WO2002031149A2 - Polyvalent cation-sensing receptor proteins in aquatic species - Google Patents

Abstract

The present invention provides nucleic acid sequences encoding a polyvalent cation-sensing receptor in aquatic species, wherein the receptor regulates adaptation to environmental salinity and body composition. Partial nucleotide and amino acid sequences of polyvalent cation receptor proteins in cod, haddock, hake, halibut, mackeral, pollock, sea bass, swordfish, winter flounder, tilapia, tuna, catfish, seabream, turbot and salmonid fish such as atlantic salmon, chum salmon, coho salmon, king salmon, pink salmon, sockeye salmon, rainbow trout, and arctic char, are also provided.

Description

POLYVALENT CATION-SENSING RECEPTOR PROTEINS IN AQUATIC SPECIES

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/240,392, filed on October 12, 2000, and U.S. Provisional Application No. 60/240,003, filed on October 12, 2000. The entire teachings ofthe above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Aquaculture is of great importance for the production of food as well as other commercial products such as pearls. Examples of major difficulties encountered in aquaculture include bacterial, parasitic, and chemical contaminants in seawater, as well as the sensitivity offish to changes in salinity. Unfortunately, finding solutions to these difficulties are hampered by the limited understanding of biological processes offish. Therefore, a need exists to gain a better understanding ofthe biological processes of fish that are related to adaptation to varying salinities, or ridding fish of bacteria or parasites, or chemical contaminants. In particular, a need exists to isolate genes that play an important role in these areas.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a polyvalent cation-sensing receptor protein (PVCR) in aquatic species which allows the successful adaptation offish to their environmental salinity.

The present invention encompasses nucleic acid sequences encoding PVCR proteins of aquatic species, hi one embodiment, the present invention is an isolated nucleic acid molecule having at least 70% identity with SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

In another embodiment, the present invention is an isolated nucleic acid molecule having at least 70% identity with a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; encoding a PolyValent Cation-sensing Receptor (PVCR) polypeptide, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species. h another embodiment, the present invention is an isolated PVCR polypeptide having at least 70% similarity with SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50; wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

In another embodiment, the present invention is an isolated nucleic acid ^• molecule comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; and a complementary strand of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

In yet another embodiment, the present invention is an isolated RNA molecule encoded by a nucleic acid molecule of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

In yet an additional embodiment, the present invention is an isolated RNA molecule encoded by a nucleic acid molecule of a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

In a further embodiment, the present invention is an isolated RNA molecule encoded by a nucleic acid molecule of a complementary strand of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

In an additional embodiment, the present invention is an isolated nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ DD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. In another embodiment, the present invention is an isolated nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50, wherein the nucleic acid molecule is RNA. In a further embodiment, the present invention is an isolated nucleic acid molecule that hybridizes under high stringency conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; a coding sequence of SEQ ID NOS: 1, 3, 5, 1, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; a complementary strand of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; and a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.

In an additional embodiment, the present invention is an isolated nucleic acid molecule that hybridizes under high stringency conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; a complementary strand of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; and a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50, wherein the probe hybridizes to a nucleic acid molecule that senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species. hi yet another additional embodiment, the present invention is a plasmid or vector containing a nucleic acid molecule comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

In still another embodiment, the present invention is a plasmid or vector containing a nucleic acid molecule comprising a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. In another embodiment, the present invention is a plasmid or vector containing a nucleic acid molecule encoding an amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

In an additional embodiment, the present invention is a host cell transformed with a nucleic acid molecule selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49.

In yet an additional embodiment, the present invention is a host cell transformed with a nucleic acid molecule selected from the group consisting of a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49. hi a further embodiment, the present invention is a host cell transformed with a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. In yet another further embodiment, the present invention is a host cell expressing a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. The polypeptide is expressed in a cell selected from the group consisting of a fish cell, a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell. hi another embodiment, the present invention is a cDNA from an ATCC

Nos.: PTA-2538; PTA-2539; PTA-2541; PTA-2542; PTA-2543; PTA-2544; PTA- 2545; PTA-2546; PTA-2547; PTA-2548; or PTA-2549.

