CA2509373A1 - Fluorescent proteins from aquatic species - Google Patents

Abstract

Provided are four new fluorescent proteins. The proteins were derived from t wo wild-type fluorescent proteins: a red fluorescent protein (RFP) that was isolated from Actinodiscus or Discosoma sp. 1 and a green fluorescent protei n (GFP) isolated from Montastraea cavernosa. Two mutant forms were generated from each wild-type protein. Each of the mutated forms has a higher fluorescence intensity than the respective wild-type form. The mutant forms of the fluorescent proteins allow for more sensitive detection of the fluorescence emitted by the proteins. Additionally, one of the mutant protei ns is more resistant to photobleaching than its wild-type protein. The inventio n also encompasses isolated nucleic acids encoding the mutant forms of the wil d- type RFP and GFP.

Description

638.008 FLUORESCENT PROTEINS FROM AQUATIC SPECIES
BIBLIOGRAPHY
Complete bibliographic citations of the references referred to herein by the first author's last name in parentheses can be found in the Bibliography section, immediately preceding the claims.
REFERENCE TO GOVERNMENT GRANT
This invention was made with United States government support awarded by the National Institute of Environmental Health Sciences-MFBSC, Contract # ES05705, NIH-National Institute of Neurological Disorders and Stroke, Contract # NS36998, and National Institute of General Medicine, Contract # GM 57505. The United States has certain rights in this invention.
FIELD OF THE INVENTION
The invention relates to the field of biochemical assays and reagents. More specifically, this invention relates to fluorescent proteins and to methods for their use.
DESCRIPTION OF THE RELATED ART
The number of available fluorescent reporter genes has increased as researchers have isolated genes encoding fluorescent proteins from an increasing variety of organisms and included the genes in cloning cassettes. For example, fluorescent proteins from sea creatures have been used as reporter genes capable of integration into DNA via cloning cassettes. Products of these genes fluoresce under certain wavelengths of light, pernzitting the tracking of proteins in, e.g., heterologous cells, such as dog and monkey cells. The most commonly used proteins of this nature fluoresce green, and were obtained from the jellyfish, Aequorea victoria, and sea pansy, Re~2illa ~eniforrnis.
Additionally, a red fluorescent protein (RFP), known as drFP583, and a turquoise fluorescent protein, known as dsFP483, have been isolated from the IndoPacific mushroom corals (Discosonza sp. "red" and Discosonaa stniata, respectively).
Both Discosoma and Actinodiscus are mushroom corals, soft bodied anthozoans that do not produce an external skeleton. It should be noted that the relationship between the genus Discosonaa and the genus Actiyaodiscus is not well understood. Both Acti~zodiscus and {0004G4G3.DOC /) Discosoma are members of the Actifaodiscidae Family, which is a member of the Coralli~apof plaaria (mushroom) Order. The taxonomy of the CorallimpoYplaania is poorly defined, and therefore, the nature of the relation of Actinodiscus to Discosonaa is uncertain. Discosoriaa and Actiraodiscus are believed to be different genera of the same family, but they could be more closely or distantly related.
The availability of a variety of fluorescent proteins and reagents has enhanced the opportunities for researchers employing reporter proteins in their work.
Isolated DNAs encoding fluorescent proteins have been mutated to alter their optical properties. For instance, mutating tyrosine at amino acid 66 to histidine in the amino acid composition of the Aequof°ea green fluorescent protein (GFP) changes this protein to one that fluoresces blue. Changing amino acid 64 from phenylalanine to leucine, amino acid 65 from serine to threonine, and amino acid 145 from tyrosine to phenylalanine generates a GFP that fluoresces at a brighter intensity than the parent molecule and has a shifted excitation optima. In addition, by genetically modifying the amino acid composition of GFP, researchers have been able to change its light absorption/emission characteristics, creating yellow fluorescent proteins.
Fluorescent proteins can be used in a number of assays. In one example, fluorescent proteins can be used in Fluorescence Resonance Energy Transfer (FRET) assays. FRET occurs with fluorophores for which the emission spectrum of one overlaps with the excitation spectrum of the second. When the fluorophores are brought into close proximity, excitation of the "donor" results in emission from the "acceptor."
Pairs of such fluorophores are thus useful for monitoring molecular interactions.
Fluorescent proteins, such as GFP, are useful for analysis of protein:protein interactions either ifz vivo or iia vit~~o if their fluorescent emission and excitation spectra overlap to allow FRET.
The donor and acceptor fluorescent proteins may be produced as fusion proteins with the proteins being analyzed for interactions. These types of applications of GFPs are particularly appealing for high throughput assays, because the readout is direct and independent of subcellular localization.
Because of its easily detectable green fluorescence, GFP from Aequorea has been used widely to study gene expression and protein localization. Furthermore, GFP, like other fluorescent proteins, does not require a substrate or cofactor to fluoresce; hence, it is possible to directly express GFP and use it as a reporter in numerous species and in a wide variety of cells. However, factors other than fluorescence color and intensity affect the utility of a protein in research. The stability of many fluorescent proteins makes them {00046463.DOC /} 2 undesirable reporters to use if one seeks to determine short term or repetitive events.
Moreover, accumulated protein can be toxic to some mammalian cells. For example, certain forms of GFP from Aequo~ea have been demonstrated to induce apoptosis (Liu, et al.). Although the inventors do not wish to be limited to a single explanation of the toxicity of GFP from AequoYea, it is believed that this is probably due to free radical (HZOz) formation which occurs in a 1:1 stoichiometry with GFP production, making high ' levels of GFP expression particularly toxic. This is believed to be a direct result of chromaphore maturation and is believed to occur in every known GFP.
Photobleaching is another concern with previous fluorescent proteins.
Photobleaching is a light induced change in a fluorophore, resulting in the loss of absorption of light of a particular wavelength by the fluorophore. This results in loss of fluorescence of the fluorophore. Many fluorescent proteins rapidly photobleach under excitation. This process is usually reversible but can limit the usefulness of GFP
expression, e.g. by reducing time available to photograph specimens. However, where the photobleaching is rapidly reversible, this property makes the fluorescent protein useful for certain applications.
Thus, the need remains for easily expressible fluorescent proteins that have a range of spectral outputs and low toxicity to maximize research capabilities.
Such research applications include, but are not limited to, short-lived fluorescent proteins as genetic reporters, which would enable monitoring of short-lived activities.
Such research applications further include, but are not limited to, low toxicity fluorescent proteins that would allow long-term monitoring or stable transfection of cells or organisms.
Furthermore, the need also remains for fluorescent proteins having novel emission spectra. This would permit monitoring multiple processes simultaneously and could minimize background fluorescence. It would also increase the choices available for FRET analysis systems. Moreover, fluorescent proteins having brighter relative fluorescence are needed to permit detection of low level expression of the fluorescent proteins.
SUMMARY OF THE INVENTION
The invention, which is defined by the claims set out at the end of this disclosure, is intended to solve at least some of the problems noted above. The invention provides improved fluorescent proteins with enhanced properties e.g., substantially enhanced {0004G4G3.DOC /}

