Methods to identify signal sequences
OwnerJonathan M. Graff, Matthew Muensterinventors
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Abstract
The present invention provides screening methods using bacterial cells to identify nucleic acid sequences encoding eukaryotic proteins comprising signal sequences and/or transmembrane sequences. Provided are several breast cancer and adipose tissue nucleic acid and proteins sequences that encode proteins comprising signal sequences and/or transmembrane sequences. Also provided are diagnostic methods and kits that utilize the proteins identified by the present methods to diagnose and detect diseases, physiological states and conditions, including cancer and those associated with fat metabolism.
Claims113
A method of screening candidate eukaryotic nucleic acid for one or more nucleic acid sequence encoding a signal sequence and/or a transmembrane sequence comprising:
- a) providing a bacterial cell;
- b) contacting the bacterial cell with at least one plasmid comprising a candidate eukaryotic nucleic acid segment and a marker gene comprising a mutation in a region comprising a signal sequence and/or a transmembrane sequence of the marker gene;
- and
- c) screening for function of the marker gene;
- wherein function of the marker gene indicates that the candidate nucleic acid segment comprises a sequence that encodes a signal sequence and/or a transmembrane sequence.
A method of screening candidate nucleic acid for one or more nucleic acid sequences encoding a polypeptide comprising a signal sequence and/or a transmembrane sequence comprising:
- a) providing a bacterial cell;
- b) contacting the bacterial cell with at least one plasmid comprising a candidate nucleic acid segment and a marker gene comprising a mutation in a region comprising a signal sequence and/or a transmembrane sequence of the marker gene;
- and
- c) screening for function of the marker gene;
- wherein function of the marker gene indicates that the candidate nucleic acid segment comprises a sequence that encodes a polypeptide comprising a signal sequence and/or a transmembrane sequence.
A method of screening candidate nucleic acid for one or more nucleic acid sequences encoding a polypeptide comprising a signal sequence and/or a transmembrane sequence comprising:
- a) providing a bacterial cell;
- b) contacting the bacterial cell with at least one construct comprising a candidate nucleic acid segment and a mutated selectable marker gene comprising a mutation in a region comprising a signal sequence and/or a transmembrane sequence of the marker gene;
- and
- c) screening for survival of the cell on selectable media;
- wherein survival of the cell or its progeny cells on the selectable media indicates that the candidate nucleic acid segment comprises a sequence encoding a polypeptide comprising a signal sequence and/or a transmembrane sequence.
A construct for screening for nucleic acid sequences encoding a polypeptide comprising a signal sequence and/or a transmembrane sequence comprising:
- a) a replication system functional in a bacterial host cell;
- b) at least a first marker gene;
- and
- c) a candidate nucleic acid sequence;
- wherein expression of the marker gene in a bacterial cell indicates that the candidate nucleic acid sequence encodes a polypeptide comprising signal sequence and/or a transmembrane sequence.
Dependencies are inferred from the claim text.