USOO6146826A Patent (19) 11 Patent Number: 6,146,826 Chalfie et al. (45) Date of Patent: *Nov. 14, 2000

54 GREEN FLUORESCENT PROTEIN Inouye, S. And Tsuji, F.I., (1994) “ green fluores cent protein -Expression of the gene and 75 Inventors: , New York, N.Y.; characteristics of the recombinant protein,” FEBS Letters Douglas Prasher, East Falmouth, Mass. 341:277-280. Perozzo, et al., (1987), “X-ray Diffraction and Time-re 73 Assignees: The Trustees of Columbia University Solved Fluorescence Analyses of Aequorea Green Fluores in the City of New York, New York, cent Protein Crystals' The Journal of Biological Chemistry N.Y.; Woods Hole Oceanographic 7713-7716. Institution, Woods Hole, Mass. Prasher, D.C., (1995) “Using GFP to see the light” Science 11:320-323. * Notice: This patent issued on a continued pros Prendergast, F.G. and Mann, K.G., (1978) “Chemical and ecution application filed under 37 CFR physical properties of and the green fluorescent 1.53(d), and is subject to the twenty year protein isolated from , patent term provisions of 35 U.S.C. 17:3448-3453. 154(a)(2). Ward, W.W. and Bokman, S.H., (1982) “Reversible dena 21 Appl. No.: 08/367,236 turation of Aequorea green-fluorescent protein: Physical Separation and characterization of the renatured protein,” 22 PCT Filed: Sep. 9, 1994 Biochemistry 21:4535–4544. 86 PCT No.: PCT/US94/101.65 “Glowing method is found to tag work of genes,” (1994) Columbia University Record, 19:1 and 6. S371 Date: Jun. 18, 1996 Chalfie, M. et al. (1994) “Green Fluorescent Protein as a Marker for Gene Expression” Science, vol. 263: 802-805. S 102(e) Date: Jun. 18, 1996 Fire, A. et al. (1990) “A modular set of lacZ fusion vectors 87 PCT Pub. No.: WO95/07463 for Studying gene expression in ', Gene, vol. 93: 189-198. PCT Pub. Date: Mar. 16, 1995 Morise, H. et al. (1974) “Intermolecular Energy Transfer in Related U.S. Application Data the Bioluminescent System of Aequorea’ Biochemistry, vol. 13, No. 12: 2656-2662. 63 Continuation-in-part of application No. 08/192.274, Feb. 4, Shimomura, O. (1979) “Structure of the Chromophore of 1994, abandoned, which is a continuation-in-part of appli Aequorea Green Fluorescent Protein', FEBS Letters, vol. cation No. 08/119,678, Sep. 10, 1993, Pat. No. 5,491,084. 104, No. 2: 220-222. 51 Int. Cl." ...... C12O 1/68; C12N 1/15; Webster's II New Riverside University Dictionary, (1994) C12N 1/21; C12N 5/10 Soukhanov et al. eds. p. 775. 52 U.S. Cl...... 435/6; 435/69.1; 435/252.3; Primary Examiner Robert A. Schwartzman 435/254.11; 435/254.2; 435/325; 435/419; Attorney, Agent, or Firm John P. White; Cooper & 435/455; 435/471 Dunham LLP 58 Field of Search ...... 435/6, 69.1, 69.7, 435/69.8, 189, 240.2, 240.4, 172.3, 320.1, 57 ABSTRACT 253, 325, 419,455, 471, 252.3, 254.11, 254.2; 530/350, 536/23.1, 23.2, 23.4, 23.5, This invention provides a cell comprising a DNA molecule 24.1 having a regulatory element from a gene, other than a gene encoding a green fluorescent protein operatively linked to a 56) References Cited DNA sequence encoding the green fluorescent protein. This invention also provides living organisms which comprise the U.S. PATENT DOCUMENTS above-described cell. This invention also provides a method 5,268,463 12/1993 Jefferson ...... 536/23.7 for Selecting cells expressing a protein of interest which 5,491,084 2/1996 Chalfie et al...... 435/189 comprises: a) introducing into the cells a DNAI molecule having DNA sequence encoding the protein of interest and FOREIGN PATENT DOCUMENTS DNAII molecule having DNA sequence encoding a green O540064 5/1993 European Pat. Off.. fluorescent protein; b) culturing the introduced cells under WO9101305 2/1991 WIPO. conditions permitting expression of the green fluorescent WO9417208 8/1994 WIPO. protein and the protein of interest; and c) selecting the WO9423039 10/1994 WIPO. cultured cells which express green fluorescent protein, thereby Selecting cells expressing the protein of interest. OTHER PUBLICATIONS Finally, this invention provides various uses of a green Cubitt, A.B. et al., (1995) “Understanding, improving and fluorescent protein. using green fluorescent proteins,” TIBS Trends in Biochemi calSciences, 20:448-455. 26 Claims, 3 Drawing Sheets U.S. Patent Nov. 14, 2000 Sheet 1 of 3 6,146,826

U.S. Patent Nov. 14, 2000 Sheet 2 of 3 6,146,826

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O.O - s 300 400 5OO 600 WAVELENGTH (nm) U.S. Patent Nov. 14, 2000 Sheet 3 of 3 6,146,826

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6,146,826 1 2 GREEN FLUORESCENT PROTEIN mitting expression of the fused protein; c) detecting the location of the fused protein product, thereby localizing the This application is a continuation-in-part of U.S. Ser. protein of interest. Nos. 08/119,678, U.S. Pat. No. 5,491,084, and 08/192,274, abandoned, International Applicaton No. PCT/US94/101.65 BRIEF DESCRIPTION OF FIGURES filed Sept. 10, 1993 and Feb. 4, 1994, Sep. 9, 1994 FIG. 1 Expression of GFP in E. coli. The bacteria on the respectively, the contents of which are hereby incorporated right side of the figure have the GFP expression plasmid. by reference. This photograph was taken while irradiating the agar plate The invention disclosed herein was made with Govern with a hand-held long-wave UV source. ment support under NIH Grant No. 5R01 GM30997 from the FIG. 2 Excitation and Emission Spectra of E. coli Department of Health and Human Services. Accordingly, the generated GFP (solid lines) and purified A. victoria GFP (L U.S. Government has certain rights in this invention. form; dotted lines). BACKGROUND OF THE INVENTION FIG. 3 Expression of GFP in a first stage Caenorhabditis 15 elegans larva. Two touch receptor neurons (PLML and Throughout this application various references are ALML) and one other neuron of unknown function (ALNL) referred to within parenthesis. Disclosures of these publica are indicated. Processes can be seen projecting from all three tions in their entireties are hereby incorporated by reference cell bodies. The arrow points to the nerve ring branch from into this application to more fully describe the State of the art the ALML cell (out of focus). The background fluorescence to which this invention pertains. Full bibliographic citation is due to the 's autofluorescence. for these references may be found at the end of this application, preceding the Sequence listing and the claims. DETAILED DESCRIPTION OF THE Several methods are available to monitor gene activity INVENTION and protein distribution within cells. These include the Throughout this application, the following Standard formation of fusion proteins with coding Sequences for 25 abbreviations are used to indicate Specific nucleotides: f-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 expres C = cytosine A = adenosine Sion and protein localization in living cells that requires no T = thymidine G = guanosine exogenously-added compounds. This method uses a cDNA encoding the Green fluorescent This invention provides a cell comprising a DNA mol Protein (GFP) from the jelly fish (3). In A. ecule having a regulatory element from a gene, other than a victoria, GFP absorbs energy generated by aequorin upon gene encoding a green fluorescent protein operatively linked the Stimulation by calcium and emits a green light. 