The work resulting in this invention was funded in part by NIEHS-MFBSC grant no. ES05705 and NIH-NINDS grant no. NS36998. The U. S. government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to fluorescent proteins, genes encoding the proteins, and methods for their use.

2. Background Information Genes encoding fluorescent proteins (FPs) have been isolated from a wide variety of cnidarian species and have been useful in a variety of contexts, including the tracking of proteins in heterologous cells, production of transgenic animals, and other purposes. The products of these genes fluoresce under certain wavelengths of light, allowing efficient measurement under a wide variety of in vivo and in vitro conditions. For example, such genes have been operably linked to genes encoding proteins of interest and used to study gene expression and protein localization, as fluorescent tags to aid in visualizing protein trafficking, and as components of indicator systems that allow fluorescent sensing of small molecules and pH.

Transgenic animals containing such genes are useful, for example, in environmental applications, and are becoming popular as pets.

In spite of the many fluorescent proteins and genes isolated thus far, a need remains for new fluorescent proteins with several favorable properties, including better stability of fluorescence, lower toxicity, and better resistance to photobleaching.

SUMMARY OF THE INVENTION The invention provides an isolated green fluorescent protein identified as "Mick", having an excitation maximum of about 492 nm and an emission maximum of about 502 nm. The invention also provides a nucleic acid encoding Mick. Mick can be isolated from the scleractinian coral Montastraea cavernosa, and is highly resistant to photobleaching.

Mick has exceptional long-term stability and resistance to photobleaching compared to EGFP (the most widely used FP on the market, available through ClonTech) and other available FPs. Bacterial cells expressing Mick will remain fluorescent for long periods of time while the fluorescence of EGFP-expressing cells will diminish rapidly after c. a. 48 hours.

The excitation and emission spectra of Mick were obtained as described below and are shown in Figure 3. Amino acid and nucleic acid sequences of Mick are shown in Figure 4.

The invention further provides an isolated cyan fluorescent protein identified as"Pdl", having an excitation maximum of about 482 lun and an emission maximum of about 494 nm. The invention also provides a nucleic acid encoding Pdl. Pdl can be isolated from the scleractinian coral Pocillopora damicornis. The excitation and emission spectra of Pdl were obtained as described below and are shown in Figure 5.

Amino acid and nucleic acid sequences are shown in Figure 6.

The invention further provides an isolated blue-cyan fluorescent protein identified as"Dstl", having an excitation maximum of about 438 nm and an emission maximum of about 482 nm. The invention also provides a nucleic acid encoding Dstl. Dstl can be isolated from the non-scleractinian anthozoan Discosoma striata.

The excitation and emission spectra of Dstl were obtained as described below and are shown in Figure 7. Amino acid and nucleic acid sequences are shown in Figure 8.

In addition, included in the invention are fragments and variants of Mick, Pdl and Dstl that exhibit similar fluorescent properties, and nucleic acids encoding such fragments and variants. Suitable fragments and variants can be obtained through routine experimentation by methods known to persons of skill in the art. Variants include 1-20 (preferably no more than 10) amino acid deletions, substitutions or insertions, preferably conservative substitutions, as will be known to those of skill in the art. It is expected that fragments of close to full length (i. e. containing at least about 90%, preferably 95%, and most preferably 98 or 99% of the original amino acid sequence) will retain the properties of the protein. Such functional fragments can be discovered by routine experimentation using methods described herein and those known in the art. For example, Dopf J, Horiagon TM (1996) Deletion mapping of the Aequorea victoria green fluorescent protein. Gene 173: 39-44, describes suitable techniques.

The invention also provides an isolated nucleic acid sequence that encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide or protein that is responsive to a chemical, biological, electrical or physiological parameter and a fluorescent protein moiety, wherein the sensor polypeptide is operatively linked to the fluorescent protein moiety, and wherein the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.

Techniques for obtaining such indicators and constructs are known to those of skill in the art, as described, for example in Halloran MC, et al. (2000) Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127 (9): 1953-1960.

