Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—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
  • 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
  • C12N15/8286—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 insect resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the invention relates to the efficient expression in plants of AT-rich genes, especially Bacillus thuringiensis (Bt) genes encoding insecticidal crystal proteins (ICP).
• the invention thus relates to a process that comprises the RNA polymerase II independent production of predominantly uncapped, non-polyadenylated RNA transcripts of the native coding sequences of AT-rich genes, preferably Bt ICP genes, said transcripts comprising translation enhancing sequences, particularly those derived from the 5' region and 3' region of positive-stranded RNA plant viruses, preferably of necroviruses, that enable efficient cap- and poly(A)- independent translation of the RNA transcripts in plant cells to yield high levels of proteins specified by the AT-rich genes, more particularly insecticidal levels of Bt ICPs.
• nucleic acid sequences were provided that encode a Bt ICP with essentially the same amino acid sequence as an existing Bt ICP but wherein one or more of the following modifications were included: the nucleic acid sequence surrounding the translation initiation codon was changed to resemble more the translation initiation sequences preferably used by plants. - the overall codon usage was modified to better reflect the preferred codon usage of a particular plant species. cryptic promoter signals were removed.
• nucleic acid sequences that target the hnRNA into an abortive splicing pathway were eliminated.
• - potential termination signals for DNA-dependent RNA polymerase II within the coding sequence were removed.
• putative mRNA destabilizing sequences were replaced. presumptive alternative polyadenylation sites were avoided.
• ICP coding sequence under control of a T7 promoter or a plastid expression signal in the chloroplasts of tobacco plants in an attempt to circumvent the problem of poor expression of full-length protoxin genes from the nucleus of plants, particularly those with a high AT-content.
• the regenerated plants from these transplastomic lines were reported to express Bt ICP at a high level in mature leaves using the prokaryotic-like transcriptional and translational machinery of the plastid (Mc Bride et al., Bio/Technology 13: 362-365 (1995); WO 95/24492, WO 95/24493).
• eukaryotic mRNA unique features of eukaryotic mRNA are the presence of the m ⁇ G cap at its 5' end and a 3' poly(A) tract.
• Several functions at different stages of gene expression have been attributed to the cap at the 5' end, which is added shortly after transcription elongation has started, including a role in RNA stabilization, splicing, transport and translation.
• the cap structure supposedly binds to the translation initiation factor elF-4F, allowing the ribosomal subunits and proper factors to bind and initiate at the first AUG codon in a favourable sequence context. Absence of this 5' cap structure in naturally capped plant viral RNA or cellular mRNA decreases the translational efficiency substantially [Fletcher er al, J. Biol. Chem.
• poly(A) tail found at the 3' end of most eukaryotic mRNAs has been implied in mRNA stability, its transport into the cytoplasm, and its efficient translation [Jackson and Standart, Cell, 62: 15- 24,1990].
• the poly(A) tail, complexed with poly(A)-binding protein is believed to enhance the formation of 40S translational initiation complexes, presumably through promoting some sort of interaction between 5' and 3'- proximal elements of the mRNA [Tarum and Sachs, Genes and Dev. 9: 2997-3007 (1995)].
• RNAs of plant viruses often lack one or both.
• the RNAs are translated early upon infection, even though cellular templates are prevalent. It is often due to the presence of alternative terminal structures that viral RNA templates exhibit high translational efficiency.
• US Patent (US) 4,820,639 describes a process and means for increasing production of protein translated from eukaryotic messenger ribonucleic acid comprising transferring a regulatory nucleotide (nt) sequence from a viral coat protein mRNA to the 5' terminus of a gene or complementary deoxyribonucleic acid (cDNA) encoding the protein to be produced to form a chimeric DNA sequence.
• nt regulatory nucleotide
• cDNA complementary deoxyribonucleic acid
• EP 0589841 provides a dual method for producing male-sterile plants, as well as compositions and methods for high level expression of a coding region of interest in a plant by expression of a T7 RNA polymerase in a plant cell that contains a second expression cassette comprising a T7
• chimeric genes comprise: a.) a first promoter recognized by a DNA-depende ⁇ t RNA polymerase different from a eukaryotic RNA polymerase II, particularly a T3 or T7 RNA polymerase specific promoter; b.) a DNA region encoding a chimeric RNA which comprises a 5" UTR, a heterologous coding sequence, preferably an AU- rich coding sequence, and a 3' UTR; and optionally c.) a terminator sequence recognized by said RNA polymerase wherein the chimeric RNA, produced by the RNA polymerase, is uncapped and comprises: i) a first translation enhancing sequence derived from the 5' region of genomic or subgenomic RNA of a positive stranded RNA plant virus, preferably a necrovirus, especially
• STNV-2 or TNV-A located in the 5' region of the chimeric
• RNA ii) a second translation enhancing sequence derived from the 3' region of genomic or subgenomic RNA of a positive-stranded RNA plant virus, preferably a necrovirus, especially STNV-2 or TNV-A, located in the 3' region of the chimeric RNA; and which is capable of being translated in the cytoplasm of a plant cell, to produce the protein or polypeptide.
• the transcribed uncapped RNA coding sequence may be polycistronic.
• plant cells and plants particularly corn plant cells and plants, comprising these chimeric genes, integrated in their nuclear DNA, whereby the plant cell produces the RNA polymerases corresponding to the used promoters and terminators.
• the first promoter is a single subunit bacteriophage RNA polymerase specific promoter, such as a T3 or T7 RNA polymerase specific promoter, and wherein such plant cells or plants further comprise a chimeric polymerase gene including: a.) a second plant-expressible promoter; b.) a DNA sequence encoding a single subunit bacteriophage RNA polymerase such as a T3 or T7 RNA polymerase functionally linked to a nuclear localization signal; operably linked so that upon expression of the chimeric polymerase gene a functional and properly located RNA polymerase is produced.
• the first promoter is a single subunit bacteriophage RNA polymerase specific promoter, such as a T3 or T7 RNA polymerase specific promoter
• plant cells or plants further comprise a chimeric polymerase gene including: a.) a second plant-expressible promoter; b.) a DNA sequence encoding a single subunit bacter
• the invention further provides a process for producing a plant expressing a protein or polypeptide encoded by a heterologous gene, preferably an AT-rich gene, especially a Bt ICP encoding gene, which comprises the steps of: a.) transforming the nuclear genome of a plant cell with the above- mentioned chimeric genes; and b.) regenerating a transformed plant from the transformed cell.
• a heterologous gene preferably an AT-rich gene, especially a Bt ICP encoding gene
• Figure 1A schematically represents the relative protein accumulation profiles in plant protoplasts obtained by translation of a capped chimeric RNA comprising the translation enhancing sequences of the invention, in reference to an efficiently translated capped and polyadenylated RNA.
• Figure 1B schematically represents the relative protein accumulation profiles in plant protoplasts obtained by translation of a uncapped chimeric RNA comprising the translation enhancing sequences of the invention, in reference to the capped version of the same chimeric RNA comprising the translation enhancing sequences of the invention.
• Figure 2A depicts schematically different possible locations of first and second translation enhancing sequences with regard to the homologous coding sequence and untranslated regions of a viral genomic or subgenomic RNA.
• Figure 2B is a schematic representation of different possible locations of first and second translation enhancing sequences with regard to the heterologous coding sequence and untranslated regions of the chimeric RNAs encoded by the cap-independently expressed chimearic genes of the invention.
• AT-rich genes have an enhanced probability of harbouring cryptic signals interfering with efficient transcription and translation in plant cells, especially in monocotyledonous cells, such as corn cells.
• Expression problems are magnified when the AT content of the coding region of the heterologous gene surpasses significantly the mean AT content of the coding regions of the host plant in which expression is attempted. These expression problems might already arise when the coding sequence of the gene of interest, although not particularly AT-rich when taken as a whole, contains an AT-rich nucleotide-stretch of about 400 residues.
• the present invention provides a new method to promote expression to a high level, of coding sequences, preferably coding sequences of AT- rich genes such as Bt ICP genes, particularly native coding sequences of Bt ICP genes which are integrated in the plant's nuclear genome. It was realized that problems associated with the expression of coding sequences of heterologous AT-rich genes at the transcriptional and/or post- transcriptional level can be overcome by using an RNA polymerase different from the eukaryotic DNA-dependent RNA polymerase II, to produce uncapped RNAs encoding the protein or polypeptide of interest. These uncapped RNAs are then efficiently translated into the desired protein or polypeptide, by using the translation enhancing sequences provided in this invention.
• the invention is based on the realization that transciption by an RNA polymerase different from the eukaryotic DNA dependent RNA polymerase II, of AT-rich genes such as Bt ICP genes, particularly native coding sequences of Bt ICP genes, integrated in the nuclear genome of a plant, generates sufficiently large amounts of RNA, without suffering from the mentioned transcriptional and post-transcriptional problems.
• the resulting RNA is however uncapped and non-polyadenylated.
• the invention is further based on the finding by the applicants, that when uncapped RNAs comprising native coding sequences of heterologous genes and suitable translation enhancing sequences derived from 5' and 3' regions of the genomic RNA coding for the coat protein of a necrovirus, such as STNV-2, are introduced in plant cells, these RNAs are translated efficiently.
• the invention thus provides the means and methods to transcribe AT rich genes by an RNA polymerase different from the eukaryotic DNA dependent RNA polymerase II, to produce uncapped RNAs encoding the protein or polypeptide of interest, which are efficiently translated by the inclusion of translation enhancing sequences from 5' and 3' regions of RNA viruses which allow efficient translation of uncapped RNAs in a cap- independent manner.
• cap-independently expressed chimeric genes are provided comprising an AT-rich coding sequence and DNA encoding translation enhancing sequences of a necrovirus, under control of a promoter recognized by an RNA polymerase different from eukaryotic RNA polymerase II. Integration of such chimeric genes in a plant cell expressing the alternative RNA polymerase results in the production of
• RNA transcripts 5 predominantly uncapped and non-poiyadenylated RNA transcripts which are translated efficiently due to the presence of the translation enhancing sequences.
• leader and 5'UTR refer to the part of a protein-encoding RNA molecule, preceding the initiation codon of the 0 coding sequence. These terms are employed interchangeably and may also be used to refer to a DNA, encoding such a leader.
• trailer and 3'UTR refer to the part of a protein-encoding RNA molecule, downstream of the stop codon of the coding sequences. Again, these terms are employed interchangeably and may also be used to refer to a s DNA encoding such a trailer.
• the 5'UTR and 3'UTR of an RNA plant virus mentioned in this specification flank the coding sequence of the coat protein of that virus.
• the "5' region" of a protein-encoding RNA molecule refers to the extreme 5' end of that RNA and comprises at least o the 5'UTR of that RNA but may include several nucleotides extending immediately downstream of the initiation codon of the homologous coding region.
• the "3' region” of a protein-encoding RNA molecule refers to the extreme 3' end of that RNA and comprises at least the 3'UTR of that RNA but may include several nucleotides extending immediately upstream 5 of the stop codon of the homologous coding region.
• coding region refers to an RNA molecule or sequence which can be translated into a continuous sequence of amino acids of a biologically active protein or peptide (e.g., an enzyme or a protein toxic to insects) or to the DNA molecule or sequence o encoding such an RNA. Whether the "coding region” refers to a RNA or DNA molecule will be readily understood by the context.
• a coding sequence to be utilized in a cap-independently expressed chimeric gene will be generally derived from the coding region of a heterologous gene, and an appropriate initiation codon has to be provided, if necessary.
• a "DNA region encoding an RNA region” may refer to any part of a
• DNA molecule that is transcribed can relate to the entire transcribed region of a gene, but also to parts thereof, e.g., part of a coding sequence, a DNA-region corresponding to a first or second translation enhancing sequence, a 5' or 3' UTR, or a 5' or 3' region.
• expression of a gene refers at least to the combination of phenomena (transcriptional, post-transcriptional and translational events) which result in the production of the primary translation product , i.e., a protein or a polypeptide.
• the term also relates to the effect the translation product or its derivative may have on the phenotype of the cell or of the plant.
• a cap-independently-expressed chimeric gene (CIG) of this invention generally comprises : a) a first promoter recognized by a DNA-dependent RNA polymerase, different from eukaryotic DNA-dependent RNA polymerase II, b) a DNA encoding an RNA molecule which comprises :
• an untranslated leader sequence 1) an untranslated leader sequence; 2) a coding region encoding a heterologous protein or polypeptide, preferably an AU-rich coding region; and 3) an untranslated trailer sequence, and, optionally, c) a terminator sequence recognized by the same RNA polymerase which recognizes the first promoter.
• the CIGs of this invention are further characterized in that they comprise DNAs encoding first and second translation enhancing sequences.
• the first translation enhancing sequence is generally located in the untranslated leader sequence, but it may overlap with the coding region, i.e., it may extend downstream of the initiation codon of the coding region. Preferably, the first translation enhancing sequence is located around that translation initiation codon.
