Cytosolic forms of beta-lactamase and uses thereof
AssigneeThe Regents of the University of California
InventorsRoger Y. Tsien, Gregor Zlokarnik
Life of this patent
Estimating the term…
Abstract
The present invention is directed to nucleic acid molecules that encode a cytosolic form of beta-lactamase and cells that include such nucleic acid molecules. These cells can be used in a variety of methods, such as methods for monitoring the expression of a gene.
Claims24
A method of determining the amount of beta-lactamase activity in a cell, comprising the steps of:
- a) providing a mammalian host cell transfected with a recombinant nucleic acid molecule comprising an expression control sequence adapted for function in a mammalian cell operatively linked to a nucleic acid sequence coding for the expression of a cytosolic form of beta-lactamase,
- wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion,
- b) contacting a sample comprising said mammalian host cell or an extract of said mammalian host cell with a substrate for beta-lactamase, and
- c) determining the amount of said substrate for beta-lactamase cleaved, whereby the amount of said substrate for beta-lactamase cleaved is related to the amount of beta-lactamase activity.
A method for monitoring the expression of a mammalian gene, comprising:
- a) providing a mammalian host cell transfected with a recombinant nucleic acid molecule comprising an expression control sequence adapted for function in said mammalian host cell operatively linked to a nucleic acid sequence coding for the expression of a cytosolic form of beta-lactamase, wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion,
- b) contacting a sample comprising said mammalian host cell or an extract of said mammalian host cell with a substrate for beta-lactamase, and
- c) determining cleavage of the substrate, whereby the cleavage of substrate indicates expression.
A method for determining whether a test compound alters the expression of a gene operably linked to a set of expression control sequences, comprising:
- a) providing a cell transfected with a recombinant nucleic acid construct comprising an expression control sequence adapted for function in said cell operably linked to a nucleic acid sequence coding for the expression of a cytosolic form of beta-lactamase, wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion,
- b) contacting the cell with the test compound,
- c) contacting a sample comprising said cell or an extract of said cell with a beta-lactamase substrate, and
- d) determining cleavage of said beta-lactamase substrate, whereby the cleavage of said beta-lactamase substrate indicates whether the test compound alters expression.
A recombinant nucleic acid molecule, comprising:
- an expression control sequence adapted for function in a vertebrate cell operably linked to a nucleotide sequence encoding a cytosolic form of beta-lactamase,
- wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion.
A recombinant nucleic acid molecule, comprising:
- an expression control sequence adapted for function in an eukaryotic cell operably linked to a polynucleotide comprising a nucleotide sequence encoding a cytosolic beta-lactamase,
- wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion.
A cell, comprising a mammalian host cell and a recombinant nucleic acid molecule, comprising:
- an expression control sequence adapted for function in a mammalian cell and operably linked to a polynucleotide comprising a nucleotide sequence encoding a cytosolic form of beta-lactamase,
- wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion.
A recombinant nucleic acid molecule, comprising:
- a nucleic acid molecule encoding a cytosolic beta-lactamase operatively linked to a mammalian expression control sequence,
- wherein said nucleotide sequence encoding a cytosolic form of beta-lactamase lacks a nucleotide sequence encoding an amino acid sequence for secretion.
A mammalian cell, comprising:
- a recombinant nucleic acid molecule encoding a cytosolic form of a beta-lactamase operatively linked to a mammalian expression control sequence, wherein said beta-lactamase is functional and localized in the cytosol of said mammalian cell.
A recombinant nucleic acid molecule, comprising a nucleotide sequence encoding a non-secreted form of beta-lactamase,
- wherein said nucleotide sequence encoding a non-secreted form of beta-lactamase is operatively linked to a mammalian expression control sequence that is responsive to substances that modulate cell surface receptors.
A mammalian expression vector, comprising a recombinant nucleic acid molecule, comprising
- a nucleotide sequence encoding a non-secreted form of beta lactamase wherein said nucleotide sequence encoding a non-secreted form of beta lactamase is operatively linked to a mammalian expression control sequence.
Dependencies are inferred from the claim text.