| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 23, 2001 |
| PATENT TITLE |
Rapidly degrading GFP-fusion proteins and methods of use |
| PATENT ABSTRACT | Green fluorescent protein (GFP) is widely used as a reporter in determining gene expression and protein localization. The present invention provides fusion proteins with a half life of ten hours or less with several embodiments having half lives of 4 hours or less. Such proteins may be constructed by fusing C-terminal amino acids of the degradation domain of mouse ornithine decarboxylase (MODC), which contains a PEST sequence, to the C-terminal end of an enhanced variant of GFP (EGFP). Fluorescence intensity of the fusion protein in transfected cells is similar to that of EGFP, but the fusion protein, unlike EGFP, is unstable in the presence of cycloheximide. Specific mutations in the MODC region have resulted in mutants with varying half lives, useful for a variety of purposes. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | July 30, 1999 |
| PATENT REFERENCES CITED |
Rechsteiner et al., Cell Biology, vol. 1, pp. 435-440, 1990.* Loetscher et al., The Journal of Biological Chemistry, vol. 266, No. 17, pp. 11213-11220, 1991.* Li et al., The Journal of Biological Chemistry, vol. 273, No. 52, pp. 34970-34975, Dec. 25, 1998.* Moradpour et al., Characterization of Cell Lines Allowing Tightly Regulated Expression of Hepatitis C Virus Core Protein, Virology vol 222, p. 51-63, 1996. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of assaying the positive or negative regulatory function of a transcriptional or translational control sequence which controls or regulates the expression of a coding sequence in a host cell with a transient fluorescent reporter protein, comprising the steps of: a) transfecting cells with an expression vector comprising a DNA sequence that codes for a fluorescent fusion protein comprising a Green Fluorescent Protein (GFP) and a PEST sequence, wherein said fusion protein has a half life of no more than about ten hours, and wherein said expression vector contains said transcriptional or translational control sequence that may affect the expression of said fluorescent fusion protein; b) transfecting cells with a control expression vector comprising a DNA sequence that codes for said fluorescent fusion protein, wherein said control expression vector does not contain said transcriptional or translational control sequence; and c) detecting a presence, absence or amount of fluorescence in said cells, wherein an increase or decrease in fluorescence in cells transfected with the vector containing the control sequence compared to cells transfected with the vector not containing the control sequence indicates a positive or negative regulatory function, respectively for said transcriptional or translational control sequence. 2. The method of claim 1, wherein said transcriptional or translational control sequences are selected from the group consisting of a promoter, an enhancer, a transcription terminator, and a polyadenylation signal. 3. The method of claim 1 wherein the GFP is wild type GFP, GFPS65T, humanized GFP, EGFP, blue-emitting GFP, cyan-emitting GFP or yellow-emitting GFP. 4. The method of claim 1 the PEST sequence is selected from the group consisting of a murine ornithine decarboxylase (MODC) PEST sequence, amino acids 376-461 of MODC, amino acids 376-456 of MODC and amino acids 422-461 of MODC. 5. The method of claim 1 wherein the PEST sequence is selected from the group consisting of variants of MODC.sub.376-461, MODC.sub.376-456, and MODC.sub.422-461, containing amino acid substitutions selected from the group consisting of P26A/P427A, P438A, E428A/E430A/E431A, E444A, S440A, S445A, T436A, D433A/D434A and D448A. 6. A method of assaying the positive of negative effect of a test compound on the regulatory function of a transcriptional or translational control sequence which controls or regulates the expression of a coding sequence in a host cell with a transient fluorescent reporter protein, comprising the steps of: a) transfecting cells with an expression vector comprising a DNA sequence that codes for a fluorescent fusion protein comprising a Green Fluorescent Protein (GFP) and a PEST sequence and having a half life of no more than about ten hours, wherein said expression vector contains said transcriptional or translational control sequence that may affect the expression of said fluorescent fusion protein; b) treating said transfected cell with said test compound or a control compound that has no effect on the expression of said fluorescent fusion protein; and c) detecting an amount of fluorescence in said cells, wherein an increase or decrease in fluorescence in cells treated with said test compound compared to cells treated with said control compound indicates a positive or negative effect respectively of said test compound on the regulatory function of said transcriptional or translational control sequence. 