The invention also includes transgenic nonhuman animals having a nucleic acid sequence that encodes a fluorescent protein of the invention. Techniques for making such transgenic animals are also known in the art. Methods for making transgenic zebrafish can be found, for example, in Gibbs PDL and Schmale MC (2000) GFP as a genetic marker scorable throughout the life cycle of transgenic zebrafish. Marine Biotechnology 2 (2): 107-125 and Gibbs PDL, Peek A, and Thorgaard G (1994). An in vivo screen for the luciferase transgene in zebrafish.

Molecular Marine Biology and Biotechnology 3: 307-316.

The fluorescent peptides of the invention can be used as markers to detect expression of a gene of interest, for example by inserting the gene of interest along with DNA encoding the fluorescent protein into a vector. Such vectors can be transfected into eukaryotic or prokaryotic host cells, including, for example, bacterial cells, insect cells, yeast cells, and mammalian cells. Techniques useful for carrying out this and other aspects of the invention can be found in Sambrook et al. (Molecular Cloning. A Laboratory Manual, 3rd ed. (2001), Cold Spring Harbor Laboratory Press).

Fluorescent proteins of the invention can also be used in biochemical assays and as reagents, for example to monitor fermentation processes or quantify gene expression and to label cells for tracking purposes.

The fluorescent proteins of the invention can also be used in a range of applications including use as easily scorable transgenic markers and as taxonomic markers for genetic studies of corals and other cnidarians. They can also be incorporated into diagnostic kits as a color change indicators or used in FRET systems. Other uses include food colorants, cosmetics, paints and dyes.

Other aspects of the invention include luminescent ornamental fish (e. g. for hobbyists) and a wide variety of other living organisms, including, for example, bacteria, fungi, viruses, animals and plants, into which the nucleic acids and proteins can be incorporated for recreational, medical, research and other purposes.

The invention provides an isolated protein comprising the amino acid sequence identified herein as Mick (SEQ ID NO : 2), or a fragment or variant thereof having the excitation and emission properties shown in Figure 1, and an isolated nucleic acid encoding the protein.

The invention also provides an isolated nucleic acid comprising the nucleic acid sequence identified herein as Mick (SEQ ID NO : 1).

The invention provides an isolated protein comprising the amino acid sequence identified herein as Pdl (SEQ ID NO : 4) or a fragment or variant thereof having the excitation and emission properties shown in Figure 4, and an isolated nucleic acid encoding Pdl.

The invention further provides an isolated nucleic acid comprising the nucleic acid sequence identified herein as Pdl (SEQ ID NO : 3).

The invention also provides an isolated protein comprising the amino acid sequence identified herein as Dstl (SEQ ID NO : 6), or a fragment or variant thereof having the excitation and emission properties shown in Figure 5, and an isolated nucleic acid encoding Dstl.

The invention further provides an isolated nucleic acid comprising the nucleic acid sequence identified herein as Dstl (SEQ ID NO : 5).

It will be appreciated by the skilled practitioner that the invention also includes nucleic acids and proteins that consist essentially of, or consist of, SEQ ID NOS: 1, 2,3, 4,5, and6.

The invention also provides a vector comprising one of any of the nucleic acids of the invention, a host cell comprising one of the nucleic acids of the invention linked to an operable promoter, a transgenic animal comprising one of the nucleic acids of the invention. Preferably the transgenic animal is a fish, more preferably, a zebrafish.

The invention further provides a nucleic acid construct comprising i) a coding sequence encoding a polypeptide or protein of interest, and

ii) a nucleic acid of one of claims 3,4, 7, 8, 11 or 12 encoding a fluorescent protein, wherein the coding sequence and the nucleic acid are linked in such a manner that expression of the fused sequence yields a fluorescent hybrid protein in which the protein of interest is fused to the fluorescent protein. Also included in the invention is a cell comprising this nucleic acid construct. For example, Mick, Pdl or Dstl can be linked to myc or other genes of interest and analyzed as described by Langenau DM, et al. (2003) Myc-induced T cell leukemia in transgenic zebrafish. Science 299 (5608): 887-890.

