Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-03-24
2001-06-26
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S325000, C435S320100
Reexamination Certificate
active
06251676
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to molecular biology.
BACKGROUND OF THE INVENTION
One approach for elucidating protein-protein binding in cells is the yeast-based two-hybrid system (Fields and Song (1989) Nature 340:245). That system utilizes chimeric genes and detects protein-protein interactions via the activation of reporter-gene expression. Reporter-gene expression occurs as a result of reconstitution of a functional transcription factor caused by the association of fusion proteins encoded by the chimeric genes. Typically, polynucleotides encoding two-hybrid proteins are constructed and introduced into a yeast host cell. The first hybrid protein consists of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein (often referred to as the “bait”). The second hybrid protein consists of the Gal4 activation domain fused to a polypeptide sequence of a second protein (often referred to as the “prey”). Binding between the two-hybrid proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacZ or HIS3), which is operably linked to a Gal4 binding site.
SUMMARY OF THE INVENTION
In a first method, expression of a reporter gene that encodes a fluorescent polypeptide is used to indicate that an interaction has occurred between a bait and a prey protein. An advantage of using a fluorescent reporter polypeptide is that an interaction between a bait and prey in a mammalian cell can be readily detected, e.g., within 96 hours. In addition, the use of a fluorescent reporter polypeptide allows the identification of a single fluorescing mammalian cell without further manipulation or damage to the cell. For example, a cell that fluoresces can be identified under a fluorescent microscope. To determine the sequence of the prey protein, total DNA from a fluorescing cell is prepared, and the DNA sequence that encodes the prey amplified and sequenced.
In a second method, a prey plasmid containing an Epstein-Barr virus origin of replication (ori-P) and a bait plasmid are transfected into a mammalian cell that expresses Epstein-Barr virus nuclear antigen-1 (EBNA-1). The oriP allows the prey plasmid to replicate episomally and indefinitely in the cell. Since the prey plasmid is maintained episomally in a closed circular form, the prey plasmid can be readily introduced and recovered from a bacterial host cell.
In one aspect, the invention features a method for detecting an interaction between a bait and a prey in a mammalian cell. The method includes: (a) providing a mammalian cell containing: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence containing a DNA binding site for a DNA-binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein, including a DNAbinding domain and the bait; (iii) a prey nucleotide sequence encoding a prey fusion protein including a transcriptional activation domain and a prey; (b) incubating the cell for 96 hours or less; e.g., 72, 48, 24, or 16 hours; (c) detecting reporter gene expression, if present, thereby detecting an interaction between the bait and the prey.
The method can further include isolating total DNA from a cell expressing the reporter gene, and amplifying the nucleotide sequence that encodes the bait or prey. In one embodiment, the reporter gene is integrated into a chromosome of the cell. In another embodiment, the bait or prey is encoded by a nucleotide sequence from a nucleic acid library. The cell can be any mammalian cell, e.g., a primary, secondary or an immortalized cell such as a CV-1 cell. The reporter gene can be a fluorescent polypeptide such as a green fluorescent protein (GFP) or a blue fluorescent protein (BFP).
In another aspect, the invention features a method for detecting an interaction between a bait and a prey in a mammalian cell. The method includes: (a) providing a mammalian cell containing (i) an Epstein-Barr virus nuclear antigen-1 (EBNA-1); (ii) a reporter gene operably linked to a transcriptional regulatory sequence containing a DNA binding site for a DNA-binding domain, (iii) a bait nucleotide sequence encoding a bait fusion protein, including a DNA-binding domain and the bait (e.g., a known protein), (iv) a prey nucleotide sequence including an origin of replication for the Epstein-Barr virus nuclear antigen-1 (oriP) and encoding a prey fusion protein including a transcriptional activation moiety and the prey (e.g., an unknown protein); and (b) detecting reporter gene expression, if present, thereby detecting an interaction between the bait and the prey. The method can further include (c) isolating DNA from a cell expressing the reporter gene; and (d) recovering the nucleotide sequence including the oriP sequence, which encodes the prey fusion protein. An example of a suitable reporter gene includes a reporter gene that encodes a fluorescent protein such as a green fluorescent protein or a blue fluorescent protein. In some embodiments, the reporter gene is integrated into a chromosome of the cell. The cell can be any mammalian cell that expresses EBNA-1 (or manipulated to express EBNA-1). An example of a mammalian cell includes a cell derived from a primate or a canine. The cell can be a primary, secondary or an immortalized cell. An example of an immortalized cell is a CV-1 cell. The bait and/or the prey can be encoded by a nucleotide sequence from a nucleic acid library.
The invention also features a kit for detecting an interaction between a bait and prey in a mammalian cell. The kit includes: (a) a first gene construct which includes a regulatory sequence operably linked to a nucleotide sequence encoding a DNA-binding domain, and wherein the first gene construct includes a cloning site for inserting a nucleotide sequence encoding the bait into the first gene construct such that the bait is expressed in frame with the DNA-binding domain; (b) a second gene construct which includes: an oriP sequence, a regulatory sequence operably linked to a nucleotide sequence encoding a transcriptional activation domain, and wherein the second gene construct includes a cloning site for inserting a nucleotide sequence encoding the prey into the second gene construct such that the prey is expressed in frame with the transcriptional activation domain; (c) a mammalian cell that expresses an EBNA-1, including a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for the DNA-binding domain, wherein the reporter gene expresses the fluorescent polypeptide when the bait and prey interact; and (d) instructions for use.
Also within the scope of the invention is a method of identifying an agent that disrupts interaction between a bait and a prey, including: (a) providing a mammalian cell having: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for a DNA-binding domain, (ii) a first nucleotide sequence encoding a bait fusion protein, including a DNA-binding domain and the bait, (iii) a second nucleotide sequence encoding a prey fusion protein including a transcriptional activation domain and the prey; (b) contacting the mammalian cell with a test agent; (c) incubating the cell for 96 hours or less, e.g., 72, 48, 24 or 16 hours; and (d) detecting a decrease in expression of the reporter gene compared to the level of expression of the reporter gene in a mammalian control cell, if present, thereby detecting an agent that disrupts interaction between the bait and the prey.
The invention further features a method of identifying an agent that enhances interaction between a bait and a prey, including: (a) providing a mammalian cell having: (i) a reporter gene encoding a fluorescent polypeptide operably linked to a transcriptional regulatory sequence including a DNA binding site for a DNA-binding domain, (ii) a bait nucleotide sequence encoding a bait fusion protein includ
Isselbacher Kurt J.
Shioda Toshihiro
Fish & Richardson P.C.
Lu Frank
The General Hospital Corporation
Whisenant Ethan
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