7. The method of claim 6 wherein the GFP is wild type GFP, GFPS65T, humanized GFP, EGFP, blue-emitting GFP, cyan-emitting GFP or yellow-emitting GFP. 8. The method of claim 6 wherein the PEST sequence is selected from the group consisting of a murine ornithine decarboxylase (MODC) PEST sequence, amino acids 376-461 of MODC, amino acids 376-456 of MODC and amino acids 422-461 of MODC. 9. The method of claim 6 wherein the PEST sequence is selected from the group consisting of variants of MODC.sub.376-461, MODC.sub.376-456, and MODC.sub.422-461, containing amino acid substitutions selected from the group consisting of P26A/P427A, P438A, E428A/E430A/E431A, E444A, S440A, S445A, T436A, D433A/D434A and D448A. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to the field of biochemical assays and reagents. More specifically, this invention relates to modified fluorescent proteins and to methods for their use. 2. Description of the Related Art Because of its easily detectable green fluorescence, green fluorescent protein (GFP) from the jellyfish Aequorea Victoria has been used widely to study gene expression and protein localization. GFP fluorescence does not require a substrate or cofactor; hence, it is possible to use this reporter in numerous species and in a wide variety of cells. GFP is a very stable protein which can accumulate and thus is often toxic to mammalian cells. Recently, crystallographic structures of wild-type GFP and the mutant GFP S65T reveal that the GFP tertiary structure resembles a barrel (Ormo et al. (1996) Science 273: 1392-1395; Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nature Biotech 14: 1246-1251). The barrel consists of beta sheets in a compact antiparallel structure. In the center of the barrel; an alpha helix containing the chromophore is shielded by the barrel. The compact structure makes GFP very stable under diverse and/or harsh conditions, such as protease treatment, making GFP an extremely useful reporter in general. On the other hand, its stability makes it difficult to determine short-term or repetitive events. A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful for a variety of research purposes. New versions of GFP have been developed via mutation, including a "humanized" GFP DNA, the protein product of which has increased synthesis in mammalian cells (see Cormack, et al., (1996) Gene 173, 33-38; Haas, et al., (1996) Current Biology 6, 315-324; and Yang, et al., (1996) Nucleic Acids Research 24, 4592-4593). One such humanized protein is "enhanced green fluorescent protein" (EGFP). Other mutations to GFP have resulted in blue-, cyan- and yellow-fluorescent light emitting versions. Ornithine decarboxylase (ODC) is an enzyme critical in the biosynthesis of polyamines. Murine ornithine decarboxylase is one of most short-lived proteins in mammalian cells, with a half life of about 30 minutes (Ghoda, et al., (1989) Science 243, 1493-1495; and Ghoda, et al. (1992) Mol. Cell. Biol. 12, 2178-2185). Rapid degradation of murine ornithine decarboxylase has been determined to be due to the unique composition of its C-terminus, a portion of which has a PEST sequence--a sequence which has been proposed as characterizing short-lived proteins. The PEST sequence contains a region enriched with proline (P), glutamic acid (E), serine (S), and threonine (T), often flanked by basic amino acids, lysine, arginine, or histidine (Rogers, et al., (1989) Science 234:364-68; Reichsteiner, M. (1990) Seminars in Cell Biology 1:433-40). The ornithine decarboxylase of Trypanosoma brucei (TbODC) does not have a PEST sequence, and is long-lived and quite stable when it is expressed in mammalian cells (Ghoda, et al. (1990) J. Biol. Chem. 265: 11823-11826); however appending the C terminus of murine ornithine decarboxylase to TbODC makes TbODC become unstable. Moreover, deletion of the C-terminal, PEST-containing region from murine ornithine decarboxylase prevents its rapid degradation (Ghoda, L., et al. (1989) Science 243: 1493-1495). The prior art is deficient in a destabilized or short-lived GFP. The present invention fulfills this need in the art. SUMMARY OF THE INVENTION A rapid turnover or destabilized GFP can be used in research applications where prior art GFPs cannot. Such applications include using the destabilized GFP as a genetic reporter for analyzing transcriptional regulation and/or cis-acting regulatory elements, or as a tool for studying protein degradation. Further, a rapid turnover GFP permits easier development of stable cell lines which express the GFP gene, since toxic levels of GFP are avoided because the GFP protein is degraded quickly. The present invention provides a fusion protein with a half life decreased markedly from that of wildtype GFP. In one embodiment, there is provided a fusion protein comprising an EGFP fused to a peptide producing a destabilized protein. In another embodiment, there is provided a fusion protein with a half life of about ten hours or less, preferably with a half life of about 4 hours or less, more preferably with a half life of 2 hours or less and even more preferably with a half life of 1 hour or less. A preferred embodiment of this aspect of the invention includes EGFP, and/or a PEST sequence-containing portion of a C-terminus of murine ornithine decarboxylase (MODC). Specific preferred embodiments of the present invention include EGFP-MODC.sub.376-461 ; EGFP-MODC.sub.376-456 ; EGFP-MODC.sub.422-461 ; P426A/P427A; P438A; E428A/E430A/E431A; E444A; S440A; S445A; T436A; D433A/D434A; and