Microbial β-glucuronidase genes, gene products and uses thereof
AssigneeCambia
InventorsRichard A. Jefferson, Jorge E. Mayer
Life of this patent
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Abstract
Genes encoding microbial β-glucuronidases and proteins and their uses are provided.
Claims19
An isolated nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:14) or a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.
An isolated nucleic acid molecule that encodes one of the amino acid sequences of SEQ ID NOs.:
- 19-21, or a variant thereof wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.:
- 19-21 and which encodes a functional β-glucuronidase.
An expression vector comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the β-glucuronidase is encoded by a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:
- 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase.
An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises one of the amino acid sequences of SEQ ID NOs.:
- 19-21, or variant thereof, wherein the variant has at least 75% amino acid identity to one of SEQ ID NOs.:
- 19-21, and which encodes a functional β-glucuronidase.
A method for monitoring expression of a 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 a nucleic acid molecule comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:
- 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:
- 14) and which encodes functional β-glucuronidase and a nucleic acid molecule encoding a product of the gene of interest;
- wherein the β-glucuronidase and the gene of interest are co-expressed;
- (b) detecting the presence of the microbial β-glucuronidase, thereby monitoring expression of the gene of interest.
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 a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:
- 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase, and a nucleic acid sequence encoding a product of the gene of interest, such that the vector construct integrates into the genome of the host cell;
- wherein the β-glucuronidase and the gene of interest are co-expressed;
- (b) detecting the presence of the microbial β-glucuronidase, thereby establishing that the host cell is transformed.
A method for positive selection for a transformed cell, comprising:
- (a) introducing into a host cell a vector construct, the vector construct comprising a nucleic acid sequence comprising nucleotides 1-1689 of FIGS. 4I-J (SEQ ID NO:
- 14) or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1-1689 of FIG. 4I-J (SEQ ID NO:14) and which encodes a functional β-glucuronidase;
- (b) exposing the host cell to a sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that an aglycone is released, wherein the aglycone is required for growth of the host cell;
- wherein a host cell that expresses the β-glucuronidase grows, thereby positively selecting a transformed cell.
An isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:
- 22, or a variant thereof wherein the variant has at least 90% amino acid identity to SEQ ID NO:
- 22 and which encodes a functional β-glucuronidase.
An expression vector, comprising a nucleic acid sequence encoding a microbial β-glucuronidase in operative linkage with a heterologous promoter, wherein the microbial β-glucuronidase comprises the amino acid sequence of SEQ ID NO:
- 22, or variant thereof, wherein the variant has at least 90% amino acid identity to SEQ ID NO:
- 22, and which encodes a functional β-glucuronidase.
Dependencies are inferred from the claim text.