35 to a DNA sequence encoding the green fluorescent protein. This invention discloses that GFP expressed in prokary This invention provides a cell comprising a DNA mol otic and eukaryotic cells is capable of producing a Strong ecule having a regulatory element from a gene, other than a green fluorescence when excited with near UV or blue light. gene encoding a green fluorescent protein operatively linked Since this fluorescence requires no additional gene products to a DNA sequence encoding the green fluorescent protein, from A. victoria, chromophore formation is not Species 40 wherein the cell is Selected from a group consisting essen Specific. tially of bacterial cell, yeast cell, fungal cell, insect cell, nematode cell, plant or animal cell. SUMMARY OF THE INVENTION Suitable animal cells include, but are not limited to Vero This invention provides a cell comprising a DNA mol cells, HeLa cells, CoS cells, CVI cells and various vertebral, ecule having a regulatory element from a gene, other than a 45 invertebral, mammalian cells. gene encoding a green fluorescent protein operatively linked In an embodiment, the bacterial cell is . to a DNA sequence encoding the green fluorescent protein. AS used herein, “a regulatory element” from a gene is the This invention also provides living organisms comprising DNA sequence which is necessary for the transcription of the above-described cell. 50 the gene. This invention provides a method for Selecting cells In this invention, the term “operatively linked' means that expressing a protein of interest which comprises: a) intro following Such a link the regulatory element can direct the ducing into the cells a DNAI molecule having DNA transcription of the linked protein-coding DNA sequence. Sequence encoding the protein of interest and DNAII mol The gene encoding a green fluorescent protein includes ecule having DNA sequence encoding a green fluorescent 55 DNA molecules coding for polypeptide analogs, fragments protein; b) culturing the introduced cells in conditions or derivatives of antigenic polypeptides which differ from permitting expression of the green fluorescent protein and naturally-occurring forms in terms of the identity or location the protein of interest; and c) selecting the cultured cells of one or more amino acid residues (deletion analogs which express green fluorescent protein, thereby Selecting containing less than all of the residues Specified for the cells expressing the protein of interest. 60 protein, Substitution analogs wherein one or more residues This invention also provides a method for localizing a Specified are replaced by other residues and addition analogs protein of interest in a cell: a) introducing into a cell a DNA where in one or more amino acid residues is added to a molecule having DNA sequence encoding the protein of terminal or medial portion of the polypeptides) and which interest linked to DNA sequence encoding the green fluo share Some or all properties of naturally-occurring forms. rescent protein such that the protein produced by the DNA 65 These DNA molecules include: the incorporation of molecule will have the protein of interest fused to the green codons "preferred” for expression by Selected mammalian or fluorescent protein; b) culturing the cell in conditions per non-mammalian hosts, the provision of Sites for cleavage by 6,146,826 3 4 restriction endonuclease enzymes, and the provision of further embodiment, the bacterial cell is an E. coli cell. In a additional initial, terminal or intermediate DNA sequences still further embodiment, this E. coli cell is designated that facilitate construction of expression vectors. SMC1 (ATCC Accession No. 69554). As an example, plasmid pCFP10.1 codes for a mutated This SMC1 bacterial cell was deposited on Feb. 4, 1994, GFP protein having the 80th amino acid residue as an 1993 with the American Type Culture Collection (ATCC), arginine rather than a glutamine predicted to be in native 10801 University Boulevard, Manassas, Va. 20110, U.S.A. GFP from A. Victoria. This mutated protein retains the under the provisions of the Budapest Treaty for the Inter property to fluoresce like the natural protein. national Recognition of the Deposit of Microorganism for In an embodiment, the regulatory element is a promoter. the Purposes of Patent Procedure. Bacterial cell SMC1 was In a further embodiment, the promoter is activated by a accorded ATCC Accession Number 69554. heavy metal. Such promoters are well-known in the art (J. H. This invention further provides an isolated green fluores Freedman, L. W. Slice, A. Fire, and C. S. Rubin (1993) cent protein produced from the above-described cells which Journal of Biological Chemistry, 268:2554). comprise a DNA molecule having a regulatory element from In another embodiment, the promoter is that from a a gene, other than a gene encoding a green fluorescent cytochrome P450 gene. Cytochrome P450 is well-known in 15 protein operatively linked to a DNA sequence encoding the the art and there are a number of P450 promoters known. green fluorescent protein. This isolated green fluorescent In still another embodiment, the promoter is that from a protein can then be further modified in vitro for various uses. StreSS protein gene. Such StreSS proteins are well-known in This invention disclose an efficient method for expression the art (E. G. Stringham, D. K. Dixon, D. Jones and E. D. of green fluorescent protein Such that large amount of the Candido (1992) of the Cell, 3:221; and protein could be produced. Methods to isolate expressed William J. Welch (May, 1993), Scientific American, page protein have been well-known and therefore, green fluores 56). In a further embodiment, the stress protein is a heat cent protein may be isolated easily. Shock protein. This invention provides a living organism comprising the This invention provides a cell comprising a DNA mol 25 cell comprising a DNA molecule having a regulatory ele ecule having a regulatory element from a gene, other than a ment from a gene, other than a gene encoding a green gene encoding a green fluorescent protein operatively linked fluorescent protein operatively linked to a DNA sequence to a DNA sequence encoding the green fluorescent protein, encoding the green fluorescent protein. wherein the promoter is from a gene necessary for the In another embodiment, the living organism is human. In viability of a cell. another embodiment, the living organism is a mouse. The In another embodiment, the regulatory element is an living organism may be other mammals. In addition, this enhancer. Enhancers are well-known in the art. invention is applicable to other vertebrates, non-vertebrates This invention provides a cell comprising a DNA mol and living organisms. ecule having a regulatory element from a gene, other than a In an embodiment, the living organism is C. elegans. In gene encoding a green fluorescent protein operatively linked 35 Still another embodiment, the living organism is Drosophila, to a DNA sequence encoding the green fluorescent protein, Zebra fish, Virus or bacteriophage. wherein the DNA sequence encodes the Aequorea Victoria A bacteriophage carrying the green fluorescent protein green fluorescent protein. gene can infect a particular type of