fluorescence and reduced toxicity. The improved fluorescent proteins are useful in research and can be used, e.g., to determine or detect gene expression, e.g., up- or down-regulation, to monitor promoter activity, to allow longer term monitoring, and to localize proteins.
A new, wild-type red fluorescent protein (RFP) was isolated from an aquatic species believed to be either an Actinodiscus or Discosorraa species. This protein is referred to hereinafter as AclDsRFP. The invention provides two protein mutants derived from AclDsRFP. The mutant RFPs of the invention are referred to herein as Red I and Red II. An isolated nucleic acid encoding each of these and their associated amino acid sequences are also included.
In addition, a novel green fluorescent protein (GFP) was isolated from Moratastr°aea caver~raosa. This protein is referred to herein as McGFP.
The invention provides two novel proteins derived from McGFP. Preferred novel proteins are referred to herein as Green I and Green II. It was also discovered that certain mutants of McGFP
photobleach very quickly.
The invention also provides a nucleic acid construct that includes a first coding sequence that encodes a selected polypeptide and a second coding sequence that encodes a fluorescent protein of the invention. The first coding sequence is fused to the second coding sequence such that expression of the fused sequence yields a fluorescent hybrid protein in which the polypeptide encoded by the first coding sequence is fused to the polypeptide encoded by the second coding sequence.
Vectors including the various isolated nucleic acids of the invention are also provided. Vectors'of varying capacities are well known to molecular biologists and can be used to transform a eukaryotic or prokaryotic cell. They can also be used with irz vivo and in vitYO expression systems.
A method of detecting expression of a nucleic acid encoding a fluorescent protein is also provided. The method includes introducing the nucleic acid of the invention into a cell or organism. In a preferred embodiment, a promoter controls expression of the nucleic acid. Expression of the nucleic acid is detected by emission of fluorescent light, allowing detection of expression of the nucleic acid. In a preferred embodiment, the cell is a eulcaryotic cell. In another preferred embodiment, the cell is a prokaryotic cell. The expression of the nucleic acid can be detected in vivo. It can also be detected irr vitr~a and in fixed cells, such as formalin fixed cells.
{00046463.DOC /} 4, In a preferred embodiment of the method of detecting expression of a nucleic acid, a gene of interest is fused to nucleic acid encoding a fluorescent protein.
The fusion protein may include a subcellular specific locator signal, allowing measurement of expression from the promotor and/or subcellular localization. Expression of the gene of interest is detected by emission of fluorescent light.
Also provided is a cell including a nucleic acid of the invention. In a preferred embodiment, the nucleic acid is integrated into a genome of the cell. In another preferred embodiment, the nucleic acid is not integrated into a genome of the cell. For example, the nucleic acid can exist extrachromosomally.
An animal having an isolated nucleic acid of the invention is additionally provided. In a preferred embodiment, the animal is a zebrafish.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred exemplary embodiments of the invention are illustrated in the accompanying drawings in which:
FIGS. lA-1B are a sequence alignment of the DNA encoding two mutant red fluorescent proteins: Red I (SEQ. ID. NO:1) and Red II (SEQ. ID. NO: 3), each of which was generated from a protein originally isolated from ActinodiscuslDiscosoma sp. In this and all other alignments, the differences between the sequences being aligned are indicated by a missing monomer in the "consensus" line.
FIG. 2 is an alignment of the amino acids encoded by the DNA sequences of Red I (SEQ. ID. NO: 2) and Red II (SEQ. ID. NO: 4).
FIG. 3 is a graph of a spectral analysis of Red I.
FIG. 4 is a graph of a spectral analysis of Red II.
FIG. 5 is a graph of spectral analyses of Red I and Red II.
FIGS. 6A-6B show a sequence alignment of the DNA encoding two mutant green fluorescent proteins: Green I (SEQ. ID. NO:S) and Green II (SEQ. ID. N0:7), each of which was generated from a protein originally isolated from M. cave~raosa.
FIG. 7 is an amino acid alignment of the amino acids encoded by the DNA
sequences of Green I (SEQ. ID. NO: 6) and Green II (SEQ. ID. N0:8).
FIG. 8 is a graph of a spectral analysis of Green I.
FIG. 9 is a graph of a spectral analysis of Green II.
FIG. 10 is a graph of spectral analyses of Green I and Green II.
{00046463.DOC /}

Before explaining embodiments of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
DETAILED DESCRIPTION
Definitions:
For purposes of the present invention, the following definitions apply:
The standard, one-letter codes "A," "C," "G," "T," and "U" are used herein for the nucleotides adenine, cytosine, guanine, thymine, and uracil, respectively. "N"
designates any nucleotide. Oligonucleotide or polynucleotide sequences are written from the 5'-end to the 3'-end.
As used herein, "amino acids" are described in keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3557-59, (1969). All amino acid residues identified herein are in the natural L-configuration. In keeping with standard polypeptide nomenclature, abbreviations for amino acid residues are as shown in the following Table of Correspondence.
TABLE OF CORRESPONDENCE
1-Letter 3-Letter AMINO ACID
Y Tyr L-tyrosine G Gly glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine {000464G3.DOC /} 6 H His L-histidine Q Gln L-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine D Asp L-aspartic acid N Asn L-asparagine C Cys L-cysteine By "protein" and "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. The synthetic genes of the invention may also encode a variant of a naturally-occurring protein or polypeptide fragment thereof. Preferably, such a protein polypeptide has an amino acid sequence that is at least 85%, preferably 90%, and most preferably 95% or 99% identical to the amino acid sequence of the naturally-occurring (native) protein from which it is derived.
The term "isolated" when used in relation to a nucleic acid, as in "isolated nucleic acid" or "isolated polynucleotide," refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, e.g., DNA and RNA, are found in the state they exist in nature. For example, a given DNA
sequence, e.g., a gene, is found on the host cell chromosome in proximity to neighboring genes;
RNA sequences, e.g., a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins.
However, isolated nucleic acid includes, by way of example, such nucleic acid in cells ordinarily expressing that nucleic acid where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanlced by a different nucleic acid sequence than that found in nature. The isolated nucleic acid may be present in single-stranded or double-stranded form. When an isolated nucleic acid is to be utilized to express a protein, the oligonucleotide contains at a minimum, the sense or coding strand, i.e., the oligonucleotide may be single-stranded, but may contain both the sense and anti-sense strands, i.e., the oligonucleotide may be double-stranded.
{00046463.DOC l) 7 The term "isolated" when used in relation to a polypeptide, as in "isolated protein"
or "isolated polypeptide" refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source.
Thus, an isolated polypeptide is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated polypeptides, e.g., proteins and enzymes, are found in the state in which they exist in nature.
The term "purified" or "to purify" means the result of any process that removes some of a contaminant from the component of interest, such as a protein or nucleic acid.
The percent of a purified component is thereby increased in the sample.
With reference to nucleic acids of the invention, the term "nucleic acid"
refers to DNA, genomic DNA, cDNA, RNA, mRNA and a hybrid of the various nucleic acids listed. The nucleic acid can be of synthetic origin or natural origin. A
nucleic acid, as used herein, is a covalently linked sequence of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next, and in which the nucleotide residues (bases) are linked in specific sequence, i.e., a linear order of nucleotides.
The term "wild-type" as used herein, refers to a gene or gene product that has the characteristics of that gene or gene product isolated from a naturally occurring source. A
wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "wild-type" form of the gene. In contrast, the teml "mutant"
refers to a gene or gene product that displays modifications in sequence andlor functional properties, i.e., altered characteristics, when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
As used herein, "higher relative fluorescence intensity" or "increased brightness"
refers to fluorescence intensity or brightness that is greater than that exhibited by a wild-type fluorescent protein under a given set of conditions. Generally, an increase in fluorescence intensity or brightness means that fluorescence of a variant is at least 25% or more, and preferably greater than 50%, or more, and more preferably greater than 100%
or more intense or bright than the wild-type fluorescent protein under a given set of conditions.
{0004G4G3.DOC /}
g The term "nucleic acid construct" denotes a nucleic acid that is composed of two or more nucleic acid sequences that are derived from different sources and that are ligated together using methods laiown in the art.
The term "host cell" as used herein, refers to a cell from any organism.
Preferred host cells are derived from plants, bacteria, yeast, fungi, insects, or other animals.
Methods for introducing polynucleotide sequences into various types of host cells are well known in the art. Host cell includes progeny or potential progeny of these designations.
The term "vector" is used in reference to nucleic acid molecules into which fragments of DNA may be inserted or cloned and can be used to transfer DNA
segments) into a cell and capable of replication in a cell. Vectors may be derived from plasmids, bacteriophages, viruses, cosmids, and the like.
The term "expression vector" as used herein refer to DNA or RNA sequences containing a desired coding sequence and appropriate DNA or RNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
Prokaryotic expression vectors typically include a promoter, a ribosome binding site, an origin of replication for autonomous replication in a host cell and possibly other sequences, e.g. an optional operator sequence, optional restriction enzyme sites. A
promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. Eukaryotic expression vectors typically include a promoter, optionally a polyadenlyation signal and optionally an enhancer sequence.
The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements, e.g., promoters, enhancers, and termination elements, in an expression vector.
In the present invention, there may be employed conventional molecular biology and microbiology within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch ~z Maniatis, Molecular Cloning: A
Laboratory Manual, Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
In accordance with the invention, new wild-type fluorescent proteins have been isolated. The wild-type fluorescent proteins have been mutated to create mutant forms of (00046463.DOC /}

