Claims for Patent: 5,952,547

Summary for Patent: 5,952,547

Title:	Modified Bacillus thuringiensis genes with improved expression in plant cells, methods of production on and use
Abstract:	The invention relates to modified Bacillus thurigiensis genes with improved expression in plant cells, their preparation and uses. The invention relates more particularly to DNA fragments encoding all or part of a Bt insecticidal crystal protein, modified by translationally neutral modification(s) in cryptic promoter(s) and/or abortive intron(s). The invention also discloses method of preparing such modified DNAs, and methods of protecting plants from an insect pest.
Inventor(s):	Cornelissen; Marc (Heusden, BE), Soetaert; Piet (Laarne, BE), Stam; Maike (Al Amstelveen, NL), Dockx; Jan (Turnhout, BE), Van Aarssen; Roel (Gent, BE)
Assignee:	Plant Genetic Systems, N.V. (Brussels, BE)
Application Number:	08/232,016
Patent Claims:	1. A process for modifying the coding region of a Bacillus thuringiensis insecticidal crystal protein (Bt ICP) gene to be expressed in a plant cell; which process comprises the following steps: identifying in the coding region of said Bt ICP gene at least one DNA sequence encoding an abortive intron; and modifying said coding region in a translationally neutral manner to obtain a modified coding region in which said at least one DNA sequence encoding an abortive intron has been inactivated, wherein said DNA sequence encoding an abortive intron can be identified by a process comprising the steps of: (i) introducing said coding region under control of a plant-expressible promoter in plant cells, (ii) amplifying DNA fragments obtained from mRNA transcribed from said coding region in said plant cells, (iii) localizing a gap in said DNA fragments with a size smaller than the size expected for a mature mRNA by aligning said DNA fragments with the DNA sequence encoding a full-size unprocessed RNA; wherein said gap is identified as a DNA sequence encoding an abortive intron when inactivation of splice sites surrounding the abortive intron results in an increase of the level of proper sized mRNA upon transcription in plant cells. 2. The process of claim 1, wherein the coding region of said Bt ICP gene is modified in a translationally neutral manner by introducing at least one of the following modifications in said coding region; a) introducing a DNA encoding an intron, spliced in the plant cell; and b) replacing amino acid codons in the coding region by other codons encoding the same amino acids. 3. The process of claim 2, wherein said coding region encodes a toxic portion of said ICP. 4. The process of claim 2, wherein the ATG translation initiation codon of said coding region has been replaced by the following sequence: AAAACCATGGCT. 5. The process of claim 4, wherein the coding region which is modified is that of the cryItb14 gene. 6. The process of claim 2, wherein said coding region encodes a Bt2 ICP and wherein the first 28 N-terminal codons of said coding region have been replaced by an ATG codon. 7. The process of claim 2, wherein said Bt ICP gene is a bt2 gene, a bt884 gene, a cryIAb6 gene, a cryIAb22 gene, a bt14 gene, a bt15 gene, or a bt18 gene. 8. The process of claim 7, wherein said Bt ICP gene is a bt2, a bt884 or a cryIAb22 gene and wherein a cryptic promoter is inactivated by translationally neutral modifications between nucleotide positions 742 and 813 in the coding region of bt884. 9. The process of claim 7, wherein said Bt ICP gene is a bt2, bt884, cryIAb22, or cryIAb6 gene and wherein said at least one DNA sequence encoding an abortive intron is inactivated by translationally neutral modifications in a region within 10 nucleotides of one or more of the following sequences in the coding region: AAG:GTGGGG, AGT:GTAAGT, and CGG:GTAAGA. 10. The process of claim 1, wherein said coding region after modification differs from a native coding region of a Bt ICP gene by having less than 3% of the nucleotides in said native coding region changed from A or T to G or C nucleotides. 11. The process of claim 1, wherein said coding region after modification differs from a native coding region of a Bt ICP gene by having less than 1% of the nucleotides in said native coding region changed from A or T to G or C nucleotides. 12. The process of claim 1, wherein the abortive intron is inactivated by translationally neutral modification in said coding region in about 3 to about 10 nucleotides on either side of the 5' and 3' abortive intron splice sites. 