In an additional embodiment, the present invention is an isolated polypeptide molecule comprising SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, wherein the polypeptide is all or a portion of a polypeptide which senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species. h another embodiment, the present invention is an isolated polypeptide molecule comprising a polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species. i a further embodiment, the present invention is a composition comprising a polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; and a pharmaceutically-acceptable carrier. In an additional embodiment, the present invention is a composition comprising a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32,

34, 36, 38, 40, 42, 44, 46, 48 or 50; and a pharmaceutically-acceptable carrier.

In another embodiment, the present invention is an antibody specific for a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, wherein the polypeptide senses ion concentrations; alters water intake; alters water absoφtion; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

In yet another embodiment, the present invention is an antibody specific for a polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33,

35, 37, 39, 41, 43, 45, 47, or 49, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

In a further embodiment, the present invention is a method for detecting an aquatic PVCR polypeptide in a sample comprising the steps of combimng a sample to be tested with a PVCR-specific antibody, under suitable conditions for formation of a complex between the antibody and PVCR; and detecting the formation ofthe complex.

The present invention also encompasses a labeled antibody, h a particular embodiment, the antibody is radioactively labeled, hi another particular embodiment, the complex is detected or measured using a second antibody comprismg a detectable label.

In another embodiment, the present invention is a method for detecting an aquatic PVCR nucleic acid molecule in a sample comprising the steps of combining a sample to be tested with a PVCR-specific hybridization probe, under suitable conditions for specific hybridization ofthe PVCR-specific probe and the sample and detecting the PVCR nucleic acid molecule in the sample, wherein the PVCR nucleic acid detected in the sample encodes a polypeptide that senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species. h another embodiment, the present invention is directed toward a kit for detecting the presence or absence of a PVCR, comprismg an anti-PVCR antibody. In another embodiment, the present invention is directed toward a kit for detecting the presence or absence of a PVCR, comprising nucleic acid sequences having a detectable label that can hybridize to a nucleic acid of an aquatic PVCR. The present invention also encompasses the nucleic acids encoding aquatic

PVCR proteins, their complements and fragments thereof. The nucleic acids encoding PVCR proteins can be derived from aquatic species including, for example, teleost fish and elasmobranch fish. Additionally, the aquatic species can include cod, haddock, hake, halibut, mackeral, pollock, sea bass, swordfϊsh, tilapia, winter flounder, tuna, catfish, seabream, turbot and sahnonid fish such as atlantic salmon, chum salmon, coho salmon, king salmon, pink salmon, sockeye salmon, rainbow trout, and arctic char. Further, the amino acid sequences of aquatic PVCR proteins and fragments thereof are provided. Also, encompassed in the present invention is the RNA transcribed by the nucleic acids which encode the aquatic PVCR proteins. The present invention also provides antibodies specific for the aquatic PVCR proteins and fragments. DNA probes specific for aquatic PVCR nucleic acid sequences, complements and fragments are provided. The present invention encompasses plasmids or vectors containing the nucleic acid sequences of the aquatic PVCR genes, their complement, and fragments thereof. The present invention also provides host cells contaimng the aquatic PVCR nucleic acids, their complement, and fragments thereof, and/or nucleic acids encoding the aquatic PVCR proteins. The present invention also encompasses kits for the isolation of aquatic PVCR homologs, and detection of aquatic PVCR nucleic acid and or protein expression in various fish tissues. The identification of polyvalent cation-sensing receptor proteins in aquatic species provides several advantages over current aquaculture methods. First, the present invention increases the efficiency of aquaculture by decreasing the loss of fish during the fresh to seawater transition. Altering the expression of PVCR offish spares them the osmotic shock of this transaction and provides greater resistance to bacteria, parasites and other contaminants. The present invention improves the health, size and flavor ofthe cultured fish.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and IB illustrate the annotated partial nucleotide sequence (SEQ ID NO: 1) and the deduced amino acids sequence (SEQ ID NO: 2) ofthe cod polyvalent cation-sensing receptor protein. Figures 2A and 2B illustrate the annotated partial nucleotide sequence (SEQ

ID NO: 3) and the deduced amino acids sequence (SEQ ID NO: 4) ofthe haddock polyvalent cation-sensing receptor protein.