• the second translation enhancing sequence is generally located in the untranslated trailer sequence, but it may also overlap with the coding region, i.e., it may extend upstream of the stop codon of the coding region. Preferably, the second translation enhancing sequence is located around that stop codon.
• Preferred cap-independently expressed chimeric genes of the invention are CIGs as described above, wherein the DNA encoding a heterologous protein or polypeptide is AT-rich.
• "AT-rich" DNA coding sequences as referred to herein are those coding DNA sequences, comprising a continuous nucleotide sequence of at least 400 nucleotides, preferably of a least 600 nucleotides in length, with an AT content of at least 55%, preferably of at least 57.5%, particularly of at least 60%, more particularly of at least 62 %.
• AT rich coding sequences also include those coding sequences, where the entire coding sequence has an AT content of at least 55%, preferably of at least 57.5%, particularly of at least 60%, especially of at least 62 %.
• coding sequences smaller than 400 nucleotides are considered AT-rich when the entire coding sequence has an AT content of at least 55%, preferably of at least 57.5%, particularly of at least 60%, especially of at least 62 %.
• AT rich coding sequences thus include but are not limited to e.g., coding sequences of Bt ICP genes, but also sequences encoding fusion proteins between an Bt ICP and a protein encoded by a GC-rich coding sequence.
• AU rich is defined by the same criteria as an "AT rich DNA", except that thymine (T) is replaced by uracil (U).
• Another class of preferred CIGs are those CIGs wherein the first and second translation enhancing sequences are derived from a TNV strain, particularly from TNV-A, especially from TNV sg RNA 2.
• the CIGs are integrated in the nuclear genome of cells of a host plant.
• these genes contain promoters recognized by the endogenous RNA polymerase I or III of the host, or recognized by a bacteriophage single subunit RNA polymerase.
• the gene encoding the single subunit RNA polymerase is also introduced and expressed in a functional and properly located form in the same plant cell. It goes without saying that the choice of the RNA polymerase will depend on the particular promoter of the CIG and vice versa.
• heterologous refers to any coding sequence which is different from the coding sequence naturally associated with a 5' UTR or 3' UTR from a viral RNA from which the first or second translation enhancing sequences are derived.
• a heterologous coding region does not contain a region of more than 20, preferably not more than 15 codons of the viral RNA ccoding region.
• heterologous on the contrary means that such a coding sequence is naturally associated with a 5 * UTR or 3" UTR from a viral RNA from which the first or second translation enhancing sequences are derived
• a heterologous, respectively homologous protein is thus a protein encoded by a heterologous, respectively homologous coding sequence.
• necrovirus refers to any plant virus isolate normally included in this taxonomic group, as well as their satellite viruses, exemplified by, but not limited to, tobacco necrosis virus strains, satellite tobacco necrosis virus strains, chenopodium necrosis virus, carnation yellow stripe virus, and lisianthus necrosis virus.
• the term "native DNA” or “native DNA sequence” refers to a DNA as found in its natural state, as well as a DNA containing small modifications whereby the overall AT content of that DNA is essentially retained, and the amount of modified bases, preferably of modified adenine or thymine, is limited to maximally 3%, particularly less than 1 %.
• a native DNA with small modifications should have at least 95%, preferably 99% sequence identity with respect to that native DNA without such modifications. Examples of such modifications include, but are not limited to, the modification of the nucleotide sequence to introduce or remove a restriction enzyme recognition site or to change one or more amino acids in order to make a protein protease-resistant.
• native DNA will be used predominantly with regard to all or part of the heterologous coding sequence encoding a biologically functional protein or polypeptide, such as a BT ICP coding region.
• native Bt ICP encoding sequence may thus be a truncated version comprising the minimal toxic fragment.
• RNA as used herein designates any genomic or subgenomic RNA of, or produced by a positive stranded RNA plant virus in nature.
• RNA polymerase that generates uncapped, non-polyadenylated RNA transcripts of a CIG.
• the nature of the RNA polymerase evidently determines the first promoter to be included in the CIG and vice versa.
• a useful RNA polymerase is a bacteriophage single subunit RNA polymerase such as the RNA polymerases derived from the E.
• the T3 RNA polymerase and the T7 RNA polymerase are especially preferred.
• the first promoter should be a T3 RNA polymerase specific promoter and a T7 RNA polymerase specific promoter, respectively.
• a T3 RNA polymerase specific promoter and a T7 RNA polymerase specific promoter are referred to as a T3 promoter and a T7 promoter, respectively.
• a T3 promoter to be used as a first promoter in the CIG can be any promoter of the T3 genes as described by McGraw et al, Nucl. Acid Res. 13: 6753-6766 (1985).
• a T3 promoter may be a T7 promoter which is modified at nucleotide positions -10, -11 and -12 in order to be recognized by T3 RNA polymerase [(Klement et al., J. Mol. Biol. 215, 21- 29(1990)].
• a preferred T3 promoter is the promoter having the "consensus" sequence for a T3 promoter, as described in US Patent 5,037,745.
• a T7 promoter which may be used according to the invention, in combination with T7 RNA polymerase, comprises a promoter of one of the T7 genes as described by Dunn and Studier, J. Mol. Biol. 166: 477-535 (1983).
• a preferred T7 promoter is the promoter having the "consensus" sequence for a T7 promoter, as described by Dunn and Studier (supra).
• T3 or T7 promoters as described above include nucleotides immediately downstream of the transcription initiation site. At the 3' end of the described T3 or T7 promoter for use in this invention, up to six nucleotides can be removed to prevent the incorporation of additional nucleotides in the 5' UTR of the transcripts from the CIGs. Particularly preferred are the T3 promoter of SEQ ID No.18 between the nucleotide positions 14 and 32 and the T7 promoter of SEQ ID No.30 between nucleotide positions 22 and 39. Another particularly preferred promoter is the T7 promoter of SEQ ID No. 30 between nucleotide positions 22 and 39 followed by 4 nucleotides of the consensus sequence (i.e., GGAG) as described by Dunn and Studier (supra).
• RNA polymerase I Another useful RNA polymerase for application in this invention is RNA polymerase I.
• the CIG of this invention may comprise a RNA polymerase I promoter.
• RNA polymerase I normally transcribes the tandemly repeated rRNA genes in eukaryotic cells such as plant cells, and the promoter signals are located in the intergenic spacer sequences between the rRNA gene repeats. It is preferred that the RNA polymerase I promoter used in the CIG of this invention originates or is derived from the plant species to be transformed with the CIG, although this is not required.
• RNA polymerase I specific rRNA promoter region from corn derived from the 3 kb intergenic spacer as described for Black Mexican Sweet Maize [McMullen et al., Nucl. Acids Res. 14: 4953-4968 (1986)] is used.
• a preferred promoter region comprises the nucleotide sequence of the EMBL nucleotide sequence database under accession number X03990 (EMBL X03990, which is herein incorporated by reference) between nucleotide positions 2160 and 2296, particularly a promoter region including all subrepeats of the intergenic spacer, such as a promoter region comprising the nucleotide sequence of EMBL X03990 between nucleotide positions 154 and 3118.
• a promoter region wherein some of the subrepeats have been deleted such as a promoter region comprising the nucleotide sequence of EMBL X03990 between nucleotide positions 939 and 3118.
• promoter regions wherein some or all of the nucleotides downstream of the transcription initiation point have been deleted such as a promoter region comprising the nucleotide sequence of EMBL X03990 between nucleotide positions 154 and 2590 or a promoter region comprising the nucleotide sequence of EMBL X03990 between nucleotide positions 2160 and 2296.
• a promoter region comprising the nucleotide sequence of EMBL X03990 between nucleotide positions 154 and 2590
• a promoter region comprising the nucleotide sequence of EMBL X03990 between nucleotide positions 2160 and 2296.
• RNA polymerase I promoter regions derived from the 3 kb intergenic region of the maize line B73.
• rRNA intergenic spacers comprising RNA polymerase I promoters which may be used according to the invention, are known in the art for rye [Appels et al, Can J Genet Cytol 28:673-685 (1986)], wheat [Barker et al, J. Mol. Biol. 201: 1-17 (1988)], radish [Delcasso- Tremousaygue et al., Eur. J. Biochem 172: 767-776 (1988)], rice [Takaiwa er al., Plant Mol. Biol.
• RNA polymerase III RNA polymerase III
• the cap-independently expressed chimeric gene of this invention may comprise a RNA polymerase III promoter.
• RNA polymerase III normally transcribes the majority of small RNAs, such as tRNAs, 5S RNAs and small nuclear RNAs (snRNAs) involved in mRNA processing, in eukaryotic cells such as plant cells.
• Suitable promoters for this invention recognized by RNA polymerase III are the promoters transcribing snRNAs of plants such as U3 or U6 snRNA from Arabidopsis thaliana [Waibel and Filipowicz, Nucl. Acids Res. 18: 3451-3458 (1990), Marshallsay et al., Nucl. Acids Res. 18: 3459-3466 (1990)] or the promoter transcribing tRNAs of plants such as tRNA met from soybean [Bourque and Folk, Plant Mol. Biol. 19: 641-647(1992)].
• the transcribed region of a CIG comprises a heterologous AT-rich coding sequence, as defined above.
• the transcribed region comprises a sequence encoding a Bt ICP having insecticidal activity to at least one insect species.
• a transcribed region comprising a sequence encoding a truncated Bt ICP, which lacks nucleotides either at the 5' end or the 3' end of the coding sequence, or both, but still comprises the sequence coding for the minimal toxic fragment.
• Particularly preferred Bt ICP encoding sequences for use in this invention are cry1Ab5, cry9C , crylBa, cry3C, cry3A, cryl Da and cryl Ea.
• cry1Ab5 represents the cn/IAb gene described by Hofte et al, Eur. J. Biochem. 161 : 273-280 (1986); cry9C represents the crylH gene described by Lambert et al., Appl. and Env. Microbiol. 62: 80-86 (1996); crylBa represents the c/ylB described by Brizzard and Whitely, Nucl. Acid Resarch 16: 4168-4169 (1988); cry3C represents the crylllD gene described by Lambert et al., Gene 110: 131-132 (1992); cry3A represents the c/ylllA gene described by H ⁇ fte et al., Nuc. Acids Res.
• cryl Da and cryl Ea represent the bt4 and bt18 genes, respectively, described in WO 90/02801 , according to the classification proposed by Crickmore et al, Abstract presented at the 28th annual meeting of the Society for Invertebrate Pathology, 16-21 July 1995.
• CIGs of the invention may further include the use of genes encoding a Bt ICP fused to a protein allowing selection, e.g., gentamycin acetyl transferase (GAT) encoded by aac(6') or phosphinotricin acetyl transferase (PAT) encoded by bar.
• GAT gentamycin acetyl transferase
• PAT phosphinotricin acetyl transferase
• CIGs encoding chimeric toxins wherein a domain of the toxic BT ICP fragment has been exchanged for a similar domain of another BT ICP, as described by Bosch et al. [BIO/TECHNOLOGY 12, 915-918(1994)] are also encompassed by the invention.
• the CIGs according to the invention may be polycistronic, comprising between the first and second translation enhancing sequence at least 2 and up to 5 cistrons, although more cistrons may be possible.
• polycistronic CIG Transcription of such a polycistronic CIG yields polycistronic RNA that should preferably comprise an internal ribosome entry site [Jackson and Kaminski, RNA 1 : 985-1000 (1995); Levis and Astier-Monifacier, Virus Genes 7: 367-379 (1993); Basso et al. J. Gen. Virology 75: 3157-3165 (1994)] between the cistrons.
• at least one cistron is AT-rich.
• the CIGs used in the invention may further include a terminator recognized by the RNA polymerase which is used to enable transcription of the CIG.
• Suitable terminators are known in the art and should preferably be chosen according to the specific promoter that is used. For instance, when a T3 promoter is used, a T3 specific terminator such as described by Sengupta er al., J. Biol. Chem. 264: 14246-14255 (1989), preferably in a duplicated form, can be used. Since a T7 RNA polymerase terminates as efficiently on a T3 terminator (T3-T ⁇ ) as on a T7 terminator (T7-T ⁇ ) [Macdonald er al., J. Mol. Biol.
• a terminator region comprising T3-T ⁇ may be used as well for CIGs containing a T3 promoter as for those containing a T7 promoter.
• the terminator regions used should comprise the corresponding species-specific RNA polymerase I terminators which are present in the intergenic regions between the rRNA repeats [Reeder and Lang, Molecular Microbiology 12: 11-15 (1994)].
• the terminator regions used may comprise the corresponding trailer sequences associated with genes normally transcribed by RNA polymerase III, such as the genes encoding U3 or U6 snRNA from Arabidopsis thaliana [Waibel and Filipowicz, supra, Marshallsay et al. supra] or the gene encoding tRNA met from soybean [Bourque and Folk, supra].
