dissimilar.
• Any one polyribozyme of the invention may include a mixture of "long arm” and “short arm” ribozymes, and also of “symetric” and asymetric” ribozymes. Alternatively, it may include only long arm ribozymes, or only short arm, or only asymetric, etc... The structure of the ribozyme can therefore be optimised, and tested.
• the hybridising arms of the polyribozyme may be derived from at least two different types of virus.
• Such polyribozymes are referred to hereinafter as "chimeric polyribozymes".
• chimeric polyribozymes As an example of this type of construction, mixtures of TLCV and TYLCV derived sequences can be cited, for example : the Sardinian, Israeli or Tunisian strain.
• This type of polyribozyme ensures the inactivation of both types of virus, if present together, or allows the use of a single construction in different geographical areas.
• the polyribozymes may include, between two consecutive "R” units, a non-complementary sequence (A) which has a length of at least 4 bases, and which is non- complementary to the (-)sense RNA transcript or DNA genome.
• A non-complementary sequence
• the non-complementary sequence of the polyribozyme may have a precise function, for example, it may be constituted by a coding sequence which can be used to select transformants or also a sequence containing a ribozyme which acts on a substrate other than the primary target of the DNA virus or which is cis acting on a part of the polyribozyme. It typically contains a polylinker for cloning, or an adaptor to facilitate construction of chimeric polyribozyme.
• the non-complementary sequence usually has a length comprised between 4 and 500 bases, for example 20 to 100 bases. When there is a plurality of non- complementary sequences, they may together constitute as much as about 90% of the length of the polyribozyme, for example 50%.
• These non-complementary sequences normally replace portions of the complementary sequences X and X' , but leave at least 4 complementary bases immediately next to the catalytic domain.
• the ribozymes of the invention are constructed so that the hybridising arms are complementary to a region or to regions which is (are) highly conserved between variants or mutants of the same strain, between strains of the same virus type or even between viruses of different types.
• amino-acid identity is given in percent derived with the program BestFit of the UWGCG-Sequence analysis package.
• the word size (K- tuple) was 2, the gap weight was 2.0 and gaplength weight was 0.1.
• the program FastA yielded the same relationship.
• amino-acid identity was calculated using PALIGN program of the PC/GENE sequence analysis package using the structure-genetic comparison matrix (open gap cost value, 10 ; unit gap cost value, 10) .
• ABMVA Abutilon Mosaic Virus DNA A ;
• ACMVA African Cassava Mosaic Virus DNA A ;
• BGMVA Bean Golden Mosaic Virus DNA A ;
• ICMV Indian Cassava Mosaic Virus DNA A ;
• SqlCV Squash Leaf Curl Virus DNA A ;
• PYMVA Potato Yellow Mosaic Virus DNA A ;
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TGMVA Tomato Golden Mosaic Virus DNA A
• TYLCV-I Tomato Yellow Leaf Curl Virus Israeli isolate
• TYLCV-S Tomato Yellow Leaf
• the degree of sequence diversity between the 3 isolates of TYLCV and TLCV is in pronounced constrast to the usual very high sequence similarity between different geminivirus isolates or strains, for example : the DNA sequences of the Kenyan and Nigerian isolates of ACMV show 96 % identity and Kenyan, Nigerian and South African isolates of MSV vary maximally by about 2 %.
• the present inventors carried out a comparison of the Cl RNA transcript and identified three very highly conserved regions, two of which are shown in figure 9. These regions are the following (the following numerotation is the TLCV numerotation of the complementary sense transcript) :
• the nucleotide sequences of the Cl gene in three TLCV isolates are shown in Figure 1.
• the complete nucleotide sequence of the Cl gene from TLCV is disclosed in Mullineaux et al.. Virology, 193 : 414-423, 1993 and Dry et al. J. Gen. Virol., 74 : 147-151, 1993.
• the Israeli isolate is disclosed by Navot et al in Virology, 185 : 151-161, 1991 and the Sardinian isolate is disclosed by Khyer-Pour et al, Nucl. Acid. Res. 19 : 6763-6769, 1991.
• Polyribozymes constructed with target sites in these conserved regions may therefore be used to ensure that all related strains or variants are inactivated.
• the polyribozyme is the deleted short arm version, whose hybridising arms correspond exclusively to the conserved regions, wide sequence diversity between the conserved regions does not affect the efficient hybridisation of the polyribozyme to the substrate.
• a "conserved region” is considered as having a 100 % homology over a distance of about 20 bases. Generally speaking, the homology may be around 90 % over 30 bases.
• Ribozymes Rl, R4, R5 and R2 are designed so that the target sites lie within these conserved regions, as shown in the examples below.