Figures 3A and 3B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 5) and the deduced amino acids sequence (SEQ ID NO: 6) ofthe hake polyvalent cation-sensing receptor protem.

Figures 4A and 4B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 7) and the deduced amino acids sequence (SEQ ID NO: 8) ofthe halibut polyvalent cation-sensing receptor protein.

Figures 5 A and 5B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 9) and the deduced amino acids sequence (SEQ ID NO: 10) ofthe mackeral polyvalent cation-sensing receptor protein.

Figures 6A and 6B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 11) and the deduced amino acids sequence (SEQ ID NO: 12) ofthe pollock polyvalent cation-sensing receptor protein. Figures 7A and 7B illustrate the annotated partial nucleotide sequence (SEQ

ID NO: 13) and the deduced amino acids sequence (SEQ ID NO: 14) ofthe sea bass polyvalent cation-sensing receptor protein.

Figures 8A and 8B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 15) and the deduced amino acids sequence (SEQ ID NO: 16) ofthe swordfish polyvalent cation-sensing receptor protein. Figures 9A-C illustrate the annotated partial nucleotide sequence (SEQ ID NO: 17) ofthe tilapia polyvalent cation-sensing receptor protein.

Figures 10A-D illustrate the annotated partial nucleotide sequence (SEQ ID NO: 19) and the deduced amino acids sequence (SEQ ID NO: 20) o the winter flounder polyvalent cation-sensing receptor protein.

Figures 11 A and 1 IB illustrate the annotated partial nucleotide sequence (SEQ ID NO: 23) and the deduced amino acids sequence (SEQ ID NO: 24) ofthe tuna polyvalent cation-sensing receptor protein.

Figures 12A and 12B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 25) and the deduced amino acids sequence (SEQ ID NO: 26) ofthe catfish polyvalent cation-sensing receptor protein.

Figures 13 A-13C illustrate the annotated partial nucleotide sequence (SEQ ID NO: 31) and the deduced amino acids sequence (SEQ ID NO: 32) ofthe atlantic salmon polyvalent cation-sensing receptor protein. Figures 14A and 14B illustrate the annotated partial nucleotide sequence

(SEQ ID NO: 33) and the deduced amino acids sequence (SEQ ID NO: 34) ofthe chum salmon polyvalent cation-sensing receptor protein.

Figures 15A and 15B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 35) and the deduced amino acids sequence (SEQ ID NO: 36) ofthe coho salmon polyvalent cation-sensing receptor protein.

Figures 16A and 16B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 37) and the deduced amino acids sequence (SEQ ID NO: 38) ofthe king salmon polyvalent cation-sensing receptor protein.

Figures 17A and 17B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 39) and the deduced amino acids sequence (SEQ ID NO: 40) of the pink salmon polyvalent cation-sensing receptor protein.

Figures 18A and 18B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 41) and the deduced amino acids sequence (SEQ ID NO: 42) ofthe sockeye salmon polyvalent cation-sensing receptor protein. Figures 19A and 19B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 43) and the deduced amino acids sequence (SEQ ID NO: 44) ofthe rainbow trout polyvalent cation-sensing receptor protein.

Figures 20A and 20B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 45) and the deduced amino acids sequence (SEQ ID NO: 46) ofthe arctic char polyvalent cation-sensing receptor protein.

Figures 21 A-C shows aligned nucleotide sequence of 594 nucleotides for 8 fish species, each of which codes for an identical sequence of an aquatic PVCR protein. Figures 22 A and 22B shows aligned corresponding predicted amino acid sequences derived from nucleotide sequences displayed in Figure 21.