D448A. In yet another aspect of the invention, there is provided an isolated DNA molecule encoding a fluorescent fusion protein with a half life that is markedly decreased from that of wildtype GFP. In one embodiment of this aspect of the invention, there is provided a n isolated DNA molecule encoding a fluorescent fusion protein with a half life of about ten hours or less, preferably with a half life of about 4 hours or less, more preferably with a half life of 2 hours or less and even more preferably with a half life of 1 hour or less. In a preferred embodiment of this aspect of the invention, the isolated DNA molecule encoding the fluorescent fusion protein is a synthetic GFP gene containing codons preferentially found in highly expressed human proteins. Further, the present invention provides a vector capable of expressing the isolated DNA molecule encoding a GFP fusion protein with a decreased half life. In one embodiment of the vector, the vector contains an inducible promoter. In another aspect of the invention, there is provided a method of labeling cells with a transient GFP reporter. In this method, a DNA vector comprising an inducible promoter and the isolated DNA encoding a GFP fusion protein with a decreased half life is utilized. This vector is transfected into cells which are cultured under conditions in which the promoter induces transient expression of the GFP fusion protein of the present invention, which provides a transient fluorescent label. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic map of EGFP and EGFP-MODC fusion proteins. EGFP is fused with a region of the C terminus of MODC, including amino acids from 376 to 461, 376 to 456 or 422 to 461. The fusion proteins were expressed in CHO K1 Tet-off cells and their fluorescence intensities were compared under a fluorescence microscope. FIG. 2 shows the fluorescence stabilities of EGFP and EGFP-MODC.sub.422-461 in cells in the presence of cycloheximide and examined with a fluorescence microscope. CHO K1 Tet-off cells were transfected with vectors expressing these two proteins. After 24 hours, the transfected cells were treated with 100 mg/ml cycloheximide for 0, 1, 2, 3, and 4 hours. FIG. 3 shows flow cytometric analysis of the fluorescence stabilities of EGFP and EGFP-MODC.sub.422-461. CHO K1 Tet-off cells were transfected with EGFP and EGFP-MODC.sub.422-461. After 24 hours, the transfected cells were treated with 100 .mu.g/ml cycloheximide for 0, 1, 2, and 3 hours. The treated cells were collected with EDTA and 10,000 cells were subjected to FACS analysis FIG. 4 is a graph summarizing the flow cytometric data from FIG. 3, demonstrating that EGFP-MODC.sub.422-461 -transfected cells rapidly lose fluorescence after cycloheximide treatment, whereas EGFP cells maintain fluorescence. FIG. 5 is a photograph of western blot analysis of protein stabilities of EGFP and EGFP-MODC.sub.422-461. Cells collected during flow cytometry were used for preparing cell lysates. The cell lysates were subject to SDS gel electrophoresis and transferred onto a membrane. EGFP and the EGFP fusion protein were detected with a monoclonal antibody against GFP. FIG. 6 is a schematic map of the PEST sequence of the fusion EGFP-MODC.sub.422-461 indicating the position of the mutations. FIG. 7 is a table summarizing the results obtained measuring persistence of fluorescent signal in transfected CHO K1 Tet-off cells expressing EGFP, EGFP-MODC.sub.422-461, and the PEST mutants. Transfection was performed in CHO/tTA cells using the procedure given in Example 2. After 24 hours, cells were treated with cycloheximide for 0, 2, and 4 hours, and analyzed for fluorescence by FACS Caliber. FIG. 8 shows a schematic illustration of d2EGFP, dECFP and dEYFP. FIG. 9 shows the construction of destabilized EGFP Variants. FIG. 10 shows the fluorescence stabilities of EGFP and dEGFP Variants. FIG. 11 shows the increase in induction by CRE-d1EGFP and CRE-d2EGFP. DETAILED DESCRIPTION OF THE INVENTION The invention describes a genetically-engineered fluorescent protein that is destabilized, having a rapid turnover in a cell. More specifically, this fusion protein comprises a fluorescent protein which has a half life of no more than about ten hours and most preferably with a half life of no more than 2 hours. Preferably, the fluorescent protein is selected from the group consisting of EGFP, ECFP and EYFP. In one embodiment, the engineered GFP is a fusion protein of EGFP and a peptide the inclusion of which produces a destabilized protein. An example of such a peptide is the C-terminal region of murine ornithine decarboxylase (MODC). In a specific, illustrative case, the degradation domain of murine ornithine decarboxylase from amino acids 422 to 461 was appended to the C-terminal end of an enhanced variant of GFP (EGFP). The fluorescence intensity of the EGFP-MODC.sub.422-461 fusion protein in transfected cells was similar to that of EGFP, but the fusion protein, unlike EGFP, dissipated over time in cells treated with cycloheximide. The half-life of the fluorescence of the EGFP-MODC.sub.422-461 fusion protein was about 2 hours, while that of EGFP was more than 24 hours. The ornithine decarboxylase degradation