WO1991013994A1 - Expression de genes - Google Patents

Expression de genes Download PDF

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Publication number
WO1991013994A1
WO1991013994A1 PCT/AU1991/000088 AU9100088W WO9113994A1 WO 1991013994 A1 WO1991013994 A1 WO 1991013994A1 AU 9100088 W AU9100088 W AU 9100088W WO 9113994 A1 WO9113994 A1 WO 9113994A1
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Prior art keywords
nucleic acid
promoter
acid sequence
sub
heterologous gene
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PCT/AU1991/000088
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English (en)
Inventor
Wayne Lyle Gerlach
Mark Jefferson Young
Joanne Julie Fillatti
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Commonwealth Scientific And Industrial Research Organisation
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Publication of WO1991013994A1 publication Critical patent/WO1991013994A1/fr

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    • 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
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation

Definitions

  • This invention broadly relates to gene expression and in particular relates to nucleic acid sequences capable of providing amplified levels of heterologous gene transcripts both in-vivo and in-vitro. This invention further relates to host cells, plants and animals containing said nucleic acid sequences.
  • Transcribed mRNA of a desired gene or genes may be translated into desirable polypeptide products, useful, for example, as therapeutics, biological reagents, antibiotics, antivirals and the like.
  • RNA transcripts of a desired gene may encode polypeptides which modify phenotype, for example, by affecting sterility, salt tolerance, viral susceptibility, drought tolerance, acidity, colour and the like.
  • RNA transcripts of a desired gene or genes may be translated into polypeptide products having a host of phenotypic actions/effects, these being dependent upon the nature of the polypeptide product.
  • RNA transcripts may have activity in their own right, such as antisense reagents which block translation or inhibit RNA function, or as endoribonucleases (sometimes known as ribozymes) which can effect catalytic cleavage of target RNA molecules.
  • antisense reagents which block translation or inhibit RNA function
  • endoribonucleases sometimes known as ribozymes
  • nucleic acid sequence characterised in that it comprises:
  • nucleic acid sequence of this invention is generally in the form of DNA or derivatives thereof, and may be single or double-stranded.
  • DNA "derivatives" are well known in the art and include base modifications, modifications of the sugar moiety, modifications of the phosphodiester linking groups, and modifications of the 5' and 3' termini.
  • the oligonucleotide backbone (that is the phosphodiester linkages) may be modified in a variety of ways, for example, in the same manner as for DNA anti- sense oligonucleotides.
  • Methylphosphonate or phosphorothioate linkages may be used to replace conventional phosphodiester linkages.
  • Nucleotide bases and sugar moieties may be substituted with one or more substituents such as halogen, alkyl, alkoxy, and the like.
  • Carbon atoms, nitrogen atoms or oxygen atoms forming part of the nucleotide base or sugar moiety may be replaced with different atoms such as carbon, sulphur, nitrogen, halogen and the like.
  • Deoxynucleotides may be replaced with ribonucleotides. As previously mentioned, the above modifications are well known in the art, and are described, for example in Buck et al. (Science 248: 208-212 (1990)). Where nucleic acid sequences are double-stranded, the complementary strand is selected to have complementary sequence.
  • Nucleic acid sequences may additionally comprise a selectable marker, such as an antibiotic or herbicide resistance gene, and may further include nucleotide sequences to facilitate integration of said nucleic acid sequences into chromosomal DNA of eukaryotic or prokaryotic cells, or poly adenylation sequences.
  • the nucleic acid sequences of this invention may contain T-DNA of the Ti-plasmid of Agrobacterium.
  • other types of nucleotide sequences can be used to facilitate integration into the genome of eukaryotic or prokaryotic cells.
  • viral sequences such as long terminal repeats (LTR's) may be employed.
  • the heterologous gene sequence is in an orientation (inverted) which gives rise to a negative sense RNA on transcription (primary transcript), also known as a minus (-) strand, whose nucleotide sequence corresponds to the non-coding strand of a heterologous gene sequence.
  • primary transcript also known as a minus (-) strand
  • This minus strand is not capable of being translated to the desired protein product, as it is in the wrong orientation and would give a nonsensical product if translated.