• the CIG integrated in the nuclear genome of a plant cell is transcribed in an RNA polymerase II independent manner.
• This can be achieved in accordance with the invention by incorporating in the CIG a promoter and terminator as described above.
• the transgenic plant cells do not naturally contain the RNA polymerase required for the recognition of the promoter and transcription of the CIG, these cells need to comprise a second chimeric gene encoding that RNA polymerase, further referred to as the chimeric polymerase gene.
• RNA polymerase e.g., T7 or T3 promoters
• a chimeric polymerase gene encoding a T7 or T3 RNA polymerase [US 5,102,802] should also be incorporated in the nuclear DNA of the host plant cell.
• mutant bacteriophage RNA polymerases as exemplified for T7 RNA polymerase by McDonalds et al., J. Mol. Biol. 238: 145-148 (1994) may be used in this invention.
• Such mutant bacteriophage T7 RNA polymerases no longer recognize the rare termination signals encountered in heterologous genes under control of a T7 promoter, while still terminating at bona fide T7 RNA polymerase termination signals.
• hybrid bacteriophage RNA polymerases as described by Joho et al., J. Mol. Biol. 215: 31-39 (1990), with altered specificity and promoter preference, may be used according to the invention.
• the chimeric polymerase gene comprises a 5' regulatory region, i.e. the promoter region, necessary for expression in plant cells.
• This plant- expressible promoter may be a constitutive promoter, such as a CaMV35S promoter [Odell et al. Nature 313, 810-812] or may be regulated in a tissue- specific way, such as the promoters disclosed in WO 92/13957, WO 92/13956 or EP 0344029.
• Another suitable regulated promoter is a light- inducible promoter such as the promoter of the small subunit of Rubisco.
• the expression of the single subunit bacteriophage RNA polymerase may also be temporarily regulated using promoters which are only expressed at a certain developmental state, or are induced by external stimuli such as nematode-feeding (WO 92/215757), or fungus-infection (WO 93/19188).
• Further suitable promoters are plant-expressible promoters regulated by the presence of plant-growth regulators such as abscisic acid, steroid- inducible promoters or copper-inducible promoters.
• the spatial or temporal regulation of the promoter used in the chimeric polymerase gene will of course be reflected in the expression pattem of the single subunit bacteriophage RNA polymerase in the transformed plants of this invention, and ultimately in the expression pattern of the CIG comprising the corresponding promoter.
• the single subunit bacteriophage RNA polymerase should be operably linked to a nuclear localization signal (NLS) [Raikhel, Plant Physiol. 100: 1627-1632 (1992) and references therein], such as the NLS of SV40 large T-antigen [Kalderon er al. Cell 39: 499-509 (1984)].
• NLS nuclear localization signal
• the NLS can be operably linked to the polymerase in different ways.
• the NLS is joined to the amino-terminus of the polymerase, or located within the N-terminal region of the polymerase, particularly within the first 20 amino acids of the polymerase, more particularly between amino acid 10 and 11 of the T7 polymerase.
• the chimeric polymerase gene may further include any other necessary regulatory sequences such as terminators [Guerineau et al, Mol. Gen. Genet. 226:141-144 (1991), Proudfoot Cell, 64:671-674 (1991 ), Safacon er al., Genes Dev 5: 141-149 (1991); Mogen et al., Plant Cell, 2: 1261-1272 (1990); Munroe et al., Gene, 91: 151-158 (1990); Ballas er al., Nucleic Acids Research 17: 7891-7903 (1989); Joshi et al., Nucleic Acid Research 15: 9627-9639 (1987)], plant translation initiation consensus sequences [Joshi, Nucleic Acids Research 15: 6643-6653 (1987)], introns (Luehrsen and Walbot, Mol.
• terminators [Guerineau et al, Mol. Gen. Genet. 226:141-144 (1991), Proudfoot Cell
• the first and second translation enhancing sequences which may be used are preferably derived from positive- stranded RNA viruses.
• Preferred translation enhancing sequences are derived from necroviruses, preferably from STNV or TNV strains, especially from STNV-2 or TNV-A sgRNA2.
• VA/lnl see end of this section for the mathematical formula allowing estimation of functional half-life of the RNA (t 7 J and translation efficiency (A)] of the mentioned protein of at least 20%, preferably at least 25%, of the peak level resulting from in vivo translation of similar capped, non-polyadenylated first reference RNA (i.e., a first reference RNA identical to the uncapped RNA but with a cap- structure).
• the peak level resulting from in vivo translation of the capped non-polyadenylated first reference RNA should be at least 10% of the peak level resulting from in vivo translation of a second reference RNA which is capped and polyadenylated and comprises the ⁇ leader of TMV [Gallie et al.
• RNAs Methods to generate capped and uncapped RNAs in vitro, for the introduction of such RNAs in plant protoplasts and to compare the translation efficiencies and functional half-lives of RNAs are described at the end of this section, as well as in Examples 2, 3 and 4.
• the translation enhancing sequences are largely derived from sequences comprised in the leaders and trailers of genomic or subgenomic viral RNAs (e.g., Fig 2A (1 ), (5), (3) and (7).
• first translation enhancing sequence comprising nucleotide sequences extending immediately downstream of the initiation codon of the homologous protein (i.e., comprising nucleotides of the 5' end of the viral homologous coding sequence; e.g., Fig 2A (2) and (4)), or to use a second translation enhancing sequence comprising nucleotide sequences extending immediately upstream of the stop codon of the homologous 9814 PC17EP97/02832
• Figure 2A schematically summarizes the different possible positions of nucleotide sequences comprising translation enhancing sequences (indicated by the thin lines ) with reference to the homologous coding sequence (CDS; indicated as a solid black bar) and 5' and 3' untranslated region (5'UTR and 3'UTR; indicated as open bars) of a viral genomic or subgenomic RNA .
• First translation enhancing sequences include those indicated by 1-4, second translation enhancing sequences include those indicated by 5-8.
• Satellite tobacco necrosis virus (STNV) and tobacco necrosis virus (TNV) are plant viruses belonging to the necrovirus group.
• STNV is a satellite virus, that relies upon the viral RNA replicase of the helper virus (TNV) for its replication, but codes for its own coat protein (CP).
• CP coat protein
• the genome consists of one single-stranded RNA strand with positive polarity, and the nucleotide sequence is known for several strains.
• the nucleotide sequence consists of a leader sequence or 5' untranslated region ("UTR") of 29-32 nucleotides (nt), a CP encoding region of 588-597 nt, and a trailer sequence or 3' UTR of 616-622 nt [Ysenbaert et al. J. Mol. Biol. 143: 273-287 (1980), Danthinne et al, Virology 185, 605-614 (1991)].
• the 5' UTRs of the STNV strains are nearly identical and can fold into a hairpin structure with a stem of 6 or 7 bp enclosing a loop of seven residues.
• the trailer sequences which exhibit 64 % sequence identity between the nucleotide sequence of STNV-1 and STNV-2, can fold into a secondary structure consisting of three (or four) pseudo knots flanked by two hairpins, ending with an extended double helix that spans the last 350 residues of the sequence and includes several internal loops, bulged out nucleotides, and bifurcations. [Danthinne et al, (1991) supra].
• the STNV RNA does not contain a m 7 G cap structure, nor a covalently linked virus-encoded protein at the 5' end . Neither does it contain a poly(A) tail at the 3' end [Horst et al. Biochemistry 10: 4748-4752 (1971 ); Smith and Clark, Biochemistry 18: 1366-1371(1976)]. Yet, STNV RNA is translated efficiently in vitro.
• TED enhances in vitro translation when fused to a heterologous coding sequence (encoding -glucoronidase), but the level of enhancement depends on the nature of the 5' UTR and is larger in combination with the STNV 5 * terminally located 173 nucleotides [Danthinne et al., supra (1993)].
• Preferred first translation enhancing sequences comprise the leader of STNV-2, especially preferred is a first translation enhancing sequence comprising the nucleotide sequence between nucleotide positions 1 and 32 of SEQ ID No.2 , particularly preferred is a first translation enhancing sequence comprising the nucleotide sequence between nucleotide positions 1 and 38 of SEQ ID No.2 comprising an initiation codon and the second codon of the coat protein coding sequence.
• Preferred second translation enhancing sequences comprise portions effective in enhancing translation of uncapped RNAs, derived from the trailer sequence of STNV-2, particularly the nucleotide sequence between nucleotide positions 632 and 753 of SEQ ID No.2, quite particularly the nucleotide sequence of SEQ ID No. 2 between nucleotide positions 632 and 760.
• TNV is a small icosahedral plant virus, with a single genomic RNA of about 3.7 kb.
• the nucleotide sequence of different isolates has been published (except for some terminal nucleotides) [Meulewaeter et al. Virology 177:699-709 (1990); Coutts et al., J. Gen. Virol. 72: 1521-1529 9814 PC17EP97/02832
• TNV specific RNAs Upon infection of plant cells, six TNV specific RNAs are produced: the genomic RNA, two subgenomic (sg) RNAs of 1.5 kb (sgRNAI ; starts at nt 2184 of TNV-A) and 1.2 kb (sgRNA2 ; starts at nt 2461) which are 3' co- terminal, and the corresponding minus-strand RNAs.
• the RNA of TNV strain A contains six major open reading frames (ORFs) and most likely serves as mRNA for the synthesis of a 23-kDa protein and a 82-kDa read-through protein, which are encoded by ORFs 1 and 2.
• the internal cistrons are most probably expressed from the two 3'-co-terminal subgenomic RNAs.
• the 5' ends of the largest and smallest subgenomic RNAs are located upstream of ORFs 3 and 5, respectively [Meulewaeter et al., J. Virology 66: 6419-6428 (1992)].
• a very similar genome organization was proposed for TNV-D and for the carmovirus melon necrotic spot virus [Riviere and Rochon, J. Gen. Virol. 71: 1887-1896 (1990)].
• the smallest subgenomic RNA probably directs the synthesis of the viral coat protein [Meulewaeter et al., J.
• the coat protein gene is followed by a trailer sequence of 241 nucleotides.
• first translation enhancing sequences comprise portions derived from the 5' regions of TNV-A sgRNA2, such as the nucleotide sequence of SEQ ID No.1 between nucleotide positions 2461 and 2619, which still comprises 7 nucleotides of the coat protein coding sequence.
• a first translation enhancing sequence comprising the nucleotide sequence between nucleotide positions 2461 and 2612 of SEQ ID No.1 , particularly the nucleotide sequence between nucleotide positions 2461 and 2603 of SEQ ID No.
• Preferred second translation enhancing sequences comprise portions effective in enhancing translation of uncapped RNAs, derived from the 3' region sequence of the TNV sgRNA2, particularly the nucleotide sequence between positions 3399 and 3684 of SEQ ID No.1 , which still comprises 41 nucleotides upstream of the stop codon of the coat protein coding sequence, preferably the nucleotide sequence between nucleotide positions 3429 and 3611 of SEQ ID No.1 , especially the nucleotide sequence between nucleotide positions 3472 and 3611 of SEQ ID No.1.
• the translation enhancing sequences as derived from the 5' regions or 3' regions of an RNA plant virus can be modified by small insertions, deletions or substitutions, so that their capacity to enhance cap- independent translation or their synergistical interaction is not negatively affected. Such variants are referred to herein as "derivatives" and their use as enhancers for cap-independent translation form part of the invention. Generally, it is preferred that such a derivative has at least 90 % sequence identity to the natural translation enhancing sequence.
• % sequence identity of two related nucleotide or amino acid sequences refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared.
• a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
• nucleotide stretches which allow interactions between a pair of first and second translation enhancing sequences or between one or both of the translation enhancing sequences and the 3' end of the 18S rRNA are left unchanged.
• first translation enhancing sequence the nucleotide sequence of SEQ ID No. 1 between nucleotide positions 2461 and 2619
• second translation enhancing sequence the nucleotide sequence of SEQ ID No. 1 between nucleotide positions 3399 and 3684
• nucleotide positions 2464 and 2479 between nucleotide positions 2563 and 2567, between nucleotide positions 2571 and 2574, between nucleotide positions 2576 and 2586, between nucleotide positions 3449 and 3463, between nucleotide positions 3465 and 3472, and between nucleotide positions 3475 and 3482 are left unchanged.
• sequences of SEQ ID No. 2 between nucleotide positions 9 and 19, between nucleotide positions 24 and 30, between nucleotide positions 33 and 37, between nucleotide positions 636 and 640, between nucleotide positions 646 and 652, and between nucleotide positions 692 and 698 are left unchanged. Nevertheless, if one of these regions are changed, it is important to make the corresponding mutations in the appropriate complementary region.
• first and second translation enhancing sequences may be derived from a different RNA virus, or from different genomic or subgenomic RNAs from the same virus. However, due to the fact that the first and second translation enhancing sequences often interact in enhancing cap-independent translation (e.g., when derived from STNV or TNV strains), it is preferred that first and second translation enhancing sequences are derived from the same genomic or subgenomic viral RNA.