domain dramatically decreases EGFP stability. The rapid turnover version of EGFP has at least four advantages over EGFP. The rapid turnover of the EGFP-MODC fusion causes less toxicity to cells expressing the fusion protein. Thus, one advantage is the feasibility of establishing stable cell lines using DNA coding for EGFP-murine ornithine decarboxylase. Further, the destabilized EGFP-MODC decreases EGFP accumulation. Accumulation of fluorescent protein can interfere with the sensitivity of analysis. Thus, the destabilized, rapid turnover fusion protein renders more sensitive results. Additionally, destabilized EGFP can be used as a transient reporter to study transcriptional regulation and/or action of cis-acting regulatory elements. Finally, the EGFP-MODC fusion protein can be used to study processes involving multiple gene expression. Moreover, the EGFP-MODC fusion protein has the advantages inherent to use of EGFP. For example, the use of EGFP in drug screening assays is particularly advantageous because GFP fluorescence can be detected intracellularly without performing additional expensive steps; e.g. lysing cells, adding exogenous substrates or cofactors, fixing the cell preparation, etc. A single illustration of such an assay is screening test compounds for interruption of the TNF activation pathway, a pathway which ultimately affects apoptosis. Compounds identified in the assay would be useful in controlling the cellular processes involved in cancer and inflammation. Further, the reporter gene of the present invention can be linked with different enhancer elements and used to monitor diverse biological processes such as heat response, response to heavy metals, glucocorticoid activation or response to cAMP. In particular, destabilized EGFP is useful for studying developmental processes where genes are transiently expressed, dynamics of protein transport, localization of proteins within cells, and periodic and cyclical expression of genes that control unique biological phenomena such as circadian rhythms. Indeed, other applications of the EGFP-MODC fusion protein in screening assays would be appreciated readily by those having ordinary skill in this art. Moreover, by using an inducible promoter, expression of the EGFP-MODC fusion protein is activated or deactivated at will, making a construct expressing the protein useful in cell lineage studies. Prior art GFP models express GFP at levels that are toxic and interfere with cell development, thus making cell lineage studies impossible. Additionally, destabilized EGFP can be used as a reporter to study the kinetics of mRNA transcription from a regulated promoter, because the fluorescence intensity of destabilized EGFP is a direct measure of the level of gene expression at any given time point. As used herein, the term "GFP" refers to the basic green fluorescent protein from Aequorea Victoria, including prior art versions of GFP engineered to provide greater fluorescence o r fluoresce in different colors. The sequence of A. Victoria GFP has been disclosed in Prasher D.C. et al. (1992) Gene 111:229-33. As used herein, the term "EGFP" refers to GFP which has been "humanized", as reported in Kain et al. (1995) Biotechniques 19(4):650-55. "Humanized" refers to changes made to the GFP nucleic acid sequence to optimize the codons for expression of the protein in human cells. As used herein, the term "peptide which produces a destabilized protein" refers to a sequence of amino acids or a peptide which promotes destabilization or rapid turnover of the protein of which it is a part; i.e., by inducing protein degradation. The PEST sequence described herein is one such sequence. Other sequences known in the art are those peptides that promote phosphorylation and protein-protein interactions. As used herein, the term "EGFP-MODC" refers to EGFP fused at its C terminus to murine ornithine decarboxylase sequences. As used herein, the term "P438A" refers to an EGFP-MODC fusion protein in which the proline at position 438 in the murine ornithine decarboxylase sequence (a proline residing in the PEST portion of the sequence) has been replaced with alanine. The same nomenclature is used for EGFP-MODC mutants P426A/P427A; E428A/E430A/E431A; E444A; S440A; S445A; T436A; D433A/D434A; and D448A. Further elucidation is shown in FIG. 6. As used herein, the term "half life" refers to the period of time in which half of the fluorescent signal from a fluorescent protein expressed in cells disappears and half remains. As used herein, the term "Tc" refers to tetracycline. As used herein, the term "CHX" refers to cycloheximide. In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds. (1985)); "Transcription and Translation" (B. D. Hames & S. J. Higgins eds. (1984)); "Animal Cell Culture" (R. I. Freshney, ed. (1986)); "Immobilized Cells and Enzymes" (IRL Press, (1986)); B. Perbal, "A Practical Guide To Molecular Cloning" (1984). A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. A DNA "coding sequence" is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. A polyadenylation signal and transcription termination sequence may be located 3' to the coding sequence. Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for and/or regulate expression of a coding sequence in a host cell. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various vectors of the present invention. As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence. A cell has been "transformed" or "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. A "heterologous" region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, heterologous DNA includes coding sequence in a construct where portions of genes from two different sources have been brought together so as to produce a fusion protein product. Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. As used herein, the term "reporter gene" refers to a coding sequence attached to heterologous promoter or enhancer elements and whose product may be assayed easily and quantifiably when the construct is introduced into tissues or cells. Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, which provide for the expression of a coding sequence in a host cell. The amino acids described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH.sub.2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence: Y Tyr tyrosine G Gly glycine F Phe Phenylalanine M Met methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine Q Gln glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cys cysteine It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein. Thus, the present invention is directed to a fusion protein comprising GFP so that the resulting fusion protein has a half life of no more than about ten hours and as little as less than one hour. In a preferred form, the GFP is EGFP. Preferably, the fusion protein comprises EGFP fused to a PEST sequence-containing portion of a C-terminus of murine ornithine decarboxylase (MODC). Representative examples of PEST sequence-containing portion of a C-terminus of murine ornithine decarboxylase include MODC.sub.376-461, MODC.sub.376-456, MODC.sub.422-461, P426A/P427A, P438A, E428A/E430A/E431A, E444A, S440A, S445A, T436A, D433A/D434A and D448A. One example of the GFP fusion protein of the present invention has the sequence shown in SEQ ID No. 1. The present invention is also directed to an isolated DNA molecule encoding the fusion protein which comprises a fluorescent protein selected from the group consisting of EGFP, ECFP and EYFP. One example of the isolated DNA of the present invention has the sequence shown in SEQ ID No: 2. The present invention is also directed to a vector capable of expressing this isolated DNA molecule. In one form, the vector contains a inducible promoter and is a tetracycline-regulated expression vector. The present invention is also directed to a method of producing a stable cell line that expresses a fluorescent protein, e.g., GFP, comprising the step of transfecting cells with a vector disclosed herein. In addition, the present invention is directed to a method of assaying activation or deactivation of promoters or other transcriptional or translational elements with a transient fluorescent reporter protein, comprising the steps of transfecting cells with an expression vector comprising a fusion protein having a half life of no more than about ten hours, preferably less than four hours and most preferably less than one hour, wherein the fusion protein is under the influence of the promoter, transcriptional or translational element, and detecting the presence, absence or amount of fluorescence in said cells. In this method, the amount of fluorescence present in the cell is a measure of the fluorescent protein that is being expressed. Detecting differences in fluorescence intensity between cells expressing the fluorescent protein under different transcriptional or translational elements of interest is a rapid and straightforward procedure to measure effects of these transcriptional or translational elements. Further, an additional step may be performed wherein transfected cells are treated with a compound of interest to determine the effect of the compound of interest on the transcriptional or translational elements. Detecting a change in fluorescence upon treatment of the cells with the compound of interest is a rapid and straightforward procedure to measure the effects of the compounds on interest on the transcription or translation of the expressed fusion protein. In addition, the present invention is directed to methods of studying cell lineage comprising the steps of transfecting undifferentiated cells with a vector capable of expressing the destabilized fluorescent fusion protein of the present invention, growing the undifferentiated cells under conditions in which the undifferentiated cells become differentiated cells, and detecting an absence or presence of fluorescence in the differentiated cells. Further, the present invention provides a method of using a fusion protein described herein in cell localization studies, comprising the steps of transfecting cells with an expression vector comprising a fluorescent fusion protein having a half life of no more than ten hours and preferably less than four hours wherein the fusion protein is linked to a putative cell localization element, growing the cell and detecting a location of fluorescence in the cells. |
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