• the present invention further relates to a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least one part of a target mRNA sequence encoding the Cl gene of at least two variants of particular geminivirus and in particular at least two variants of TLCV, and wherein said catalytic region is capable of cleaving said target mRNA sequence from each of said variants thereby susbtantially reducing replication of said geminivirus (e.g. TLCV, TYLC and their mutants, derivatives and variants) .
• a geminivirus e.g. TLCV, TYLC and their mutants, derivatives and variants
• the polyribozymes of the invention are usually constituted of RNA. Nonetheless, it is possible to replace some parts of the polyribozyme by DNA, for example the hybridizing arms or parts of these arms, or also a part of the catalytic region, in particular the "loop", provided that the catalytic activity is maintained (see, for example, the substitution of the RNA by DNA described in the international patent application wo-9119789) .
• the ribozymes of the present invention can be represented as a molecule of formula (I) :
• (X) n and (X) n ' represent ribonucleotide sequences substantially complementary to a mRNA sequence of a DNA virus; the sum of n and n 1 may be the same or different; an (*) represents a base pair between complementary ribonucleotides ; Y represents any ribonucleotide and each Y may be the same or different ; Y' and Y" represent ribonucleotides of complementary sequence along at least part of their length to allow base pairing between the oligoribonucleotides, or Y' and Y" together form a single RNA sequence wherein at least part of said sequence comprises a stem formed by base pairing between complementary nucleotides; and optionally, an additional nucleotide selected from any one of A, G, C or U is inserted after 1 A.
• the ribozyme is as set forth in formula (II) : 3 I
• (X) n and (X) n ' represent ribonucleotide sequences substantially complementary to a mRNA of a DNA virus and in particular a geminivirus such as TLCV ;
• Y represents any ribonucleotide and each Y residue may be the same or different ;
• an (*) represents a base pair between complementary ribonucleotides; n and n' are as previously defined;
• B represents a bond, a base pair, a ribonucleotide, or an oligoribonucleotide containing at least 2 ribonucleotides ;
• m and m' are 1 or more and may be the same or different ; and optionally, an additional nucleotide selected from any one of A, G, C or U is inserted after 1 A.
• the ribozyme is as set forth in formula (III) :
• (X) n and (X) n ' represent a ribonucleotide sequence substantially complementary to a mRNA of a DNA virus and in particular a geminivirus such a TLCV ;
• Y represents any ribonucleotide and each Y residue may be the same or different an (*) represents a base pair between complementary ribonucleotides ;
• n and n' are as previously defined.
• the DNA virus is a single stranded DNA virus.
• the single stranded DNA virus is a geminivirus.
• the geminivirus is TLCV and (X) n and (X) n - represent a sequence capable of hybridising to a region of Cl gene mRNA.
• Particulary preferred ribozymes of the present invention are specifie to the Cl gene of TLCV and its variants and comprise the nucleotide • sequences set forth below :
• the catalytic regions illustrated above and in Figure 7 have a conserved structure and sequence, it has been observed that some nucleotides may be deleted, inserted, substituted or modified without prejudice to the activity of the ribozyme.
• the invention comprises the use of these modified catalytic regions in the polyribozyme provided that their catalytic activity is conserved. This activity can be verified by using the tests described below.
• one or more nucleotides of the catalytic region II illustrated in formula I, II and III may be replaced by nucleotides containing bases such as adenine, guanine, cytosine, methylcytosine, uracil, thymine, xanthine, hypoxanthine, inosine or other methylated bases.
• the "conserved" bases C-G which together form the first base pair of the catalytic loop, can be replaced by U-A (Koizumi et al. , FEBS Letts. 228, 2, 228-230,
• the nucleotides of the catalytic region illustrated in Figure 7 can also be modified chemically.
• the nucleotides are composed of a base, a sugar and a monophosphate group. Each of these groups can thus be modified. Such modifications are described in "Principles of Nucleic Acid Structure” (Ed. Wolfram Sanger, Springer Verlag, New York, 1984) .
• the bases may bear substituents such as halogeno, hydroxy, amino, alkyl, azido, nitro, phenyl groups, etc..
• the sugar moiety of the nucleotide may also be subjected to modifications such as the replacement of the secondary hydroxyl groups by halogeno, amino or azido groups or even to 2* methylation.
• the phosphate group of the nucleotides may be modified by the replacement of an oxygen by N, S or C, giving rise to a phosphoramidate, phosphorothioate and phosphonate, respectively. These latter may exhibit useful pharmacokinetic properties.
• bases and/or the nucleotides of the catalytic region may also bear substituents such as amino acids, for example, tyrosine or histidine.
• the ribozyme may comprise as catalytic region one or more structures such as those illustrated in Figure 17.
• This structure called “minizyme”, is described in the international patent application WO-A-9119789. It represents a catalytic region of the "hammerhead” type, the "loop” of which has been replaced by a "P” group.
