Claims for Patent: 5,599,670

Summary for Patent: 5,599,670

Title:	.beta.-glucuronidase and glucuronide permease gene system
Abstract:	The present invention relates to the .beta.-glucuronidase (GUS) gene fusion system, and to the cloning and characterization of the .beta.-glucuronidase and glucuronide permease genes of Escherichia coli. It is based on the surprising discovery that gene fusions comprising the .beta.-glucuronidase gene may be effectively expressed in a wide variety of organisms to produce active .beta.-glucuronidase enzyme. Because of the abundance and availability of useful substrates for .beta.-glucuronidase enzyme, GUS gene fusions may serve as a superior reporter gene system as well as an effective means of altering cellular phenotype. In conjunction with recombinant glucuronide permease, which may be used to render host cells permeable to .beta.-glucuronidase substrates, the GUS gene fusion system offers almost unlimited applications in the fields of plant and animal genetic engineering.
Inventor(s):	Jefferson; Richard A. (Canberra, AU)
Assignee:	Cambia Biosystems. L.L.C. (Canberra, AU)
Application Number:	08/329,701
Patent Claims:	1. A method of introducing a gene of interest or portion thereof into a host cell, comprising introducing into the host cell a DNA construct, the DNA construct comprising DNA encoding beta-glucuronidase and DNA encoding a product of the gene of interest or portion thereof, wherein the DNA encoding beta-glucuronidase (a) encodes a protein comprising the amino acid sequence as depicted in FIG. 2 or (b) hybridizes under stringent conditions to the complement of nucleotides 300 to 2105 as depicted in FIG. 2 and which encodes a functional beta-glucuronidase. 2. The method according to claim 1, wherein the host cell is a plant cell. 3. The method according to claim 1, wherein the host cell is an animal cell. 4. The method according to claim 1, wherein the host cell is a fungal cell. 5. The method according to claim 1, wherein the host cell is a bacterial cell. 6. The method according to claim 1, wherein the DNA construct is a plasmid vector. 7. The method according to claim 6, wherein the plasmid vector is a binary Agrobacterium vector. 8. The method according to claim 1, wherein the DNA construct further comprises a selectable marker. 9. The method according to claim 8, wherein the selectable marker confers resistance to kanamycin. 10. The method according to claim 1, wherein the product is a protein. 11. A method of introducing a controller element into a host cell, comprising introducing into the host cell a DNA construct, the DNA construct comprising DNA encoding beta-glucuronidase and a DNA sequence of the controller element, wherein the DNA encoding beta-glucuronidase (a) encodes a protein comprising the amino acid sequence as depicted in FIG. 2 or (b) hybridizes under stringent conditions to the complement of nucleotides 300 to 2105 as depicted in FIG. 2 and which encodes a functional beta-glucuronidase, and wherein the DNA encoding beta-glucuronidase is under direction of controller element. 12. The method according to claim 11, wherein the host cell is a plant cell. 13. The method according to claim 11, wherein the host cell is an animal cell. 14. The method according to claim 11, wherein the host cell is a fungal cell. 15. The method according to claim 11, wherein the host cell is a bacterial cell. 16. The method according to claim 11, wherein the DNA construct is a plasmid vector. 17. The method according to claim 16, wherein the plasmid vector is a binary Agrobacterium vector. 18. The method according to claim 11, wherein the DNA construct further comprises a selectable marker. 19. The method according to claim 18, wherein the selectable marker confers resistance to kanamycin. 20. The method according to claim 11, wherein the controller element is selected from the group consisting of a promoter, an enhancer, an operator, a ribosome binding site, a signal peptide sequence, a chloroplast targeting sequence, a mitochondrial localization sequence, a nucleus targeting sequence and an intron. 21. The method according to claim 11, wherein the controller element is a plant promoter. 22. The method according to claim 11, wherein the controller element is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter. 23. A method of monitoring expression of a gene of interest or a portion thereof in a host cell, comprising: (a) introducing into the host cell a DNA construct, the DNA construct comprising DNA encoding beta-glucuronidase and DNA encoding a product of the gene of interest or a portion thereof; wherein the DNA encoding beta-glucuronidase (a) encodes a protein comprising the amino acid sequence as depicted in FIG. 2 or (b) hybridizes under stringent conditions to the complement of nucleotides 300 to 2105 as depicted in FIG. 2 and which encodes a functional beta-glucuronidase, and (b) detecting the presence of beta-glucuronidase activity, thereby monitoring expression of the gene of interest. 