• FIG. 2B Different possible positions of the first and second translation enhancing sequences in the chimeric RNAs encoded by the cap- independently expressed chimearic genes, with respect to the heterologous coding sequence and untranslated regions(indicated i to iv), are schematically represented in Figure 2B.
• the heterologous coding sequence is indicated by a dotted bar.
• Translation enhancing sequences are indicated by the same bracketted arabic numbers as in Figure 2A, and the portions of 5'UTR and 3' UTR and/or homologous coding sequence are indicated using the same color code as in Figure 2B.
• Thick black lines refer to unrelated sequences, such as the intervening sequences between a first or a second translation enhancing sequence and the heterologous coding sequence.
• a first translation enhancing sequence is located in the 5' region of the chimeric RNA transcribed from the CIG, particularly in the 5' UTR of the chimeric RNA(e.g. Fig 2B i, ii and iii) or in a region surrounding the translation initiation codon of the heterologous sequence; in other words, the translation initiation codon may be comprised within the first translation enhancing sequence (e.g., Fig 2B iv) .
• a second translation enhancing sequence is located in the 3' region of the chimeric RNA transcribed from the CIG, particularly in the 3' UTR of the chimeric RNA(e.g., Fig 2B i,ii and iii) or in a region surrounding the translation stop codon of the heterologous sequence; in other words the translation stop codon of the heterologous sequence may be comprised within the second translation enhancing sequence (e.g., Fig 2B iv).
• the first translation enhancing sequence may be located immediately upstream of the initiation codon of the coding sequence or it may be spaced therefrom by an intervening sequence of up to 100 nt, preferably up to 50 nt (see e.g., Fig 2b ii and iii).
• the second translation enhancing sequence may be located immediately downstream of the stop codon of the coding sequence or it may be spaced therefrom by an intervening sequence of up to 100 nt, preferably up to 50 nt (see e.g., Fig 2B ii and iii).
• a translational fusion between a first translation enhancing sequence comprising nucleotide sequences extending immediately downstream of the initiation codon of the homologous coding sequences, and the coding sequence of interest e.g., Fig 2B iv.
• a second translation enhancing sequence including nucleotide sequences extending immediately upstream of the initiation codon of the homologous coding sequences, and the coding sequence of interest (e.g., Fig 2B iv).
• translational enhancing sequence refers to a part of an RNA molecule or RNA sequence, but may also be used to refer to a DNA molecule encoding such part.
• the DNA regions encoding the translational enhancers used in this invention may be directly derived from a cDNA copy of the RNA from positive-stranded RNA viruses, but may also be partly or completely synthesized chemically.
• RNA molecules As leaders and trailers evidently are parts of RNA molecules, while the sequences in the sequence listing refer to DNA molecules, it is clear that when it is stated in the description or the claims that a leader or trailer or translation enhancing sequence in an RNA comprises a nucleotide sequence as in the sequence listing, the nucleotide sequence referred to is actually the non-transcribed strand of the double-stranded DNA molecule presented in the sequence listing, which can be transcribed into the mentioned leader or trailer RNA. In other words, the actual base-sequence of the leader or trailer RNA molecule is identical to the base-sequence of the DNA molecule represented in the SEQ ID No referred to, except that thymine is replaced by uracil.
• 5' regions and 3' regions derived from plant viruses known in the art to stimulate translation of uncapped RNA in vitro include a leader and trailer from barley yellow dwarf virus serotype PAV [ Wang and Miller J. Biol. Chem. 22: 13446-13452 (1995)].
• Translation enhancing sequences derived from these 5' UTR and 3' UTR may also be used according to the invention.
• first translation enhancing sequences of TNV-AC36 comprise the nucleotide sequence of SEQ ID No. 40, particularly the nucleotide sequence of SEQ ID N° 40 between nucleotide positions 1 and 90.
• Preferred second translation enhancing sequences comprise the nucleotide sequence of SEQ ID N° 41 , particularly the nucleotide sequence of SEQ ID N° 41 between nucleotide positions 102 and 227.
• CIGs of the invention encode an RNA comprising first and second translational enhancing sequences in their 5' and 3' regions, but these regions may include additional sequence elements.
• the region surrounding the initiation codon of the CIG may be adapted to include e.g., plant translation initiation consensus sequences [Joshi, Nucleic Acids Research 15: 6643-6653 (1987)].
• the CIGs of the invention can further comprise one or more functional elements that can increase expression of the CIG, 5 particularly increase the transcription of the CIG.
• Such functional elements include DNA sequences which enhance the accessibility of the promoter of the CIG for the cognate polymerase, such as DNA sequences influencing the local chromatin structure (scaffold attachment regions, matrix attachment regions as e.g., described by Breyne et al. [The Plant Cell 4:
• the invention is especially useful for the efficient expression of AT- rich coding sequences, especially those encoding Bt ICPs, particularly native coding regions encoding Bt ICPs, integrated in the nuclear DNA of
• this invention can be used for the efficient expression of any gene, in this regard, the use of first and second translation enhancing sequences derived from TNV sgRNA2 to increase 0 the production of heterologous gene products in plant cells, when combined with the efficient production of predominantly uncapped, non- polyadenylated transcripts by a bacteriophage single subunit RNA polymerase, such as T3 or T7 RNA polymerase, is particularly important.
• the present invention can therefore be used for the efficient production of 5 any protein or polypeptide of interest by the use of a CIG comprising a suitable promoter such as T3 or T7 promoter, a DNA encoding a first translation enhancing sequence derived from STNV-2 or TNV sgRNA2, a DNA region encoding a heterologous protein or polypeptide of interest, a DNA encoding a second translation enhancing sequence derived from 0 STNV-2 or TNV sgRNA2, and a terminator recognized by the used bacteriophage RNA polymerase.
• a suitable promoter such as T3 or T7 promoter
• a DNA encoding a first translation enhancing sequence derived from STNV-2 or TNV sgRNA2 a DNA region encoding a heterologous protein or polypeptide of interest
• a DNA encoding a second translation enhancing sequence derived from 0 STNV-2 or TNV sgRNA2 and a terminator recognized by the used bacteriophage RNA poly
• RNA-polymerase such as T3 or T7 RNA polymerase
• a wide variety of peptides or proteins can be produced in plants using genes such as those coding for peptides or proteins with pharmaceutical interest, for seed proteins modified so as to enhance nutritional value or to include peptides of interest, for chaperonins, for bactericidal or bacteriostatic peptides.
• genes which upon expression lead to plants having an increased resistance to herbicides e.g., phosphinotricin, glyphosate, triazines
• plants that can better withstand adverse environmental factors e.g., high salt concentrations in the soil, extreme temperatures etc.
• adverse environmental factors e.g., high salt concentrations in the soil, extreme temperatures etc.
• the invention may also be used to express to a high level inhibitors to proteases, amylases or RNases (e.g., barnase-inhibiting barstar).
• the recombinant DNA comprising the CIGs also comprises a conventional chimeric marker gene.
• the chimeric marker gene can comprise a marker DNA that is under the control of, and operatively linked at its 5' end to, a promoter, preferably a constitutive plant- expressible promoter, such as a CaMV 35S promoter, or a light inducible promoter such as the promoter of the gene encoding the small subunit of Rubisco; and operatively linked at its 3' end to suitable plant transcription termination and polyadenylation signals.
• the marker DNA preferably encodes an RNA, protein or polypeptide which, when expressed in the cells of a plant, allows such cells to be readily separated from those cells in which the marker DNA is not expressed.
• the choice of the marker DNA is not critical, and any suitable marker DNA can be selected in a well known manner.
• a marker DNA can encode a protein that provides a distinguishable color to the transformed plant cell, such as the A1 gene (Meyer et al.
• the marker gene could be operably linked to similar expression controls, i.e., promoter, first and second translation enhancing sequences and terminator as used for the CIG, thereby allowing direct selection for transgenic cell lines wherein cap- independent translation occurs very efficiently.
• the chimeric polymerase gene is preferably in the same genetic locus as the CIG so as to ensure their joint segregation. This can be obtained by combining both chimeric genes on a single transforming DNA, such as a vector or as part of the same T-DNA. However, a joint segregation is not always desirable. Therefore both constructs can be present on separate transforming DNAs, so that transformation might result in the integration of the two constructs at different locations in the plant genome, or even in seperate lines, which subsequently have to be crossed to yield a hybrid plant whereby the CIG and chimeric polymerase are joined in a single cell.
• a plant expressing a chimeric gene in a cap-independent manner can be obtained from a single plant cell by transforming the cell in a known manner, resulting in the stable incorporation of a cap-independently expressed chimeric gene of the invention into the nuclear genome.
• a recombinant DNA of the invention i.e., a recombinant DNA comprising a CIG, a chimeric polymerase gene and/or a chimeric marker gene can be incorporated in the nuclear DNA of a cell of a plant, particularly a plant that is susceptible to Agrobacterium-me ⁇ ated transformation.
• Gene transfer can be carried out with a vector that is a disarmed Ti-plasmid, comprising the recombinant DNA of the invention, and carried by Agrobacterium. This transformation can be carried out using the procedures described, for example, in EP 0116718.
• Ti-plasmid vector systems comprise the recombinant DNA of the invention between the T- DNA border sequences, or at least to the left of the right T-DNA border.
• any other type of vector can be used to transform the plant cell, applying methods such as direct gene transfer (as described, for example, in EP 0233247), pollen-mediated transformation (as described, for example, in EP 0270356, WO85/01856 and US 4,684,611), plant RNA virus-mediated transformation (as described, for example, in EP 0067553 and US 4,407,956), iiposome-mediated transformation (as described, for example, in US 4,536,475), and the like.
• Other methods such as microprojectile bombardment as described, for example, by Fromm et al. [(1990), Bio/Technology 8: 833] and Gordon- Kamm et al.
• Cells of monocotyledonous plants such as the major cereals, can also be transformed using wounded or enzyme-degraded intact tissue (such as immature seedlings in corn) or the embryogenic callus obtained therefrom (such as type I callus of corn), as described in WO 92/09696.
• Corn protoplasts can be transformed using the methods of EP 0469273. The resulting transformed plant cell can then be used to regenerate a transformed plant in a conventional manner.
• the obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the cap-independently expressed chimeric gene or the chimeric polymerase gene of the invention, or both in other varieties of the same or related plant species.
• Seeds obtained from the transformed plants contain the CIG of the invention as a stable genomic insert.
• the transgenic plant according to the invention may be a dicotyledonous or a monocotyledonous plant.
• Preferred dicotyledonous plants are potato, tomato, cotton, selected Brassica species such as oilseed rape, tobacco, soybean.
• Preferred monocotyledonous plants are corn, wheat, rice and barley.
• RNAs were produced by in vitro transcription of linear DNA templates (either plasmids treated with restriction enzymes, or polymerase chain reaction (PCR) fragments) containing the appropriate promoter region, using T7 RNA polymerase (Pharmacia, Upsala Sweden) or T3 RNA polymerase (Pharmacia), essentially as described by Krieg and Melton, Nucl. Acid Res 12:7057-7070 (1984), modified in that after 90 min of incubation at 37°C , extra
• RNA polymerase 0.3U/ ⁇ l
• NTPs 0.5mM
• RNA polymerase 0.3U/ ⁇ l
• DNA template was removed by adding 1.5U/ ⁇ l DNasel (Pharmacia, Upsalla, Sweden) and incubating further for 10 min at 37°C. Subsequently, the mixture was purified by phenol extraction, and passed through a Sephadex G-50 column (Pharmacia, Upsalla, Sweden). RNA was precipitated in 0.09 M K-acetate and 66% ethanol, and resuspended in RNase-free H2O.
• RNA concentration was determined by measuring OU260- The integrity of the transcripts was verified by formaldehyde-agarose gel- electrophoresis. Capped RNAs were obtained by modifying the reaction conditions to include 0.5 mM m ⁇ GpppG and 0.05mM GTP, during the first 30 minutes of incubation.
• RNA transcripts were synthesized in vitro synthesized in vitro synthesized in vitro synthesized RNA transcripts in a wheat germ extract prepared according to Morch et al., Methods. Enzymol 118:154-164 (1986), using final concentrations of 1 mM Mg 2+ t and 110 mM K + . Reactions were performed with 3 pmol of transcript, in a total volume of 75 ⁇ l in the presence of [ 35 S] methionine. To determine protein accumulation profiles, aliquots were taken at 6 to 8 different time points, and reaction products were separated on 0.1 %SDS-12.5% polyacrylamide gells as described by Laemmli, Nature 227: 680-685, (1970). After electrophoresis, gels were fixed overnight at 4°C in a wheat germ extract prepared according to Morch et al., Methods. Enzymol 118:154-164 (1986), using final concentrations of 1 mM Mg 2+ t and 110 mM K +
• RNA degradation (chemical half-life of RNA) was analyzed and quantified as described by Danthinne et al., Mol. Cell. Biol. 13: 3340-3349 (1993).