• P may be a covalent link between G and 1 A, one or more nucleotides (RNA or DNA, or a mixture, or even derivatives described above) or any atom or group of atoms other than a nucleotide which does not- affect the catalytic activity.
• P represents a plurality of nucleotides, it may contain internal base pairings.
• the sequence and the . number of nucleotides constituting the group "P” is not critical and may vary from 1 to 20 nucleotides for example, and preferably from 1 to 6. It is preferable to select a sequence lacking internal base pairings of the Watson-Crick type.
• the catalytic activity of the ribozymes of the invention may be verified in vitro by placing the ribozyme, or a sequence which after transcription will give rise to the ribozyme, in contact with the substrate, followed by demonstration of the cleavage.
• the experimental conditions for the in vitro cleavage reaction are the following : a temperature comprised between 4 and 60°C, and preferably between 20 and 55 ⁇ C, a pH comprised between about 7.0 and 9.0, in the presence of divalent metals, such as Mg 2+ , at a concentration of 1 to 100 mM (preferably 1 to 20 mM) .
• the polyribozyme is usually present in an equimolar ratio with the substrate, or in excess.
• the in vivo cleavage conditions are those existing naturally in the cell.
• the ribozyme is said to "hybridise” to a target mRNA sequence or to have “complementarity” hybridising arms.
• hybridisation and complementarity is definied herein as an extent of duplex formation sufficient to form a stable enough structure so that cleavage with the target triplet can occur.
• the ribozyme and the secondary structure of the mRNA preferably less than about 5-10, more preferably less than about 3 to 5 and even more preferably less than 1 to 3 bases would be mismatched between the target mRNA sequence and one or both hybridising arms of the ribozyme. Over the whole length of the hybridising arm, not more than 10 % of mismatches should occur. Accordingly, the present invention extends to ribozymes described herein and in particular Rzl, Rz2, Rz3, Rz4 and Rz5, to the polyribozymes containing them, and to any or all functional mutants, derivatives, parts, homologues or analogues thereof.
• mutants include single or multiple nucleotide substitutions, deletions and/or additions to the hybridising regions and/or catalytic regions of the ribozymes provided such modified ribozymes are still functional in cleaving the target mRNA sequence.
• the present invention extends to a ribozyme having a hybridising domain and a catalytic domain, wherein the hybridising domain comprises a sequence of nucleotides substantially complementary to a sequence of nucleotides in a mRNA molecule encoding the Cl gene of TLCV or TYLCV or which hybridises to said TLCV or TYLCV mRNA sequence in vitro under high stringency conditions and wherein said catalytic region is capable of cleaving said target mRNA sequence.
• This aspect of the present invention is conveniently determined by the extent of cleavage of the target mRNA sequence in vitro.
• the stringency conditions set forth in Sambrook et al, Molecular Cloning : A laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA, pages 387-389 at paragraph 11 are considered high stringent conditions.
• medium stringency may be used involving 0.25-0.5 % w/v SDS at 45° C for 2-3 hours.
• the alternative conditions are applicable depending on concentration, purity and source of nucleic acid molecules.
• the present invention extends to genetic constructs comprising the ribozymes herein described.
• These genetic constructs generally comprise a vector sequence as a vehicle for transferring the ribozyme to a host cell and a promoter sequence to direct transcription of the ribozyme and a terminator.
• a preferred promoter includes but is not limited to a 35S Cauliflower Mosaic Virus (CaMV) or to a double 35S CaMV promoter.
• CaMV 35S Cauliflower Mosaic Virus
• the CaMV promoter is described by Odell et al, Nature 313 : 810-812, 1985.
• the ribozymes are generally in the complementary form such that upon transcription, the ribozyme is produced.
• the genetic constructs of the present invention are used to generate transgenic plants capable of constitutively, developmentally or inducibly (e.g. in response to certain stimuli) expressing the ribozymes herein described. Most preferred transgenic cells are rendered substantially resistant to TLCV infection.
• the ribozymes and genetic constructs of the present invention can be conveniently tested in vivo using virus-sensitive plants.
• a particularly useful indicator plant is the tobacco plant.
• the analysis may be qualitative such as by screening for severity of symptoms or quantitative where samples are analysed, for example, by measuring the level ribozyme expression or level of target mRNA. A combination of qualitative or quantitative analyses may also be employed.
• All of the known means for introducing foreign DNA into plants may be used, for example Aqrobacterium, electroporation, protoplast fusion, bombardment with a particle gun, or penetration of DNA into cells such as pollen, the microspore, the seed and the immature embryo, viral vectors such as the Geminiviruses or the satellite viruses.
• Aqrobacterium tumefaciens and rhizo enes constitute the preferred means.
• the sequence coding for the polyribozyme is introduced into a suitable vector together with all of the regulatory sequences necessary such as promoters, terminators, etc....as well as any sequence necessary for selecting the transfor ants.