The invention also provides a method of detecting expression of a nucleic acid comprising introducing a nucleic acid construct, as described above, into a cell or organism, and detecting expression of the nucleic acid by emission of fluorescent light.

The term"isolated"is intended to refer to any compound or agent that is in a form not found in its original environment or in nature, e. g., more concentrated, more purified, separated from at least one other component with which it is naturally associated.

The terms"peptide", "polypeptide", and"protein"as used herein, are intended to refer to amino acid sequences of various sizes, and are not considered to imply any size limitation on the compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 a : Photobleaching of Montastraea green FPs. Two FPs are shown, Mick and a second FP, MfaGl, cloned independently by Gibbs and Carter. Note the lack of photobleaching in Mick compared to the strong reduction in MfaGl fluorescence under the same conditions. MfaGl is more representative of the currently known Montatraea green FPs. Measurements were made in a SPEX spectrofluorometer by exposing diluted aqueous stock solutions to low intensity excitation light. Bleaching was induced by continual exposure of each sample to the maximum available excitation light. The rebound in MfaGl fluorescence occurred when the continual excitation light was shut off.

Figure lb : Photobleaching of Mick and EGFP under identical conditions. Dilute samples of Mick and EGFP were mixed with 0.5% low melt agarose and poured into polycarbonate cuvettes. Each cuvette was placed into the excitation beam of a SPEX spectrofluorometer and fluorescence was measured each second for 120 seconds.

Shown is a representative bleaching curve of three samples of each FP under low light exposure. Time-based photobleaching in EGFP is more than twice as fast as Mick.

This relationship held true for the entire range of light intensities that could be produced by the spectrofluorometer.

Figure 2: Bacterial streaks under blue light excitation: a) EGFP (left) and Mick (right) after 24 hours at 37 °C b) Same two streaks (slightly different location) after an additional four days at room temperature Bacterial plates after one day at 37 °C and four additional days at room temperature (photos taken with identical exposure settings): c) EGFP colonies d) Mick colonies Transgenic and wild type fish : e) Three days old, bright field f) Three days old, fluorescence g) Five days old, bright field h) Five days old, fluorescence i) Three months old (photo taken under blue light with no additional color modification or filtering) Figure 3: Excitation and Emission Spectra of Mick. The excitation spectrum was obtained using a SPEX spectrofluorometer by setting the emission wavelength to 520 nm and scanning all excitation wavelengths at 2 nm increments between 200 and 510 nm. The emission spectrum was obtained by setting the excitation light to 420 nm and scanning all emission wavelengths at 2 nm intervals between 440 and 700 nm.

Figure 4: Nucleotide (SEQ ID NO : 1) and Amino Acid (SEQ ID NO : 2) Alignment of Mick

Figure 5: Excitation and Emission Spectra of Pdl Figure 6: Nucleotide (SEQ ID NO : 3) and Amino Acid (SEQ ID NO : 4) Alignment of Pdl Figure 7: Excitation and Emission Spectra of Dstl Figure 8: Nucleotide (SEQ ID NO : 5) and Amino Acid (SEQ ID NO : 6) Alignment of Dstl DETAILED DESCRIPTION OF THE INVENTION We have cloned several new fluorescent proteins from three anthozoan species. Each has its own set of useful and unique attributes. They are as follows: 1. Mick: a new green FP from the Caribbean coral, Montastraea cavernosa. This FP expresses at a high level in both E. coli and zebrafish. It is also highly resistant to photobleaching. In vivo fluorescence does not decrease even after prolonged exposure to intense UV light. In aqueous solution, other FPs (including other green FPs from Montastraea spp.) photobleach rapidly. Compared to EGFP, the most popular FP available on the market today, Mick produces more stable fluorescence in E. coli and can be visualized in zebrafish under room lighting.

2. Pdl: a new cyan fluorescent protein from the Pacific coral, Pocillopora damicornis. This cyan FP expresses well in E. coli and the Pdl sequence is dissimilar from most other anthozoans.