• Protein accumulation (P) in function of time (t) was analyzed using the mathematical description described by Danthinne et al (1993; supra) in which T corresponds to the time point at which the first translation product is completed, A is the translation efficiency of the mRNA and is the functional half-life of the mRNA. From this formula, it can be deduced that
• RNA in tobacco protoplasts by electroporation.
• RNA into the protoplasts was carried out by electroporation in the presence of 10-15 pmol of RNA per 10 protoplasts in 300 ⁇ l. Electroporation was performed immediately after the addition of the protoplasts to the RNA.
• C Capacitance
• EQ initial field strength
• RNA from protoplasts was prepared as described by Denecke et al
• RNA quantification was performed by densitometric scanning of the autoradiograph resulting from the Northern hybridization using a DT120 laser scanner and analysing the data with the Molecular
• Proteins were isolated from tobacco protoplasts by 10 seconds sonication (using a Soniprep 150, MSE Scientific Instruments, Crawley, England) in an extraction buffer consisting either of 50 mM Tris/HCI, 2mM EDTA, 0.15 ⁇ g/ ⁇ l DTT, 0.15 ⁇ g/ ⁇ l BSA and 30 ⁇ g/ ⁇ l
• PMSF for protoplasts wherein PAT and chloramphenicol acetyltransferase (CAT) encoding transcripts were introduced
• 50 mM Tris/HCI 5% glycerol
• 100 mM KCI 1 mM benzamidine HCl
• 5 mM ⁇ -amino-n-caproic acid 10 mM EDTA
• 10 mM EGTA 1 ⁇ g/ml antipain
• 1 ⁇ g/ml leupeptin 14 mM ⁇ -mercapto-ethanol and 1 mM
• (dP/dt)/(dR/dt) can be estimated by non-linear regression using GraphPad PrismTM software version 1.02.
• Example 1 Plasmid constructions used for in vitro transciption to generate the test RNAs used for the in vitro and in vivo translation experiments.
• pFM20, pFM21 , pFM23 and pFM24 are in vitro transcription plasmids containing original TNV-A cDNA fragments cloned in the Smal site of pGEM®-3Z (Promega Biotec, Madison, Wise.) as described by Meulewaeter et al., supra (1990).
• pFM20 contains the nucleotide sequence between nucleotide 1763 and 3660 of SEQ ID No.1 ;
• pFM21 contains a cDNA corresponding to the nucleotide sequence between nucleotide 20 and 2619 of SEQ ID No.1 ;
• pFM23 contains a cDNA corresponding to the nucleotide sequence between nucleotide 2593 and 3510 of SEQ ID No.1 ;
• pFM24 contains a cDNA corresponding to the nucleotide sequence between nucleotide 19 and 1632 of SEQ ID No.1.
• pFM33 is a 3'-terminal TNV-A cDNA clone in the Seal site of pAT153.
• the cDNA was synthesized on TNV dsRNA as described by Danthinne et al., supra (1991).
• the cDNA clone contains the nucleotide sequence between 3334 and 3684 of SEQ ID No.1 , followed by three A- residues.
• pAT153 is a derivative of pBR322 lacking the 0.62 kb Haell B- fragment [Twiggs and Sheratt, Nature 283:216-218, (1980)].
• pFM136 [(Meulewaeter et a/., supra (1992)] contains the car coding sequence of Tn9, flanked by additional nucleotides on a fragment having the sequence of SEQ ID No.3, cloned as an Xbal, filled-in Cla ⁇ fragment between the Xbal and trimmed Kpn ⁇ sites of pGEM®-3Z .
• pFM133 and pFM134 were made by insertion of the bar coding region as a filled-in BamHI fragment from pGEMBAR into the trimmed Sacl site of pFM23 and pFM20, respectively, in such a way that upon transcription with T7 RNA polymerase an RNA encoding PAT is produced.
• pGEMBAR is a clone of a modified BamHI fragment of pGSR1 (EP 242236), comprising the coding sequence of the bar gene, wherein the sequence around the initiation codon (CCATGA) has been changed into a ⁇ /col restriction recognition sequence (CCAJ_G_G). This BamHI fragment has been cloned into the BamHI site of pGEM®-1.
• pFM140 Insertion of the 1426-bp blunt-ended EcoRI-FVtvl fragment of pFM134 into the blunt-ended Sacl fragment of pFM136 resulted in plasmid pFM140.
• pFM139 was obtained by the insertion of the car gene, as a Pstl, blunt-ended Sacl fragment from pFM136, between the Pstl and blunt- ended Mlu ⁇ sites of pFM134.
• a translational fusion between the TNV coat protein and the cat open reading frames was made by transfer of the 830-bp filled-in BamHI fragment from pFM21 into the trimmed Sacl site of pFM136.
• a 1371 bp Pst ⁇ -Nsi ⁇ fragment from the resulting plasmid was inserted between the Pstl and ⁇ /s/l sites of pFM134 in such a way that both sites are restored, resulting in plasmid pFM138.
• pXD324 contains downstream of the T7 promoter : the -fragment of tobacco mosaic virus, the bar coding region, a poly(dA/dT) track of about 100 residues, and the SP6 promoter.
• This plasmid is composed of the following nucleotide sequence: from nucleotide 1 to 790 it contains the nucleotide sequence of SEQ ID No.4; from nucleotide 791 to 1221 it contains the sequence complementary to the sequence between nucleotides 2865 and 2435 of pGEM®-1 (Promega Biotec, Madison, Wise); from nucleotide 1222 to 3696 it contains the nucleotide sequence between the nucleotide at position 269 and the nucleotide at position 2743 of pGEM®-3Z.
• pFM108 is pGEM®-3Z derivative that, by deletion of the sequence between the nucleotide at position 2 and the nucleotide at position 17, contains a Kpn ⁇ site at the start of transcription of the T7 promoter [Danthinne et a/.,supra (1993)].
• pXD535 is an in vitro transcription plasmid that contains a full-length STNV-2 cDNA clone except for the first nucleotide (sequence as in SEQ ID No.2 between the nucleotide at position 2 and the nucleotide at position 1245, downstream of the T7 promoter [Danthinne et al.supra (1993)].
• the STNV-2 cDNA was cloned between the Smal and trimmed Kpn ⁇ sites of a plasmid obtained by cloning of the 515-bp long Aat ⁇ -Pst ⁇ fragment of pFM 108 between the Aat ⁇ I and Pstl sites of pAT153.
• pGEM4N is a derivative of pGEM®-4 (Promega Biotec, Madison, Wise.) obtained by digestion with H/r/dlll, filling-in, and religating. In this way, an Nhe ⁇ site is created.
• pGEM9C1 A Kpn ⁇ -Nhe ⁇ fragment containing codons 44 to 666 of the cry9C coding region flanked by translation initiation and termination sites (nucleotide sequence between nucleotide 6 and 1892 of SEQ ID No.5), was cloned between the Kpn ⁇ and Nhe ⁇ sites of pGEM4N, resulting in plasmid pGEM9C1.
• pGEM9C2 is a similar plasmid containing a synthetic coding region for the codons 44 to 666 of cry9C flanked by translation initiation and termination sites.
• the cry9C encoding Nco ⁇ -Nhe ⁇ fragment of pGEM9C1 has been exchanged for the Nco ⁇ -Nhe ⁇ fragment comprising the synthetic coding region, which has the nucleotide sequence between nucleotide 8 and 1888 of SEQ ID No. 34).
• a Nco ⁇ -Nhe ⁇ fragment containing codons 29 to 616 of the cry1Ab5 coding region flanked by transation initiation and termination sites was cloned between the ⁇ /col and Nhe ⁇ sites of pGEM9C1 , resulting in plasmid pGEM1Ab1.
• Plasmid pAB02 was constructed as follows: a PCR fragment, obtained with primers FM10 and FM11 having the nucleotide sequences of SEQ ID No.7 and SEQ ID No.8, using plasmid pFM20 as template, was digested with BamHI (in first primer) and Bsml and cloned between the Bsml and BamHI sites of pFM20, resulting in plasmid pFM187. This plasmid now contains a Bsal site at the 5' end of the TNV sgRNA2 sequence.
• the 5' end of the subgenomic RNA2 was fused to the T7 promoter by cloning the 1224-bp Bsal(filled-in)-Psfl fragment of pFM187 between the Kpn ⁇ (blunted) and Pst ⁇ site of pFM108, resulting in plasmid pFM187B.
• the 3' end of TNV sgRNA2 was reconstructed by PCR using primers FM8 and FM9 having the nucleotide sequences of SEQ ID No.9 and SEQ ID No.10 with pFM33 as template.
• the amplified fragment was digested with Pstl and Bsu36I and cloned between the Psfl and Bsu36l sites of pFM20 and pFM187B, resulting in plasmids pFM20C and pAB02, respectively.
• pRD01 was created by restricting pAB02 with EcoRl, followed by filling-in the protruding termini with Klenow polymerase and religation. This creates a new stop codon at nucleotide 735 of the TNV-A CP mRNA (nucleotide 3195 of SEQ ID No. 1 ).
• RNA specified by this plasmid encodes a C-terminally truncated CP protein of 21-kDa.
• Plasmids pRD02, pRD06, pRD03, pRD04, and pRD05 were created as follows.
• pRD01 contains a unique BsfBI site immediately downstream of the newly introduced stop codon.
• pRD01 was restricted by Bs/BI and respectively one of the following enzymes: Asp718, Nhel, BsaAI, Bsu36l, and BamHI. The linearized DNA fragments were treated with Klenow polymerase and religated.
• Plasmid pAB01 was constructed by cloning the 592-bp Nde ⁇ -Bsm ⁇ fragment of pFM23 between the Ndel and Bsml sites of pAB02.
• Plasmid pMA300 [Andriessen et al., Virology 212: 22-224 (1995)] was constructed in two steps starting with plasmid pFM24.
• the intact 5'end of the TNV-A sequence was reconstructed using complementary oligomers encoding the first 35 nucleotides of TNV-A (nucleotide sequence between nucleotide 1 and 35 of SEQ ID No.1) to create plasmid pFM39.
• a fragment from plasmid pFM21 containing TNV-A residues 311 to 2619 was inserted in pFM39.
• pTNV was constructed as follows: the 1636-bp Nsil - Hindlll fragment of pFM20C was cloned between the Nsil and Hindlll sites of pMA300, resulting in plasmid pTNV.
• pTNV contains the full-length TNV-A sequence under control of a T7 promoter.
• T7 RNA polymerase directs the synthesis of a transcript that differs from the natural RNA only by the addition at the 5'-end of an extra G residue.
• Plasmids to obtain chimeric TNV-cat RNAs were constructed as follows. A PCR fragment obtained with primers FM10 and FM12 having the nucleotide sequences of SEQ ID No.7 and SEQ ID No.11 , using plasmid pFM140 as template, was digested with BamHI (present in the first primer) and BspEI (present in the cat gene) and cloned between the BspEI and BamHI sites of pFM140, resulting in plasmid pFM188. This plasmid contains a Bsal site at the 5' end of the TNVsgRNA2 leader sequence.
• the 5'end of the TNVsgRNA2 was fused to the T7 promoter by cloning the 929-bp Bsal(filled-in)-Psrl fragment of pFM188 between the Kpnl (blunted) and Psfl site of pFM108. This resulted in plasmid pFM188B.
• the 1335-bp Nsil-Xbal fragment of pFM138 was ligated to the 5097- bp Nsil-Nhel fragment of pTNV, resulting in plasmid pFM216.
• the 1155-bp Pvul-Pstl fragment of pFM216 was ligated to the 2830- bp Pvul (partially digested)-Psfl fragment of pAB02, resulting in plasmid pFM188G.
• the 891-bp Ncol-Ndel fragment of pFM188B was ligated to the 3072-bp Ncol-Ndel fragment of pFM216, resulting in plasmid pFM188H.
• a PCR fragment was obtained with primers FM23 and FM24 having the nucleotide sequences of SEQ ID No.18 and SEQ ID No.19, using plasmid pFM188C as a template, digested with EcoRl and Ndel and cloned between the EcoRl and Ndel sites of pFM188C, resulting in plasmid pVE190. In this way the T7 promoter of pFM188C was exchanged for a T3 promoter.
• DNA fragments were PCR-amplified with primers FM16 and FM17 having the nucleotide sequences of SEQ ID No.12 and SEQ ID No.13, and with primers FM18 and FM19 having the nucleotide sequences of SEQ ID No.14 and SEQ ID No.15. Both fragments were then used in an overlap extension PCR with primers FM16 and FM19, having the nucleotide sequences of SEQ ID No.12 and SEQ ID No.15 to amplify a DNA fragment containing an Nhel site just downstream of the cat stop codon. The amplified fragment was digested with Ncol and BamHI and cloned between the ⁇ /col and BamHI site of pFM188C, resulting in plasmid pVE192.