13. The process of claim 1, which further comprises the steps of: identifying in the coding region of said Bt ICP gene, at least one cryptic promoter in either a sense or an anti-sense orientation; and modifying said coding region in a translationally neutral manner to obtain a modified coding region in which said at least one cryptic promoter has been inactivated, wherein said cryptic promoter can be identified by a process comprising the steps of: (i) identifying a pausing region in said coding sequence inhibiting elongation of transcription by RNA polymerase II by means of run-on analysis on plant cell nuclei containing said coding sequence in their nuclear genome; (ii) cloning said pausing region or a part thereof in two orientations in a transcription initiation-probe vector downstream of an enhancer of a plant promoter and upstream of a DNA sequence that can be transcribed; (iii) introducing said vector into plant cells; (iv) detecting the accumulation of RNA species which is initiated from the pausing region; and (v) localizing the transcription initiation site in said RNA species; wherein said pausing region is identified as a cryptic promoter when said RNA is produced from a transcription initiation site located in said pausing region or the part thereof. 14. The process of claim 13, wherein said cryptic promoter comprises a CCAAT sequence or a TATA sequence. 15. The process of claim 13, wherein the abortive intron is inactivated by translationally neutral modification in said coding region in about 3 to about 10 nucleotides on either side of the 5' and 3' abortive intron splice sites, and wherein the cryptic promoter is inactivated by translationally neutral modification in said coding region of 20 to 90 nucleotides upstream of the experimentally determined transcription initiation site of said cryptic promoter. 16. A method of improving the production level of a Bt ICP protein in a plant cell, wherein said method comprises: identifying, in a coding region of a nucleic acid encoding said Bt ICP protein, at least one DNA sequence encoding a plant abortive intron; modifying said coding region in a translationally neutral manner to obtain a modified coding region in which said at least one DNA sequence encoding a plant abortive intron is inactivated; and introducing said modified coding region into said plant cell under the control of a promoter functional in a plant cell. 17. A process for protecting a plant from an insect pest, comprising the steps of: transforming the genome of the plant with a chimeric gene which comprises the following operably-linked DNA fragments: (1) a promoter for directing transcription in plant cells; (2) a modified coding region obtained by the process of claim 1; (3) a transcript 3' end formation and polyadenylation region functional in plant cells. 18. The process of claim 17, wherein said promoter is a 35S promoter, a TR1' promoter, or a TR2' promoter. 19. The process of claim 17, wherein said transcript 3' end formation and polyadenylation region is from the chalcone synthase gene. 20. A modified Bt ICP region, obtained from a native Bt ICP coding region by the process of claim 1 wherein said coding region of said Bt ICP gene is modified by making translationally neutral replacements in the DNA sequence encoding a splice site of said abortive intron. 21. A modified Bt ICP coding region of claim 20, wherein said modified Bt ICP coding region encodes a protein which is truncated at its N-and/or C-terminal end. 22. A modified Bt ICP coding region obtained from a native Bt ICP coding region by the process of claim 13 wherein the coding region of said Bt ICP gene is modified by making translationally neutral nucleotide replacements in said cryptic promoter and making translationally neutral nucleotide replacements in the DNA sequence encoding a splice site of said abortive intron. 23. A modified Bt ICP coding region obtained from a native Bt ICP coding region by the process of claim 1, wherein said at least one DNA sequence encoding an abortive intron is inactivated by making translationally neutral nucleotide replacements in said native Bt ICP coding region in about 3 to about 10 nucleotides on either side of a 5' and a 3' abortive intron splice site. 24. A modified Bt ICP coding region obtained from a native Bt ICP coding region by the process of claim 13, wherein said at least one cryptic promoter is inactivated by making translationally neutral modifications in said native Bt ICP coding region of 20 to 90 nucleotides upstream of the experimentally determined transcription initiation site of said cryptic promoter and said abortive intron is inactivated by making translationally neutral nucleotide replacements in a DNA sequence encoding a splice site of said abortive intron. 25. A modified Bt ICP coding region, obtained from a native Bt ICP coding region by making translationally neutral nucleotide replacements in the abortive intron 5' splice site motifs AGT:GTAAGT, CGG:GTAAGA and AAG:GTGGGG. 26. A plant cell, transformed to comprise the modified Bt ICP coding region of claim 20. 27. A plant, modified to comprise the modified Bt ICP coding region of claim 20.

Details for Patent 5,952,547

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2016-09-14
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2016-09-14
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2016-09-14