Claims for Patent: 5,861,277

Summary for Patent: 5,861,277

Title:	Methods and compositions for enhancing the expression of genes in plants
Abstract:	Disclosed are methods of increasing exogenous protein expression in a cell or a transgenic plant. Constructs, i.e., vectors, DNA fusions and polynucleotides, for use in conjunction with the methods to cause increased exogenous protein expression are also disclosed. These constructs generally include intron 1 and/or intron 2 of the PAT1 gene. Additionally disclosed are cells, including recombinant cells, and plant lines transformed with the described constructs. In particular, a cultivated, transgenic food plant, the genome of which has been augmented through the genomic introduction of a preselected exogenous protein gene not found in the genome of non-transformed parentage of the plant is described. Also described are seed, progeny and cells of the described transgenic food plant.
Inventor(s):	Rose; Alan B. (Davis, CA), Last; Robert L. (Ithaca, NY)
Assignee:	Boyce Thompson Institute for Plant Research, Inc. (Ithaca, NY)
Application Number:	08/723,624
Patent Claims:	1. A method of increasing exogenous protein expression in a cell, said method comprising the steps of: a) constructing a DNA fusion comprising intron 1 or intron 2 of the PAT1 gene operatively linked to a DNA segment from a gene other than the PAT1 gene encoding the exogenous protein one desires to express in the cell; and b) introducing said fusion into a host cell so that the cell expresses the fusion. 2. The method of claim 1, wherein said cell is a plant cell. 3. The method of claim 1, wherein said DNA fusion comprises intron 1 of the PAT1 gene. 4. The method of claim 1, wherein said DNA fusion comprises intron 2 of the PAT1 gene. 5. The method of claim 1, wherein said DNA fusion comprises intron 1 and intron 2 of the PAT1 gene. 6. The method of claim 1, wherein said DNA fusion comprises a modified intron 1 of the PAT1 gene having the sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. 7. A method of increasing exogenous protein expression in a transgenic plant, said method comprising the steps of: a) constructing a DNA fusion comprising intron 1 or intron 2 of the PAT1 gene operatively linked to a DNA segment from a gene other than the PAT1 gene encoding the exogenous protein one desires to express in b) the transgenic plant; and expressing said fusion in said plant. 8. The method of claim 7, wherein said DNA segment encodes a reporter gene in accordance with Table 1. 9. The method of claim 7, wherein said DNA segment encodes a protein of pharmacological interest. 10. The method of claim 9, wherein said DNA segment encodes an animal vaccine gene in accordance with Table 2. 11. The method of claim 9, wherein said DNA segment encoding exogenous protein encodes a human vaccine gene in accordance with Table 2. 12. The method of claim 11, wherein said DNA segment encodes Hepatitis B surface antigen. 13. The method of claim 7, wherein said plant is further defined as a tobacco plant, a potato plant, a banana plant, a tomato plant, or any member of the plant genus Brassica. 14. The method of claim 13, wherein said plant is further defined as a potato plant. 15. The method of claim 7, wherein said DNA fusion is introduced into said plant by Agrobacterium tumefaciens-mediated transformation by vacuum filtration. 16. The method of claim 7, wherein said DNA fusion further comprises a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3, and intron 1 or intron 2 of the PAT1 gene. 17. The method of claim 16, wherein said DNA fusion further comprises a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3, and intron 1 of the PAT1 gene. 18. The method of claim 16, wherein said DNA fusion further comprises a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3, and intron 2 of the PAT1 gene. 19. The method of claim 16, wherein said DNA fusion further comprises a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3 and introns 1 and 2 of the PAT1 gene. 20. The method of claim 16, wherein said DNA segment encodes a reporter gene. 21. The method of claim 20, wherein said DNA fusion further comprises a DNA segment is further defined as encoding .beta.-glucuronidase (GUS) reporter gene. 22. The method of claim 21, wherein said GUS reporter gene is derived from pBI101, pBI121, pBI221, or pCTGus. 23. The method of claim 7, wherein said DNA segment encodes a protein of pharmacological interest. 24. The method of claim 23, wherein said DNA segment encodes an animal vaccine gene in accordance with Table 2. 25. The method of claim 23, wherein said DNA segment encodes a human vaccine gene in accordance with Table 2. 26. The method of claim 25, wherein said DNA segment encodes Hepatitis B surface antigen. 