24. The method according to claim 23, wherein the host cell is a plant cell. 25. The method according to claim 23, wherein the host cell is an animal cell. 26. The method according to claim 23, wherein the host cell is a fungal cell. 27. The method according to claim 23, wherein the host cell is a bacterial cell. 28. The method according to claim 23, wherein the DNA construct is a plasmid vector. 29. The method according to claim 28, wherein the plasmid vector is a binary Agrobacterium vector. 30. The method according to claim 23, wherein the DNA construct further comprises a selectable marker. 31. The method according to claim 30, wherein the selectable marker confers resistance to kanamycin. 32. The method according to claim 23, wherein the product is a protein. 33. The method according to claim 23, wherein the DNA construct further comprises a promoter. 34. The method according to claim 33, wherein the DNA encoding the product and the DNA encoding beta-glucuronidase are under control of the same promoter. 35. The method according to claim 34, wherein the DNA encoding the product and the DNA encoding beta-glucuronidase are transcribed on a same RNA molecule. 36. The method according to claim 33, wherein the order of the DNA construct is promoter sequence, gene of interest, and beta-glucuronidase. 37. A method of monitoring activity of a controller element in a host cell, comprising: (a) introducing into the host cell a DNA construct, the DNA construct comprising DNA encoding beta-glucuronidase and a DNA sequence of the controller element; wherein the DNA encoding beta-glucuronidase (a) encodes a protein comprising the amino acid sequence as depicted in FIG. 2 or (b) hybridizes under stringent conditions to the complement of nucleotides 300 to 2105 as depicted in FIG. 2 and which encodes a functional beta-glucuronidase, and wherein the DNA encoding beta-glucuronidase is under direction of the controller element; and (b) detecting the presence of beta-glucuronidase activity, thereby monitoring activity of the controller element. 38. The method according to claim 37, wherein the host cell is a plant cell. 39. The method according to claim 37, wherein the host cell is an animal cell. 40. The method according to claim 37, wherein the host cell is a fungal cell. 41. The method according to claim 37, wherein the host cell is a bacterial cell. 42. The method according to claim 37, wherein the DNA construct is a plasmid vector. 43. The method according to claim 42, wherein the plasmid vector is a binary Agrobacterium vector. 44. The method according to claim 37, wherein the DNA construct further comprises a selectable marker. 45. The method according to claim 44, wherein the selectable marker confers resistance to kanamycin. 46. The method according to claim 37, wherein the controller element is selected from the group consisting of a promoter, an enhancer, an operator, a ribosome binding site, a signal peptide sequence, a chloroplast targeting sequence, a mitochondrial localization sequence, a nucleus targeting sequence and an intron. 47. The method according to claim 37, wherein the controller element is a plant promoter. 48. The method according to claim 37, wherein the controller element is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter. 49. A method for transforming a host cell with a gene of interest or portion thereof, comprising: (a) introducing into the host cell a DNA construct, file DNA construct comprising DNA encoding beta-glucuronidase mad DNA encoding a product of the gene of interest or a portion thereof, such that the DNA construct integrates into the genome of the host cell; wherein the DNA encoding beta-glucuronidase (i) encodes a protein comprising the amino acid sequence as depicted in FIG. 2 or (ii) hybridizes under stringent conditions to the complement of nucleotides 300 to 2105 as depicted in FIG. 2 and which encodes a functional beta-glucuronidase; and (b) detecting the presence of beta-glucuronidase activity, thereby establishing that the host cell is transformed. 50. The method according to claim 49, wherein the host cell is a plant cell. 51. The method according to claim 49, wherein the host cell is an animal cell. 52. The method according to claim 49, wherein the host cell is a fungal cell. 53. The method according to claim 49, wherein the host cell is a bacterial cell. 54. The method according to claim 49, wherein the DNA construct is a plasmid vector. 55. The method according to claim 49, wherein the plasmid vector is a binary Agrobacterium vector. 