• DNA fragments were amplified with primers FM16 and FM21, having the nucleotide sequences of SEQ ID No.12 and of SEQ ID No.17, and with primers FM20 and FM19, having the nucleotide sequences of SEQ ID No.16 and SEQ ID No.15. Both fragments were then used in an overlap extension PCR with primers FM16 and FM19, having the nucleotide sequences of SEQ ID No.12 and SEQ ID No.15 to amplify a DNA fragment containing an Nhel site at nucleotide963-968 of TNV sgRNA2 (nucleotides 3423-3428 of SEQ ID No.1 ). The amplified fragment was digested with ⁇ /col and BamHI and cloned between the ⁇ /col and BamHI sites of pFM188C, resulting in plasmid pVE193.
• pVE192 The 1037-bp Ndel-Nhel fragment of pVE192 was cloned between the Ndel and Nhel sites of pVE193, resulting in plasmid pVE195.
• pVE192 was digested with Nhel and ⁇ s ⁇ /361, blunted, and religated, resulting in plasmid pVE196.
• Plasmids to obtain chimeric STNV-cat RNAs were constructed in the following way.
• a mutant STNV leader (designated STNV * ) was cloned downstream of the T7 promoter by insertion of the annealed oligodeoxyribonucleotides FM14 and FM15, having the nucleotide sequences of SEQ ID No.22 and SEQ ID No.23 between the Smal and trimmed Kpnl sites of pFM108, resulting in plasmid pFM184A.
• the STNV * leader was subsequently fused to the cat coding region by insertion of the 520-bp ⁇ /col(filled-in)- ⁇ /del fragment of pFM184A between the Ndel and blunted BssHII sites of pFM139, resulting in plasmid pFM189.
• pFM191 the cat coding region was placed upstream of the TED of STNV-2 (TED2) by insertion of the 900-bp Na ⁇ -NlalV fragment of pFM189 between the ⁇ /arl and blunted Ncol sites of pFM175.
• pFM169 was made by inserting the cat coding region, as a Pstl-Nrul fragment of pFM136 between the Psfl and filled-in Xbal sites of pXD324. Insertion of the 430-nt-long Ncol-Sphl fragment of pFM191 between the ⁇ /col and Sphl sites of pFM169 yielded plasmid pFM191A.
• pFM179 A derivative of pXD324, named pFM179, was made by religating blunt-ended Hindlll- digested plasmid. Upon linearization of the resulting plasmid with Nhel, RNA is synthesized which has GCUAG downstream of the poly(A) tail. The poly(dA:dT)-track of pFM179 was placed downstream of TED by inserting the 1100-nt-long Spel-Ndel fragment of pFM191A between the Xbal and Ndel sites of pFM179. The resulting plasmid was named pFM209. The length of the poly(dA:dT) track of pFM191A and pFM209 was estimated by polyacrylamide gel electrophoresis to be about 100 bp.
• pFM191B was made by inserting the 430-nt long Ncol-Sphl fragment of pFM191 between the Nco ⁇ and SpM sites of pFM136.
• a fragment containing the T7 promoter fused to the first 38 nucleotides of the STNV-2 cDNA was amplified by PCR on pFM175 using primers FM1 and FM13, having the nucleotide sequences of SEQ ID No.20 and SEQ ID No.21. After digestion with Mlul and Ndel, this fragment was cloned between the BssHII and Ndel sites of pFM189 and pFM191 , resulting in plasmids pFM189A and pFM191E, respectively.
• Plasmid pFM207E was constructed by ligating the 726 bp Pvull-Afllll fragment from pFM191E and the 615 bp long Pvull-EcoRI fragment of pFM191 in the 2556 bp EcoRl-Afllll vector fragment from pFM191E.
• Plasmids to obtain chimeric STNV-cry RNAs were obtained in several steps as outlined.
• the 1496-bp long Ndel-Hindll fragment of pXD535 was cloned between the Ndel and Eco47lll sites of pXD324, resulting in plasmid pFM214.
• a synthetic DNA fragment consisting of the annealed oligodeoxyribonucleotides FM4 and FM5, having the nucleotide sequences of SEQ ID No.26 and SEQ ID No.27, was cloned between the BsaAI and ⁇ /col sites of pFM214C, resulting in plasmid pFM214A.
• pFM214A was used as template in a PCR reaction with the primers FM1 and FM7, having the nucleotide sequence of SEQ ID No.20 and SEQ ID No.28 and the resulting fragment was digested with Ndel and ⁇ /col.
• pRVL11 was obtained by the same strategy except that the Ncol-Nhel fragment of pGEM9C2, comprising a synthetic coding region of cry9C was used.
• STNV-2 5'UTR and TED2 cooperate in stimulating cap- independent translation of heterologous mRNAs in vivo.
• the first set of experiments demonstrate that 5' information affecting translation is contained within the 5'-terminal 38 nt of STNV-2, comprising the full sequence complementarity with TED2.
• Translation of an RNA which has the STNV-2 leader plus the first two codons of the CP coding region (further named STNV-2 leader) translafionally fused to the cat coding region was compared to that of an analogous RNA with a mutated leader (STNV* leader) which has a reduced complementarity with TED2.
• STNV* leader an analogous RNA with a mutated leader
• Translation of the RNA with the STNV-2 leader was not affected by the presence of a cap structure, whereas the RNA with the STNV * leader required the cap to maintain its functional stability (Table 1 ).
• RNAs were translated in a wheat germ extract. CAT protein accumulation was quantified after 18, 25, 32, 40, 50, 65, 80, and 100 min of incubation.
• TED2 second translation enhancing sequence from STNV-2
• STNV-2 second translation enhancing sequence from STNV-2
• Control 3'UTR is a 120 nt plasmid derived sequence; translation stimulation has been normalized to the corresponding RNA construct without TED2, for each case separately.
• TED2 stimulates translation in vivo about 7-fold.
• the stimulatory effect was about 4-fold.
• TED2 did not increase translation of capped and polyadenylated cat RNA.
• RNA transcripts comprising cat (summarized in Table 3), were introduced by electroporation in tobacco protoplasts. Samples for protein extraction were taken 6 hrs after RNA introduction, and the levels of CAT protein accumulated was determined. RNA level determination revealed that 90 mm after electroporation the cat mRNA levels varied less than two-fold, indicating an RNA delivery with similar efficiency between the separate introduced RNAs After 256 min, the cat mRNA levels were 3-5 fold lower in all experiments, indicating similar chemical half-lives for the different mRNAs
• ND not determined
• BB below background level (which is 2pg)
• control refers to a 120 nt unrelated plasmid derived sequence
• CAT accumulation from uncapped RNAs was about five-fold higher in tobacco protoplasts expressing the STNV-2 5'UTR, than when a mutant 5'UTR of the similar length was used (STNV*). (A similar enhancement was observed in other independent experiments). Additionally, CAT protein accumulation profiles in tobacco protoplasts electroporated in the presence of uncapped TED2 containing cat RNAs with the STNV* and the STNV-2 5'UTR were determined (Table 4). The STNV-2 leader fusion RNA encoded a higher peak level than the STNV* fusion RNA. The main difference between the profiles was that the initial rate of CAT accumulation was much greater for the STNV-2 leader fusion RNA than for the STNV* fusion RNA.
• Example 3 Determination of the nucleotide sequences from TNV sgRNA2 leader and trailer that synergistically stimulate translation in vitro and in vivo.
• TNV sgRNA2 contains translation enhancing sequences which allow uncapped TNV sgRNA2 to be translated in vitro to a coat protein peak level of 83 % of the level obtained after in vitro translation of capped TNV sgRNA2.
• RNAs were synthesized on the indicated plasmid DNA using T7 RNA polymerase. Samples were taken after 20, 30, 45, 60, 80, and 100 min of incubation at 25°C.
• TNV sgRNA2 The elements of the TNV sgRNA2 that are required for an efficient translation were determined by comparison of translation of full-length TNV sgRNA2 with translation of deletion mutants in a wheat germ translation system. RNAs were synthesized in vitro from the DNA templates summarized in Table 6, using T7 RNA polymerase. Translation of these RNAs, which differ in the presence or absence of the sgRNA2 5' UTR or 3' UTR sequences, was compared in a wheat germ translation system (Table 6). The indicated nucleotides remaining are the 3' nucleotides for the 5' UTR and the 5' nucleotides for the 3' UTR.
• the 3' UTR In the absence of the 5' UTR sequence, the 3' UTR increased the protein peak level only 1.5-fold, exclusively due to a longer functional half- life.
• the 5' UTR stimulated translation in the absence of the trailer about 3- fold.
• translation stimulation by the 5' UTR and 3' UTR is much higher than stimulation by the individual elements, indicating that the TNV sgRNA2 5' UTR and 3' UTR stimulate translation synergistically in vitro.
• the TNV sgRNA2 thus contains both a 5' and 3' translational enhancing sequence. Table 6. Effect of leader and trailer on translation of TNV sgRNA2 in vitro
• pi refers to a 23 nucleotide long polylinker sequence.
• the 3' border of the translation stimulating region in the trailer was determined by translation in a wheat germ extract of 3' deletion mutants of TNV sgRNA2 (Table 7). These mutant RNAs were synthesized in vitro using T7 RNA polymerase and pAB02 plasmid DNA that was linearized with different restriction enzymes. Translation of the RNA that lacks the 3'- terminal 73 nucleotides was comparable to that of the full-length sgRNA. Deletion of the next 49 nucleotides resulted in a two-fold decrease of translation. Further deletion of the trailer resulted in a further, gradual decrease in translation. These data allow to conclude that the 3' border of the second translation enhancing sequence lies between nucleotide 1102 and 1151 of sgRNA2. Table 7. Determination of the 3' border of the 3' translation stimulating region of TNV sgRNA2.
• RNA specified by the resulting plasmid encodes a C-terminally truncated CP protein of 21-kDa.
• Translation of this RNA in the wheat germ extract was comparable to translation of the wild-type sgRNA2 (Table 8). This shows that the location of the translation termination site is not crucial for translation stimulation by the second translation enhancing sequence.
• TNV-A was determined by comparison of the translation in vitro of the RNA comprising the newly introduced stop codon with translation of internal deletion mutants RNAs were synthesized from the plasmids linearized with ⁇ sal listed in Table 9, using T7 RNA polymerase, and translated in a wheat germ cell free extract The data, summarized in Table 9, demonstrated that nucleotides 738 to 1011 of sgRNA2 could be deleted without affecting translation of the mutant RNA in vitro.
• nucleotide 1044 extended to nucleotide 1044 caused a drop in translation of more than 10- fold, resulting in the same level of translation as for an RNA lacking the 3' UTR Conclusively, the 5' border of the second translation enhancing sequence is located between nucleotides 101 1 and 1044 of sgRNA2
• the data also prove that the 5' and 3' translation stimulating regions are distinct domains, with the second translation enhancing sequence located between nucleotides 1011 and 1151 of sgRNA2
• RNA levels in the transfected protoplasts were determined by quantitative Northern blot analysis to estimate the efficiency of RNA introduction.
• CAT protein levels (Table 10) revealed that the RNA which comprised only TNV 3' UTR specified low levels of CAT.
• the RNAs with both 5' and 3' UTR sequences from TNV directed the synthesis of levels of CAT which were 25- to 35-fold higher as compared to the RNA lacking TNV 5' UTR sequences.
• Similar levels of CAT protein resulted from the translation of the TNV-cat RNAs differing in the length of the 5' and 3' UTR sequence.
• Efficiency of uncapped RNA translation is only four fold lower than translation efficiency of capped RNA and only two-fold lower than for a very efficiently translated mRNA (pFM169f-) inc jj
• RNA was synthesized on the indicated plasmid DNAs using T7 RNA polymerase and introduced in tobacco protoplasts by electroporation.
• Total RNA was isolated from the protoplasts 140 mm after electroporation.
• the cat RNA levels are in amol/ ⁇ g of total RNA
• the CAT protein level (pg/mg of soluble protein) was determined 340 mm after RNA introduction, in duplo.
• RNA was synthesized, using T3 RNA polymerase from Ssal-, and
• RNAs were introduced into tobacco protoplasts. CAT accumulation was monitored, at least 5 hours after RNA introduction This revealed that the minimal 3' TNV sequences required for an efficient translation of an uncapped cat mRNA are located between nt 1012 and 1151 of TNV-A sgRNA2 (see Table 10 bis) Table 10bis Translation of chimeric TNV-caf RNAs in tobacco protoplasts 3
• RNA was syntesized on the indicated plasmid DNAs using T7 RNA polymerase and introduced in tobacco protoplasts by electroporation.
• Total RNA was isolated from the protoplasts 130 min after electroporation. The cat RNA levels are in amol/ ⁇ g of total RNA.
• the CAT protein level (pg/40 ⁇ g of soluble protein) was determined 5 hours after RNA introduction, in duplo.