  • transcription driven from the sub- genomic promoter located on the primary transcript at the 3' end thereof produces a positive sense RNA transcript which may then be translated to give a desired protein product.
  • the primary RNA transcript is in a positive (+) sense. Transcription from the sub-genomic sequence produces a negative (-) sense RNA (secondary transcript) which may then act as an anti-sense RNA or an endoribonuclease.
  • Nucleotide sequences of this invention may comprise all or part of a transfer vector such as a plasmid or phage transfer vector.
  • Transfer vectors may contain nucleotide sequences encoding selectable markers, such as antibiotic resistance and may include nucleotide sequences to facilitate integration of said nucleic acid sequences into chromosomal DNA of eukaryotic or prokaryotic cells.
  • Plasmids containing the nucleic acid sequences of this invention would comprise an origin of replication to enable plasmid replication inside host cells.
  • Transcriptional promoter sequences may be selected from any nucleotide sequences which bind or interact with DNA dependent RNA polymerase to effect transcription of DNA sequences operably linked to said promoter.
  • said promoters are of eukaryotic origin, and in a preferred embodiment of this invention, are of plant origin and may, for example, be plant viral promoters. Suitable promoters are well known in the art and are described for example in 0'Neal et al. ( ucleic Acids. Res. 15: 8661 (1988)) and Harpster et al. (Mol. Gen. Genet. 212: 189-190 (1988)).
  • Transcriptional promoters may be induceable by various stimuli, such as temperature, salinity, moisture, stress, viral infection and the like.
  • Transcriptional promoter sequences may contain more than one promoter, to enable transcriptional induction under various stimuli or conditions.
  • the heterologous gene sequence may encode any desired peptide product, such as hormones (e.g. insulin, relaxin, growth hormone and LHRH), toxins, antibiotics, such as proteins which interfere with virus infection, biological reagents, proteases which degrade cellular x proteins, or two or more such peptide products.
  • said heterologous nucleic acid sequences may encode polypeptides which modify phenotype, sterility, salt tolerance, fruit ripening viral susceptibility, drought tolerance, acidity, colour, and the like, or proteases which degrade cellular proteins. It is important to appreciate that the precise nature of the polypeptide product(s) encoded by a heterologous nucleotide sequence is not of importance and may be selected according to a particular desired purpose.
  • the protein product encoded by a heterologous gene is a protein which disrupts viral replication in plant or animal cells.
  • the nucleic acid sequence of this invention is generally integrated into the chromosomal DNA of a plant or animal cell by methods well known in the art.
  • the heterologous gene sequence encoding the protein product is present in an inverted form to produce a primary transcript corresponding to the non- coding strand of the heterologous gene sequence.
  • the heterologous gene sequence may encode RNA transcripts which function as antisense RNAs, that is, are of complementary sequence to specific cellular RNAs in eukaryotic or prokaryotic cells, which may, for example, be concerned with the translation of protein products, viral replication, cell division, metabolism, differentiation, division, sterility, phenotypic traits or a host of cellular processes.
  • the nucleotide sequence of the heterologous gene sequence is, of course, selected to be substantially complementary thereto, and therefore capable of hybridization thereto, thus inactivating a desired exogenous/endogenous/pathogenic RNA sequence within a cell.
  • Heterologous nucleotide sequences may encode one or more endoribonucleases, such as described, for example, by Haseloff and Gerlach (Nature 334: 585-591 (1988)) and Cech et al. (U.S. Patent No. 4,987,071).
  • endoribonucleases are capable of site specific cleavage of target RNAs, and therefore may be used to inactivate any desired cellular RNA, which may, for example, be involved in viral replication, or cellular regulation or processes (such as division, differentiation, and the like).
  • the heterologous gene sequence encoding an anti-sense RNA or endoribonuclease directs transcription of a positive sense (+) transcript, which on transcription from the sub-genomic promoter produces a negative sense (-) secondary transcript.
  • the sub-genomic promoter is operably linked to the heterologous gene sequence such that on transcription of the nucleic acid sequence to give a primary transcript, the sub-genomic promoter then drives transcription of the heterologous nucleic acid sequence to produce a secondary amplified transcript.
