Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-06-18
2000-11-14
Schwartzman, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 4352523, 43525411, 4352542, 435325, 435419, 435455, 435471, C12Q 168, C12N 115, C12N 121, C12N 510
Patent
active
061468266
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
Several methods are available to monitor gene activity and protein distribution within cells. These include the formation of fusion proteins with coding sequences for .beta.-galactosidase (22), and luciferases (22). The usefulness of these methods is often limited by the requirement to fix cell preparations or to add exogenous substrates or cofactors. This invention disclose a method of examining gene expression and protein localization in living cells that requires no exogenously-added compounds.
This method uses a cDNA encoding the Green fluorescent Protein (GFP) from the jelly fish Aequorea victoria (3). In A. victoria, GFP absorbs energy generated by aequorin upon the stimulation by calcium and emits a green light.
This invention discloses that GFP expressed in prokaryotic and eukaryotic cells is capable of producing a strong green fluorescence when excited with near UV or blue light. Since this fluorescence requires no additional gene products from A. victoria, chromophore formation is not species specific.
SUMMARY OF THE INVENTION
This invention provides a cell comprising a DNA molecule having a regulatory element from a gene, other than a gene encoding a green fluorescent protein operatively linked to a DNA sequence encoding the green fluorescent protein. This invention also provides living organisms comprising the above-described cell.
This invention provides a method for selecting cells expressing a protein of interest which comprises: a) introducing into the cells a DNAI molecule having DNA sequence encoding the protein of interest and DNAII molecule having DNA sequence encoding a green fluorescent protein; b) culturing the introduced cells in conditions permitting expression of the green fluorescent protein and the protein of interest; and c) selecting the cultured cells which express green fluorescent protein, thereby selecting cells expressing the protein of interest.
This invention also provides a method for localizing a protein of interest in a cell: a) introducing into a cell a DNA molecule having DNA sequence encoding the protein of interest linked to DNA sequence encoding the green fluorescent protein such that the protein produced by the DNA molecule will have the protein of interest fused to the green fluorescent protein; b) culturing the cell in conditions permitting expression of the fused protein; c) detecting the location of the fused protein product, thereby localizing the protein of interest.
BRIEF DESCRIPTION OF FIGURES
FIG. 1 Expression of GFP in E. coli. The bacteria on the right side of the figure have the GFP expression plasmid. This photograph was taken while irradiating the agar plate with a hand-held long-wave UV source.
FIG. 2 Excitation and Emission Spectra of E. coli-generated GFP (solid lines) and purified A. victoria GFP (L form; dotted lines).
FIG. 3 Expression of GFP in a first stage Caenorhabditis elegans larva. Two touch receptor neurons (PLML and ALML) and one other neuron of unknown function (ALNL) are indicated. Processes can be seen projecting from all three cell bodies. The arrow points to the nerve ring branch from the ALML cell (out of focus). The background fluorescence is due to the animal's autofluorescence.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this application, the following standard abbreviations are used to indicate specific nucleotides:
______________________________________ C = cytosine A = adenosine
T = thymidine G = guanosine
______________________________________
This invention provides a cell comprising a DNA molecule having a regulatory element from a gene, other than a gene en
REFERENCES:
patent: 5268463 (1993-12-01), Jefferson
patent: 5491084 (1996-02-01), Chalfie et al.
Cubitt, A.B. et al, (1995) "Understanding, improving and using green fluorescent proteins," TIBS Trends in BiochemicalSciences, 20:448-455.
Inouye, S. And Tsuji, F.I., (1994) "Aequorea green fluorescent protein -Expression of the gene and fluorescence characteristics of the recombinant protein," FEBS Letters 341:277-280.
Perozzo, et al., (1987), "X-ray Diffraction and Time-resolved Fluorescence Analyses of Aequorea Green Fluorescent Protein Crystals" The Journal of Biological Chemistry 7713-7716.
Prasher, D.C., (1995) "Using GFP to see the light" Science 11:320-323.
Prendergast, F.G. and Mann, K.G., (1978) "Chemical and physical properties of Aequorin and the green fluorescent protein isolated from Aequorea forskalea," Biochemistry 17:3448-3453.
Ward, W.W. and Bokman, S.H., (1982) "Reversible denaturation of Aequorea green-fluorescent protein: Physical separation and characterization of the renatured protein," Biochemistry 21:4535-4544.
"Glowing method is found to tag work of genes," (1994) Columbia University Record, 19:1 and 6.
Chalfie, M. et al. (1994) "Green Fluorescent Protein as a Marker for Gene Expression" Science, vol. 263: 802-805.
Fire, A. et al. (1990) "A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans", Gene, vol. 93: 189-198.
Morise, H. et al. (1974) "Intermolecular Energy Transfer in the Bioluminescent System of Aequorea" Biochemistry, vol. 13, No. 12: 2656-2662.
Shimomura, O. (1979) "Structure of the Chromophore of Aequorea Green Fluorescent Protein", FEBS Letters, vol. 104, No. 2: 220-222.
Webster's II New Riverside University Dictionary, (1994) Soukhanov et al. eds. p. 775.
Chalfie Martin
Prasher Douglas
Schwartzman Robert A.
The Trustees of Columbia University in the City of New York
White John P.
Woods Hole Oceanographic Institution
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