• RNA transcripts comprising first and second translation enhancing sequences from STNV-2, using as templates the DNA listed in Table 12, were introduced in tobacco protoplasts by electroporation (together with TNV RNA to supply the RNA-dependent RNA polymerase in trans). These transcripts contain either native or synthetic coding regions of a Bt ICP gene. After 48 hrs, the amount of synthesized protein and positive-strand RNA was determined. Table 12 summarizes the ratios of synthesized protein over synthesized RNA (normalized to the value obtained for native coding sequence).
• the ratio of accumulated protein/ accumulated RNA after 48 hrs was higher when native coding sequences were utilized than when synthetic coding regions, with codon preferences closer to that of plants, were used.
• RNA fmol/0.5 ⁇ g total RNA
• P protein (ng/mg soluble protein)
• t time(hours)
• Example 5 stimulates autonomously the translation of dicistronic RNAs in vitro.
• pFM203 and PFM203B were based on pMA442, which is an in vitro transcription plasmid containing the nptll coding region between the first 173 nucleotides and the trailer of the STNV-2 RNA. It consists of the following sequences: from nucleotide 1 to 1003 it has the nucleotide sequence of SEQ ID No.38; from nucleotide 1004-1616 it has the nucleotide sequence between 633 and 1245 of SEQ ID No.
• pFM203 was obtained by cloning of the 1246-bp long Xho ⁇ -Nsil fragment of pMA442 between the Sail and Psfl sites of pFM189.
• pFM203B To construct pFM203B, the ⁇ /s/l-bluntcd-/4sp718l 1077 bp fragment of pMA442 was first cloned between the Psfl and blunted Xbal sites of pFM189, resulting in pFM211A. Religation of blunted A/col-£coRI-digested pXD324 DNA resulted in pFM170D. To obtain pFM170, the npfll coding region was inserted as an EcoRI-BsfBI fragment (SEQ ID No. 39 between the nucleotides at position 3 and 818) between the EcoRl and Accl sites of pFM170D.
• pFM203 A 260-nt-long Psfl-filled-in-BamHI fragment of pFM170 was inserted between the Psfl and trimmed Kpnl sites of pFM211A, resulting in plasmid pFM203B.
• pFM203 and pFM203B can be represented as follows: pFM203:T7-STNV * leader-caNSTNV2(1-173)-npfll(transl.fusion)-TED pFM203B : T7-STNV*leader-caf-TMVIeader-npfll-TED
• RNA polymerase of BspHI- or Spel- digested plasmid pFM203 or pFM203B DNA resulted in the synthesis of dicistronic RNAs lacking or including TED, respectively.
• Capped and uncapped RNA transcripts were translated in vitro in a wheat germ extract. Protein accumulation profiles were determined and translation efficiencies as well as functional half-lives were deduced, allowing calculation of the peak levels.
• TED2 stimulates autonomously the translation of dicistronic RNAs in vitro.
• cassettes which are vomitily under the control of a T3 or T7 promoter, comprise: (i) a terminator sequence for T3 and T7 RNA polymerases,(ii) Bt ICP encoding genes, flanked by appropriate DNA regions encoding the first and second translation enhancing sequences of TNV-A or STNV-2, (iii) marker genes which are either under the control of a plant-expressible promoter, or are under control of T3 or T7 promoters and are further flanked by appropriate DNA regions encoding first and second translation enhancing sequences of TNV-A or STNV-2, and (iv) a T3 or T7 RNA polymerase encoding gene under control of a plant-expressible promoter, whereby the RNA polymerase is joined to a nuclear localisation signal of SV40 T-antigen.
• a synthetic DNA fragment comprising the T3 terminator sequence, flanked by unique restriction sites was cloned as a Pstl-Hindlll downstream of the TNV trailer, between the Psfl and Hindlll sites of pVE190 (see Example 1 ), resulting in plasmid pVE198.
• the terminator fragment was then duplicated by ligating the terminator-containing EcoRI-Xbal and EcoRI-Spel fragments of pVE198 or the terminator-containing Ndel-Xbal and Ndel-Spel fragments, resulting in plasmid pVE199.
• the duplicated terminator fragment of pVE199 was fused to the ApaLI site of the TNV trailer by cloning of the 631-bp >ApaLI(blunted)-EcoRI fragment of pVE195 (see Example 1 ) between the EcoRl and trimmed Psfl sites of pVE199, yielding plasmid pFM500.
• a fragment was amplified by PCR on plasmid pRVL11 (see Example 1 ) with primers FM22 and FM25 having the nucleotide sequences of SEQ ID No.30 and SEQ ID No.31 , digested with Hindlll and Ndel, and cloned between the Hindlll and Ndel sites of pRVL.11 , resulting in plasmid pRVL17.
• the cry9C-containing Ndel-Spel fragment of pRVI_17 was cloned between the Ndel and Spel sites of pFM500, resulting in plasmid pFM407.
• cry1A(b)-containing Ncol-Nhel fragment of pGEM1Ab1 (see example 1 )is fused to the 310-bp Aatll-Ncol and the 2554-bp Nhel-Aatll fragments of pFM407, resulting in plasmid pFM408.
• a PCR fragment is was amplified with primers FM22 and FM6 having the nucleotide sequence of SEQ ID No.30 and SEQ ID No.29 using plasmid pAB02 (see Example 1) as a template, digested with Nhel and Ndel and cloned between the Nhel and Ndel sites of pFM500, resulting in plasmid pFM401.
• a PCR fragment was amplified with primers FM26 and FM6 having the nucleotide sequence of SEQ ID No.32 and SEQ ID No.29 using plasmid pVE190 (see example 1) as a template, digested with Nhel and Ndel and cloned between the Nhel and Ndel sites of pFM500, resulting in plasmid pFM501.
• pFM402 is then digested with Nhel and Bsu36l, blunted and ligated, resulting in plasmid pFM403.
• cry1A(b)-containing is cloned between the Ncol and Nhel sites of pFM401 , resulting in pFM404.
• the cry-containing Ncol-Eagl fragments of pFM402, pFM403, and pFM404 are then cloned between the ⁇ /col and Eagl sites of pFM501 , resulting in plasmids pFM502, pFM503, and pFM504, respectively.
• plasmids pFM502 and pFM504 were constructed by cloning the Ncol-Nhel fragment of pGEM9C1 , respectively the Ncol-Nhel fragment of pGEM1Ab1 in Ncol-Nhel digested pFM501.
• Marker gene cassettes (iii) Marker gene cassettes.
• Plasmid pDE110 is a pUC-derivative containing the bar coding region under the control of the 35S promoter and the 3' end formation signal of Cauliflower mosaic virus. It comprises the followings fragments: from nucleotide 1 to nucleotide 401 it equals nucleotide 1 to nucleotide 401 of pUC19 (Yanisch-Perron er a/., 1985); from nucleotide 402 to nucleotide 1779 it comprises a promoter region of the Cauliflower mosaic virus 35S RNA (Odell et al.
• nucleotide 1781 to nucleotide 2332 it comprises the coding region of the bialaphos resistance (bar) gene from Streptomyces hygroscopicus (Thompson et al., 1987); from nucleotide 2351-2614 it comprises a fragment containing the 3'-end formation signal of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982); and from nucleotide 2615 to nucleotide 4883 it equals nucleotide 418 to nucleotide 2686 of pUC19.
• bar bialaphos resistance
• the bar-gene containing ⁇ /col-filled- in-Mlu ⁇ fragment of pFM133 was cloned between the ⁇ /col and filled-in Nhel sites of pFM401 and pFM501 , resulting in plasmids pFM405 (T7-promoter) and pFM505 (T3-promoter), respectively.
• plasmid pFM406 was obtained by fusing the the bar- gene containing Nhel-Ncol fragment of pFM405 to the 1.2 kb Bgll-Ncol fragment and the 1.8 kb Nhel-Bgll fragment of pFM407.
• T7 RNA polymerase coding region is present on a DNA fragment which has the following sequence: from nucleotide 1 to 35: the nucleotide sequence as in SEQ ID No.36 (comprising the coding sequence for the nuclear localisation signal of the SV40 large T-antigen); from nucleotide 36 to nucleotide 2684: the sequence of Genbank Accession No.
• T3 RNA polymerase coding region is comprised within a similar DNA fragment in which the sequence between the nucleotide at position 36 and the nucleotide at position 2684 are replaced with the sequence of Genbank Accession No. X02981 (incorporated herein by reference) between the nucleotide at position 144 and the nucleotide at position 2795.
• Such fragments can be obtained by PCR using appropriate primers and plasmids pAR1173 (ATCC 39562) or the T7 genome; and plasmid pCM56 (ATCC 53202) or the T3 genome.
• pFM409 is a pUC19-derivative containing four unique 8-base cutters (Sse8387l, Ascl, Notl, Sgfl), wherein between the Sse8387l and Ascl sites a gene cassette is inserted which consists of: a CaMV35S promoter, the leader sequence of the cab22L gene from Petunia, the 5' region of the crylA(b)5 coding region and a 3'-end formation signal of CaMV.
• nucleotide 1 to nucleotide 186 it equals the nucleotide sequence of pUC19 from nucleotide position 1 to nucleotide position 186; from nucleotide position 187 to nucleotide position 1220 it has the nucleotide sequence of SEQ ID No.35; from nucleotide position 1221 to nucleotide position 3460 it has the nucleotide sequence of pUC19 between the nucleotides at position 447 and 2686 of pUC19.
• the T7 RNA polymerase coding region is placed under the control of a 35S promoter of CaMV by cloning as a Ncol-Nhel fragment of the above mentioned DNA between the ⁇ /col and Nhel sites of pFM409 , resulting in plasmid pFM410.
• T3 RNA polymerase coding region is cloned as an Ncol-Nhel fragment of the above mentioned DNA between the ⁇ /col and Nhel sites of pFM409, resulting in plasmid pFM510.
• V Assembly of the plant transformation vectors.
• the major plasmids, used for the assembly of the plant transformation vectors have the following schematized structure:
• pFM402 T7p-TNVIeader-cry9C-TNVtrailer(1 )-T3term(2x)
• pFM403 T7p-TNVIeader-cry9C-TNVtrailer(2)-T3term(2x)
• pFM404 T7p-TNVIeader-cry1 Ab5-TNVtrailer(1 )-T3term(x2)
• pFM503 T3p-TNVIeader-cry9C-TNVtrailer(2)-T3term(2x)
• pFM504 T3p-TNVIeader-cry1 Ab5-TNVtraiier(1 )-T3term(2x)
• pFM405 T7p-TNVIeader-bar-TNVtrailer(1 )
• the DNA encoding the translation enhancing sequence indicated as TNV trailer (1 ) has the sequence of SEQ ID No.1 between the the nucleotides at position 3429 and 3611 ; the one indicated as TNV trailer (2) has the sequence of SEQ ID No.1 between the nucleotides 3472 and 3611.
• TED refers to the DNA encoding a STNV second translation enhancing sequence corresponding to SEQ ID No.2 between nucleotides at position 632 and 753;
• P35S refers to a CaMV35S promoter;
• TNV leader refers to the DNA encoding first translation enhancing sequence corresponding to the nucleotide sequence of SEQ ID No.1 between the nucleotides at positions 2461 and 2603;
• STNV leader refers to the DNA encoding a first translation enhancing sequence corresponding to SEQ ID No. 2 between nucleotides at position 1 and 38;
• cab22L leader refers to the DNA sequence encoding the leader sequence from cab22L gene of Petunia, having the nucleotide sequence complementary to the nucleotide (A
• T7p refers to the T7 promoter having the sequence of SEQ ID No.30 between nucleotides 22 and 39
• T3p refers to the T3 promoter having the sequence of SEQ ID No.18 between nucleotides 14 and 32
• 3' nos and 3' 35S refer to the 3' region of the nopaline synthase gene and the CaMV 35S transcript (having the complementary nucleotide sequence of SEQ ID No.
• T3 term refers to the terminator region of phage T3 having the nucleotide sequence of SEQ ID No.24
• cry 9C refers to the native nucleotide sequence encoding a truncated toxic fragment of CRY9C as indicated in SEQ ID No. 5 between nucleotide positions 6 and 1892
• cry 1A(b) refers to the native nucleotide sequence encoding a truncated toxic fragment of CRY1Ab5 as indicated in SEQ ID No. 6 between nucleotide positions 8 and 1783 .
• pTFM600 was derived from plasmid pGSC1700 [Comelissen and Vandewiele (1989), Nucl. Acids Res. 17: 833] but differs from the latter in that it does not contain a beta-lactamase gene and that its T-DNA is characterized by the sequence of SEQ ID No.37.
• PGVS20 was derived from pTFM600 by removal of the Spbl site, followed by introduction of a DNA fragment derived from the nptl gene (Genbank Accesion No. V00359 between nucleotides 787 and 2308 wherein nucleotides 1592 and 1593 were removed) in the vector-part outside the T-DNA region, using standard recombinant DNA procedures.