the fluorescent proteins that have, e.g., a higher relative fluorescence intensity or a greater ability to resist photobleaching than their respective wild-type fluorescent proteins.
I. Isolation of Wild-Type Fluorescent Proteins briefly, tissue from an organism of interest was collected and homogenized by methods described in detail below. RNA was isolated, and RT-PCR was performed.
Size-selected DNA was recovered from an agarose gel, and then ligated into a vector suitable for bacterial selection. The vector can include an inducible or constitutive promoter. In a preferred embodiment, plasmids were used as the vectors.
Plasmids were electroporated into competent bacteria. Bacteria were incubated on LB-ampicillin plates (100 ~g/ml). Clones of interest were isolated based upon qualitative determination of fluorescence intensity and/or color quality.
Using this procedure, which is explained in detail in the Examples below, a cDNA
encoding a wild-type red fluorescent protein was isolated from an aquatic species believed to be Actiraodiscus or Discosoma. Because of the uncertainty in the relationship of the genera Actifaodiscus or Discosoma, which is discussed above in the Description of the Related Art section, this protein is referred to hereinafter as AclDsRFP.
The isolated cDNA encoding AclDsRFP was further mutated, and isolates were selected based upon properties of interest, as is detailed below. Additionally, using the methods described herein, a cDNA encoding a wild-type green fluorescent protein (GFP) was isolated from Moutasty-aea cave~yaosa and is referred to herein as McGFP. As is described below, the isolated cDNA encoding McGFP was further mutated, and selected based upon properties of interest.
II. Creation of Mutated Fluorescent Proteins For each of the wild-type genes isolated, random mutations in the coding sequences were induced in each through PCR conducted under low stringency conditions (low annealing temperature, excessively long extension times, extra cycles, as described below) to induce mutations during the polymerization. The resulting PCR
products were cloned into bacterial expression vectors. Clones were screened for increased relative fluorescence, colors of interest, and decreased photobleaching when compared to the corresponding wild-type fluorescent protein. The vector DNA was then purified from selected clones and sequenced to determine the relevant PCR-induced mutational changes. This process was then reiterated several times.
{00046463.DOC !} 1 ~

