Methods and means for obtaining modified phenotypes
AssigneeCommonwealth Scientific and Industrial Research Organisation
InventorsPeter Michael Waterhouse, Ming-Bo Wang
Life of this patent
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Abstract
Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells, particularly in plant cells, by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell. Preferably, the unpolyadenylated target-specific RNA is provided by transcription of a chimeric gene comprising a promoter, a DNA region encoding the target-specific RNA, a self-splicing ribozyme and a DNA region involved in 3′ end formation and polyadenylation.
Claims37
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, said method comprising the step of providing to the nucleus of said plant cell aberrant RNA comprising a target specific nucleotide sequence.
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, said method comprising the step of introducing into the nuclear genome of said plant cell a chimeric DNA to generate a transgenic plant cell, said chimeric DNA comprising the following operably linked parts:
- a) a plant-expressible promoter region;
- b) a target-specific DNA region;
- c) a DNA region encoding a self-splicing ribozyme;
- and
- d) a DNA region involved in 3′ end formation and polyadenylation wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed.
A chimeric DNA molecule for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, said chimeric DNA molecule comprising
- a) a plant-expressible promoter region;
- b) a target-specific DNA region;
- c) a DNA region encoding a self-splicing ribozyme;
- and
- d) a DNA region involved in 3′ end formation and polyadenylation;
- wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed.
A plant comprising the plant cell of claim 29.
A method for identifying a phenotype associated with the expression of a nucleic acid of interest in a plant cell, said method comprising:
- a) selecting within said nucleic acid of interest a target sequence of at least 10 consecutive nucleotides;
- b) introducing a chimeric DNA into the nucleus of a suitable plant host cell comprising said nucleic acid of interest, said chimeric DNA comprising the following operably linked DNA fragments:
- i) a plant-expressible promoter region;
- ii) a target-specific DNA region comprising a nucleotide sequence of at least about 70% to about 100% sequence identity to said target sequence or to the complement of said target sequence;
- followed by
- iii) a DNA region encoding a self-splicing ribozyme located immediately upstream of P2 iiv) a DNA region involved in 3′ end formation and polyadenylation;
- c) observing the phenotype by a suitable method.
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, said method comprising the step of providing to the nucleus of said eukaryotic cell aberrant RNA comprising a target specific nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of said nucleic acid of interest.
A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, said method comprising the step of introducing into the nuclear genome of said eukaryotic cell a chimeric DNA to generate a transgenic plant cell, said chimeric DNA comprising the following operably linked parts:
- a) a promoter region functional in said eukaryotic cell;
- b) a target-specific DNA region comprising nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of said nucleic acid of interest;
- c) a DNA region encoding a self-splicing ribozyme;
- and
- d) a DNA region involved in 3′ end formation and polyadenylation wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed.
A eukaryotic cell comprising a nucleic acid of interest, normally capable of being phenotypically expressed, further comprising a chimeric DNA comprising the following operably linked parts:
- a) a promoter region functional in said eukaryotic cell;
- b) a target-specific DNA region comprising nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of said nucleic acid of interest;
- c) a DNA region encoding a self-splicing ribozyme;
- and
- d) a DNA region involved in 3′ end formation and polyadenylation wherein said chimeric DNA when transcribed in said eukaryotic cell produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3′ end of the first RNA molecule comprising the polyadenylation site has been removed.
Dependencies are inferred from the claim text.