bacteria. The infection In an embodiment, the Aequorea Victoria green fluores may be easily detected via the expression of the green cent protein is cloned in a plasmid. This plasmid is a 40 fluorescent protein. Therefore, by using appropriate modification of the pBS(+) (formerly called Bluescribe+) bacteriophages, the presence of that particular type of bac vector (Stratagene(R) which has inserted within it an EcoRI teria may be detected. fragment containing the cDNA sequence of the Aequorea Similarly, a eucaryotic virus carrying the green fluores Victoria green fluorescent protein (as modified herein). The cent protein gene may infect a Specific cell type. The fragment was obtained from JFP10 (Prasher, D. C., 45 Eckenrode, V. K., Ward, W. W.; Prendergast, F. G., and infection may be easily detected by monitoring the expres Cormier, M. J., (1992) Primary structure of the Aequorea Sion of the green fluorescent protein. victoria green fluorescent protein. Gene, 111:229–233) by Methods to introduce exogenous genetic material into a amplification using the polymerase chain reaction (Saiki, R. cell are well-known in the art. For example, exogenous DNA K., Gelfand, D. H., Stoffel, S., Sharf, S.J., Higuchi, G. T., 50 material may be introduced into the cell by calcium phos Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Primer phate precipitation technology. Other technologies, Such as directed enzymatic amplification of DNA with a thermo the retroviral vector technology, electroporation, lipofection stable DNA polymerase. Science, 239:487-491) with prim and other viral vector Systems. Such as adeno-associated erS flanking the EcoRI sites and Subsequent digestion with Virus System, or microinjection may be used. EcoRI. The sequence of the cDNA in pCFP10.1 differs from 55 The above-described cells and living organisms are useful the published sequence (5) by a change of the 80th codon of to detect effects of external Stimulus to the regulatory the coding Sequence from CAG to CGG, a change that element. The Stimulus may have direct or indirect effects on replaces a glutamine with arginine in the protein Sequence. the regulatory element. Such effects will be detectable This pCFP10.1 plasmid was deposited on Sep. 1, 1993 through either the induction of expression and production of with the American Type Culture Collection (ATCC), 10801 60 the green fluorescent protein or Switching off the expression University Boulevard, Manassas, Va. 20110, U.S.A. under of the green fluorescent protein. the provisions of the Budapest Treaty for the International Cells expressing the green fluorescent proteins may be Recognition of the Deposit of Microorganism for the Pur conveniently Separated by a fluorescence-activated cell poses of Patent Procedure. Plasmid pCFP10.1 was accorded SOrter. ATCC Accession Number 75547. 65 These cells and organisms may be used to detect the In another embodiment, this invention provide a bacterial presence of different molecules in various kinds of biologi cell which is expressing the green fluorescent protein. In an cal Samples Such as blood, urine or Saliva. By operatively 6,146,826 S 6 linking a regulatory element of the gene which is affected by initiation Sequences for ribosome binding. For example, a the molecule of interest to a green fluorescent protein, the bacterial expression vector includes a promoter Such as the presence of the molecules will affect the regulatory element lac promoter and for translation initiation the Shine which in turn will affect the expression of the green fluo Dalgarno Sequence and the Start codon ATG. Similarly, a rescent protein. Therefore, the above-described cells are eukaryotic expression vector includes a heterologous or useful for the detection of molecules. Such detection may be homologous promoter for RNA polymerase II, a down used for diagnostic purposes. An example of Such a mol Stream polyadenylation signal, the Start codon ATG, and a ecule is a hormone. termination codon for detachment of the ribosome. Such This invention provides a living organism comprising the cell comprising a DNA molecule having a regulatory ele vectors may be obtained commercially or assembled from ment from a gene, other than a gene encoding a green the Sequences described by methods well-known in the art, fluorescent protein operatively linked to a DNA sequence for example the methods described above for constructing encoding the green fluorescent protein, wherein the regula vectors in general. tory element is for a StreSS protein. To maximize the expression of the green fluorescent This invention provides a living organism comprising the protein, the Sequence flanking the translation initiation cell comprising a DNA molecule having a regulatory ele 15 codon may be modified (reviewed by Kozak, 1984), com ment from a gene, other than a gene encoding a green pilation and analysis of Sequences upstream from the trans fluorescent protein operatively linked to a DNA sequence lation start site in eucaryotic mRNAS. Nucl. Acids. Res. encoding the green fluorescent protein, wherein the StreSS 12:857-872). A sequence may then be generated to produce protein is a heat-shock protein. higher amounts of the GFP protein. This invention provides a method to produce green fluo In addition, artificial introns may be introduced So as to rescent protein comprising a) culturing the above-described increase the production of the protein. cells comprising a DNA molecule having a regulatory Other special targeting Sequences may be inserted into the element from a gene, other than a gene encoding a green GFP gene. One Such targeting Sequence is the nuclear fluorescent protein operatively linked to a DNA sequence localization signal (Such as the SV40 nuclear localization encoding the green fluorescent protein; and b) isolating and 25 Signal). purifying the green fluorescent protein produced from the The host cell of the above expression system may be cells. Standard methods for isolating and purifying proteins Selected from the group consisting of the cells where the are well-known in the art. In an embodiment, the cells used protein of interest is normally expressed, or foreign cells for production of green fluorescent proteins are E. coli cells. Such as bacterial cells (Such as E. coli), yeast cells, fungal In a further embodiment, the E. coli cells are cultured cells, insect cells (such as Sf9 cell in the baculovirus aerobically. expression System), nematode cells, plant or animal cells, This invention provides a method to Synthesize green where the protein of interest is not normally expressed. fluorescent protein comprising a) culturing the cell desig Suitable animal cells include, but are not limited to Vero nated SMCl; and b) isolating and purifying the green fluo 35 cells, HeLa cells, Cos cells, CV1 cells and various primary rescent protein produced from the cell. mammalian cells. This invention provides a method for Selecting cells In an embodiment of the method for localizing a protein expressing a protein of interest which comprises: a) intro of interest in a cell, the DNA encoding the green fluorescent ducing into the cells a DNAI molecule having DNA protein is from Aequorea victoria. Sequence encoding the protein of interest and DNAII mol 40 This invention