Figures 23A and 23B illustrate the annotated partial nucleotide sequence (SEQ ID NO: 47) and the deduced amino acids sequence (SEQ ID NO: 48) ofthe seabream polyvalent cation-sensing receptor protein. Figures 24A and 24B illustrate the annotated partial nucleotide sequence

(SEQ ID NO: 49) and the deduced amino acids sequence (SEQ ID NO: 50) ofthe turbot polyvalent cation-sensing receptor protein.

DETAILED DESCRIPTION OF THE INVENTION Aquatic species are able to "smell" or otherwise sense the ion concentrations and/or salinity in their environment. The present invention relates to the identification and characterization of polyvalent cation-sensing receptor (PVCR) proteins in aquatic species. Aquatic PVCR proteins play a role in the adaptation of fish to marine and freshwater environments. Additionally, PVCR proteins influence the growth and development offish.

Aquatic PVCR proteins have been found in teleosts and elasmobranchs. Teleosts are boney fish found in marine, freshwater, or euryhaline environments such as summer and winter flounder, arctic char, cod, trout, killifish and salmon. Elasmobranchs are cartilaginous fish, such as sharks, rays and skates, and are predominately marine. Marine teleost fish live in seawater possessing a high osmolality (1,000 mos ) that normally contains 10 millimolar (mM) Ca2⁺, 50 mM Mg2+ and 450 mM NaCl. Since their body fluids are 300-400 mosm, these fish are obligated to drink seawater, absorb salts through their intestine and secrete large quantities of NaCl through their gills and Mg^⁺ and Ca^⁺ through their kidneys. Their kidneys produce only small amounts of concentrated urine. hi contrast, freshwater teleost fish possess body fluids of 300 mosm and normally live in water of less than 50 mosm containing 5-20 mM NaCl and less than 1 mM Ca^⁺ and Mg2⁺. These fish drink little, but absorb large amounts of water from their dilute environment. As a result, their kidneys produce copious dilute urine to maintain water balance. Freshwater fish gill tissue has a low permeability to ions and gill epithelial cells extract NaCl from water.

Euryhaline fish acclimate to various salinities by switching back and forth between these two basic patterns of ion and water transport. For example, when freshwater adapted teleost fish are challenged with high salinities, their gill epithelia rapidly alter net NaCl flux such that NaCl is secreted rather than reabsorbed. Reduction of extracellular Ca2⁺ from 10 mM to 100 micromolar profoundly inhibits this transport process. In flounder species, transfer to seawater activates a series of changes in the kidney allowing for secretion of large quantities of Ca^⁺ and Mg2⁺ by renal epithelia and recovery of water via a thiazide sensitive NaCl cotransporter in the urinary bladder. hi a similar fashion, adaption of marine euryhaline fish to freshwater is possible because of a net reversal of epithelial ionic gradients such that NaCl is actively reabsorbed and divalent metal ion secretion ceases. These changes are a direct result of a fish "sensing" an increased or decreased environmental ion concentration which result in physiological responses described above. These changes are mediated by alterations in hormones, especially prolactin, cortisol and arginine vasotocin. These alterations in a cluster of critical hormones and functional changes in epithelial transport in gill, intestine, bladder and kidney are vital not only to rapid euryhaline adaption, but also throughout development offish embryos, larvae and during metamorphosis. In one embodiment, the present invention is directed to an isolated nucleic acid molecule encoding a Polyvalent Cation-sensing Receptor (PVCR) protein in an aquatic species, wherem the PVCR protein allows fish to sense ion concentrations; alter water intake, water absorption or urine output; or modulate the percentage of fat, protein or moisture of muscle.