3. Dstl : a new blue-cyan fluorescent protein from the Pacific soft coral, Discosoma striata.

Table 1: Comparison of Characteristics of Mick, Pdl and Dstl Mick Pdl Dstl Excitation Maximum: 492 nm 482 run 438 nm Emission Maximum: 502 nm 494 nm 482 nm Nucleotides: 681 666 681 Amino Acids: 227 222 227

Materials and Methods Species collection and animal husbandry Single, bright green colonies of Montastraea cavernosa and Montastraea faveolata colonies were collected from reefs in the South Florida area. A single branch from a large colony of Pocillopora damicornis was collected from the Pacific coast of Panama. Colony of Discosoma striata was purchased at a local aquarium store. All corals were maintained in flow through aquariums using filtered sea water at the Rosenstiel School of Marine and Atmospheric Sciences (University of Miami) until tissue processing could occur.

Isolation of total RNA RNA was isolated using the Totally RNA kit (Ambion, Inc.) from a cellular mass lightly airbrushed from the underlying skeleton of each colony. Briefly, the tissue was homogenized and mixed by tube inversion with approximately 10 volumes of denaturation solution and extracted with 1 volume of phenol/chloroform/isoamyl alcohol. 1/10 volume of 3M sodium acetate was added to the supernatant which was extracted an additional time with 1 volume of acid-phenol/chloroform. One volume of isopropanol was added to the supernatant and total RNA was precipitated by centrifugation, the pellet washed with 75% ethanol and resuspended in 50 uL RNAse- free water. Yield was measured by spectrophotemetry and quality was assessed by gel electrophoresis.<BR> <P>Reverse transcription, amplification and recovery of cDNAs The RLM-RACE kit (Ambion, Inc,) was used to create cDNAs using the "small reaction"protocol with 1 ug of total RNA. Calf intestinal phosphatase was used to remove 5'phosphates from degraded and non-capped RNA. Tobacco acid pyrophosphatase was then used to remove the cap from mRNAs.

A 5'RNA adaptor:

5'-<BR> GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3' (SEQ ID NO : 7) was ligated to total decapped mRNAs with T4 RNA ligase.

Reverse transcription was accomplished using MMLV reverse transcriptase and a polyT primer: 5'-CTCGAGAAGCTTGAATTCGGATCCTTTTTTTTTTTTTTTTT-3' (SEQ ID NO : 8) Initial cDNA amplification was performed using the Outer RNA Adapter Primer supplied in the kit: 5'-GCTGATGGCGATGAATGAACACTG-3' (SEQ ID NO : 9) and the polyT primer described above. The PCR conditions were: 94°C/5 min, 80°C hold, add polymerase, 34 cycles of 94°C/30 sec, 60°C/1 min, 72°C/1 min and a final 10 min of 72°C. Amplified cDNAs were gel purified. The area between 500-1200 bp was removed from the gel with a clean razor blade and recovered by slow speed centrifugation through siliconized glass wool followed by isopropanol precipitation.

Cloning of PdaCl and DstCl One microliter of the recovered PCR amplified cDNA was ligated into the PCR 2.1 TOPO vector (Invitrogen). The resulting plasmids were used to electrotransform Top-10 E. coli cells (Invitrogen), plated out on 100 llg/ml LB-Amp medium and incubated overnight.

Clonirzg of Mick Secondary amplification of the Montastraea cavernosa cDNA was done using SuperTaq polymerase (Ambion, Inc.) and two gene-specific primers we had previously designed for another fluorescent protein gene, McaGl. The primers were initially designed for cloning McaGl into the plasmid vector pBS II KS+ [It had originally been cloned into the pCR 2.1 TA vector (Invitrogen)]. The upper primer included a Kpn I restriction site, both Shine-Dalgarno and Marilyn Kozak

upregulation sequences (SD-MK) and 16 bases of the coding region, starting with the initial start codon, ATG. The lower primer included 13 bases of the 3'untranslated region (UTR) from McaGl, a stop codon, and a Xba I restriction site.