provides a method for localizing a protein ecule having DNA sequence encoding a green fluorescent of interest in a cell which comprises: a) introducing into a protein; b) culturing the introduced cells in conditions cell a DNA molecule having DNA sequence encoding the permitting expression of the green fluorescent protein and protein of interest linked to DNA sequence encoding a green the protein of interest; and c) selecting the cultured cells fluorescent protein Such that the protein produced by the which express green fluorescent protein, thereby Selecting 45 DNA molecule will have the protein of interest fused to the cells expressing the protein of interest. green fluorescent protein; b) culturing the cell in conditions This invention also provides the above method, wherein permitting expression of the fused protein; and c) detecting the cells are Selected from a group consisting essentially of the location of the fused protein composed of green fluo bacterial cells, yeast cells, fungal cells, insect cells, nema rescent protein in the cell, thereby localizing a protein of tode cells, plant or animal cells. Suitable animal cells 50 interest in a cell, wherein the cell normally expressing the include, but are not limited to Vero cells, HeLa cells, Cos protein of interest. cells, CV1 cells and various primary mammalian cells. This invention provides a method for detecting expression In an embodiment, DNAI and DNAII are linked. In of a gene in a cell which comprises: a) introducing into the another embodiment, the DNA encodes the Aequorea Vic cell a DNA molecule having DNA sequence of the gene toria green fluorescent protein. 55 linked to DNA sequence encoding a green fluorescent pro This invention provides a method for localizing a protein tein Such that the regulatory element of the gene will control of interest in a cell which comprises: a) introducing into a expression of the green fluorescent protein; b) culturing the cell a DNA molecule having DNA sequence encoding the cell in conditions permitting expression of the gene; and c) protein of interest linked to DNA sequence encoding a green detecting the expression of the green fluorescent protein in fluorescent protein Such that the protein produced by the 60 the cell, thereby indicating the expression of the gene in the DNA molecule will have the protein of interest fused to the cell. green fluorescent protein; b) culturing the cell in conditions This invention provides a method for indicating expres permitting expression of the fused protein; and c) detecting Sion of a gene in a Subject which comprises: a) introducing the fused protein composed of the green fluorescent protein into a cell of the subject a DNA molecule having DNA in the cell, thereby localizing a protein of interest in a cell. 65 Sequence of the gene linked to DNA sequence encoding a Regulatory elements required for expression include pro green fluorescent protein Such that the regulatory element of moter Sequences to bind RNA polymerase and translation the gene will control expression of the green fluorescent 6,146,826 7 8 protein; b) culturing the cell in conditions permitting expres expressed in prokaryotic (Escherichia coli) or eukaryotic Sion of the fused protein; and c) detecting the expression of (Caenorhabditis elegans) cells. Because exogenous Sub the green fluorescent protein in the cell, thereby indicating Strates and cofactors are not required for this fluorescence, the expression of the gene in the cell. GFP expression can be used to monitor gene expression and In an embodiment of the above methods, the green protein localization in living organisms. fluorescent protein is the Aequorea victoria green fluores Light is produced by the bioluminescent Aequo cent protein. rea victoria when calcium binds to the photoprotein This invention provides a method for determining the aequorin (1). Although activation of aequorin in vitro or in tissue-specificity of transcription of a DNA sequence in a heterologous cells produces blue light, the jellyfish produces Subject which comprises: a) introducing into a cell of the green light. This latter light is the result of a Second protein subject a DNA molecule having the DNA sequence linked to DNA sequence encoding a green fluorescent protein Such in A. victoria that derives its excitation energy from aequorin that the DNA sequence will control expression of the green (2), the green fluorescent protein (GFP). fluorescent protein; b) culturing the Subject in conditions Purified GFP, a protein of 238 amino acids (3), absorbs permitting the expression of the green fluorescent protein; blue light (maximally at 395 nm with a minor peak at 470 and c) detecting the expression of the green fluorescent 15 nm) and emits green light (peak emission at 509 nm with a protein in different tissue of the Subject, thereby determining shoulder at 540 nm) (2,4). This fluorescence is very stable; the tissue-specificity of the expression of the DNA sequence. virtually no photobleaching is observed (5). Although the This invention provides a method for determining the intact protein is needed for fluorescence, the same absorp presence of heavy metal in a Solution which comprises: a) tion spectral properties found in the denatured protein are culturing the cell comprising a DNA molecule having a found in a hexapeptide that starts at amino acid 64 (6, 7). The promoter from a gene, other than agreen fluorescent protein GFP chromophore is derived from the primary amino acid operatively linked to a DNA sequence encoding the green Sequence through the cyclization of Ser-dehydroTyr-Gly fluorescent protein, wherein transcription at the promoter is within this hexapeptide (7). The mechanisms that produce activated by a heavy metal in the Solution; and b) detecting the dehydrotyrosine and cyclize the polypeptide to form the 25 chromophore are unknown. To determine whether additional expression of the green fluorescent protein, the expression of factors from A. victoria were needed for the production of the green fluorescent protein indicates the presence of heavy the fluorescent protein, applicants tested GFP fluorescence metal. in heterologous systems. Here applicants show that UFP This invention provides a method for detecting pollutants expressed in prokaryotic and eukaryotic cells is capable of in a Solution which comprises: a) culturing the cell com producing a strong green fluorescence when excited by blue prising a DNA molecule having a promoter from a gene, light. Because this fluorescence requires no additional gene other than a green fluorescent protein operatively linked to products from A. Victoria, chromophore formation is not a DNA sequence encoding the green fluorescent protein, Species Specific and occurs either through the use of ubiq wherein the promoter is activated by a heavy metal or a toxic uitous cellular components or by autocatalysis. organic compound or the promoter is for a StreSS protein in 35 Expression of GFP in Escherichia coli (8) under the the Solution; and b) detecting expression of the green control of the T7 promoter results in a readily detected green fluorescent protein, the expression of the green fluorescent fluorescence (9) that is not observed in control bacteria. protein indicates the presence of