Nucleic acids encoding aquatic PVCR are identified by screening a cDNA library with a PVCR-specific probe under conditions known to those of skill in the art to identify homologous receptor proteins. For example, a partial winter flounder PVCR cDNA was isolated by screening a winter flounder urinary bladder cDNA library with a probe consisting of a fragment of a cDNA encoding a highly conserved sequence ofthe shark kidney calcium receptor. Techniques for the preparation and screening of a cDNA library are well-known to those of skill in the art. For example, techniques such as those described in Riccardi, et al., Proc. Nat. Acad. Sci. USA, 92:131-135 (1995), can be used. Positive clones can be isolated, subcloned and their sequences determined. Using the sequences of either a full length or several over-lapping partial cDNAs, the complete nucleotide sequence of the winter flounder PVCR cDNA can be obtained and the encoded amino acid sequence deduced. The sequences ofthe aquatic PVCR can be compared to other aquatic PVCRs to determine differences and similarities. Alternatively, partially aquatic PVCR genes were isolated by Polymerase Chain Reaction (PCR) of genomic DNA with degenerate primers (SEQ ID NOS: 21 and 22) specific to a highly conserved sequence of calcium receptors which do not contain introns. For example, partial aquatic PVCR clones were obtained for cod (SEQ ID NO: 1), haddock (SEQ ID NO: 3), hake (SEQ ID NO: 5), halibut (SEQ ID NO: 7), mackeral (SEQ ID NO: 9), pollock (SEQ ID NO: 11), sea bass (SEQ ID NO: 13), swordfish (SEQ ID NO: 15), tilapia (SEQ ID NO: 17), tuna (SEQ ID NO: 23), catfish (SEQ ID NO: 25), atlantic salmon (SEQ ID NO: 31), chum salmon (SEQ ID NO: 33), coho salmon (SEQ ID NO: 35), king salmon (SEQ ID NO: 37), pink salmon (SEQ ID NO: 39), sockeye salmon (SEQ ID NO: 41), rainbow trout (SEQ ID NO: 43), arctic char (SEQ ID NO: 45), seabream (SEQ ID NO: 47), turbot (SEQ ID NO: 49) by using degenerate primers that permit selective amplification of a sequence (nucleotides 2279-2934 of shark kidney calcium receptor) that is highly conserved in both mammalian and shark kidney calcium receptors (see Example 1). The degenerate primers (SEQ ID NOS: 21, and 22) amplify a sequence of 653 base pairs that is present in the extracellular domain of calcium receptors. The resulting amplified 653 bp fragment was ligated into a cloning vector and transformed into bacterial cells for growth, purification and sequencing. Additionally, aquatic PVCR genes can be isolated by Reverse Transcriptase-Polymerase Chain Reaction (RT- PCR) after isolation of poly A+ RNA from aquatic species with the same or similar degenerate primers. Methods of PCR and RT-PCR are well characterized in the art (See generally, PCR Technology: Principles and Applications for DNA

Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al, Academic Press, San Diego, CA, 1990); Mattila, et al, Nucleic Acids Res., 19:4967 (1991); Eckert, et al, PCR Methods and Applications, 1:17 (1991); PCR (eds. McPherson, et al, IRL Press, Oxford); and U.S. Patent 4,683,202. Poly A+ RNA can be isolated from any tissue which contains PVCR by standard methods as described. Preferred tissue for polyA+RNA isolation can be determined using an antibody which is specific for the highly conserved sequence of calcium receptors, by standard methods. The partial genomic or cDNA sequences derived from a PVCR gene of fish are unique and, thus, can be used as a unique probe to isolate the full-length cDNA from each species. Moreover, this DNA fragment can form the basis for specific assay kits for detection of PVCR expression in various tissues of fish.

As shown in Figures 1-20 and 23-24, partial aquatic PVCR clones have been obtained from: cod (SEQ ID NO: 1), haddock (SEQ ID NO: 3), hake (SEQ ID NO: 5), halibut (SEQ ID NO: 7), mackeral (SEQ ID NO: 9), pollock (SEQ ID NO: 11), sea bass (SEQ ID NO: 13), swordfish (SEQ ID NO: 15), tilapia (SEQ ID NO: 17), tuna (SEQ ID NO: 23), catfish (SEQ ID NO: 25), winter flounder (SEQ ID NO: 19), atlantic salmon (SEQ ID NO: 31), chum salmon (SEQ ID NO: 33), coho salmon (SEQ ID NO: 35), king salmon (SEQ ID NO: 37), pink salmon (SEQ ID NO: 39), sockeye salmon (SEQ ID NO: 41), rainbow trout (SEQ ID NO: 43), arctic char (SEQ ID NO: 45), seabream (SEQ ID NO: 47), turbot (SEQ ID NO: 49). These sequences were determined using methods described herein and known in the art.

