Claims for Patent: 6,391,547

Summary for Patent: 6,391,547

Title:	Microbial .beta.-glucuronidase genes, gene products and uses thereof
Abstract:	Genes encoding microbial .beta.-glucuronidase and protein that is secreted and its uses are provided.
Inventor(s):	Jefferson; Richard A. (Googong, AU), Harcourt; Rebecca Louise (Sydney, AU), Kilian; Andrzej (Kaleen, AU), Wilson; Katherine Joanna (Townsville, AU), Keese; Paul Konrad (Curtin, AU)
Assignee:	Center for the Application of Molecular Biology to International Agriculture (Canberra, AU)
Application Number:	09/149,727
Patent Claims:	1. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a secreted form of microbial Bacillus .beta.-glucuronidase. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises nucleotides 1662-3467 of FIG. 1 (SEQ ID NO: 1) or hybridizes under stringent conditions to the complement of the sequence comprising nucleotides 1662-3467 of FIG. 1 (SEQ ID NO: 1), and which encodes a functional .beta.-glucuronidase. 3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes the amino acid sequence of FIG. 3 (SEQ ID NO: 2), or a variant thereof, and which encodes a functional .beta.-glucuronidase. 4. The nucleic acid molecule of claim 3, further comprising a second nucleic acid molecule that encodes the amino acid sequence of FIG. 4A (SEQ ID NO: 4), or a variant thereof, and wherein the second nucleic acid molecule is fused to the 5' end of the nucleic acid molecule encoding .beta.-glucuronidase. 5. An isolated nucleic acid molecule encoding a membrane-bound form of Bacillus .beta.-glucuronidase. 6. The nucleic acid molecule of claim 5, further comprising a sequence encoding an outer membrane-spanning peptide fused to the sequence encoding .beta.-glucuronidase. 7. A vector, comprising a nucleic acid molecule encoding a secreted form of a microbial Bacillus .beta.-glucuronidase, wherein the .beta.-glucuronidase sequence is in operative linkage with a promoter element. 8. The vector of claim 7, wherein the vector is a binary Agrobacterium tumefaciens plasmid vector. 9. The vector of claim 7, wherein the promoter 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. 10. The vector of claim 9, wherein the promoter element is selected from a group consisting of a promoter functional in a plant cell, a promoter functional in a bacterium, a promoter functional in an animal cell and a promoter functional in a fungal cell. 11. The vector of claim 7, wherein the vector is functional in a bacterium. 12. The vector of claim 7, further comprising a nucleic acid sequence encoding a product of a gene of interest or portion thereof. 13. The vector of claim 12, wherein the product is a protein. 14. A host cell containing the vector according to claim 7. 15. The host cell of claim 14, wherein the host cell is selected from the group consisting of a plant cell, an insect cell, a fungal cell, an animal cell and a bacterial cell. 16. A method of introducing a controller element into a host cell, comprising introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial .beta.-glucuronidase and a nucleic acid sequence controller element, wherein the nucleic acid sequence encoding the .beta.-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 (SEQ ID NO: 2) or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 (SEQ ID NO: 1), and which encodes a functional beta-glucuronidase, and wherein the nucleic acid sequence encoding .beta.-glucuronidase is in operative linkage with the controller element. 17. The method according to claim 16, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell. 18. The method according to claim 16, wherein the vector construct is a binary Agrobacterium vector. 19. The method according to claim 16, 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. 20. The method according to claim 19, wherein the controller element is functional in a plant cell. 21. The method according to claim 19, 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. 22. A method of monitoring expression of gene of interest or a portion thereof in a host cell comprising: (a) introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial .beta.-glucuronidase and nucleic acid sequence encoding a product of the gene of interest or a portion thereof; wherein the nucleic acid sequence encoding the microbial .beta.-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 (SEQ ID NO: 2) or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 (SEQ ID NO: 1) and which encodes a functional .beta.-glucuronidase, and (b) detecting the presence of secreted microbial .beta.-glucuronidase, thereby monitoring expression of the gene of interest. 23. The method according to claim 22, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell. 24. The method according to claim 22, wherein the product is a protein. 25. The method according to claim 22, wherein the vector construct further comprises a promoter. 26. The method according to claim 22, wherein the nucleic acid sequence encoding the product and the nucleic acid sequence encoding .beta.-glucuronidase are in operative linkage with the same promoter. 27. A method of monitoring activity of a controller element in a host cell, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial .beta.-glucuronidase and a nucleic acid sequence of the controller element; wherein the nucleic acid sequence encoding the .beta.-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 (SEQ ID NQ:2) or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 (SEQ ID NO:1) and which encodes a functional .beta.-glucuronidase, and wherein the nucleic acid sequence encoding .beta.-glucuronidase is in operative linkage with the controller element; and (b) detecting the presence of secreted .beta.-glucuronidase, thereby monitoring activity of the controller element. 28. The method according to claim 27, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell. 29. The method according to claim 27, wherein the vector construct is a binary Agrobacterium vector. 30. The method according to claim 27, 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. 31. The method according to claim 27, wherein the controller element is a promoter functional in a plant cell. 32. A method for transforming a host cell with a gene of interest or portion thereof, comprising: (a) introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial .beta.-glucuronidase and nucleic acid sequence encoding a product of the gene of interest or a portion thereof, such that the vector construct integrates into the genome of the host cell; wherein the nucleic acid sequence encoding .beta.-glucuronidase (i) encodes a protein comprising the amino acid sequence of FIG. 3 (SEO ID NO: 2) or (ii) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 (SEQ ID NO: 1) and which encodes a functional .beta.-glucuronidase; and (c) detecting the presence of secreted .beta.-glucuronidase, thereby establishing that the host cell is transformed. 33. The method according to claim 32, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell. 34. The method according to claim 32, wherein the vector construct is a binary Agrobacterium vector. 35. The method according to claim 32, wherein the product is a protein. 36. The method according to claim 32, wherein the vector construct further comprises a promoter. 37. The method according to claim 32, wherein the gene of interest and .beta.-glucuronidase are under control of the same promoter.

Details for Patent 6,391,547

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		2017-06-25
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2017-06-25
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2017-06-25