27. The method of claim 7, wherein said DNA fusion further comprises a DNA segment encoding an exogenous protein operatively linked to exon 3 and intron 2 of the PAT1 gene. 28. The method of claim 7, wherein said DNA fusion further at least one intron and a DNA segment encoding the entire chloroplast transit peptide of the PAT1 gene. 29. The method of claim 28, wherein said DNA fusion further comprises a DNA segment encoding chloroplast transit peptide of PAT1 is further defined as comprising exon 1 and at least 10 codons of exon 2 of the PAT1 gene. 30. The method of claim 7, wherein said DNA fusion further comprises a DNA segment comprising at least one intron and a DNA segment comprising at least 60 codons of the chloroplast transit peptide of the PAT1 gene. 31. A recombinant vector comprising a DNA segment encoding an exogenous protein gene, other than the product of the PAT1 gene, operatively linked to a gene segment comprising intron 1 or intron 2 of the PAT1 gene. 32. The vector of claim 31, wherein said DNA segment encoding exogenous protein encodes a reporter gene in accordance with Table 1. 33. The vector of claim 32, wherein said DNA segment encoding reporter gene is further defined as encoding .beta.-glucuronidase (GUS) reporter gene. 34. The vector of claim 33, wherein said GUS reporter gene is derived from the vector pBI101, pBI121, pBI221 or pCTGus. 35. A DNA fusion comprising a DNA segment encoding an exogenous protein, other than the product of the PAT1 gene, operatively linked to exons 1 and 2, 8 codons of exon 3, and intron 1 or intron 2 of the PAT1 gene. 36. The DNA fusion of claim 35, comprising a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3, and intron 1 of the PAT1 gene. 37. The DNA fusion of claim 35, comprising a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3, and intron 2 of the PAT1 gene. 38. The DNA fusion of claim 35 comprising a DNA segment encoding an exogenous protein operatively linked to exons 1 and 2, 8 codons of exon 3 and introns 1 and 2 of the PAT1 gene. 39. The DNA fusion of claim 35, wherein said DNA segment encodes a reporter gene. 40. The DNA fusion of claim 39, wherein said DNA segment is furter defined as encoding .beta.-glucuronidase (GUS) reporter gene. 41. The DNA fusion of claim 40, wherein said GUS reporter gene is derived from pBI101, pBI121, pBI221, or pCTGus. 42. The DNA fusion of claim 35, wherein said DNA segment encodes a protein of pharmacological interest. 43. The DNA fusion of claim 42, wherein said DNA segment encodes an animal vaccine gene in accordance with Table 2. 44. The DNA fision of claim 42, wherein said DNA segment encodes a human vaccine gene in accordance with Table 2. 45. The DNA fusion of claim 44, wherein said DNA segment encodes Hepatitis B surface antigen. 46. A DNA fusion comprising a DNA segment encoding an exogenous protein, other than the product of the PAT1 gene, operatively linked to exon 3 and intron 2 of the PAT1. 47. A DNA fusion consisting essentially of at least one intron and a DNA segment encoding the entire chloroplast transit peptide of the PAT1 gene. 48. The DNA fusion of claim 47, wherein said DNA segment encoding chloroplast transit peptide of PAT1 is further defined as comprising exon 1 and at least 10 codons of exon 2 of the PAT1 gene. 49. A DNA fusion consisting essentially of at least one intron and a DNA segment comprising at least 60 codons of the chloroplast transit peptide of the PAT1 gene. 50. An isolated polynucleotide of the PAT1 gene consisting essentially of exon 1, 2 or 3 and intron 1 or intron 2 of the PAT1 gene. 51. The polynucleotide of claim 50, wherein said polynucleotide comprises intron 1 of the PAT1 gene. 52. The polynucleotide of claim 51, wherein said polynucleotide further comprises exon 1 of the PAT1 gene. 53. The polynucleotide of claim 50, wherein said polynucleotide comprises intron 2 of the PAT1 gene. 54. The polynucleotide of claim 53, wherein said polynucleotide farther comprises exon 3 of the PAT1 gene. 55. The polynucleotide of claim 50, wherein said polynucleotide comprises intron 1 and intron 2 of the PAT1 gene. 56. The polynucleotide of claim 55, wherein said polynucleotide further comprises exon 3 of the PAT1 gene. 57. An expression vector comprising a polynucleotide in accordance with any one of claims 51-56. 58. A cell transformed with the expression vector of claim 57. 59. The cell of claim 58, wherein said cell is further defined as a plant cell. 60. The cell of claim 59, wherein said cell is further defined as a wild-type Arabidopsis cell. 61. A construct that increases the expression of exogenous protein RNA in transgenic plants, said construct comprising exons 1 and 2, introns 1 and 2, and 8 codons of exon 3 of the PAT1 gene operatively linked to a DNA segment encoding the exogenous protein one desires to express, wherein the exogenous protein is a protein other than the PAT1 gene product. 