pollutants in the Solution. Upon illumination with a long-wave UVSource, fluorescent Finally, this invention provides a method for producing bacteria were detected on agar plates containing the inducer fluorescent molecular weight markers comprising: a) linking 40 isopropyl-3-D-thiogalactoside (IPTG) (FIG. 1). When GFP a DNA molecule encoding a green fluorescent protein with was partially purified from this strain (10), it was found to a DNA molecule encoding a known amino acid Sequence in have fluorescence excitation and emission spectra indistin the same reading frame; b) introducing the linked DNA guishable from those of the purified native protein (FIG. 2). molecule of step a) in an expression System permitting the The spectral properties of the recombinant GFP Suggest that expression of a fluorescent protein encoded by the linked 45 the chromophore can form in the absence of other A. victoria DNA molecule; and c) determining the molecular weight of products. the expressed fluorescent protein of step b), thereby produc Transformation of the nematode Caenorhabditis elegans ing a fluorescent molecular weight marker. also resulted in the production of fluorescent GFP (11) (FIG. Various expression Systems are known in the art. The E. 3). GFP was expressed in a small number of neurons under coli expression System, one of the commonly used System is 50 the control of a promoter for the mec-7 gene. The mec-7 described in the following Section. aene encodes a f-tubulin (12) that is abundant in six touch The determination of molecular weight may be done by receptor neurons in C. elegans and less abundant in a few comparing the expressed fluorescent protein of step b) with other neurons (13, 14). The pattern of expression of GFP was known molecular weight markers. Alternatively, the similar to that detected by MEC-7 antibody or from mec molecular weight can be predicted by calculation Since the 55 7lacZ fusions (13–15). The strongest fluorescence was seen linked DNA sequence is known (and So is the amino acid in the cell bodies of the four embryonically-derived touch Sequence being encoded). In an embodiment, the expressed receptor neurons (ALML, ALMR, PLML, PLMR) in fluorescent protein is purified. The purified fluorescent pro younger larvae. The processes from these cells, including tein can be conveniently used as molecular weight markers. their terminal branches, were often visible in larval . This invention will be better understood from the Experi 60 In some newly hatched animals, the PLM processes were mental Details which follow. However, one skilled in the art Short and ended in what appeared to be prominent growth will readily appreciate that the Specific methods and results cones. In older larvae, the cell bodies of the remaining touch discussed are merely illustrative of the invention as cells (AVM and PVM) were also seen; the processes of these described more fully in the claims which follow thereafter. cells were more difficult to detect. These postembryonically Experimental Details 65 derived cells arise during the first of the four larval Stages A cDNA for the Aequorea Victoria green fluorescent (16), but their outgrowth occurs in the following larval protein (GFP) produces a fluorescent product when Stages (17), with the cells becoming functional during the 6,146,826 9 10 fourth larval stage (18). GFP's fluorescence in these cells is 2. J. G. Morin and J. W. Hastings, J. Cell. Physiol. 77,313 consistent with these previous results: no fluorescence was (1971); H. Morise, O. Shimomura, F. H. Johnson, J. detected in these cells in newly hatched or late first-stage Winant, Biochemistry 13, 2656 (1974). larvae, but it was seen in four often late Second-stage larvae, all nine early fourth-Stage larvae, and Seven of eight young 3. D. C. Prasher, V. K. Eckenrode, W. W. Ward, F. G. adults (19). In addition, moderate to weak fluorescence was Prendergast, M. J. Cormier, Gene 111, 229 (1992). seen in a few other neurons (FIG. 3) (20). The details of the 4. W. W. Ward, C. W. Cody, R. C. Hart, M. J. Cormier, expression pattern are being examined. Photochem. Photobiol. 31, 611 (1980). Like the native protein, GFP expressed in both E. coli and 5. F. G. Prendergast, personal communication. C. elegans is quite stable (lasting at least ten minutes) when 6. O. Shimomura, FEBS Lett. 104, 220 (1979). illuminated with 450-490 nm light. Some photobleaching occurs, however, when the cells are illuminated with 7. C. W. Cody, D. C. Prasher, W. M. Westler, F. G. 340–390 nm or 395-440 nm light (21). Prendergast, W. W. Ward, Biochemistry 32, 1212 Several methods are available to monitor gene activity (1993). and protein distribution within cells. These include the 8. Plasmid pCFP10.1 contains the EcoRI fragment encod formation of fusion proteins with coding Sequences for 15 ing the GFP cDNA from gfp 10 (3) in pBS(+) B-galactosidase, firefly luciferase, and bacterial luciferase (Stratagene(R). The fragment was obtained by amplifi (22). Because Such methods require exogenously-added cation with the polymerase chain reaction PCR; R. K. Substrates or cofactors, they are of limited use with living Saiki et al., Science 239, 487 (1988) with primers tissue. Because the detection of intracellular GFP requires flanking the EcoRI sites and Subsequent digestion with only irradiation by near UV or blue light, it is not substrate EcoRI. DNA was prepared by the Magic Minipreps limited. Thus, it should provide an excellent means for procedure (Promega) and Sequenced (after an addi monitoring gene expression and protein localization in liv tional ethanol precipitation) on an Applied BioSystems ing cells (23, 24). Because it does not appear to interfere DNA Sequencer 370A at the DNA Sequencing facility with cell growth and function, GFP should also be a con at Columbia College of Physicians and Surgeons. The Venient indicator of transformation and one that could allow 25 sequence of the cDNA in pCFP10.1 differs from the cells to be separated using fluorescence-activated cell Sort published Sequence by a change in codon 80 within the ing. Applicants also envision that GFP can be used as a vital marker So that cell growth (for example, the elaboration of coding Sequence from CAG to CGG, a change that neuronal processes) and movement can be followed in situ, replaces a glutamine residue with arginine R. Heim, S. especially in animals that are essentially transparent like C. Emr, and R. Tsien (personal communication) first elegans and Zebrafish. The relatively small size of the alerted us to a possible Sequence change in this clone protein may facilitate its diffusion throughout the cytoplasm and independently noted the same change. This of extensively branched cells like neurons and glia. Since the replacement has no detectable effect on the spectral GFP fluorescence persists after treatment with formaldehyde properties of the protein (FIG. 2). (9), fixed preparations can also be examined. In addition, An E. coli expression construct was made with PCR to absorption of appropriate laser light by GFP-expressing cells 35 generate a fragment with an Nhe Site at the Start of (as has been done for lucifer yellow-containing cells) (25), translation and an EcoRI site 5' to the termination signal of could result in the