a. Red I and Red II
The DNA encoding the AclDsRFP, the wild-type RFP from Discosonaa, was mutated as indicated above with several rounds of low stringency PCR. After cloning into a vector and visual screening of transformed bacteria for fluorescent properties, the proteins encoded by Red I and Red II were selected based upon higher relative fluorescence intensity when compared to the parent AclDsRFP wild type. Red II
has a fluorescent intensity that is at least 50% greater than the intensity of Red I, as determined qualitatively.
The DNA sequences of Red I (SEQ. ID. NO:1) and Red II (SEQ. ID. N0:2) are shown in FIGS. lA-1B. Compared to the DNA encoding Red I, the DNA encoding Red II contains a single nucleotide difference: at position 694: Red I has a G, and Red II has an A. The amino acid sequences of Red I (SEQ. ID. N0:2) and Red II (SEQ. ID.
N0:4) are shown in FIG. 2. This figure indicates that at position 232, Red I has a D, whereas Red II has an N.
Referring now to FIG. 3-5, spectral analyses of Red I and Red II arid for both Red I and Red II are shown.
b. Green I and Green II
The DNA sequence encoding, McGFP, the wild-type GFP from M. cave~nosa, was mutated using PCR under low stringency conditions, as described herein. A
mutant GFP with brighter fluorescence was isolated and is indicated as Green I. A
second round of low stringency PCR was performed on Green I, yielding a second mutant GFP
with high resistance to photobleaching and is indicated as Green II. When compared to the DNA sequence encoding the Green I, the DNA encoding Green II contains a single nucleotide change: a cytosine to thymine mutation at nucleotide 527, as shown in FIGS.
6A-6B as SEQ.ID.NO:S and SEQ.ID.N0:7 . The amino acid sequences are shown in FIG. 7 as SEQ. ID. N0:6 and SEQ. ID. N0:8. This figure indicates an S at position 176 in Green I, and an F at the same position in Green II.
Green I had higher relative fluorescence intensity when compared to the McGFP.
Green II also higher relative fluorescence but also has a high resistance to photobleaching, a property not evident in the Green I protein. Using a mercury arc laser commonly employed in fluorescence microscopy, manipulated by an AttoarcTM HBO
100W variable power supply, Green I was found to photobleach under appropriate {0004G4G3.DOC /} 11 conditions. Low laser intensity is considered to be less than 25% intensity, and high laser intensity is considered to be greater than 75% for this instrument. Cells were visualized using a Zeiss Axiovert S 100 Microscope. Under low laser intensity, such as 10 %, both derivative proteins appear the same. However, after several seconds under high laser intensity, such as 80%, Green I light is not visible. After several minutes in the dark followed by re-illumination, the fluorescence of Green I is again evident.
Green I is also unusual in that excitation with 420 nm light rapidly reverses photobleaching.
Green II
does not undergo this photobleaching response.
Referring to FIGS. 8-10, spectral analyses of Green I (FIG. S) and Green II
(FIG.
9) and for both Green I and Green II (FIG. 10) are shown.
IV. Exemplary Uses of the Fluorescent Proteins All of the fluorescent proteins described herein can be used as markers to detect expression of a gene of interest such as by inserting the gene of interest and DNA
encoding a fluorescent protein into a vector. The vector can be transformed into a eukaryotic or prokaryotic host cells, such as, e.g., bacterial cells, insect cells, yeast cells, and mammalian cells. As is known in the art, host cells are competent or can be rendered competent by a variety of techniques, including, but not limited to, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAF
dextran, receptor-mediated endocytosis, electroporation, and micro-injection of the DNA
directly into the cells. The DNA can be integrated into the chromosomal DNA of the host cell or it can exist extrachromosally, such as via a plasmid.
Fluorescent proteins of the invention can also be used in biochemical assays and as reagents. For example, the fluorescent proteins can be used as a reporter to monitor fermentation processes and to quantify gene expression.
Additionally, the fluorescent proteins of the invention provide additional fluorescent proteins that can be used in multiple labeling systems. For example, one of the GFPs of the invention can be used to label a first population of cells and one of the RFPs of the invention can be used to label a second population of cells such that two different populations of cells can be tracked, e.g., during fluorescence activated cell sorting. Similarly, Red I and Red II can be used, e.g., to label and track different populations of cells because Red I and Red II fluoresce at different wavelengths.
{00046463.DOC /} 12 Further, the inventive fluorescent proteins can also be used in prokaryotic and eukaryotic expression systems. For instance, fusion proteins can be generated to contain coding sequences for a fluorescent protein and another gene. A typical fusion protein of the invention includes a first coding sequence that encodes a selected polypeptide and a second coding sequence that encodes a fluorescent protein of the invention.
The first coding sequence is fused to the second coding sequence such that expression of the fused sequence yields a fluorescent hybrid protein in which the polypeptide encoded by the first coding sequence is fused to the polypeptide encoded by the second coding sequence. It is believed that the fluorescent proteins of the invention can be fused either at the 3' or 5' end of the coding sequence. Furthermore, unlike fusion proteins with coding sequences for proteins such as ~3-galactosidase, fusion proteins containing coding sequences for a fluorescent protein do not require exogenously added substrates or cofactors.
This beneficially permits the fluorescent proteins to be used in living cells.
The fluorescent proteins provided herein can also be used as irr vivo markers, such as in mRNA microinjection assays. Other examples of uses of fluorescent proteins are as an in vivo marker in transgenic mice, Caerahor~babditis elegaras, Drosophila rnelanogasten, and Zebrafish.
In addition, fluorescent proteins of the invention can be used as taxonomic markers for studies of cnidarian genetics, color indicators in diagnostic kits, colored food additives, and cosmetic ingredients.
EXAMPLES
The following Examples are provided for illustrative purposes only. The Examples are included herein solely to aid in a more complete understanding of the presently described invention. The Examples do not limit the scope of the invention described or claimed herein in any fashion.
Example 1. Species Collection and Animal Husbandry:
Coral colonies were obtained from two sources. First, a solid, brick-red colony consisting of several ActinodiscuslDiscosorna sp, polyps was obtained from a local aquarium store. Second, Montastr-aea caverraosa colonies were collected from reefs in the South Florida area. Both species were maintained in small aquariums with flow-through, filtered sea water.
{00046463.DOC /} _ 13 Example 2. Isolation of Total RNA, Reverse Transcription, Amplification and Recovery of cDNAs:
RNA was isolated using the Totally RNA kit (Catalog # 1902, Ambion, Inc., Austin, TX) from a single polyp (Actinoc~iscuslDiscosofna sp.) or from a cellular mass lightly airbrushed from the underlying skeleton (M. caverrt.osa). 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. The aqueous supernatant was transferred to a clean vessel. One-tenth volume of 3M sodium acetate was added to the supernatant, which was then extracted an additional time with 1 volume of acid-phenol/chloroform. Again, the aqueous supernatant was transferred to a clean vessel. One volume of isopropanol was added to the supernatant, and total RNA
was precipitated by centrifugation. The pellet was washed with 75% ethanol and resuspended in 50 uL RNAse-free water. Yield was measured with a spectrophotometer, and quality was assessed by gel electrophoresis.
The RNA thus isolated was used in a First Choice TM RLM-RACE kit (catalog #
1700, Ambion, Inc.) to create and amplify cDNAs. The "small reaction"
protocol, as per RLM-RACE protocol version 0010, was followed. Briefly, total RNA is treated with Calf intestinal phosphatase (CIP) to remove S' phosphates from degraded and non-capped RNA, and then phenol:chloroform extracted. Tobacco acid pyrophosphatase (TAP) was then used to remove the cap from full-length mRNA. An included 5' RACE adaptor (5'-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3') (SEQ. ID. N0:9) 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:10).
Amplification of the resultant cDNA was done using the same polyT primer, the 5' RACE Outer Primer (5'-GCTGATGGCGATGAATGAACACTG-3') (SEQ. ID.
NO:11) and SuperTaq Polymerase (Ambion, Inc., catalog 2050).
PCR was conducted as follows: 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 fraction from about 500-1200 base pair (bp) was collected and recovered by slow speed centrifugation through siliconized glass wool followed by isopropanol precipitation.
{0004G4G3.DOC /} 14 Example 3. Transformation and Selection of Bacterial clones, Plasmid Preparation, and DNA Sequencing of Wild-Type Fluorescent Proteins:
The gel purred cDNA fraction was ligated into the pCR II cloning vector (Invitrogen, Carlsbad, CA), with resultant plasmids electrotransformed into Top 10 E.
coli (Invitrogen). Transformed bacteria were grown on LB-ampicillin (100 ~g/ml) plates and colonies were screened for fluorescence using a Leica MZFLIII fluorescence stereo dissection microscope. Single fluorescent colonies were picked, restreaked for several rounds to resolve mosaicism, and grown in liquid culture. Plasmid DNA was prepared using the Qiagen Midi kit (Qiagen, Valencia, CA).
A colony expressing a wild-type red fluorescent protein (RFP) was isolated from the bacterial colonies generated from the ActiraodiscuslDiscosonaa sp. 1 RNA.
This is herein referred to as AclDsRFP.
In addition, a colony expressing a wild-type green fluorescent protein (GFP) was isolated from the bacterial colonies generated from the M. caveryaosa RNA and was named McGFP.
Example 4. Generation of Mutations of Fluorescent Proteins:
Each of the genes coding for the wild-type proteins was subject to error-prone PCR. The PCR products were cloned, and were selected qualitatively based on expression of proteins encoded by the PCR products.
Specifically, a 5' "upper" primer containing a Kpn I restriction site, Shine-Dalgarno and Kozak consensus uptranslation sequences, the starting ATG of the coding region, and approximately 10 by of downstream homology was designed for both AclDsRFP and McGFP. The upper primer for AclDsRFP was:
5'-CCGGTACCTAAGGAGGCCACCATGAGTTGTTCC-3' (SEQ. ID. N0:12) and the upper primer for McGFP was:
5'-CCGGTACCTAAGGAGGCCACCATGAGTGTGATAAAAC-3' (SEQ. ID. N0:13).
A 3' "lower" primer containing an Xba I restriction site, the stop codon of the coding region, and approximately 10 by of upstream homology was also designed for each of the fluorescent protein isolates. The lower primer for AclDsRFP was:
5'-CCACTAGTCTAGATCATTACCGCTC-3' (SEQ. ID. N0:14) and the lower primer for McGFP was:
5'-GGTCTAGATTACTTGGCCTGCCTC-3' (SEQ. ID. NO:15).
{00046463.DOC !} 15 Low-stringency PCR protocol was performed on the fluorescent protein plasmid DNA using the above primers. The conditions were: 94°C/5 min, ~0°C/hold, add polymerase, 10 cycles of 94°C/1 min, 42°C/2 min, 72°C/3 min, 30 cycles of 94°C/30 sec, 55°C/1 min, 72°C/1 min and a final 10 min of 72°C. The PCR products were purified with S a phenol/chloroform extraction and isopropanol precipitation, digested with Kpn I/Xba I, gel purified, and ligated into Kpn I/Xba I digested pBS II KS+ plasmid (Stratagene, La Jolla, CA). The ligation mix was electrotransformed into Top 10 cells (Invitrogen) and colonies were screened for 1) brightness greater than parent, 2) speed of color development, 3) size, and 4) color.
Two mutants were generated from AclDsRFP, the wild-type red fluorescent protein by several rounds of low stringency PCR, as described above. After cloning into a vector and visual screening of transformed bacteria for fluorescent properties, the proteins encoded by Red I and Red II were selected based upon higher relative fluorescence intensity when compared to the parent AelDsRFP wild type. The mutant clones generated were named Red I and Red II.
In addition, the Green I mutant was generated from the McGFP, the wild-type green fluorescent protein. A second round of low-stringency PCR was performed on the DNA encoding Green I, and colonies meeting the above four criteria were isolated. The mutant clone generated from Green I was named Green II. Sequencing was performed by The University of Iowa DNA Facility (Iowa City, IA) using purified plasmid preparations. The DNA sequences of Red I and Red II are shown in FIGS. lA-1B, and the amino acid sequences are shown in FIG. 2. The DNA sequences of Green I and Green II are shown in FIGS. 6A-6B, and the amino acid sequences are shown in FIG. 7.
Example 5. Fluorescent Proteins Cloned into Other Expression Vectors and Spectral Analysis Thereof:
For mammalian expression, each of the four mutant fluorescent proteins (Red I, Red II, Green I, and Green II) was restriction digested with KpnI/XbaI, then polymerase treated to create blunt ends, and cloned into pCI-neo Mammalian Expression Vector (Promega, Madison, WI) using the blunt end SmaI restriction site to generate pCI-Neo-Red I, pCI-Neo-Red II, pCI-Neo-Green I, and pCI-Neo-Green II.
Spectral analysis was determined on the fluorescent proteins expressed in both mammalian and bacterial cells. Mammalian CHO cells were transfected with pCI-Neo-{00046463.DOC /} 16 Red I, pCI-Neo-Red II, pCI-Neo-Green I, or pCI-Neo-Green II plasmid using Trans-FastTM Transfection Product (Promega). Spectral analysis was determined on cell lysis of the transfected CHO cells using a Spex Fluorolog 1680 0.22m Double Spectrometer. Red II and Green II plasmids were also expressed in prokaryotic cells. The fragment of the appropriate plasmids was PCR amplified with oligonucleotides that generate NcoI-NotI
restriction sites. The PCR amplicons were digested with NcoI and NotI and cloned into the same sites in a prokaryotic expression vector, which was then used to transform E.
eoli JM109 bacteria. After purification of fluorescent proteins, excitation and emission spectra for some proteins were collected and other proteins were evaluated visually using the Zeiss microscope. Note that the spectra were recorded at 24 hours, however, mature fluorescence for Red II takes more than 24 hours, and by 48 hours, the emission peak is at 583 nm.
Example 6. Transgenic Animals Expressing the Fluorescent Protein:
DNA for microinjection into zebrafish embryos was prepared as described previously (Gibbs et al., 1994), with a ftnal concentration of approximately 2 ,ug/ml linear DNA and 0.05% phenol red. Under a dissecting microscope, several hundred newly fertilized eggs in embryo rearing solution (ERS - tap water with 25 mg/L
neomycin sulfate, 0.5 mg/L methylene blue, 17.5 mg/L sodium thiosulfate, and 125 ul/L
Amquel [Kordon]) were placed in a 35 mm petri dish of 1% agar containing a single depression of 1-2 mm. With the left hand, a single egg with an intact chorion was rolled into the depression using a number 5 forceps and held in place with the animal pole upright. With the right hand, the egg was microinjected with a running syringe (Gilmont S-1100 with a 5-10 micron needle tip) into the central lower region of the first cell near the yolk-cytoplasm boundary. Disruption of this boundary and leakage of the DNA
solution into the underlying yolk occurred in most eggs and did not harm the resultant embryos. After microinjection, the egg was moved with the left-hand forceps to a 100 mm petri dish containing ERS, and the next egg was rolled into position in the depression.
Using this technique 500 or more eggs could be microinjected a day. The average dose per embryo, as established by Southern blot analysis, was 50 pg of DNA. This dose was empirically chosen because it resulted in a high proportion of gene transfer. Under these conditions, approximately half of the embryos died in the first 24 hours, and survival to sexual maturity was expected from 10% to 20% of the eggs injected. However, survival of embryos that were mock injected (received no DNA) was approximately 90% that of {00046463.DOC /} 17 controls receiving no treatment.
Living embryos and whole mount tissues from ash were observed and photographed with a Leica MZFL III stereo fluorescence dissection microscope equipped with a camera. The filter sets used were an FITC filter set (excitation, 470 nm; emission 515 nm), a modified FITC filter set (Leica-GFP) (excitation, 425 nm; emission, 480 nm) or a rhodamine filter set (excitation, 546 nm; emission, 590 nm). Embryos were individually transferred by pipette in an approximately 50 ,ul drop of ERS to an inverted 100 mm petri dish lid and observed in groups of about 50 embryos. To immobilize embryos for photography or detailed observation, 1 ml of a 20x stock anesthetic was added to the approximate 20 ml of ERS in a 100 mm petri dish. The 20x stock anesthetic was 3 g/L of tricaine methanesulfonate (MS-222), 20 mM Tris, pH 8, in ERS.
Anes-thetized embryos, with or without an intact chorion, became immobile in about 2 minutes and remained viable in this solution for at least 30 minutes. After observation, the embryos were moved to a fresh dish of ERS and regained their mobility in several minutes.
Embryos microinjected with linearized fish expression DNA vectors at the one-cell stage containing either Red II or Green II could be scored as positive for expression of fluorescent proteins within 24 hours. Typically, greater than 50% of the embryos contained fluorescent cells or patches which were retained in about 10% of the individuals upon maturity. From one of the adult fish containing a fluorescent patch of Red II expression, a line of fish has been derived in which Red II is ubiquitously expressed from the embryo through the adult.
{0004G4G3.DOC /}

