Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-10-14
1999-11-16
Brusca, John S.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 697, 4352523, 43525411, 4353201, 435455, 435468, 530350, 536 234, C12Q 168, C12N 1511, C12N 1563, C07K 1400
Patent
active
059855770
ABSTRACT:
This invention provides a vector comprising an isolated nucleic acid which encodes a protein of interest linked to one or more nucleic acid segments encoding at least two Green Fluorescent Proteins. Provided is the method for detecting a protein of interest in a living cell which comprises: (a) transfecting the living cell with an isolated nucleic acid which encodes the protein of interest linked to at least two Green Fluorescent Proteins. Additional isolated nucleic acids different from the nucleic acid segment of interest may also be linked to at least two nucleic acid which encodes at least two molecules of Green Fluorescent Protein. (b) culturing the transfected cell in conditions permitting expression of Green Fluorescent Protein and the protein of interest; and (c) detecting the fluorescence of the Green Fluorescent Protein, thereby detecting a protein of interest in a cell.
REFERENCES:
Romoser et al. Detection in Living Cells of Ca2+-Dependent Changes in the Fluorescence Emission of an Indicator Composed of Two Green Fluorescent Protein Variants linked by a Calmodulin-binding Sequence. J. Biological Chemistry vol. 272, pp. 13270-1327, 1997.
Ludin et al. Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins. Gene vol. 173, pp. 107-111, 1996.
Cormack, B.P., et al. (1996) "FACS-optimized mutants of the green fluorescent protein (GFP)", Gene 173:33-38, (Exhibit 1).
Dickson, R.M.,et al. (1997) "On/off blinking behavior of single molecules of green fluorescent protein", Nature 388:355-358, (Exhibit 2).
Heim, R., Cubitt, A.B., and Tsien, R.Y. (1997) "Improved Green Fluorescence", Nature 373:663-664, (Exhibit 3).
Prasher, et al. (1992) "Primary structure of the Dequorea victoria in green Fluorescence protein", Gene 111:229-233, (Exhibit 4).
Silhavy, T.J. and Beckwith, J.R. (1985) Microbiol. Rev. 49:398, (Exhibit 5).
Walker, R.A., et al. (1988) "Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies", J. Cell Biol. 107: 1437-1448, (Exhibit 6); and.
Waterman-Storer, C.M. and Salmon, E.D. (1977) "Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling", J. Cell Biol. 139:417-434, (Exhibit 7).
Brusca John S.
The Trustees of Columbia University in the City of New York
White John P.
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