Synthetic plant genes and method for preparation
AssigneeMonsanto Technology LLC
InventorsDavid A. Fischhoff, Frederick J. Perlak
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Abstract
A method for modifying structural gene sequences to enhance the expression of the protein product is disclosed. Also disclosed are novel structural genes which encode insecticidal proteins of B.t.k. HD-1, B.t.k. HD-73, B.t. tenebrionis, B.t. entomocidus, 2 protein of B.t.k. HD-1, and the coat protein of potato leaf roll virus.
Claims103
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with a coding sequence, derived from Bacillus thuringiensis (B.t.), that encodes an insecticidal protein and that contains polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said polyadenylation signal sequences in the coding sequence by substituting sense codons for codons in the coding sequence;
- and
- (c) making a structural gene that comprises a coding sequence that includes the codons substituted according to step (b) and is characterized by the reduced number of Table II polyadenylation signal sequences, and that encodes the insecticidal protein.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with a portion of a coding sequence, wherein the coding sequence is derived from Bacillus thuringiensis (B.t.) and encodes an insecticidal protein and wherein portion contains ATTTA sequences and polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said ATTTA sequences and the number of said polyadenylation signal sequences in said portion of the coding sequence by substituting sense codons for codons in said portion, wherein said substituted sense codons maintain the original encoded amino acids;
- and
- (c) making a structural gene that comprises said portion with the substitute codons and the reduced number of ATTTA and polyadenylation signal sequences, wherein the structural gene comprises a nucleotide sequence that encodes an insecticidal protein.
A method of making a structural gene, the method comprising:
- (a) starting with a coding sequence, derived from Bacillus thuringiensis (B.t.), that encodes an amino acid sequence and that contains ATTTA sequences and polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said ATTTA sequences and the number of said polyadenylation signal sequences in a portion of the coding sequence by substituting sense codons for codons in said portion, while maintaining the amino acid sequence;
- and
- (c) making a structural gene that comprises said portion with the codons substituted according to step (b) and characterized by the reduced number of ATTTA and polyadenylation signal sequences.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with an amino acid sequence of an insecticidal protein derived from Bacillus thuringiensis (B.t.), wherein wild-type B.t. gene sequence(s) encoding insecticidal polypeptide(s) from which the insecticidal protein is derived comprise polyadenylation signal sequences listed in Table II;
- and
- (b) making a structural gene that comprises a coding sequence that:
- (i) encodes the amino acid sequence of the insecticidal protein;
- and
- (ii) contains fewer polyadenylation signal sequences listed in Table II, compared to the corresponding coding sequence(s) of the wild-type B.t. gene sequences(s).
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with an amino acid sequence of an insecticidal protein derived from Bacillus thuringiensis (B.t.);
- and
- (b) making a structural gene that comprises a coding sequence that encodes the amino acid sequence and that is devoid or substantially devoid of polyadenylation signal sequences listed in Table II.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with an amino acid sequence of a portion of an insecticidal protein derived from Bacillus thuringiensis (B.t.), wherein a wild-type B.t. coding sequence from which the portion was derived includes ATTTA sequences and polyadenylation signal sequences listed in Table II;
- and
- (b) making a structural gene that encodes an insecticidal protein and that comprises a sequence that encodes the amino acid sequence of the portion and that contains fewer ATTTA sequences and fewer of said polyadenylation signal sequences than the wildtype B.t. coding sequence from which said portion was derived.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with a sequence derived from Bacillus thuringiensis (B.t.), said sequence comprising:
- (i) a coding sequence for an insecticidal protein having an amino acid sequence, or (ii) an amino acid sequence of the insecticidal protein;
- and
- (b) making a structural gene that comprises a coding sequence that encodes the amino acid sequence and that is devoid or substantially devoid of ATTTA sequences, and devoid or substantially devoid of polyadenylation signal sequences listed in Table II.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) designing a nucleotide sequence that encodes an insecticidal protein derived from Bacillus thuringiensis (B.t.) and that contains a reduced number of polyadenylation signal sequences listed in Table II, compared to a wild type B.t. coding sequence from which the insecticidal protein was derived, wherein the number of said polyadenylation signal sequences is reduced compared to the wild type B.t. coding sequence by substituting codons while maintaining the encoded amino acids;
- and
- (b) making a structural gene that comprises the nucleotide sequence, that encodes the insecticidal protein, and that is characterized by the reduced number of said polyadenylation signal sequences, compared to the wild type B.t. coding sequence.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) designing a nucleotide sequence that encodes an insecticidal protein derived from Bacillus thuringiensis (B.t.) and that is devoid or substantially devoid of polyadenylation signal sequences listed in Table II;
- and
- (b) making a structural gene that comprises the nucleotide sequence and that encodes the insecticidal protein, wherein the structural gene is devoid or substantially devoid of said polyadenylation signal sequences.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with a coding sequence, derived from Bacillus thuringiensis (B.t.), that contains polyadenylation signal sequences listed in Table II, and that encodes an insecticidal protein having an amino acid sequence;
- (b) making a structural gene that comprises a coding sequence that:
- (i) encodes the amino acid sequence of the insecticidal protein;
- and
- (ii) contains fewer polyadenylation signal sequences listed in Table II, compared to the corresponding coding sequence derived from B.t.