BIBLIOGRAPHY
Fradkov, A.F., et al. (2000) Novel Fluorescent protein from Discosonaa coral and its mutants possesses a unique far-red fluorescence. FEBS Letters 479:127-130.
Gibbs, P.D.L., Peek, A., and Thorgaard, G. (1994) An in vivo screen for the luciferase transgene in zebrafish. Mol. Mar. Biol. Bioteclafaol. 3:307-316.
Gibbs, P.D.L. ~ Schmale, M.C. (March/April 2000) GFP as a Genetic Marker Scorable Throughout the Life Cycle of Transgenic Zebra~sh. Marine Biotechnology.
2:107-125.
Liu, H.S., et al. (1999) Is Green Fluorescent Protein Toxic to the Living Cells?
Biochemical & Biophysical Research Communications 260:712-717.
Matz, M.V., et al. (October 1999) Fluorescent proteins from nonbioluminescent Ant7ZOZOa species. Nature Biotech 17:969-973.
Ormo, M., et al. (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392-1395.
Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) The Molecular Structure of GFP.
Nature Biotech 14:1246-1251.
All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.
{00046463.DOC l} 19 SEQUENCE LISTING.ST25 SEQUENCE LISTING
<110> Gibbs, Patrick D.L.
Carter., Robert w.
schmale, Michael c.
<120> FLUORESCENT PROTEINS FROM AQUATIC SPECIES
<130> 638.008 <150> U5 10/314,936 <151> 2002-12-09 <160> 15 <170> Patentln version 3.2 <210> 1 <211> 711 <212> DNA
<213> Artificial <220>
<223> mutant red fluorescent protein <220>

<221> CDS

<222> (1). 1) . (71 <400> 1 atg agttgttccaag aatgttatc aaggagttcatg aggtttaag gtt 48 Met SerCysSerLys AsnValIle LysGluPheMet ArgPheLys Val cgt atggaag acg gtcaatg caca ttt a g g gaa atagaagJc gaa 9&
9 g ~