selective killing of the cells. the GFP coding sequence from PGFP10.1. The 5' primer Further Experiments on GFP Expression was ACAAAGGCTAGCAAAGGAGAAGAAC (Sequence The TU#58 plasmid, which contains the green fluorescent ID No. 1) and the 3' primer was the T3 primer (Stratagene(R). protein (GFP) coding Sequence in the pET3a expression 40 The Nhel-EcoRI fragment was ligated into the similarly cut vector (29) was transformed into Escherichia coli strain vector pET3a A. H. Rosenberg et al., Gene 56, 125 (1987) BLR (DE3) (A. Roca, University of Wisconsin: cited in the by Standard methods (26). The resulting coding sequence Novogen Catalogue) using procedures described previously Substitutes an Ala for the initial GFP Met, which becomes (29). The resulting strain (SMC3), because of the reduced the Second amino acid in the polypeptide. The E. coli Strain recombination of the host, was much more stable for GFP expression (all the colonies on plates with amplicillin but 45 BL21(DE3).Lys S. F. W. Studier and B. A. Moffat, J. Mol. without the IPTG inducer (29) were brightly fluorescent Biol. 189, 113 (1986) was transformed with the resulting when viewed with a hand-held UV lamp). plasmid (TU#58) and grown at 37° C. Control bacteria were A second construct (TU#147), similar to TU#58, was transformed with peT3a. Bacteria were grown on nutrient made with pET11 (A. H. Rosenberg, et al. 1987). Expression plates containing amplicillin (100 ug/ml) and 0.8 mM IPTG. in BLR (DE3) from this plasmid was more tightly con 50 Transformed bacteria from this transformation Show green trolled; expression was seen soon after IPTG was added, but fluorescence when irradiated with ultraViolet light. A recom only after Some time without inducer. binant plasmid of this bacteria was used for the experiments The SMC3 strain was used to test the requirement for described here and the experiment in FIG. 2 and the experi aerobic growth of the bacteria for the production of a ment in Note 10. Several months later, applicants noticed fluorescent product. Plates were grown under anaerobic 55 that the bacterial colonies can be divided into two groups: 1) conditions in a Gas-Pak container according to the instruc Strongly fluorescent; and 2) weakly fluorescent (applicants tions of the manufacturer (Becton Dickinson Microbiology believe that the weakly fluorescent may have mutated, Systems). Colony growth was slowed under anaerobic con disabled or partial or completely deleted TU#58). One ditions and the resulting colones were not detectably fluo Strongly fluorescent colony was picked to generate the rescent after at least 3 days of growth under anaerobic 60 bacterial strain SMC1 (ATCC Accession No. 69554). A conditions (using the hand-held UV lab). Colonies, however, Similar PCR-generated fragment (see note 11) was used in became fluorescent after a day's exposure to room air (Some applicants C. elegans construct. AS others are beginning to fluorescence was seen after a few hours). use pGFP10.1, applicants have heard that while similar PCR REFERENCES AND NOTES fragments produce a fluorescent product in other organisms 65 (R. Heim, S. Emir, and R. Tsien, personal communication; S. 1. O. Shimomura, F. H. Johnson, Y. Saiga, J. Cell. Comp. Wang and T. Hazelrigg, personal communication; L. Lanini Physiol. 59, 223 (1962). and F. McKeon, personal communication; see note 23), the 6,146,826 11 12 EcoRI fragment does not (R. Heim, S. Emr, and R. Tsien, increments. The Spectral band widths were adjusted to personal communication; A. Coxon, J. R. Chaillet, and T. 0.94 nm for all spectra. Bestor, personal communication). These results may indi 11. Wild-type and mutant C. elegans animals were grown cate that elements at the 5' end of the Sequence or at the Start and genetic Strains were constructed according to S. of translation inhibit expression. Brenner, 77, 71 (1974). 9. Applicants used a variety of microscopes (Zeiss The plasmid pCFP10.1 was used as a template for PCR Axiophot, Nikon Microphot FXA, and Olympus BH2 (with the 5' primer GAATAAAAGCTAGCAAAGATGAG RFC and BX50) equipped for epifluorescence micros TAAAG (Sequence ID No. 2) and the 3' T3 primer) to copy. Usually filter Sets for fluorescein isothiocyanate generate a fragment with a 5' NheI site (at the start of fluorescence were used (for example, the Zeiss filter Set translation) and a 3" EcoRI site (3' of the termination codon). used a BP450-490 excitation filter, 510 nm dichroic, The DNA was cut to produce an Nhel-EcoRI fragment that and either a BP515–565 or a LP520 emission filter), was ligated into plasmid pPD 16.51 (12, 27), a vector although for Some experiments filter Sets that eXcited at containing the promoter of the C. elegans mec-7 gene. lower wavelengths were used (for example, a Zeiss Wild-type C. elegans were transformed by coinjecting this filter Set with BP395-440 and LP470 filters and a 460 15 DNA (TU#64) and the DNA for plasmid pRF4, which nm dichroic or with BP340–390 and LP400 filters with contains the dominant rol-6(Sul)06) mutation, into adult C. a 395 nm dichroic). In Some instances a xenon lamp elegans gonads as described by C. M. Mello, J. M. Kramer, appeared to give a more intense fluorescence than a D. Stinchcomb, and V. Ambros, EMBO.J. 10, 3959 (1991). mercury lamp when cells were illuminated with light A relatively stable line was isolated (TU1710) and the DNA around 470 nm, although usually the results were it carried was integrated as described by Mitani et al. (15) to comparable. No other attempts were made to enhance produce the integrated elements us3 and us4 (in Strains the signal (for example, by using low intensity light TU1754 and TU1755, respectively). cameras), although this may be useful in Some Living C. elegans animals were mounted on agar (or instances. agarose) pads as described (16), often with 10 mM NaNs as Previous experiments had shown that the native protein 25 an anesthetic (28) (another nematode anesthetic, was fluorescent after glutaraldehyde fixation (W. W. Ward, phenoxypropanol, quenched the fluorescence) and examined unpub. data). S. Wang and T. Hazelrigg (personal commu with either a Zeiss universal or axiophot microScope. For C. nication; 23) have found that GFP fusion proteins in Droso elegans, a long-pass emission filter works best because the phila melanogaster are fluorescent after formaldehyde fixa animals intestinal autofluorescence, (which increases as the tion. Applicants have confirmed that fluorescence persists animal matures), appears yellow (with band-pass filters the after formaldehyde fixation with applicants C. elegans autofluorescence appears green and obscures the GFP animals and with recombinant GFP isolated from E. coli. fluorescence). The chemicals in nail polish, which is often used to Seal Because much more intense fluorescence was seen in uIS4 cover slips, however, did appear to interfere with the C. than us3 animals (for example, it was often difficult to see elegans GFP fluorescence. 