Upper Primer (McaGl U1-37) 5'-CCGGTACCTAA GGAGGCCACC ATGAGTGTGATAAAAC-3' (SEQ ID NO : 10) Kpn-SD-MK Coding Region Lower Primer (McaGl L1-24) 5'-GGTCTAGA TTA CTTGGCCTGCCTC-3' (SEQ ID NO : 11) Xba Stop 3'UTR Because we did not expect 100% similarity between the McaGl primers and the new gene sequences, we used the PCR reaction conditions for what we call"dirty PCR".

This includes a low annealing temperature and long extension times for 10 cycles plus an additional 30 cycles under more normal conditions. PCR conditions were: 94°C/5 min, 80°C hold, add polymerase, 10 cycles of 94°C/1 min, 42°C/2 min, 72°C/3 min, followed by 30 cycles of 94°C/30 sec, 55°C/1 min, 72°C/1 min and a final 10 min of 72°C.

Cloning, Plasmid preparation and DNA Sequencing The PCR products were gel purified, digested with Kpn I/Xba I, and ligated into a Kpn I/Xba I digested pBS II KS+ plasmid. Resultant plasmids were electrotransformed into Top 10 E. coli (Invitrogen). Transformed bacteria were grown on LB-ampicillin (100 ug/ml) plates and colonies were screened for fluorescence using a Leica MZFLIII fluorescence stereo dissection microscope. Fluorescent colonies were screened for brightness after 24 hours at 37°C. Single colonies were picked, restreaked for several rounds to resolve mosaicism and grown in liquid culture for plasmid preparation by the Qiagen Midi kit. Sequencing was accomplished using the"Big Dye Terminator"reaction on an Applied Biosystems Model 377 DNA Sequencer at the DNA Core Facility.

Characteristics of Mick

Figure la shows a comparison of photobleaching between Mick and MfaGl (MfaGl is a green FP with typical photobleaching characteristics and was cloned independently from the scleractinian coral Montastraeafaveolata by Gibbs and Carter. Under bright excitation light, MfaGl rapidly loses fluorescence (decending part of curve). When the excitation light is turned off, fluorescence rebounds (ascending part of curve) and remains stable as long as the excitation light remains off. This figure was generated using aqueous solutions of purified cloned FPs.

Measurements were made using a SPEX spectroflurometer.

Figure lb shows a comparison of photobleaching resistance between Mick and EGFP. Measurements were made as described above but at several different light intensities. Fluorescence decays more than twice as fast in EGFP than in Mick. This relationship was true at all light intensities tested.

Figure 2: a) 24-hour old Mick-and EGFP-expressing bacterial streaks under blue light excitation. b) the same two streaks after 96 additional hours at room temperature. Note that the EGFP cells have lost a considerable amount of fluorescence while the relative intensity of the Mick-expressing cells has incereased. c-d) single colonies of EGFP (c) and Mick (d) after 24 hours at 37 degrees C followed by 96 hours at room temperature, e-f) Mick-expressing and wild-type three-day-old zebrafish embyos under white light (e) and blue light (f). g-h) Five-day-old zebrafish embryos as in e and f. i) Mick-expressing and wild-type zebrafish under blue light.

Photos a-h were taken with a digital camera mounted on a Leica MZFLIII fluorescence stereo dissecting microscope. Photo i was taken with a digital camera in a dark room with a blue light source for illumination and no additional camera filters or post-processing of color Excitation and emission spectra of Mick are given in Figure 3. The excitation spectrum was obtained using a SPEX spectrofluometer by setting the emission wavelength to 520 nm and scanning all excitation wavelengths at 2 nm increments between 200 and 500 nm. The emission spectrum was obtained by setting the excitation light to 480 nm and scanning all emission wavelengths at 2 nm intervals between 490 and 700 nm.

Transgenic Animals Transgenic zebrafish were made according to the methods described by Gibbs and Schmale (2000), and are shown in Figure 2 (e-i). Similar techniques can be used

to obtain other types of ornamental fish, for example, goldfish, koi, angelfish, tiger barbs, etc.

The embodiments illustrated and discussed in the present specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention, and should not be considered as limiting the scope of the present invention. The exemplified embodiments of the invention may be modified or varied, and elements added or omitted, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

References cited herein are hereby incorporated by reference.