• tobacco cells are transformed using standard procedures employing Aqrobacterium with the genetic construct to be tested.
• Aqrobacterium tumefaciens mediated transformation of tobacco is carried out essentially as described by Horsch et al. , Science 227 : 1229-1231, 1985, although conveniently strain LBA 4404 is used and the plantlets are selected on Kanamycin at a concentration of 250 ⁇ l/ml.
• the transformed cells are regenerated into small plants and samples removed to screen for expression.
• the plants are allowed to self-fertilise and to seed and the transgenic seeds are then used to generate new plants.
• the transgenic plants along with suitable controls are then exposed to the virus and the degree of symptom development analysed.
• a quantitative assessment is made by biochemical analysis. This approach is particularly useful for screening for ribozymes against geminiviruses and in particular TLCV. Once suitable ribozyme genetic constructs are identified using this procedure, these particular constructs can then be used to generate, for example, in the case of TLCV and TYLCV, transgenic tomato plants. Similar transformation and screening procedures are employed.
• the resistant nature of the transgenic plants can be tested by carrying out a self-fertilisation, or a cross with a non transformed genotype, on a primary descendant to obtain T.,. Subsequently, T-, plants are inoculated with the DNA virus by Agroinoculation. Degree of resistance (i.e. "complete resistance” meaning total absence of symptoms, “tolerance” meaning that the plant can be infected and shows symptoms but recovers, "sensitive” meaning that the plant exhibits symptoms and replicates the virus) is then noted.
• the present invention is particularly directed to transgenic tomato plants which exhibit at least some resistance to TLCV and TYLCV infection.
• resistance to infection is meant a qualitative reduction in symptoms (generally due to inhibition of virus replication) by more than approximately 30 %, preferably more than approximately 50 % and even more than approximately 70 %. Due to the variability of biological systems, 100 % inhibition of TLCV replication is difficult to achieve although the effectiveness of the ribozyme can be enhanced by various molecular biological techniques.
• a method of generating transgenic tomato plants exhibiting reduced symptoms following exposure to TLCV compared to non-transgenic plants comprising transforming tomato plant cells with a genetic construct encoding a ribozyme as hereinbefore described capable of cleaving Cl mRNA from TLCV, regenerating plants from said transformed cells, self-fertilising said regenerated plants and obtaining seeds there from and then growing transgenic plants from said seeds.
• the present invention extends to transgenic plants exhibiting resistance to a plant DNA virus, more particularly a geminivirus and even more particularly TLCV.
• the transgenic plants are tomato plants.
• the subject invention also extends to reproductive material and seeds from such transgenic plants (e.g. tomato plants) .
• the ribozymes may be expressed constitutively, developmentally or inducibly.
• FIG. 1 is a representation of the nucleotide sequences of the complementary-sense DNA strands from three different isolates of TLCV : top row, TLCV (Australia) ; middle row, TLCV (Israel) ; bottom row TLCV (Sardinia) .
• the sequence is represented in a 5'->3' direction.
• FIG. 2 is a schematic representation of PRIB05BIN comprising a CaMV35S-1568bp TLCV fragment (containing Rzl, Rz2 and Rz3)-CaMVpolyA expression cassette cloned into BamHI cut Binl9.
• the insertion sites of the 3 ribozymes Rzl, Rz2 and Rz3 are indicated at positions 1111, 2056 and 2355.
• This numbering is the virion-sense numbering and corresponds to positions 1654, 711 and 412, respectively, using the (-)sense numbering as illustrated in figures 8 and 10.
• Figure 3 is a schematic representation of pRIB05BINDel comprising a CaMV35S-1346bp TMCV fragment (containing Rzl, Rz2 and Rz3)-CaMVpolyA expression cassette cloned into BamHI cut Binl9.
• Figure 4 is a schematic representation of pRIB07BIN comprising a CaMV35S-396bp TLCV fragment (containing Rzl and Rz2)-CaMVpolyA expression cassette cloned into BamHI cut Binl9.
• FIG. 5 is a schematic representation of pRIB02BIN comprising a CaV35S-30bp TLCV fragment (containing Rzl)-CaMVpolyA expression cassette cloned into BamHI cut Binl9.
• FIG. 6 is a schematic representation of the location of primers used for RT-PCR analysis of ribozyme construct expression in transgenic tobacco.
• FIG. 7 is a schematic representation of a ribozyme model (from Haselhoff and Gerlach, Nature, 1988) .
• Substrate RNA can have any sequence (X) flanking GUC at the cleavage site, as long as base pairing forms with the ribozyme as shown.
• the arrow indicates the cleavage site.
• conserveed bases in ribozymes are boxed. In naturally oc ⁇ uring hammerheads, the substrate and ribozyme portions are connected by a loop, resulting in an intramolecular cleavage.