• the chimeric bar gene under control of a CaMV35S promoter is cloned as a Stul-Xbal fragment of pDE110 between the Hpal site and the Xbal site of pFM410 (containing the chimeric T7 RNA polymerase gene) and pFM510 (containing the chimeric T3 RNA polymerase gene), resulting in plasmids pFM411 and pFM511 , respectively.
• the chimeric bar gene under control of a T7 promoter is cloned as a SssHII-Xbal fragment of pFM405 (flanked by TNV-A sequences) or pFM406 (flanked by STNV-2 sequences) between the Mlul and Xbal sites of pFM410, resulting in plasmids pFM412 and pFM413, respectively.
• the chimeric bar gene under control of a T3 promoter is cloned as a BssHII-Xba! fragment of pFM505 (flanked by TNV-A sequences) between the Mlul and Xbal sites of pFM510, resulting in plasmid pFM512.
• the chimeric cry genes under control of a T7 promoter of pFM402, pFM403, pFM404, pFM407, or pFM408 are cloned as SssHII-Eagl fragments between the Ascl and Notl sites of pFM411 , pFM412, or pFM413 to obtain the plasmids pFM414-pFM422 of Table 15.
• the chimeric cry genes under control of a T3-specific promoter of pFM502, pFM503, and pFM504 are cloned as BssHII-Eagl fragments between the Ascl and Notl sites of pFM511 and pFM512.
• pVE220 comprises the following nucleotide sequence : from nucleotide 1 to 186 : the sequence from the nucleotide at position 1 to the nucleotide at position 186 of pUC19; from nucleotide 187 to 201 : the sequence from the nucleotide at position 1 to the nucleotide at position 15 of SEQ ID No.
• nucleotide 202 to 207 CCGCTG
• nucleotide 208 to 453 the sequence from the nucleotide at position 16 to the nucleotide at position 261 of SEQ ID No. 35, the complementary sequence of which comprises the 3' end formation signal of cauliflower mosaic virus
• nucleotide 454 to 3102 the sequence complementary to Genbank Accession No. V01146 from the nucleotide at position 3174 to the nucleotide at position 5822, which comprises the T7 RNA polymerase coding region
• nucleotide 3103 to 3137 the sequence complementary to the sequence from the nucleotide at position 35 to the nucleotide at position 1 of SEQ ID No.
• nucleotide 36 which comprises the coding sequence for the nuclear localization signal of the SV40 large T-antigen; from nucleotide 3138 to 3736 : the sequence from the nucleotide at position 372 to the nucleotide at position 970 of SEQ ID No. 35, the complementary sequence of which comprises the cab22L leader sequence and a promoter of the cauliflower mosaic virus 35S RNA; from nucleotide 3737 to 3738 : AT; from nucleotide 3739 to 3752: the sequence from the nucleotide at position 971 to the nucleotide at position 984 of SEQ ID No.
• nucleotide 35 from nucleotide 3753 to 3776 : the sequence from the nucleotide at position 15 to the nucleotide at position 38 of SEQ ID No. 30, comprising the T7 RNA polymerase promoter; from nucleotide 3777 to 3919 : the sequence from the nucleotide at position 2461 to the nucleotide at position 2603 of SEQ ID No. 1 , comprising a first translation enhancing sequence of TNV; from nucleotide 3920 to 5811 : the sequence from the nucleotide at position 6 to the nucleotide at position 1897 of SEQ ID No.
• nucleotide 5812 to 5994 the sequence from the nucleotide at position 3429 to the nucleotide at position 3611 of SEQ ID No. 1, comprising a second translation enhancing sequence of TNV; from nucleotide 5995 to 6109 : the sequence from the nucleotide at position 6 to the nucleotide at position 120 of SEQ ID No. 24, comprising the T3 RNA polymerase terminator sequence; from nucleotide 6110 to 6222 : the sequence from the nucleotide at position 16 to the nucleotide at position 128 of SEQ ID No.
• nucleotide 6223 to 6244 the sequence from the nucleotide at position 988 to the nucleotide at position 1009 of SEQ ID No. 35; from nucleotide 6245 to 7918 : the sequence from the nucleotide at position 947 to the nucleotide at position 2620 of pDE110 (Sful-Xbal fragment), comprising the bar coding region under the control of a promoter and a 3' end formation signal of the cauliflower mosaic virus; from nucleotide 7919 to 7931 : the sequence from the nucleotide at position 1022 to the nucleotide at position 1034 of SEQ ID No. 35; from nucleotide 7932 to 10171 : the sequence from the nucleotide at position 447 to the nucleotide at position 2686 of pUC19.
• Plasmid pVE221 comprises the following nucleotide sequence: from nucleotide 1 to 6244 : the sequence from the nucleotide at position 1 to the nucleotide at position 6244 of pVE220; from nucleotide 6245 to 6247 : AAC; from nucleotide 6245 to 6271 : the sequence from the nucleotide at position 15 to the nucleotide at position 38 of SEQ ID No. 30, comprising the T7 RNA polymerase promoter; from nucleotide 6272 to 6414 : the sequence from the nucleotide at position 2461 to 2603 the nucleotide at position of SEQ ID No.
• nucleotide 6415 to 6421 the sequence from the nucleotide at position 6 to the nucleotide at position 12 of SEQ ID No. 5; from nucleotide 6422 to 6982 : the sequence from the nucleotide at position 1780 to the nucleotide at position 2340 of pDE110, comprising the bar coding region; from nucleotide 6983 to 6987 : CTAGC; from nucleotide 6988 to 7170 : the sequence from the nucleotide at position 3429 to the nucleotide at position 3611 of SEQ ID No.
• nucleotide 7171 to 7285 the sequence from the nucleotide at position 6 to the nucleotide at position 120 of SEQ ID No. 24, comprising the T3 RNA polymerase terminator sequence
• nucleotide 7286 to 7389 the sequence from the nucleotide at position 16 to the nucleotide at position 119 of SEQ ID No. 24, comprising the T3 RNA polymerase terminator sequence
• nucleotide 7390 to 9642 the sequence from the nucleotide at position 7919 to the nucleotide at position 10171 of pVE220.
• Plasmid pVE223 comprises the following nucleotide sequence: from nucleotide 1 to 453: the sequence from the nucleotide at position 1 to the nucleotide at position 453 of pVE220; from nucleotide 454 to 3105 : the sequence complementary to Genbank Accession No.
• Plasmid pVE224 comprises the following nucleotide sequence: from nucleotide 1 to 6226 : the sequence from the nucleotide at position 1 to the nucleotide at position 6226 of pVE220; from nucleotide 6227 to 6250 : the sequence from the nucleotide at position 988 to the nucleotide at position 1011 of SEQ ID No. 35; from nucleotide 6251 to 6256: the sequence from the nucleotide at position 14 to the nucleotide at position 19 of SEQ ID No.
• pVE236 is a plasmid analogous to pVE220 wherein the additional nucleotides of the T7 consensus promoter are incorporated.
• the plasmid has the sequence of pVE220, but for the insertion of the nucleotide sequence GGAG between nucleotide position 3377 and 3778 of pVE220.
• Example 7 Plant transformation and analysis of regenerated plants.
• the plasmids of the pFMseries of Example 5 (Table 15; preferably pFM414, pFM417, pFM514 and pFM517) and pVE236 are used for introduction in maize protoplasts [according to Wang et al. Plant Cell Tissue and Organ Culture 18: 33-46 (1989); Krens et al., Nature 296: 72-74 (1982)] for transient expression assays. Further they are used for electroporation of wounded type I callus (WO 92/09696) or they are introduced into corn protoplasts (EP 0469273) to obtain transgenic corn plants
• the plant transformation vectors of the pTFM series are each mobilized into the Agrobacterium tumefaciens strain C58C1 Rif ⁇ or LBA4011 carrying the avirulent Ti plasmid ⁇ GV2260 as described by Deblaere et al (1985).
• the respective Agrobacterium strains are used to transform oilseed rape using the method described by De Block et al (1989), while rice and corn are transformed according to WO 92/09696.
• Transformed calli are selected on medium containing phosphinotricin, and resistant calli are regenerated into plants.
• RNA from the chimeric cap-independently translated genes is found.
• Plasmids pTVE228, pTVE229, and pTVE225 were introduced into Agrobacterium tumefaciens Ach5C3 containing the helper Ti-plasmid pGV4000 by mobilization.
• the resulting transconjugant strains A3684 (comprising pTVE228), A3685 (comprising pTVE229) and A3681( comprising pTVE225) were used for rice transformation according to WO 92/09696.
• the resulting transformed individual rice plants 110 from transformation with strain A3684; 22 from transformation with strain A3685; 101 from transformation with strain A3681 ) were tested for the expression of proteins reactive in a Cry9C ELISA assay.
• Cry9C ELISA assay was performed using the following procedure: Plant material was harvested, stored at -70°C and crushed. To s extract soluble proteins, 2 volumes of PBS (0.8g/l NaCl; 0.02 g/l Kcl; 0.115g/l Na2HP04; KH2PO4 ; pH7.3) were added to one volume of plant material, mixed and centrifuged for 15 minutes in the cold room. 50 ⁇ l of supernatant was applied per well in a microtiterplate (Costar "High binding" cat. Nr 3599) coated with immuno affinity purified rabitt antibodies against 0 CRY9C. A sandwich ELISA was performed using purified goat antibodies against CRY 9C.
• Plasmid pVE223 (Table 15) was used to transform corn protoplasts as described in EP 0469273. Leaves from 8 individual regenerated 0 transgenic corn plants were assayed by CRY9C specific ELISA as described above. Samples from 3 plants clearly reacted positively, allowing estimation of levels CRY9C protein between 8-13 ng/ml plant protein extract. Table 16. Results from the ELISA assay on transformed rice leaves
• MOLECULE TYPE CDNA (VI) ORIGINAL SOURCE:
• AGTATTCATA CCAAGAATAC CAAATAGGTG CAAGGCCTTA CTCAGCTAAA GACTCTAAAA 60 TGGAGCTACC AAACCAACAC AAGCAAACGG CCGCCGAGGG TTTCGTATCT TTCCTAAACT 120
• CTAAGCCCGT CAAGGGAGCT TTTCGAACCC TTGATAAGTT TCGTGATCTC TATACTAAAA 960
• ACATCATGGT ACGCAAACCT TCTGTGGTAA CATCTAAAGA CGTCACTAGC CTTATCCCAT 1860
• AAGACTCAAC ACATTTCGAT CGCAAAATAG ACATGGCAGG AAAGAAGAAC AACAACAACG 2640
• CAAACTCCGC ACTGATTCCA CCAGCACCAT CATGGCTGGC TAGCATCGCT GATCTTTACA 3000
• CTATTCCTCT TTTTGCACTT CAAAATTATC AAGTTCCTCT TTTATCAGTA TATGTTCAAG 420
• CTGCAAATTT ACATTTATCA GTTTTGAGAG ATGTTTCAGT GTTTGGACAA AGCTGGGGAT 480
• MOLECULE TYPE other nucleic acid
• DESCRIPTION /desc - "oligonucleotide FM9"
• xi SEQUENCE DESCRIPTION: SEQ ID NO: 10:
• MOLECULE TYPE other nucleic acid
• DESCRIPTION /desc - "oligonucleotide FM12"
• MOLECULE TYPE other nucleic acid
• A DESCRIPTION. /desc » "oligonucleotide FM19"
• MOLECULE TYPE other nucleic acid
• DESCRIPTION /desc « "oligonucleotide FM24'
• CTGCAGCGGA CCGACTAGTC CACCCTGAAA GCTCGTTGTG ATTGGGATAA
• CAATCTACTA 60 ATATGCAAAC CCCTTGGGTT CCCTCTTTGG
• GACTCTCAGG CGTTTTTTGC TTTAACCCTC 120 TASAGCTCGG CCGAAGCTT 139
• MOLECULE TYPE other nucleic acid
• DESCRIPTION /desc - "oligonucleotide FM3"
• MOLECULE TYPE other nucleic acid
• DESCRIPTION /desc - "oligonucleotide FM2"
• CCGCCAAGTA CACCAACTAC TGCCACACCT GGTACAACAC CGGTCTGCAC CCCCTCAGGG 660
• AAATGAGCTC CAGTCCTCTC CAAATGAAAT GAACTTCCTT ATATAGAGGA AGGCTCTTGC 480
• CTATCAGCAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA GAGCTTGCCG CCGAATGCGC 900
• GACATAGCGT TGGCTACCCG TGATATTGCT GAAGAGCTTG GCGGCGAATG GGCTGACCGC 720 TTCCTCGTGC TTTACGGTAT CGCCGCTCCC GATTCGCAGC GCATCGCCTT CTATCGCCTT 780
• ORGANISM Tobacco necrosis virus

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