Arg MetGluG Thr ValAsnGly HisGluPheGlu IleGluGly Glu y gga gaagggaggcca tacgaaggc cacaataccgta aagcttaag gta 144 Gly GluG1yArgPro TyrGluG1y HisAsnThrVal LysLeuLys Val acc aaggggggacct ttgccattt gettgggatatt ttgtcacca caa 192 Thr LysGlyGlyPro LeuProPhe AlaTrpAspIle LeuSerPro Gln ttt cagtatggaagc aaggtatat gtcaagcatcct gccgacata cca 240 Phe GlnTyrGlySer LysValTyr ValLysHisPro AlaAspIle Pro gac tataaaaagctg tcatttcct gaaggatttaaa tgggaaagg gtc 288 Asp TyrLysLysLeu SerPhePro GluGlyPheLys TrpGluArg Val atg aactttgaagac ggtggcgtc gttactgtaacc caggattcc agt 336 Met AsnPheGluAsp GlyGlyVal ValThrValThr GlnAspSer Ser ttg caggatggctgt ttcatctac aaggtcaagttc attggcgtg aac 384 Leu GlnAspG1yCys PheIleTyr LysValLysPhe IleG1yVal Asn SEQUENCE LISTING.ST25 tttccttctgatg cctgtt atgcaaaag aagacaatgggc tgggaa432 a ~

PheProSerAspG ProVal MetGlnLys LysThrMetGly TrpGlu y gccagcactgagcgt ttgtat cctcgtgat ggcgtgttgaaa ggagag480 AlaSerThrGluArg LeuTyr ProArgAsp GlyValLeuLys GlyGlu attcataaggetctg aagttg aaagacggt ggtcattaccta gttgaa528 IleHisLysAlaLeu LysLeu LysAspGly GlyHisTyrLeu ValGlu ttcaaaactatttac atggca aagaagcct gtgcagctacca gggtac576 PheLysThrIleTyr MetAla LysLysPro ValGlnLeuPro GlyTyr tactatgttgactcc aaactg gatataaca agccacaacaaa gactat624 TyrTyrValAspSer LysLeu AspIleThr SerHisAsnLys AspTyr acaatcgttgagcag tatgaa agaaccgag ggacgccaccat ctgttc672 ThrIleValGluGln TyrGiu ArgThrGlu GlyArgHisHis LeuPhe cttaaggetgaactt ggctca gacgtgggt gagcggtaa 711 LeuLysAlaGluLeu G1ySer AspVa~lG1y GluArg <210> 2 <211> 236 <21Z> PRT
<213> Artificial <220>
<223> Synthetic Construct <400> 2 Met Ser Cys Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly His Asn Thr Val Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val SEQUENCE LISTING.ST25 Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu Phe Lys Thr Ile Tyr Met Ala Lys Lys Pro Va7 Gln Leu Pro Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Lys Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His His Leu Phe Leu Lys Ala Glu Leu Gly Ser Asp Val Gly Glu Arg <210> 3 <211> 711 <212> DNA
<213> Artificial <220>
<223> mutant red fluorescent protein <220>

<221> CDS

<222> (1).
. (711) <400> 3 atg tgttcc aagaatgttatc aaggagttc atgaggttt aaggtt 48 agt Met CysSer LysAsnValIle LysGluPhe MetArgPhe LysVal Ser 1 5 l0 15 cgt gaag9a acggtcaatg9g cacgagttt gaaatagaa ggcgaa 96 atg Gl l h l A
M
t rg u G T Va AsnGly HisGluPhe GluIleGlu GlyGlu e y r gga gggagg ccatacgaaggc cacaatacc gtaaagctt aaggta 144 gaa G1y G1yArg ProTyrGluGly HisAsnThr ValLysLeu LysVal Glu acc ggggga cctttgccattt gettgggat attttgtca ccacaa 192 aag SEQUENCE
LISTING.ST25 Thr GlyGly ProLeuProPhe AlaTrpAsp LeuSer ProGln Lys Ile ttt cagtatg9a agcaaggtatat gtcaagcat cctgccgac atacca 240 Phe GlnTyrGly SerLysValTyr ValLysHis ProAlaAsp IlePro gac tataaaaag ctgtcatttcct gaaggattt aaatgggaa agggtc 288 Asp TyrLysLys LeuSerPhePro GluGlyPhe LysTrpGlu ArgVal atg aactttgaa gacggtggcgtc gttactgta acccaggat tccagt 336 M A h l t e sn P G AspGiyGlyVal ValThrVal ThrGlnAsp SerSer e u ttg caggatggc tgtttcatctac aaggtcaag ttcattggc gtgaac 384 Leu GlnAspGly CysPheIleTyr LysValLys PheIleGly ValAsn ttt ccttctgat ggacctgttatg caaaagaag acaatgggc tgggaa 432 Phe ProSerAsp GlyProValMet GlnLysLys ThrMetGly TrpGlu gcc agcactgag cgtttgtatcct cgtgatggc gtgttgaaa ggagag 480 ~

Ala SerThrGiu ArgLeuTyrPro ArgAspGly ValLeuLys GlyGlu att cataagget ctgaagttgaaa gacggtggt cattaccta gttgaa 528 Ile HisLysAla LeuLysLeuLys AspGlyGly HisTyrLeu ValGlu ttc aaaactatt tacatggcaaag aagcctgt9 cagctacca g9gtac 576 Phe LysThrIle TyrMetAlaLys LysProVal GlnLeuPro GlyTyr tac tatgttgac tccaaactggat ataacaagc cacaacaaa gactat 624 Tyr TyrValAsp SerLysLeuAsp IleThrSer HisAsnLys AspTyr aca atcgttgag cagtatgaaaga accgagg9a cgccaccat ctgttc 672 Thr IleValGlu GlnTyrGluArg ThrGluGly ArgHisHis LeuPhe ctt aaggetgaa cttggctcaaac gtgggtgag cgg 711 taa Leu LysAlaGlu LeuGlySerAsn GlyGlu Arg Val <210> 4 .
<211> 236 <212> PRT
<213> Artificial <220>
<223> Synthetic Construct <400> 4 Met Ser Cys Ser Lys Asn Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu SEQUENCE LISTING.ST25 Gly Glu Gly Arg Pro Tyr Glu Gly His Asn Thr Val Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Giu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser loo l05 llo Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu Phe Lys Thr Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser Nis Asn Lys Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His His Leu Phe Leu Lys Ala Glu Leu Gly Ser Asn Val Gly Glu Arg <210> 5 <211> 684 <212> DNA
<213> Artificial <220>
<223> mutant green fluorescent protein SEQUENCE LISTING.ST25 <220>

<22l> CDS

<222> (1)..(684) <400> 5 atg ata aaaccagac atc aagctg atggaa 48 agt atg cgt ggt gtg aag ietSer Ile 5ysProAsp Ile LysLeu MetGlu Val Met Arg Gly Lys getgtaaac cacaagttc gt attgaa ggagacgga aaag as 96 g c g ~

AlaValAsnG HisLysPhe Va~IleGlu GlyAspGly LysG~yLyg y cctttcgagg9a aaacagact atggacctt acagtcata gaag9cgca 144 ProPheGluGly LysGlnThr MetAspLeu ThrValIle GluGlyAla cctttgcctttc gettacgat atcttgaca acagtattc gattacg9c 192 ProLeuProPhe AlaTyrAsp IleLeuThr ThrValPhe AspTyrGly aacagggtattc gccaaatac ccaaaagac ataccagac tatttcaag 240 AsnArgValPhe AlaLysTyr ProLysAsp IleProAsp TyrPheLys cagacgtttccg gaggggtac tcctgggaa tgaagcatg acatacgaa 288 GlnThrPhePro GluGlyTyr SerTrpGlu ArgSerMet ThrTyrGlu gaccagg att tgcatcgcc acaaacgac ataacaatg atgaaag c 336 c ~