• FIG. 8 shows the nucleotide sequence of the complementary-sense strand of TYLCV-S. Lower case letters indicate the ribozyme cleavage site.
• the sequence of TYLCV-S was published by Kher-Pour et al. (Nucl. Acid Res. (1991) 19 : 6763)
• Figure 9 shows the regions conserved in the Replicase gene, between TLCV and TYLCV-S :
• Figure 9(a) shows the regions highly conserved around ribozymes Rl, R4 and R5 ;
• Figure 9(b) shows the regions highly conserved around ribozyme R2.
• ":" means a mismatch, a gap means no nucleotide.
• Lower case letters indicate the ribozyme cleavage site.
• FIG. 10 illustrates the nucleotide sequence of the complementary strand of TLCV. Dry et al. (J. Gen. Virol. (1993) 74 : 147) Lower case letters indicate the ribozyme cleavage site.
• FIG. 11 shows the target sequences for polyribozymes D, F and E.
• Lower case letters indicate the ribozyme cleavage site.
• Upper case letters correspond to the complementary sequences of ribozyme arms.
• FIG. 12 is a schematic representation of ribozyme constructs.
• a hatched line represents the TYLCV-S complementary-sense strand, and a solid line represents TLCV complementary sense strand.
• R • ribozyme.
• FIG. 13 is a schematic physical map of binary vector pGAZE.
• FIG. 14 is a schematic physical map of ribozyme plasmids, pGAZE-RzA, pGAZE-polyRzC, pGAZE- polyRzD, pGAZE-polyRzE, pGAZE-polyRzF.
• FIG. 15(a) is a schematic representation of pRIBOSBS : 1690bp TLCV C1/C2/C3 complementary fragment (containing 3 ribozymes) blunted into EcoRV cut Bluescript SK+ ;
• Figure 15(b) is a schematic representation of pRIBOSBIN : 2660bp Bgl II 35S- C1/C2/C3 complementary (containing 3 ribozymes) , CaMVpolyA cassette cloned into BamHI cut BIN 19; open triangles show the position of 22bp ribozyme insertions, E : EcoRI ; H : Hindlll ; K : Kpnl ; P : PstI ; Sm : Smal ; B : BamI ; SI : Sail ; St : SstI ; X : Xhol ; Xb : Xbal ; C : Clal.
• - Figure 16 is a schematic representation of the
• FIG. 17 illustrates the "Minizyme” structure (WO-A-9119789) wherein the loop of the catalytic region is replaced by an element "P".
• P may be at least 1 nucleotide (ribonucleotides, deoxyribo- nucleotides, derivatives or a mixture) , provided that the ribonucleotides of the "P" group are not base paired by "Watson -Crick" base pairings when the sequences (X') n and (X) n . and P are constituted exclusively of ribonucleotides.
• "P” may also be a bond or any atom or group of atoms which do not affect the catalytic activity of the ribozyme.
• X has the same meaning as in formula I, II, III.
• the major mRNA transcripts were identified and mapped on the circular genome of an Australian isolate of TLCV [TLCV (Australia) , Mullineaux et al., Supra ; Dry et al, Supra] .
• One of the viral mRNAs derived from the complementary sense strand of the viral DNA occurred at a lower abundance than the other TLCV transcripts.
• This low abundance mRNA transcript was identified as encoding the Cl gene, the product of which, is essential for replication of the virus. rDavies et al. Trends in Genetics, 5 : 77-81, 1989) .
• the mRNA transcript also encodes three other gene products, designated C2, C3 and C4, whose functions are not conclusively known. Accordingly, the low abundance mRNA encoding the Cl gene was selected as the target mRNA.
• the low abundance of this transcript means a higher ratio of ribozyme target sequence, thereby potentially permitting a more substantial decrease in Cl gene product.
• TLCV The nucleotide sequences of the complementary sense strands of three TLCV isolates [TLVC (Australia) , TLCV (Israel) and TLVC (Sardinia) ] were compared in order to identify conserved regions for targeting by ribozymes. The sequence comparison is shown in figure 1. Three conserved regions were identified for design of ribozymes. Two of these conserved regions are shown in figure 1. In addition to sequence comparisons, a computer program can be used to predict secondary structure of the target site to enable greater efficacy of the ribozyme.
• TLCV A 1568 fragment of TLCV from nucleotide 1075-2643 covering the Cl, C2, C3 & C4 reading frames (ORPS) was cloned into pALTER-1 (Promega) .
• Ribozyme sequences 1, 2 & 3 (Rzl, Rz2 and Rz3, respectively) were chemically synthesised as described in Example III and introduced into the, TLCV sequence using the Altered sites in vitro mutagenesis system (Promega) .
• the integrity of introduced ribozyme sequences was confirmed by sequencing using the dideoxy chain termination method.