  • the secondary transcript is capable of translation to give a polypeptide encoded by said heterologous nucleic acid sequence.
  • the sub-genomic promoter binds or interacts with RNA dependent RNA polymerase, and thus is generally of viral origin, particularly plant or animal viral origin.
  • RNA viruses use a strategy of sub-genomic RNAs to amplify and express internal cistrons in their genomic RNA. These are transcribed from the negative strand replicating form of the virus using signals known as "sub-genomic promoters" in the RNA template to commence transcription at the defined regions. This strategy provides for production of translatable RNAs from these internal genes.
  • Sub- genomic promoters have been described for a range of plus stranded plant and animal viruses (see French and Ahlquist, J. Virol. 62: 2411-2420 (1988); Levis et al. , J. Virol. 64: 1726-1733 (1990) and scientific papers referred to therein).
  • the nature of the sub-genomic promoter will, of course, depend upon the particular circumstances in which the nucleic acid sequence of this aspect of the invention is employed.
  • the sub-genomic promoter may be selected to correspond to a sub-genomic promoter of an infecting plant virus.
  • the viral RNA polymerase of the particular virus mediates transcription from the sub-genomic promoter of the heterologous nucleotide sequence which may encode a polypeptide, endoribonuclease, or anti-sense RNA to inactivate the infecting virus.
  • sub-genomic promoter may be selected to be specific for the RNA polymerase of an infecting virus.
  • viral infection would trigger transcription, which in turn would lead to viral modulation/inactivation.
  • the sub-genomic promoter may comprise multiple promoter sequences wherein each sub-genomic promoter sequence of the multiple sub-genomic promoter sequences is recognised by a specific RNA dependent RNA polymerase.
  • promoters corresponding to various plant or animal viruses may be utilised.
  • RNA polymerases are generally promoter specific, the use of multiple promoters enables transcription of the heterologous gene sequence on viral infection with a number of viral types.
  • the heterologous nucleotide sequence may encode a cytotoxic product which causes cell death or which inhibits viral function, such as by the inhibition of reverse transcriptase, disruption of viral particle assembly, or modulation or replication.
  • the nucleic acid sequence of this invention may additionally comprise a complementary sequence of a sub- genomic promoter inserted between the transcriptional promoter and the heterologous gene sequence, such that the secondary transcripts are transcribed from the sub- genomic promoter produced on transcription of the primary transcript (from the complementary sequence of the sub- genomic promoter) to give a plurality of tertiary transcripts.
  • the heterologous gene sequence encoding a desired polypeptide would be in an orientation selected to give rise to positive sense (+) RNA. Two rounds of amplification provided by the sub-genomic promoters would produce an amplified positive sense (+) tertiary transcript which may be translated to give a desired polypeptide product.
  • a eukaryotic cell which contains a nucleic acid sequence comprising: (a) a transcriptional promoter;
  • nucleic acid sequence as hereinbefore described may be integrated into the genome of the eukaryotic cell or may be present as an extra chromosomal element, such as a plasmid.
  • Transfection of eukaryotic cells is carried out according to well known methods in the art, for example by using plant or animal viruses which integrate into chromosomal DNA, or plasmids such as the Ti-plasmid of Agrobacterium, pollen mediated transformation, in-vitro protoplast transformation, lyposome-mediated transformation, and other well known methods such as are described in
  • the eukaryotic cell is a plant or animal cell.
  • transgenic plant containing a nucleic acid sequence comprising:
  • the transgenic plant may be wheat, barley, rye, sorghum, potato, rice,, rape, apple, and the like.
  • transgenic animal containing a nucleic acid sequence:
  • Plant and animal transgenes may contain the nucleic acid sequence of the first aspect of this invention described hereinbefore in some or all of the cells thereof, integrated into the genome and/or carried on extra chromosomal elements.
  • cells, plants or animals may contain additional nucleotide sequences which encode an RNA dependent RNA polymerase which is capable of effecting transcription from the sub- genomic promoter under control of one or more promoter sequences.
  • These promoter sequences may be induced by the same stimuli which induces transcription from the transcriptional promoter.