The nucleic acid sequences, SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 are shown in Figures 1-20 and 23- 24, respectively. Clones, containing SEQ ID NO: 41 ; SEQ ID NO: 43; SEQ ID NO: 31; SEQ ID NO: 45; SEQ ID NO: 33; SEQ ID NOS: 5, 7, 11, 13; SEQ ID NOS: 1, 23, 19; SEQ ID NOS: 3, 9, 17, 25; SEQ ID NO: 35; SEQ ID NO: 37; and SEQ ID NO: 39, were deposited under the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA on October 5, 2000, under Accession Numbers PTA-2538; PTA-2539; PTA-2541; PTA-2542; PTA-2543; PTA-2544; PTA-2545; PTA-2546; PTA-2547; PTA-2548; and PTA-2549, respectively.

The present invention is intended to encompass aquatic PVCR proteins, and proteins and polypeptides having amino acid sequences analogous to the amino acid sequences of aquatic PVCR proteins. Such polypeptides are defined herein as aquatic PVCR analogs (e.g., homologues), or mutants or derivatives. "Analogous amino acid sequences" (also referred to herein as "analogous polypeptide" or "analog polypeptide") as used herein refers to amino acid sequences with sufficient identity of aquatic PVCR amino acid sequence to possess the biological activity of an aquatic PVCR. For example, an analog polypeptide can be produced with "silent" changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues ofthe aquatic PVCR protein, yet still possesses the biological activity of aquatic PVCR. Examples of such differences include additions, deletions or substitutions of residues ofthe amino acid sequence of aquatic PVCR. Also encompassed by the present invention are analogous polypeptides that exhibit greater, or lesser, biological activity ofthe aquatic PVCR proteins ofthe present invention.

The aquatic PVCR proteins and nucleic acid sequences ofthe present invention include homologues, as defined herein. The homologous proteins and nucleic acid sequences can be determined using methods known to those of skill in the art. Initial homology searches can be performed for example, at NCBI against the GenBank (release 87.0), EMBL (release 39.0) databases using the BLAST network service, (Altshul, et al, J. Mol. Biol. 215: 403 (1990)), the teachings of which are incorporated herein by reference). Computer analysis of nucleotide sequences can be performed using the MOTIFS and the FindPatterns subroutines of the Genetics Computing Group (GCG, version 8.0) software. Protein and/or nucleotide comparisons can also be performed according to Higgins and Sharp (Higgins and Sharp, Gene, 73:237-244 (1988)). Homologous proteins and/or nucleic acid sequences to the PVCR protein and/or nucleic acid sequences that encode the PVCR protein are defined as those nucleic acid or amino acid sequences with at least about 70% sequence identity and/or similarity (e.g., 75%, 80%, 85%, 90%, or 95% homology) and are well known to one of skill in the art. As defined herein, percent "identity" or percent "similarity" refers to the amount of identical nucleotides or amino acids between two nucleotide or amino acid sequences, respectfully. As used herein, "percent similarity" refers to the amount of similar or conservative amino acids between two amino acid sequences. hi one embodiment, the present invention is directed to an isolated nucleic acid molecule having at least 70% identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; encoding a PVCR polypeptide in an aquatic species, wherein the PVCR allows fish to sense ion concentrations; alter water intake, water absorption or urine output; or modulate the percentage of fat, protein or moisture of muscle.