• Rzl, Rz2 and Rz3 were shown to catalyse in vitro cleavage of a complementary-sense viral RNA transcript generated with 57 RNA polymerase at 50" C and 37" C.
• the modified 1634 bp complementary-sense TLCV fragment containing Rzl, Rz2 and Rz3 was cloned in the antisense orientation (relative to Cl ORF transcription) between a double 35S CaMV promoter and a CaMV polyA termination sequence and the whole expression cassette introduced into the BamHI site of the multiple cloning site of the binary vector Binl9.
• the resulting construct is pRIBOSBIN and is shown in figure 2.
• This construct was derived from pRIBOSBINDel (Example V) by removal of a 950 bp region from the 5•-end of the TLCV fragment (nucleotide 1075) to the Xhol site (nucleotide 2025) .
• the resulting construct is pRIB07BIN and is shown in figure 4.
• a 52 bp fragment containing Rzl was amplified by polymerase chain reaction (PCR) from pRIBOSBIN (Example IV) and cloned in the antisense orientation (relative to Cl ORF transcription) between a double 35S CaMV promoter and a CaMV polyA termination sequence and the whole expression cassette introduced into the BamHI site of the multiple cloning site of the binary vector Binl9.
• PCR polymerase chain reaction
• ribozyme constructs described in Examples 5 to 8 were tested for expression in transgenic tobacco plants using PCR analysis.
• Leaf samples (150 mg) were collected from transgenic tobacco lines prior to potting out in the glasshouse and frozen in liquid N 2 . Samples were ground to a fine powder and vortexed for 30s with 0.5 ml extraction buffer (containing 0.1 M Tris-HCl (pH 9), 0.1 M NaCl, 10 mM EDTA, 1 % w/v SDS and 1 % v/v beta-mercaptoethanol) and 0.5 ml phenol. The extract was centrifuged at 12 000 g for 10 mn and the supernatant fraction collected. The supernatant fraction was extracted a further time with phenol and then with phenol/chloroform (1:1).
• 0.5 ml extraction buffer containing 0.1 M Tris-HCl (pH 9), 0.1 M NaCl, 10 mM EDTA, 1 % w/v SDS and 1 % v/v beta-mercaptoethanol
• the total nucleic acid fraction was precipitated at -20° C with sodium acetate/ethanol and centrifuged at 12 000 g for 10 mn. The pellet was washed with 80 % v/v ethanol, dried and resuspended in 50 ⁇ l sterile water.
• First strand cDNA reactions were carried as follows. Nucleic acid samples were annealed at 65" C for 5 mn with 100 mg of CaMV Term primer (5 1 - TTTACAAATACAAATACATACTAAGGGT in a total volume of 5 ⁇ l. This primer is complementary to a region within the CaMV termination sequence present in all ribozyme construct (see figure 6) transcripts produced from these transcriptions.
• control reactions were performed in which the AMV reverse transcriptase was omitted from the reaction mM Tris-HCl (pH 9), 1.5 mM MgCl 2 , 1.5 mM MgCl 2 , 0.1 % v/v Triton X-100, 200 ⁇ M dNTPs, 250 ⁇ M CaMV Term primer (see figure 6), 0.5 units of Taq DNA polymerase and 2 ⁇ l of first strand cDNA reaction mix. Two alternative 5*-end primers were used, the positions of which are shown in the attached figure :
• RIB primer 5'-GCAAGCTTCCTGAATGTTCGGAT PCR cycling conditions were 94° C for 1 mn, 50-55" C for 1 mn, 72° C for 3 mn for 15-30 cycles. PCR products were analysed on 1.2-1.8 % w/v agarose gels.
• RT-PCR analysis of RIB05BIN and RIBOSBINDel lines using the C3/CaMV Term primer combination produces DNA products approximately the predicted sizes i.e. 1690bp and 1450bp respectively.
• RT-PCR analysis of RIB05BIN, RIBOSBINDel and RIB02BIN lines using the RlB/CaMV Term primer combination produces DNA products approximating the predicted sizes i.e. 504bp, 264bp and 235 bp, respectively.
• Predicted PCR product sizes using the C3/CaMV Term primer combination are as follows :
• Predicted PCR product sizes using the RIB/CaMV Term primer combination are as follows : 1. RIB05BIN 504 bp
• This BstBI / Eco 01091 internal fragment (nt 346 to nt 982) of TYLCV-S genome was cloned into a Dra 11/ Sma I digested pBluescript II SK+ phagemid (STRATAGENE) .
• This clone was used as template for the introduction of 2 ribozymes in the most highly conserved region. Two and three dimensional folding analyses were also made to retain the more theoretical accessible target sites for cleavage with ribozyme.