  • transcribed primary transcripts RNA encoding one or more heterologous gene products would be rapidly transcribed by constitutively produced polymerase to give a secondary transcripts or tertiary transcripts which may then be translated to give a protein product.
  • the gene encoding the RNA dependent polymerase under promoter control may be integrated into the genome or be carried as an extra-chromosomal element. It may be provided as a construct with the nucleic acid sequence of this invention, where the nucleic acid sequence and the polymerase gene plus promoter are separated by transcriptional termination sequences, such that the nucleic acid sequence and the polymerase gene are separately transcribed.
  • a process for transcriptional amplification which process comprises:
  • a method for protecting a plant or animal cell from viral infection comprises introducing into one or more cells of said plant or animal a nucleic acid sequence comprising: (a) a transcriptional promoter; (b) a heterologous gene sequence operably linked to said transcriptional promoter; and
  • a sub-genomic promoter ligated to said heterologous gene sequence such that the heterologous gene sequence and sub- genomic promoter are transcribed under the action of said transcriptional promoter to give a primary transcript, and said primary transcript is capable of transcription under the action of said sub-genomic promoter to give a secondary transcript; and wherein the primary transcript produced under control of said transcriptional promoter is transcribed from the sub-genomic promoter by an RNA polymerase to give a secondary transcript which encodes a polypeptide product, or which itself, inhibits viral replication or inactivates said virus.
  • a nucleic acid sequence comprising:
  • a sub-genomic promoter ligated to said heterologous gene sequence such that the heterologous gene sequence and sub- genomic promoter are transcribed under the action of said transcriptional promoter to give a primary transcript, and said primary transcript is capable of transcription under the action of said sub-genomic promoter to give a secondary transcript; and reacting said primary transcript produced on cellular transcription of said nucleic acid sequence with an RNA dependent RNA polymerase capable of transcription from said sub-genomic promoter to give a secondary transcript which itself may comprise said heterologous gene product, or which may be translated in the cell to give a heterologous gene product.
  • a nucleic acid sequence comprising:
  • a sub-genomic promoter ligated to said heterologous gene sequence such that the heterologous gene sequence and sub- genomic promoter are transcribed under the action of said transcriptional promoter to give a primary transcript, and said primary transcript is capable of transcription under the action of said sub-genomic promoter to give a secondary transcript; wherein the primary transcript produced under control of said transcriptional promoter is transcribed from the sub-genomic promoter by an RNA polymerase to give a secondary transcript, and wherein said secondary transcript or a translated product thereof inactivates said exogenous or endogenous cellular RNA.
  • Exogenous RNA's may be any foreign RNA such as a viral RNA or RNA of a pathogenic organism.
  • An endogenous RNA is any cellular RNA.
  • cells may be transfected with an RNA dependent polymerase under promotional control, such that the polymerase may be produced within the cells under appropriate stimuli which activate said promoter.
  • RNA polymerase recognises the sub-genomic promoter
  • transcription is effected to give a positive sense RNA, which itself, or its translated product, is effective to inhibit said virus.
  • the amplified tertiary transc-ipts or encoded polypeptide products may act to inhibit/inactivate virus, inactivate exogenous or endogenous cellular RNA, modify cellular phenotype, and the like.
  • the nucleic acid sequence of this invention amplifies transcription by employing at least two promoters.
  • the first promoter transcriptional promoter
  • Each primary transcript is then in turn transcribed using the secondary promoter located on the primary transcript (sub-genomic promoter) to give a secondary transcript which is capable of translation.
  • втори ⁇ ии For each primary transcript, many secondary transcripts may be produced resulting in significant transcriptional amplification. Where a complementary sequence of a sub-genomic promoter is inserted between the transcriptional promoter and the heterologous gene sequence further transcriptional amplification is enabled. Particularly, the active sequence of the sub-genomic promoter (derived from the complementary sequence thereof) is provided in the secondary transcript. Transcription is then driven from the amplified secondary transcript by the sub- genomic promoter present thereon.
  • heterologous gene transcripts may thus be amplified considerably, when compared to the situation in which the heterologous gene is oriented in the positive orientation, and transcription thereof is singly driven by the transcriptional promoter.