A method of making a structural gene that encodes a protein, the method comprising:
- (a) starting with a coding sequence that encodes a protein and that contains polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said polyadenylation signal sequences in the coding sequence by substituting sense codons for codons in the coding sequence;
- and
- (c) making a structural gene that comprises a coding sequence that includes the codons substituted according to step (b) and is characterized by the reduced number of Table II polyadenylation signal sequences, and that encodes the protein.
A method of making a structural gene, the method comprising:
- (a) starting with a coding sequence that encodes an amino acid sequence and that contains polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said polyadenylation signal sequences in a portion of the coding sequence by substituting sense codons for codons in said portion, while maintaining the amino acid sequence;
- (c) making a structural gene that comprises said portion with the substitute codons and the reduced number of polyadenylation signal sequences;
- and
- (d) making a DNA construct that comprises the structural gene and at least one sequence selected from the group consisting of a plant promoter or a plant virus promoter.
A method of making a structural gene that encodes a protein, the method comprising:
- (a) starting with coding sequences, from one or more structural genes that encode a protein and that contain polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said polyadenylation signal sequences in the coding sequences by substituting sense codons for codons in the coding sequences;
- and
- (c) making a structural gene that comprises the coding sequences with the codons substituted according to step (b) and characterized by the reduced number of polyadenylation signal sequences, wherein the structural gene comprises a nucleotide sequence that encodes the protein.
A method of making a structural gene that encodes a protein, the method comprising:
- (a) starting with an amino acid sequence of a protein, wherein the amino acid sequence is derived from a coding sequence that contains polyadenylation signal sequences listed in Table II and ATTTA sequences;
- (b) making a structural gene that comprises a coding sequence that encodes the amino acid sequence and that is devoid or substantially devoid of ATTTA sequences, and devoid or substantially devoid of polyadenylation signal sequences listed in Table II;
- and
- (c) making a DNA construct that comprises the structural gene and at least one sequence selected from the group consisting of a plant promoter or a plant virus promoter.
A method of making a structural gene that encodes a protein, the method comprising:
- combining coding sequences to form a structural gene that encodes an insecticidal protein derived from Bacillus thuringiensis (B.t.), wherein said coding sequences and the structural gene are devoid or substantially devoid of polyadenylation signal sequences listed in Table II.
A method of making a DNA construct for expression of an insecticidal protein derived from Bacillus thuringiensis (B.t.), the method comprising combining a 5′ nontranslated sequence, a structural gene, and a 3′ nontranslated region to form a construct for expression of the insecticidal protein,
- wherein the 5′ nontranslated sequence contains a plant promoter or a plant virus promoter,
- wherein the structural gene comprises a sequence that encodes the insecticidal protein derived from B.t. and is devoid or substantially devoid of polyadenylation signal sequences listed in Table II, and
- wherein the 3′ nontranslated sequence comprises a polyadenylation signal.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with a sequence derived from Bacillus thuringiensis (B.t.), said sequence comprising:
- (i) a coding sequence for an insecticidal protein having an amino acid sequence, or (ii) an amino acid sequence of the insecticidal protein;
- and
- (b) making a structural gene that comprises a coding sequence that encodes the amino acid sequence and that is devoid or substantially devoid of polyadenylation signal sequences listed in Table II.
A method of making a structural gene that encodes a protein, the method comprising:
- (a) starting with an amino acid sequence of a protein, wherein the amino acid sequence is derived from a coding sequence that contains polyadenylation signal sequences listed in Table II;
- (b) making a structural gene that comprises a coding sequence that encodes the amino acid sequence and that is devoid or substantially devoid of polyadenylation signal sequences listed in Table II;
- and
- (c) making a DNA construct that comprises the structural gene and at least one sequence selected from the group consisting of a plant promoter or a plant virus promoter.
In a method that involves making a structural gene that encodes an insecticidal protein derived from Bacillus thuringiensis (B.t.) and that involves starting with a wild type B.t. coding sequence or with an amino acid sequence encoded thereby, the improvement which comprises reducing the number of polyadenylation signal sequences listed in Table II when making the structural gene, compared to the number of said polyadenylation signal sequences in the wild type B.t. coding sequence, by substituting codons relative to the wild-type B.t. coding sequence while maintaining the amino acid sequence.
A method of making a structural gene that encodes an insecticidal protein, the method comprising:
- (a) starting with an insecticidal portion of a coding sequence, wherein the coding sequence is derived from Bacillus thuringiensis (B.t.) and encodes an insecticidal protein and wherein the insecticidal portion contains ATTTA sequences and polyadenylation signal sequences listed in Table II;
- (b) reducing the number of said ATTTA sequences and the number of said polyadenylation signal sequences in said insecticidal portion of the coding sequence by substituting sense codons for codons in said portion;
- and
- (c) making a structural gene that comprises said insecticidal portion with the substitute codons and the reduced number of ATTTA and polyadenylation signal sequences, wherein the structural gene comprises a nucleotide sequence that encodes an insecticidal protein.
Dependencies are inferred from the claim text.