• hammerhead-type RNA in this case satellite RNA of tobacco ringspot virus (STobRV) , was used to design ribozymes that form, with a completely unrelated substrate RNA, a hammerhead structure in trans, so that the target RNA is specifically cleaved at the predicted site (Haseloff and Gerlach (1989), Gene, 82 : 43-52).
• STobRV tobacco ringspot virus
• nucleotide 407 target site GUC
• Oligo R4-R5 carrying the 2 hammerhead-type ribozymes are identical to Oligo R4-R5 carrying the 2 hammerhead-type ribozymes.
• the underlined sequences correspond to the hammerhead structure of the satellite of ToRSV.
• Bold letters correspond to the target site.
• This oligo R4-R5 was synthesized on an Applied Biosystems Model 380B DNA synthesizer.
• Oligo T7 sequencing primer (Pharmacia) : 5' TTAATACGACTCACTAT 3'
• Oligo T3 sequencing primer (Pharmacia) : 5 » ATTAACCCTCACTAAAG 3'
• PCR I the sequence between oligo R4-R5 and oligo T3 sequencing primer was amplified.
• the amplified fragment was purified on a molecular sieving column (PrimeErase Quick TM kit, STRATAGENE) .
• PCR II the second PCR (PCR II) step, this amplified fragment was used itself as a primer in combination with oligo T7 sequencing primer.
• the PCR conditions were as follows :
• PCR was carried out in a total reaction volume of 100 ⁇ l containing 50 mM KC1, 10 mM Tris-HCl pH 8.3,1.5 mM MgCl2, 300 ⁇ M dNTP each, 2 U Taq polymerase (Perkin Elmer Cetus) , 40 pg template DNA, 100 pM primer each.
• PCR II was the same as PCR I, except that 5 ⁇ l (300 ng of amplified fragment estimated from agarose gel) assay volume of PCR I was directly used for priming. Denaturation : 2 min 94°C ; annealing 2 min 40°C ; synthesis ; 3 min 72"C with 30 cycles for each step.
• the PCR product was digested with BstBI, filled in using Klenow polymerase and finally digested with Eco0109I.
• the resulting 679 nt fragment was cloned into a Sma I /Dra II digested Bluescript SK+ vector.
• the cloning vector containing the cloned sequences was transformed into, and propagated in, the bacterial host XL1 Blue (STRATAGENE) , using techniques known in the art (Maniatis, Molecular cloning, A laboratory guide, 2nd edition, 1989, Cold Spring Harbor Laboratory publishers) .
• Plasmid pRIBOSBS shown in figure 15 was the base point for the construction of the other plasmids.
• This plasmid is a pBluescript SK+ vector carrying a 1690 bp 5 1 end - Ase I fragment which corresponds to the C1/C4/C2/C3 complementary fragment of TLCV (figure 4) bearing 3 hammerhead-type ribozymes at positions :
• Plasmid pPolyribozyme E is derived from plasmid pRIB05BS by deleting the 877bp "filled-in using Klenow Polymerase Xho I - Bel I" fragment corresponding to the region between 2 hammerhead-type sequences at positions R2 and R3, and the 264 bp "5' end - Tth 1111" fragment.
• the Tth 1111 site was filled-in using klenow polymerase.
• the "Tth III I - Hindlll (site of pBluescript SK+ vector”) fragment was isolated after separation by electrophoresis on agarose gel. This fragment was cloned into EcoRI filled-in using Klenow Polymerase and Hind III sites of pBluescript SK+ vector, deleted previously from Sac I-Xba I digested using Mung Bean Nuclease fragment.
• the polyribozyme E contains 434 bases and includes 3 ribozymes : Rl (position 28) , R2 (position 327) and R3 (position 399) as shown in figures 5 and 6 (polyR2E-R2) .
• the size of the arms flanking the catalytic domains of the ribozymes is :
• the polyribozyme F plasmid is derived from pPolyribozyme E plasmid.
• the 56bp "Smal (site in pBluescript SK + vector near 5 1 end) - Sac I" fragment of the replicase gene of TLCV containing the ribozyme Rl was replaced with the 98bp "BstBI filled-in using klenow polymerase - Sac I" fragment of replicase gene of TYLCV - Sardinian isolate bearing the two ribozymes R4 and R5 as described previously.
• the exchanged fragments possess a region which is very highly conserved between different strains of TYLCV and TLCV.
• nucleotide sequence identity corresponds to 97 % between the 365 nt to 435 nt region of TLCV and the 370 nt to 440 nt region of TYLCV-Sardinian isolate ( Figure 3) .
• the chimeric polyribozyme F contains replicase gene fragments which have two different origins : TYLCV-Sardinian isolate and TLCV-australian isolate. The assembly of these fragments was made at the Sac I site. It is also possible to assemble the two fragments using an artificial polylinker. The inclusion of this non-complementary sequence has no deleterious effect on the catalytic activity of the polyribozyme.