AspGlnG Ile CysIleAla ThrAsnAsp IleThrMet MetLysG~y y gtcgacgactgt tttgtctat aaaattcga tttgatgt tg aacttt 384 ValAspAspCys PheValTyr LysIleArg PheAspGly galAsnPhe cctgccaatggt ccagttatg cagaggaag acgctaaaa tgggagcca 432 ProAlaAsnGly ProValMet GlnArgLys ThrLeuLys TrpGluPro tccactgaaaaa atgtatgtg cgtgatggg gtactgaag ggtgatgtt 480 SerThrGluLys MetTyrVa1 ArgAspG~lyValLeuLys G1yAspVal aacatggetctg ttgcttgaa g9ag9tg9c cattaccga tgtgactcc 528 AsnMetAlaLeu LeuLeuGiu GlyGlyGly HisTyrArg CysAspSer aaaactacttac aaagetaag aaggttgtc cagttgcca gactatcat 576 LysThrThrTyr LysAlaLys LysValVal GlnLeuPro AspTyrHis tttgttgaccat cgcattgag attgtgagc cacgacaaa gattacaac 624 PheValAspHis IleGlu IleValSer HisAspLys AspTyrAsn Arg aaggttaagctg tatgagcat gccgaaget cattctg9g ctgccgagg 672 LysValLysLeu GluHis AlaGlu HisSerGly LeuProArg Tyr Ala caggccaagtaa 684 Gln Ala Lys SEQUENCE LISTING.ST25 <210> 6 <211> 227 <212> PRT
<213> Artificial <220>
<223> Synthetic Construct <400> 6 Met Ser Va7 Ile Lys Pro Asp Met Lys Ile Lys Leu Arg Met Glu Gly Ala Val Asn Gly His Lys Phe Val Tle Glu Gly Asp Gly Lys Gly Lys Pro Phe Glu Gly Lys Gin Thr Met Asp Leu Thr Val Ile Glu Gly Ala Pro Leu Pro Phe Ala Tyr Asp Ile Leu Thr Thr Val Phe Asp Tyr Gly Asn Arg Val Phe Ala Lys Tyr Pro Lys Asp Ile Pro Asp Tyr Phe Lys Gln Thr Phe Pro Glu Gly Tyr Ser Trp Glu Arg Ser Met Thr Tyr Glu Asp Gln Gly Ile Cys Ile Ala Thr Asn Asp Ile Thr Met Met Lys Gly Val Asp Asp Cys Phe Val Tyr Lys Ile Arg Phe Asp Gly Val Asn Phe Pro Ala Asn Gly Pro Val Met Gln Arg Lys Thr Leu Lys Trp Glu Pro Ser Thr Glu Lys Met Tyr Val Arg Asp Gly Val Leu Lys Gly Asp Val Asn Met Ala Leu Leu Leu Glu Gly Gly Gly His Tyr Arg Cys Asp Ser Lys Thr Thr Tyr Lys Ala Lys Lys Val Val Gln Leu Pro Asp Tyr His Phe Val Asp His Arg Ile Glu Ile Val Ser His Asp Lys Asp Tyr Asn SEQUENCE LISTING.ST25 Lys Val Lys Leu Tyr Glu His Ala Glu Ala His Ser Gly Leu Pro Arg Gln Ala Lys <210> 7 <211> 684 <212> DNA
<213> Artificial <220>
<223> mutant green fluorescent protein <220>

<22 1> CDS

<22 2> (1)..(684) <40 0> 7 atg agtgt ata aaaccagac atgaagatcaag ctgcgtatg gaaggt 48 ~

iet SerVa Ile 5ysProAsp MetLysIleLys LeuArgMet GluGly get gtaaacg cacaagttc gtgattgaagga gacggaaaa ggcaag 96 g ~

Ala ValAsnG HisLysPhe ValIleGluGly AspG1yLys G1yLys y cct ttcgaggga aaacagact atggaccttaca gtcatagaa g gca 144 c Pro PheGluGly LysGlnThr MetAspLeuThr ValIleGlu G~yAla 35 40 ~ 45 cct ttgcctttc gettacgat atcttgacaaca gtattcgat tacg 1g2 Pro LeuProPhe AlaTyrAsp IleLeuThrThr ValPheAsp TyrGly aac agggtattc gccaaatac ccaaaagacata ccagactat ttcaag 240 Asn ArgValPhe AlaLysTyr ProLysAspIle ProAspTyr PheLys cag acgtttccg gaggggtac tcctgggaacga agcatgaca tacgaa 288 Gln ThrPhePro GluGlyTyr SerTrpGluArg SerMetThr TyrGlu gac cagg att tgcatcgcc acaaacgacata acaatgatg aaaggc 336 c ~

Asp GlnG Ile CysIleAla ThrAsnAspIle ThrMetMet LysGly y gtc gacgactgt tttgtctat aaaattcgattt gatggtgt aacttt 384 Val AspAspCys PheValTyr LysIleArgPhe AspGlyVa~ AsnPhe cct gccaatggt ccagttatg cagaggaagacg ctaaaatgg gagcca 432 Pro AlaAsnGly ProValMet GlnArgLysThr LeuLysTrp GluPro tcc actgaaaaa atgtatgtg cgtgatggggta ctg,aagggt gatgtt 480 Ser ThrGluLys MetTyrVa1 ArgAspG1yVal LeuLysG~lyAspVal SEQUENCELIS TING.ST25 aacatg getctgttg cttgaaggaggt ggccat taccgatgt gacttc528 AsnMet AlaLeuLeu LeuGluGlyGly GlyHis TyrArgCys AspPhe l65 170 175 aaaact acttacaaa getaagaaggtt gtccag ttgccagac tatcat576 LysThr ThrTyrLys AlaLysLysVal ValGln LeuProAsp TyrHis tttgtt gaccatcgc attgagattgtg agccac gacaaagat tacaac624 PheVal AspHisArg IleGluIleVal SerHis AspLysAsp TyrAsn 195 200 2os aaggtt aagctgtat gagcatgccgaa getcat tctgggctg ccgagg672 LysVal LysLeuTyr GluHisAlaGlu AlaHis SerG1yLeu ProArg caggcc aagtaa 684 Gln Ala Lys <210> 8 <211> 227 <212> PRT
<213> Artificial <220>
<223> Synthetic Construct <400> 8 Met Ser Val Ile Lys Pro Asp Met Lys Ile Lys Leu Arg Met Glu Gly Ala Val Asn Gly His Lys Phe Val Ile Glu Gly Asp Gly Lys Gly Lys Pro Phe Glu Gly Lys Gln Thr Met Asp Leu Thr Val Ile Glu Gly Ala Pro Leu Pro Phe Ala Tyr Asp Ile Leu Thr Thr Val Phe Asp Tyr Gly Asn Arg Val Phe Ala Lys Tyr Pro Lys Asp Ile Pro Asp Tyr Phe Lys Gln Thr Phe Pro Glu Gly Tyr Ser Trp Glu Arg Ser Met Thr Tyr Glu Asp Gln Gly I1e Cys Ile Ala Thr Asn Asp Ile Thr Met Met Lys Gly Val Asp Asp Cys Phe Val Tyr Lys I1e Arg Phe Asp Gly Val Asn Phe SEQUENCE LISTING.ST25 Pro Ala Asn Gly Pro Val Met Gln Arg Lys Thr Leu Lys Trp Glu Pro Ser Thr Glu Lys Met Tyr Val Arg Asp Gly Val Leu Lys Gly Asp Val Asn Met Ala Leu Leu Leu Glu Gly Gly G1y His Tyr Arg Cys Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Va1 Val Gln Leu Pro Asp Tyr His Phe Val Asp His Arg Ile Glu Iie Val Ser His Asp Lys Asp Tyr Asn Lys Val Lys Leu Tyr Glu His Ala Glu Ala His Ser Gly Leu Pro Arg Gln Ala Lys <210> 9 <211> 45 <212> RNA
<213> Artificial <220>
<223> adaptor <400> 9 gcugauggcg augaaugaac acugcguuug cuggcuuuga ugaaa 45 <210> 10 <211> 41 <212> DNA
<213> Artificial <220>
<223> primer <400> 10 ctcgagaagc ttgaattcgg atcctttttt tttttttttt t 41 <210> 11.
<211> 24 <212> DNA
<213> Artificial <220>
<223> primer <400> 11 SEQUENCE LISTING.ST25 gctgatggcg atgaatgaac actg 24 <210> 12 <211> 33 <212> DNA
<213> Artificial <220>
<223> primer <400> 12 ccggtaccta aggaggccac catgagttgt tcc 33 <210> 13 <211> 37 <212> DNA
<213> Artificial <220>
<223> primer <400> 13 ccggtaccta aggaggccac catgagtgtg ataaaac 37 <210> 14 <211> 25 <212> DNA
<213> Artificial <220>
<223> primer <400> 14 ccactagtct agatcattac cgctc 25 <210> 15 <211> 24 <212> DNA
<213> Artificial <220>
<223> primer <400> 15 ggtctagatt acttggcctg cctc 24

