Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance
Reexamination Certificate
1995-10-25
2001-07-24
Schwartzman, Robert A. (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers pathogen or pest resistance
C435S091300, C435S091310, C435S419000, C435S468000, C435S469000, C536S023100, C536S024500, C800S285000, C800S301000
Reexamination Certificate
active
06265634
ABSTRACT:
The present invention relates to a nucleotide sequence, called “polyribozyme”, capable of conferring on plants resistance to viruses, as well as a process for making the plants resistant. The invention also relates to the plants expressing the polyribozyme.
Several approaches have been developed to confer on cultivated plants resistance to viruses by integrating into the genome of the plants viral nucleic acid sequences: the gene for the capsid protein, the genes for non-structural proteins, anti-sense viral RNA sequences and RNAs of satellite viruses (see, for example, Cuozzo et al., 1988, Bio/Technology 6, 549-557; Rezaian et al., 1988, Plant. Mol. Biol., 11, 463-471; Harrison et al., 1987, Nature 328, 797-802).
These publications report the production of partial resistances or tolerances. Nonetheless, in most cases there are delayed symptoms or attenuated symptoms but not complete resistance.
Furthermore, some of these procedures, for example those employing the RNAs of satellite viruses, can give rise to new problems. For example, a satellite virus which reduces symptoms in one species may become lethal for another species. Moreover, mutations in the nucleotide sequences of the satellite virus introduced into the plants may increase the severity of the infection instead of diminishing it.
Similarly, the use of the capsid protein to confer resistance has disadvantages. For example, the capsid protein of a particular strain of the virus does not necessarily protect the plant against an infection by another strain of the virus. It is difficult to use the degree of homology of the amino acid sequence of the capsid protein between different viruses or between different strains to predict the degree of tolerance allowed by the expression of the protein. Furthermore, the expression of capsid proteins to protect against viral infection presents the risk of inducing heteroencapsidation between the capsid protein expressed in the plant and other viruses infecting the transgenic plant.Although it has never been demonstrated for transgenic plants, this heteroencapsidation has already been observed between two strains of BYDV and between ZYMV and PRSV.
The use of ribozymes has also been considered for conferring on plants resistance to viruses. Ribozymes are RNA molecules which act as enzymes by specifically catalysing the cleavage of the target RNA. The first experiments with ribozymes in plant cells were described in the patent application EP-A-321021. Since then, several authors have tried to optimise the structure of the ribozyme and the operating conditions in order to obtain efficient cleavage of the viral RNA.
For example, Lamb and Hay (J. Gen. Virol., 1990, 71: 2257-2264) have demonstrated the in vitro cleavage by mono-ribozymes of the RNA of the Potato Leaf Roll virus (PLRV) in regions coding for the RNA polymerase and the capsid protein. However, the in vitro cleavage reaction only occurs at 40° C.; it has not been possible to observe any reaction at all at 0° C. Plants are usually cultivated between 10 and 30° C., depending on the species. Thus, for in vivo use, Lamb and Hay suggest that the length of the complementary arms be increased. But, if the arms are too long, the formation of a stable duplex between the target RNA and the ribozyme can be caused, preventing the dissociation of the ribozyme and making it incapable of catalysing another cleavage reaction. Furthermore, depending on the length and sequence of the complementary arms, the ribozyme itself may form secondary structures which diminish its cleavage activity.
Edinoton and Nelson (“Gene Regulation: Biology of Antisense RNA and DNA: Ed. ERICKSON and IZANT, Raven Press Ltd, New-York, 1992) have described the in vitro and in vivo use of mono-ribozymes to inactivate the polymerase gene of the Tobacco Mosaic virus (TMV). They observed that the ribozymes exhibited a very different behaviour depending on whether they were used in vitro or in vivo. The activity of a ribozyme in vitro can not thus be used to predict the activity of the same ribozyme in vivo. For example, in vitro cleavage appears to be of low efficiency and requires a ribozyme concentration 20 times higher than the concentration of the TMV genomic RNA. On the other hand, in an in vivo experiment using tobacco protoplasts infected by TMV, the ribozyme suppresses 90% of the multiplication of the viral RNA. It is interesting to note that the anti-sense RNA used as control only inhibits 20% of the viral multiplication. These workers also refer to the studies of Gerlach et al. who made use of a polyribozyme targeted against the gene for the polymerase of TMV. This polyribozyme did not function in vitro owing to the length of the duplex formed between the ribozyme and the target RNA. On the other hand, in vivo, this polyribozyme cleaved the substrate. Transgenic tobacco plants expressing either the monoribozyme or the polyribozyme have shown a delay of symptoms after infection by the TMV. Complete resistance, i.e. the definitive absence of symptoms, is not described. The authors conclude that the parameters such as the optimal length of the complementary arms, the choice of the target sequence and the choice of the promoter, enabling possible problems of “compartment-alisation” of the ribozymes to be overcome, must be determined by experiment.
EP-A-0421376 describes ribozymes directed against a non-coding RNA sequence of CMV. WO-A-9213090 describes the inactivation of the RNA of the capsid protein of the CMV by the introduction of a heterologous sequence within the sequence using a monoribozyme of the “Group I intron” type. None of these documents describes the production of complete resistance to the CMV.
The technical problem which the present invention proposes to resolve is to provide a reliable agent, devoid of disadvantages, for conferring on plants resistance to viruses.
The present inventors have resolved this problem by the conception and use of a polyribozyme directed against the capsid protein of a virus. This polyribozyme is capable of inactivating the gene coding for this protein, and of thus conferring complete resistance to viruses.
The efficiency of the polyribozyme of the invention is surprising in the light of the mediocre results obtained in the prior art with the anti-sense sequences of the gene for the capsid protein, since each of these procedures involves an inactivation of the corresponding RNA. In addition, several authors had advised against the use of trans acting polyribozymes because the ribozymes are unable to function independently of each other and because catalytic regions having identical sequences sometimes have a tendency to hybridize to each other, which leads to inactive structures (see, for example, Taira, HFSP Workshop “RNA-Editing—Plant Mitochondria”, Abstract Book, Berlin, Sep. 15-20, 1992). The results obtained according to the invention are unexpected in view of the target selected, on the one hand, i.e. the capsid protein and, on the other hand, the method used to inactivate the target, i.e. a polyribozyme.
In addition to the efficiency of the inactivation, the polyribozymes of the invention possess a number of advantages in comparison with known procedures:
The ribozymes function as enzymes, catalysing the cleavage of several viral RNAs specifically without modification of structure. This enzymatic cleavage leads to the destruction of all of the viral RNAs whereas the expression of the capsid protein which inhibits viral infection functions as an inhibitor of viral multiplication.
The ribozymes are non-coding RNA molecules which can not induce heteroencapsidations or generate new viral strains.
Whereas the specificity of the tolerance induced by the capsid protein is difficult to predict, ribozymes can be constructed in order to cleave specifically one or more viral strains, or several related viruses if the complementary arms correspond to the regions of homology conserved between the different strains or between the different related viruses.
In order to have a complete understanding of the invention, it will be
Baudot Gaelle
Gruber Veronique
Lenee Philippe
Ollivo Catherine
Perez Pascual
Cooper & Dunham LLP
Gene Shears Shears Pty. Ltd.
Larson Thomas G.
Schwartzman Robert A.
White John P.
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