56. The method according to claim 49, wherein the DNA construct further comprises a selectable marker. 57. The method according to claim 56, wherein the selectable marker confers resistance to kanamycin. 58. The method according to claim 49, wherein the product is a protein. 59. The method according to claim 49, wherein the DNA construct further comprises a promoter. 60. The method according to claim 59, wherein the gene of interest and beta-glucuronidase are under control of the same promoter. 61. The method according to claim 60, wherein the gene of interest and beta-glucuronidase are transcribed on a same RNA molecule. 62. A recombinant DNA molecule comprising DNA encoding a glucuronide permease, wherein the DNA encoding the glucuronide permease comprises the amino acid sequence as depicted in FIG. 15, or hybridizes under stringent conditions to the complement of the sequence comprising nucleotides 94-1464 as depicted in FIG. 15 and which encodes a functional glucuronide permease. 63. The recombinant DNA molecule according to claim 62, wherein the DNA encoding glucuronide permease comprises the nucleotide sequence as depicted in FIG. 15 from about nucleotide number 94 to about nucleotide number 1464. 64. The recombinant DNA molecule according to claim 62, wherein the DNA encoding glucuronide permease comprises the nucleotide sequence as depicted in FIG. 15 from about nucleotide number 106 to about nucleotide number 1464. 65. A vector, comprising a recombinant DNA molecule according to any one of claims 62, 63, or 64, wherein the glucuronide permease is under direction of a controller element. 66. The vector according to claim 65, wherein the controller element is a plant promoter. 67. The vector according to claim 65, wherein the controller element is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter. 68. A host cell comprising the vector according to claim 65. 69. The host cell according to claim 68, wherein the host cell is a plant cell. 70. The host cell according to claim 68, wherein the host cell is an animal cell. 71. The host cell according to claim 68, wherein the host cell is a fungal cell. 72. The host cell according to claim 68, wherein the host cell is a bacterial cell. 73. A method of releasing a compound from a glucuronide in a host cell, comprising: (a) introducing into the host cell a DNA construct, the DNA construct comprising DNA encoding beta-glucuronidase; wherein the DNA encoding beta-glucuronidase (a) encodes a protein comprising the amino acid sequence as depicted in FIG. 2 or (b) hybridizes under stringent conditions to the complement of nucleotides 300 to 2105 as depicted in FIG. 2 and which encodes a functional beta-glucuronidase; and (b) exposing the host cell to the glucuronide, wherein the glucuronide is cleaved by the beta-glucuronidase, such that the compound is released. 74. The method according to claim 73, wherein the compound is an auxin. 75. The method according to claim 74, wherein the auxin is indole-3-ethanol. 76. The method according to claim 73, wherein the compound is a hormone. 77. The method according to claim 73, wherein the compound is a toxin. 78. The method according to claim 73, wherein the host cell is a plant cell. 79. The method according to claim 73, wherein the host cell is an animal cell. 80. The method according to claim 73, wherein the host cell is a fungal cell. 81. The method according to claim 73, wherein the host cell is a bacterial cell. 82. The method according to claim 73, wherein the compound is a herbicide. 83. A method of releasing a compound from a glucuronide in a host cell expressing endogenous beta-glucuronidase, comprising: (a) introducing into the host cell expressing endogenous beta-glucuronidase a DNA construct, the DNA construct comprising DNA encoding glucuronide permease, wherein the DNA encoding the glucuronide permease comprises the amino acid sequence as depicted in FIG. 15, or hybridizes trader stringent conditions to the complement of the sequence comprising nucleotides 94-1464 as depicted in FIG. 15 and which encodes a functional glucuronide permease; and (b) exposing the cell to a glucuronide, wherein the glucuronide is cleaved by the beta-glucuronidase, such that the compound is released. 84. The method according to claim 83, wherein the compound is a hormone. 85. The method according to claim 83, wherein the compound is a toxin. 86. The method according to claim 83, wherein the compound is an antibiotic. 87. The method according to claim 83, wherein the compound is a drug.

Details for Patent 5,599,670

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		2014-02-04
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2014-02-04
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2014-02-04