62. A plant cell transformed with a DNA fusion in accordance with any one of claims 21-35, wherein said cell comprises an increased number of exogenous protein RNAs in comparison to a plant cell not transformed with said transgene, wherein the exogenous protein RNA is not PAT1 RNA. 63. A recombinant host cell comprising an exogenous fusion protein, wherein said fusion comprises a DNA fusion in accordance with any one of claims 35-49. 64. The recombinant host cell of claim 63, wherein said exogenous fusion protein is introduced into said cell by means of a recombinant vector. 65. A recombinant host cell comprising a DNA fusion in accordance with any one of claims 35-49. 66. The recombinant host cell of claim 65, wherein said DNA fusion is introduced into said cell by means of a recombinant vector. 67. A method of expression comprising fusing a portion of the PAT1 gene operatively linked with a DNA segment encoding an exogenous protein one desires to express, wherein said DNA segment does not encode the PAT1 gene product, and introducing said fusion into wild-type Arabidopsis. 68. The method of claim 67, wherein said fusion comprises exon 1 and exon 2, 8 codons of exon 3 and intron 1 or intron 2 of the PAT1 gene. 69. The method of claim 68, wherein said fusion comprises exon 1, intron 1, exon 2, and 8 codons of exon 3 of the PAT1 gene. 70. The method of claim 68, wherein said fusion comprises exon 1, exon 2, intron 2, and 8 codons of exon 3 of the PAT1 gene. 71. The method of claim 68, wherein said fusion comprises exon 1, intron 1, exon 2 intron 2 and 8 codons of exon 3 of the PAT1 gene. 72. The method of claim 67, wherein said fusion is introduced into said Arabidopsis by means of Agrobacterium tumefaciens-mediated vacuum filtration method. 73. The method of claim 67, wherein said fusion is introduced into said Arabidopsis by means of the root explant method. 74. A transformed plant line comprising a DNA fusion in accordance with any one of claims 35-49, wherein said line contains single and unique site of transgene insertion. 75. A transformed plant line comprising a transgene encoding the entire chloroplast transit peptide of PAT1, operatively linked to a transgene, other than the PAT1 gene. 76. The plant line of claim 75, farther comprising intron 1 of the PAT1 gene. 77. The plant line of claim 76, wherein said transgene encoding transit peptide is further defined as having the nucleotide sequence of SEQ ID NO:1. 78. The plant line of claim 76 prepared by the process of creating a translational fusion protein gene and introducing said fusion into a plant. 79. The plant line of claim 78, wherein said plant is further defined as a wild-type Arabidopsis. 80. The plant line of claim 78, wherein said plant is further defined as a food plant. 81. The plant line of claim 80, wherein said plant is further defined as a tobacco plant, potato plant, a banana plant, a tomato plant, or any member of the plant genus Brassica. 82. The plant line of claim 81, wherein said plant is further defined as a potato plant. 83. A transformed plant line comprising a transgene comprising an exogenous protein gene, other than the PAT1 gene, operatively linked to exons 1 and 2, intron 1 or intron 2 and 8 codons of exon 3 of the PAT1 gene. 84. The plant line of claim 83, prepared by the process of creating a translational fusion protein gene and introducing said fusion into a plant. 85. The plant line of claim 83, wherein said fusion is further defined as comprising intron 1 of the PAT1 gene. 86. The plant line of claim 83, wherein said fusion is further defined as comprising intron 2 of the PAT1 gene. 87. The plant line of claim 83, wherein said fusion is further defined as comprising intron 1 and intron 2 of the PAT1 gene. 88. A cultivated, transgenic food plant, the genome of which has been augmented through the genomic introduction of a preselected exogenous protein gene not found in the genome of non-transformed parentage of said plant, the plant preparable by a process that includes the steps of: a) preparing a nucleic acid composition including the exogenous protein gene one desires to introduce into the genome of a food plant, said gene operatively linked to at least one intron of the PAT1 gene; b) contacting recipient food cells with said composition under conditions allowing the uptake of the exogenous protein gene by recipient cells; c) regenerating food plants from recipient cells which have received the exogenous protein gene; and d) identifying a fertile, transgenic food plant whose genome has been augmented relative to that of the corresponding nontransgenic recipient cells through the stable introduction of said exogenous protein gene, wherein said exogenous protein is not the PAT1 gene. 89. Progeny of the plant of claim 88. 90. Seed obtained from the plant of claim 88. 91. Cells obtained from the plant of claim 88.

Details for Patent 5,861,277

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		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26