35 the processes of the ALM and PLM cells in uIs3 animals 10. In the applicants initial experiments, GFP was puri when the animals were illuminated with a mercury lamp), fied from 250 ml cultures of BL21 (DE3).Lys S bacteria the former have been used for the observations reported containing TU#58; bacteria were grown in LB broth here. The general pattern of cell body fluorescence was the (26) containing amplicillin (100 tug/ml) and 0.8 mM Same in both Strains and in the parental, nonintegrated Strain IPTG. Induction was best when IPTG was present 40 (fluorescence in this Strain was as Strong as that in the uls4 continually. Nevertheless, Subsequent experiments animals). The uls4 animals, however, did show an unusual with bacterial strain SMC1 indicate that the bacteria phenotype: both the ALM and PLM touch cells were often could not grow in the constant presence of IPTG but displaced anteriorly. The mature cells usually had processes can be induced by the IPTG during the log phase in the correct positions, although occasional cells had growth. The production of fluorescent protein is best at 45 abnormally-projecting processes. These cells could be iden room temperature. Cells were washed in 4 ml of 10 mM tified as touch receptor cells, because the fluorescence was Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl, and dependent on mec-3, a homeobox gene that specifies touch 10 mM dithiothreitol A. Kumagai and W. G. Dunphy, cell fate (13, 15, 18, 28). mec-7 expression is reduced in the Cell 64, 903 (1991) and then Sonicated (2x20 sec) in ALM touch cells of the head (but not as dramatically in the 4 ml of the same buffer containing 0.1 mM PMSF, 50 PLM touch cells of the tail) in mec-3 gene mutants (13, 15). pepstatin A (1 lug/ml), leupeptin (1 lug/ml), and aproti Applicants find a similar change of GFP expression in a nin (2 tug/ml), and centrifuged at 5,000 rpm for 5 min mec-3 mutant background for both uIs3 and uls4. Thus, GFP in the cold. The Supernatant was centrifuged a Second accurately represents the expression pattern of the mec-7 time (15,000 rpm for 15 min) and then diluted seven gene. It is likely that the reduced Staining in uIS3 animals and fold with 10 mM Tris (pH 8.0), 10 mM EDTA, and 55 the misplaced cells in us4 animals is the result of either 0.02% NaN. Corrected excitation and emission spec Secondary mutations or the amount and position of the tra were obtained with a SPEX FIT11 spectrofluorom integrated DNA. eter and compared with the purified L isoprotein form 12. C. Savage, M. Hamelin, J. G. Culotti, A. Coulson, D. of GFP from A. victoria (M. Cutler, A. Roth, and W. W. G. Albertson, M. Chalfie, Genes Dev. 3, 870 (1989). Ward, unpub. data). The excitation spectra were mea 60 Sured from 300-500 nm with a fixed emission wave 13. M. Hamelin, I. M. Scott, J. C. Way, J. G. Culotti, length of 509 nm, and the emission spectra were EMBO J. 11, 2885 (1992). measured from 410-600 nm with a fixed excitation of 14. A. Duggan and M. Chalfie, unpub. data. 395 nm. All Spectra were recorded as Signal-reference 15. S. Mitani, H. P. Du, D. H. Hall, M. Driscoll, M. data (where the reference is a direct measurement of the 65 Chalfie, Development 119, 773 (1993). lamp intensity with a separate photomultiplier tube) at 16. J. E. Sulston and H. R. Horvitz, Develop. Biol. 56, 110 room temperature with 1 Sec integration times and 1 nm (1977). 6,146,826 13 14 17. W. W. Walthall and M. Chalfie, Science 239, 643 essary posttranslational modification unique to A. vic (1988). toria or nonspecific damage within the cells. 18. M. Chalfie and J. Sulston, Dev. Biol. 82,358 (1981). 22. Reviewed in T. J. Silhavy and J. R. Beckwith, Micro 19. In adults, the thicker size of the animals and the more biol. Rev. 49,398 (1985); S. J. Gould and S. Subramani, intense autofluorescence of the intestine tend to obscure Anal. Biochem. 175, 5 (1988); and G. S. A. B. Stewart these cells. and P. Williams, J. Gen. Microbiol. 138, 1289 (1992). 20. These include several cells in the head (including the 23. R. Heim, S. Emir, and R. Tsien (personal FLP cells) and tail of newly hatched animals and the BDU cells, a pair of neurons just posterior to the communication) have found that GFP expression in pharynx. Expression of mec-7 in these cells has been 1O Saccharomyces cerevisiae can make the cells Strongly Seen previously (13,15). The Strongest staining of these fluorescent without causing toxicity. S. Wang and T. non-touch receptor neurons are a pair of cells in the tail Hazelrigg (personal communication) have found that that have anteriorly directed processes that project both C-terminal and N-terminal protein fusions with along the dorsal muscle line. It is likely that these are GFP are fluorescent in Drosophila melanogaster. L. the ALN cells, the sister cells to the PLM touch cells J. 15 Lanini and F. McKeon (personal communication) have G. White, E. Southgate, J. N. Thomson, and S. Brenner, expressed a GFP protein fusion in mammalian (COS) Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1 (1986). cells. E. Macagno (personal communication) is 21. The photobleaching with 395-440 nm light is further expressing GFP in leeches. T. Hughes (personal accelerated, to within Seconds, in the presence of 10 communication) is expressing GFP in mammalian mM NaNa, which is used as a C. elegans anesthetic HEK293 cells. (11). However, when cells in C. elegans have been 24. Applicants have generated Several other plasmid con photobleached, Some recovery is seen within 10 min. Structions that may be useful to investigators. These Further investigation is needed to determine whether include a pBluescript II KS (+) derivative (TU#65) this recovery represents de novo synthesis of GFP. containing a Kpn-EcoRI fragment encoding GFP Rapid photobleaching (complete within a minute) of 25 with an Age site 5' to the translation start and a BSmI the green product was also seen when C. elegans was Site at the termination codon. Also available are gfp illuminated with 340-390 nm light. Unlike the pho versions (TU#60 TU#63) of the four C. elegans lacZ tobleaching with 395-440 nm light, which abolished expression vectors (pPD16.43, pPD21.28, pPD22.04, fluorescence produced by the 340–390 or 450-490 nm and pPD22.11, respectively) described by Fire et al., light, photobleaching with 340-390 nm light did not 1990 (27) except that they lack the KpnI fragment appear to affect the fluorescence produced by 395-490 containing the SV40 nuclear localization Signal. or 450-490 nm light. Indeed, the fluorescence produced 25. J. P. Miller and A. Selverston, Science 206, 702 by 450-490 nm light appeared to be more intense after (1979). brief photobleaching by 340-390 nm light. This selec 35 26. J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular tive photobleaching may indicate the production of cloning. A laboratory manual, 2nd Ed. Cold Spring more than one fluorescent product in the animal. These Harbor Laboratory Press, Cold Spring Harbor, New data on GFP fluorescence within E. coli and C. elegans is in contrast to preliminary Studies that Suggest that the York, (1989). isolated native and E. coli proteins are very photo 27. A. Fire, S. W. Harrison, and D. Dixon, Gene 93, 189 stable. Applicants do not know whether this in vivo 40 (1990). Sensitivity to photobleaching is a normal feature of the 28. J. C. Way and M. Chalfie, Cell 54, 5 (1988). jellyfish protein (the fluorescence in A. victoria has not 29. Chalfie, M., Tu. Y., Euskirchen, G., Ward, W. W., been examined) or results from the absence of a nec Prasher, D. C., Science 263,802 (1994).