• the polyribozyme F contains 479 bases and includes 4 ribozymes : R4 (position 59) , R5 (position 83), R2 (position 372) and R3 (position 444), as shown in figures 5 and 6.
• the size of the arms flanking the catalytic domains of the ribozymes is :
• the chimeric pPolyribozyme D is derived from pPolyribozyme F.
• the 206bp "Sacl-Swal" fragment originating from TLCV was deleted.
• the Sac I site was destroyed by action of T4-DNA polymerase.
• the polyribozyme D contains 270 bases and includes 4 ribozymes, R4 (position 59) , R5 (position 83) , R2 (position 163) and R3 (position 235) as shown in figures 5 and 6.
• the size of the arms flanking the catalytic domains of the ribozymes is :
• polyribozyme E, F and D were verified using the T7 sequencing kit supplied by Pharmacia.
• polyribozymes E, F and D were tested in vitro in accordance with the protocol described in "Protocol and Application Book” from Promega.
• the expression cassette composed of a double 35S promoter, monoribozyme Rl and poly (A) of 35S of CaMV was isolated from pRIB02BIN plasmid described above.
• the pRIB02BIN plasmid was digested with Sac I.
• Sac I sites were made blunt-ended using T4-DNA polymerase.
• the linearized pRIB02BIN plasmid was then digested with Sal I, the "Sac I filled-in Sal I" fragment which contains the expression cassette was purified on agarose gel and cloned into pBluescript SK+ vector in "Xba I filled-in using Klenow Polymerase and Sal I" sites.
• Hind III - BamHI fragment corresponding to monoribozyme Rl was then replaced with Hindlll - BamHI fragments containing the polyribozymes E, F and D isolated from pPolyribozym.es E, F and D respectively.
• the Hind III - BamHI fragment is in inverse orientation in the expression vector.
• the expression cassette with polyribozymes E, F or D were cloned into the binary vector pGAZE (figure 7) derived from pGA492 (An, Plant Physiol. (1986) 81 : 86) .
• the pGA 492 vector was double digested with Sac I and Sea I, made blunt-ended using T4-DNA polymerase and ligated after agarose gel purification.
• the eliminated fragment corresponds to nearly the entire chloramphenicol acetyl transferase gene.
• the Hind III site was also converted into EcoRI site with MalawiI - EcoRI adaptors supplied by Stratagene.
• pGA492 contains the cis-acting elements required for plant transformation, including T-DNA border, a selectable marker expressible in plants (kanamycin resistance) , cloning sites, and a wide host range replicon.
• the kanamycin resistant marker is constructed from nopaline synthase control regions and neomycin phosphotransferase coding region of Tn5.
• the pGAZE vector was digested with EcoRI and filled-in using Klenow-polymerase.
• the "Sad filled-in - Sal I" fragments containing the expression cassettes with polyribozymes E, F or D were cloned into the. EcoRI filled-in site of pGAZE.
• ribozyme A which corresponds to monoribozyme Rl including the catalytic domainand flanking arms 15 bases long
• polyribozyme C described above (example V) expression cassettes were introduced in pGAZE and/or pGA492 as mentioned above.
• RNA Total RNA is isolated from young leaves of transgenic and non-transgenic plants grown in the greenhouse according to Chandler et al. (Plant Physiol. (1983) 74 : 47) . RNA is separated on agarose-formaldehyde gels and transferred to a nitrocellulose membrane. The blot is hybridized with labelled fragment or entire gene of interest. To check whether equal amounts of RNA were applied to each line, the nitrocellulose membrane is stained with methylene blue.
• the TYLCV-Sardinian strain genome was inserted into Sst I site of the binary plant transformation vector pBin 19 (Stanley et al., EMBO J. (1986)5 : 1761) and a BamHI-fragment of about 600 bp extending from polylinker of pBin 19 through the intergenic region of TYLCV to the BamHI site at map position 152 of TYLCV was deleted. Subsequently a complete genome unit of TYLCV was inserted into the remaining Sst I site yielding a 1,8 mer of the TYLCV-genome in pBin 19. This plasmid was introduced directly into
• Agrobacterium tumefaciens cultures were grown at 28"C for about 48 hours. The cells were pelleted and washed twice with water and resuspended in 1/10 th of the initial volume of sterile water.
• Young transformed tomato plants at the 3 to 4 leaf stage, are inoculated into the petioles of the three youngest leaves with the concentrated Agrobacteria using a 18-gauge needle. The plants are placed in a closed growth chamber with 16 h - day light at 24°C under 70 % relative humidity.

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• 1993
  • 1993-07-22 EP EP93917617A patent/EP0728199A1/de not_active Withdrawn
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  • 1993-07-22 AU AU47014/93A patent/AU694093B2/en not_active Ceased
• 1994
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