  • the sub-genomic promoter is generally recognised, as mentioned above, by a viral RNA dependent RNA polymerase (viral replicase complex) thus providing applications in- vivo (that is in eukaryotic and prokaryotic cells) as follows: (a) for the virus induced expression of particular gene sequences e.g. desirable peptide products, proteins which will interfere with virus infection e.g. virus induced expression of commercially important peptides or cell-lethal proteins which might contain spread of the virus or other pathogens;
  • nucleic acid sequences with biological activity for the virus induced expression of particular nucleic acid sequences with biological activity, from primary transcripts which contain the inactive complementary strand of that nucleic acid e.g. virus induced production of the ribozyme targeted against RNA components of the virus genome (if it is an RNA virus). or its transcripts of its genetic information;
  • RNA is not capable of translation, and hence heterologous gene products will only be produced following production of a positive transcript which occurs only when transcription is initiated from the sub-genomic promoter.
  • FIGURE 1 Structure of gene construction (top) and transcription/replication path of RNA transcript (shown by arrows) ;
  • FIGURE 2 Relative GUS activity in protoplasts 48 hours after electroporation of the following constructs (a) infectious BYDV RNA alone (b) SGP-GUS gene construct alone (c) BYDV RNA + SGP-GUS gene construct. GUS activity is determined by standard fluorometric assay.
  • the 35S promoter refers to Cauliflower mosaic virus 35S promoter.
  • the GUS ( ⁇ -glucuronidase) gene negative sense orientation refers to the structural gene for the GUS reporter protein oriented such that transcription from the 35S promoter produces transcript of the negative sense sequence of this gene.
  • the sub-genomic promoter is a 121 nucleotide intergenic sequence upstream (5' ) of coat protein gene of barley yellow dwarf virus (BYDV) genome. This region is assumed to contain a sub-genomic promoter sequence which is specifically recognised by the BYDV replicase complex during virus replication, resulting in a sub-genomic mRNA for BYDV coat protein. Production of coat protein sub- genomic mRNA is assumed to only be initiated by internal initiation by the viral replicase complex from the sub- genomic promoter sequence as it is represented on the negative sense of the viral genome.
  • BYDV barley yellow dwarf virus
  • NOS 3' refers to nopaline synthase polyadenylation signals.
  • the 121 nucleotide BYDV coat protein sub-genomic promoter is fused to the GUS reporter gene so that transcription directed by the 35S promoter directs the synthesis of a negative sense GUS-sub-genomic RNA. Recognition of the sub-genomic promoter sequence represented on the negative sense GUS-sub-genomic promoter by the viral replicase complex results in production of positive sense mRNA for GUS.
  • the above gene construction was produced and propagated in bacterial plasmid vectors using standard recombinant DNA techniques.
  • Two appropriate controls were also included: (i) The GUS-SGP plasmid construct introduced into the protoplasts alone, and (ii) the infectious BYDV RNA introduced into the protoplasts alone. Gus activity was assayed in the protoplast samples after an appropriate interval.

Abstract

Séquences d'acide nucléique comprenant: (a) un promoteur de transcription; (b) une séquence de gènes hétérologue fonctionnellement liée audit promoteur de transcription; et (c) un promoteur sub-génomique lié à ladite séquence de gènes hétérologue; de sorte que cette dernière et le promoteur génomique sont transcrits par l'action dudit promoteur de transcription afin de produire un transcrit primaire, et ledit transcrit primaire est capable d'une transcription par l'action dudit promoteur sub-génomique afin de produire un transcrit secondaire. L'invention concerne également des cellules eukaryotes ainsi que des plantes transgéniques et des animaux contenant lesdites séquences d'acide nucléique, lesquelles peuvent être modifiées de manière phénotypique et/ou protégées contre des infections virales par un ou plusieurs produits de la séquence de gènes hétérologue, ou lesquelles sont capables de produire des produits polypeptidiques codés par la séquence de gènes hétérologue.
PCT/AU1991/000088 1990-03-13 1991-03-13 Expression de genes WO1991013994A1 (fr)

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