SEQUENCE LISTING

(1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 1 : 6,146,826 15 16

-continued

ACAAAGGCTA GCAAAGGAGA AGAAC 25

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE cDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (xi). SEQUENCE DESCRIPTION: SEQ ID NO:2:

GAATAAAAGC TAGCAA AGAT GAGTAAAG 28

What is claimed is: b) culturing cells resulting from Step (a) under conditions 1. A cell comprising a DNA molecule having a regulatory 25 permitting expression of the fluorescent mutant of element from a gene, other than a gene encoding a green green fluorescent protein and the protein of interest; fluorescent protein, operatively linked to a DNA sequence and encoding a fluorescent mutant of green fluorescent protein of Aequorea victoria. c) Selecting the cultured cells which express the fluores 2. The cell of claim 1, wherein the cell is selected from the cent mutant of green fluorescent protein, thereby Select group consisting of a bacterial cell, a yeast cell, a fungal cell, ing cells expressing the protein of interest. a plant cell and an animal cell. 17. The method of claim 16, wherein the first DNA 3. The cell of claim 1, wherein the regulatory element is molecule and the second DNA molecule are linked. a promoter. 18. The method of claim 16, wherein the cells are selected 4. The cell of claim 1, wherein the regulatory element is from the group consisting of bacterial cells, yeast cells, an enhancer. 35 fungal cells, plant cells and animal cells. 5. The cell of claim 1, wherein the cell is an E. coli cell. 19. The method of claim 16, wherein the cells are insect 6. A method to produce a fluorescent mutant of green cells or nematode cells. fluorescent protein of Aequorea victoria comprising: 20. A method for localizing a protein of interest in a cell a) culturing the cell of claim 1 So that the cell produces the which comprises: fluorescent mutant; and 40 a) introducing into a cell a DNA molecule having a b) isolating and purifying the fluorescent mutant So pro Sequence encoding the protein of interest linked to a duced by the cell. DNA sequence encoding a fluorescent mutant of green 7. The cell of claim 1, wherein the cell is an insect cell or fluorescent protein of Aequorea victoria Such that the a nematode cell. protein produced by the DNA molecule will have the 8. The cell of claim 3, wherein the promoter is activated 45 protein of interest fused to the fluorescent mutant of by a heavy metal. green fluorescent protein of Aequorea victoria, 9. The cell of claim 3, wherein the promoter is that from b) culturing the cell under conditions permitting expres a P450 gene. Sion of the fused protein; and 10. The cell of claim 3, wherein the promoter is from a gene encoding a StreSS protein. 50 c) detecting the location of the fused protein, thereby 11. The cell of claim 3, wherein the promoter is from a localizing the protein of interest in the cell. gene required for cell viability. 21. The method of claim 20, wherein the cell normally expresses the protein of interest. 12. The cell of claim 5 designated pCFP10.1 (ATCC 22. A method of detecting expression of a gene in a cell Accession No. 75547). which comprises: 13. The method of claim 6, wherein the cell is an E. coli 55 cell. a) introducing into the cell a DNA molecule having the 14. The cell of claim 10, wherein the stress protein is a Sequence of the gene linked to a DNA molecule encod heat-Shock protein. ing a fluorescent mutant of green fluorescent protein of 15. The method of claim 13, wherein the cell is cultured Aequorea victoria Such that a regulatory element of the aerobically. 60 gene controls expression of the fluorescent mutant of 16. A method for Selecting cells expressing a protein of green fluorescent protein of Aequorea victoria, interest which comprises: b) culturing the cell under conditions permitting expres a) introducing into the cells a first DNA molecule having Sion of the gene; and a DNA sequence encoding the protein of interest and a c) detecting the expression of the fluorescent mutant of Second DNA molecule having a DNA sequence encod 65 green fluorescent protein of Aequorea victoria in the ing a fluorescent mutant of green fluorescent protein of cell, thereby detecting expression of the gene in the Aeguorea victoria, cell. 6,146,826 17 18 23. A method for detecting expression of a gene in a living ism; and detecting expression of the fluorescent mutant organism which comprises: of green fluorescent protein of Aequorea victoria in a) introducing into a cell of the subject a DNA molecule different tissueS of the living organism, thereby deter having the Sequence of the gene linked to a DNA mining the tissue-specificity of the transcription of the molecule encoding a fluorescent mutant of green fluo 5 first DNA sequence in the living organism. rescent protein of Aequorea victoria Such that a regul 25. A method for producing a fluorescent molecular latory element of the gene controls expression of the weight protein marker comprising: fluorescent mutant of green fluorescent protein of a) linking a first DNA molecule encoding a fluorescent Aequorea victoria, mutant of green fluorescent protein of Aequorea victo b) culturing the cell under conditions permitting expres ria with a Second DNA molecule encoding a known Sion of the gene, and amino acid Sequence which is in the same reading c) detecting the expression of the fluorescent mutant of frame as the first DNA molecule; green fluorescent protein of Aequorea victoria in the cell, thereby detecting expression of the gene in the b) introducing the linked DNA molecule of step (a) into living organism. 15 a protein expression System permitting the expression 24. A method for determining the tissue-specificity of of a fusion protein comprising the fluorescent mutant transcription of a first DNA sequence in a living organism linked to the known amino acid Sequence; which comprises: c) recovering the fusion protein expressed in Step (b): and a) introducing into a cell of the living organism a DNA d) determining the molecular weight of the fusion protein molecule which comprises the first DNA sequence from Step (c), thereby producing a fluorescent molecu linked to a Second DNA sequence encoding a fluores lar weight protein marker. cent mutant of green fluorescent protein of Aequorea 26. The method of claim 25, further comprising purifica victoria Such that the first DNA sequence controls tion of the expressed protein. expression of the fluorescent mutant of green fluores cent protein of Aequorea victoria in the living organ k k k k k