Claims (46)

What is claimed is:

1. A first mutant fluorescent protein comprising an amino acid sequence that differs from that of a corresponding wild-type red fluorescent protein by at least one amino acid substitution, wherein the first mutant fluorescent protein mutant has an optical property that differs relative to the corresponding optical property produced by the wild-type red fluorescent protein.

2. The first mutant fluorescent protein of claim 1, wherein the different optical property comprises higher relative fluorescence intensity.

3. The first mutant fluorescent protein mutant of claim 2, wherein the first mutant fluorescent protein is encoded by the amino acid sequence comprising SEQ ID
NO:1 (Red I).

4. A second mutant fluorescent protein comprising an amino acid sequence that differs from that of the first mutant fluorescent protein of claim 1 by at least one amino acid substitution, wherein the second mutant fluorescent protein has an optical property that differs relative to the corresponding optical property produced by the first mutant fluorescent protein.

5. The second mutant fluorescent protein of claim 4, wherein the different optical property comprises higher relative fluorescence intensity.

6. The second mutant fluorescent protein of claim 5, wherein the amino acid substitution is at a position corresponding to position 232 of Red I of SEQ.
ID. NO: 2.

7. The second mutant fluorescent protein of claim 5, wherein the second mutant fluorescent protein is encoded by an amino acid sequence comprising SEQ ID NO:

(Red II).

8. An isolated nucleic acid encoding a mutant fluorescent protein, the nucleic acid comprising a polynucleotide sequence, or complement thereof.

9. The isolated nucleic acid of claim 8, wherein the nucleic acid comprises SEQ. ID.
NO: 1 (Red I).

10. The isolated nucleic acid of claim 8, wherein the nucleic acid comprises SEQ. ID.
NO: 3 (Red II).

11. A vector comprising the isolated nucleic acid according to claim 8.

12. The vector of claim 11, further comprising a promoter, wherein the promoter is operably linked to the nucleic acid.

13. A nucleic acid construct comprising:
(A) a first coding sequence that encodes a selected polypeptide;
(B) a second coding sequence selected from the group consisting the nucleic acid of claim 8, wherein the first coding sequence is fused to the second coding sequence such that expression of the fused sequence yields a fluorescent hybrid protein in which the polypeptide encoded by the first coding sequence is fused to the polypeptide encoded by the second coding sequence.

14. A method of detecting expression of a gene comprising introducing the nucleic acid of claim 8, into a cell or organism, allowing the cell to replicate, and detecting expression of the nucleic acid by emission of fluorescent light.

15. The method of claim 14, wherein the nucleic acid is introduced into a eukaryotic cell.

16. The method of claim 14, wherein the nucleic acid is introduced into a prokaryotic cell.

17. The method of claim 14, wherein the expression of the nucleic acid expression is detected in vivo.

18. The method of claim 14, wherein the expression of the nucleic acid is detected in vitro.

19. A cell comprising the nucleic acid of claim 8.

20. The cell of claim 19, wherein the nucleic acid is integrated into a genome of the cell.

21. The cell of claim 19, wherein the nucleic acid is not integrated into a genome of the cell.

22. An animal comprising the isolated nucleic acid of claim 8.

23. The animal of claim 22, wherein the animal is a zebrafish.

24. A first mutant fluorescent protein comprising an amino acid sequence that differs from that of a corresponding wild-type green fluorescent protein by at least one amino acid substitution, wherein the first mutant fluorescent protein mutant has an optical property that differs relative to the corresponding optical property produced by the wild-type green fluorescent protein.

25. The first mutant fluorescent protein of claim 24, wherein the different optical property comprises higher relative fluorescence intensity.

26. The first mutant fluorescent protein mutant of claim 25, wherein the first mutant fluorescent protein is encoded by the amino acid sequence comprising SEQ ID
NO:5 (Green I).

27. A second mutant fluorescent protein comprising an amino acid sequence that differs from that of the first mutant fluorescent protein of claim 24 by at least one amino acid substitution, wherein the second mutant fluorescent protein has an optical property that differs relative to the corresponding optical property produced by the first mutant fluorescent protein.

28. The second mutant fluorescent protein of claim 27, wherein the different optical property comprises higher relative fluorescence intensity.

29. The second mutant fluorescent protein of claim 28, wherein the amino acid substitution is at a position corresponding to position 176 of Green I of SEQ.
ID. NO:6.

30. The second mutant fluorescent protein of claim 28, wherein the second mutant fluorescent protein is encoded by an amino acid sequence comprising SEQ ID NO:

(Green II).

31. An isolated nucleic acid encoding a mutant green fluorescent protein, the nucleic acid comprising a polynucleotide sequence, or complement thereof.

32. The isolated nucleic acid of claim 31, wherein the nucleic acid comprises SEQ.
ID. NO: 5 (Green I).

33. The mutant form of the nucleic acid of claim 31, wherein the nucleic acid comprises SEQ. ID. NO: 6 (Green II).

34. A vector comprising the isolated nucleic acid according to claim 31.

35. The vector of claim 34, further comprising a promoter, wherein the promoter is operably linked to the nucleic acid.

36. A nucleic acid construct comprising:
(A) a first coding sequence that encodes a selected polypeptide;
(B) a second coding sequence selected from the group consisting the nucleic acid of claim 31, wherein the first coding sequence is fused to the second coding sequence such that expression of the fused sequence yields a fluorescent hybrid protein in which the polypeptide encoded by the first coding sequence is fused to the polypeptide encoded by the second coding sequence.

37. A method of detecting expression of a nucleic acid comprising introducing the nucleic acid of claim 31 into a cell or organism, allowing the cell to replicate, and detecting expression of the nucleic acid by emission of fluorescent light.

38. The method of claim 37, wherein the nucleic acid is introduced into a eukaryotic cell.

39. The method of claim 37, wherein the nucleic acid is introduced into a prokaryotic cell.

40. The method of claim 37, wherein the expression of the nucleic acid is detected in vivo.

41. The method of claim 37, wherein the expression of the nucleic acid is detected in vitro.

42. A cell comprising the nucleic acid of claim 31.

43. The cell of claim 42, wherein the nucleic acid is integrated into a genome of the cell.

44. The cell of claim 42, wherein the nucleic acid is not integrated into a genome of the cell.

45. An animal comprising the nucleic acid of claim 31.

46. The animal of claim 45, wherein the animal is a zebrafish.