The present invention also encompasses an isolated nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the complementary strand ofthe recited nucleic acid sequence. Additionally, the present invention is directed toward an isolated RNA molecule encoded by nucleic acid molecules recited above. The "complementary strand" of a nucleic acid molecule is well known in the art and refers to a nucleic acid molecule that forms Watson and Crick base-pairs with the reference nucleic acid. The coding sequence ofthe above nucleic acid molecules are described herein. As used herein, a "coding sequence" refers to the putative open reading frame of nucleic acid molecules ofthe present invention.

Aquatic species useful in the present invention include teleost and elasmobranch fish, such as flounder, arctic char, cod, trout, killifish, salmon, sharks, rays and skates.

Additionally, the "biological activity" of an aquatic PVCR protein is defined herein to mean the osmoregulatory activity of an aquatic PVCR protein. Assay techniques to evaluate the biological activity of aquatic PVCR proteins and their analogs are described in Brown, et al, New Eng. J. Med., 333:243 (1995); Riccardi, et al, Proc. Nat. Acad. Sci USA, 92:131-135 (1995); and Sands, et al, J. Clinical Investigation 99:1399-1405 (1997).

The "biological activity" of aquatic PVCR is also defined herein to mean the ability ofthe aquatic PVCR to modulate signal transduction pathways in specific marine species cells. Thus, depending on the distribution and nature of various signal transduction pathway proteins that are expressed in cells, biologically active aquatic PVCR protein can modulate cellular functions in either an inhibitory or stimulatory manner.

The term "biologically active" also refers to the ability ofthe PVCR to sense ion concentrations in the surrounding environment. The PVCR senses various polyvalent cations including calcium, magnesium and/or sodium. The PVCR is modulated by varying ion concentrations. For instance, the PVCR may be modulated (e.g., increased expression, decreased expression and/or activation) in response to a change (e.g., increase or decrease) in ion concentration (e.g., calcium, magnesium, or sodium). Responses to changes in ion concentrations of a fish containing a PVCR include the ability for a fish to adapt to the changing ion concentration. Such responses include the amount the fish drinks, the amount of urine output, and the amount of water absorption. Responses also include changes in biological processes that affect the body composition ofthe fish and its ability to excrete contaminants. As described below, PVCR protein undergoes redistribution within epithelial cells ofthe urinary bladder in flounder adapted to brackish water as compared to full strength sea water. This directly correlates with alterations in the rate of NaCl transport by these cells. Winter flounder were adapted to live in 1/10th seawater (100 mOsm/kg) by reduction in salinity from 450 mM NaCl to 45 mM NaCl over an interval of 8 hrs. (Winter and summer flounder can be maintained in 1/10 or twice the salinity for over a period of 6 months.) After a 10 day interval where these fish were fed a normal diet, the distribution ofthe PVCR in their urinary bladder epithelial cells was examined using immunocytochemistry. PVCR immunostaining is reduced and localized primarily to the apical membrane of epithelial cells in the urinary bladder. In contrast, the distribution of PVCR in epithelial cells lining the urinary bladders of control flounders continuously exposed to full strength seawater is more abundant and present in both the apical membranes as well as in punctate sequences throughout the cell. These data are consistent with previous Northern data since more PVCR protein is present in the urinary bladders of seawater fish vs fish adapted to brackish water. These data show that PVCR protein was present in vesicles in epithelial cells ofthe urinary bladder and that in response to alterations in salinity, these vesicles move from the cell cytoplasm to the apical surface of these epithelial cells. Since these same epithelial cells possess abundant NaCl cotransporter protein that is responsible for water reabsorption in the urinary bladder, these data show that the PVCR protem modulates NaCl transport in the flounder urinary bladder by altering the proportion of NaCl cotransporter protein that is present in the apical membrane. As urinary Mg²⁺ and Ca²⁺ concentrations increase when fish are present in full strength sea water, activation of apical PVCR protein causes endocytosis and removal of NaCl cotransporter from the apical membrane and, thus, reduction in urinary bladder water transport. The invention provides methods to facilitate euryhaline adaptation offish to occur, and improve the adaption. More specifically, methods are now available to regulate salinity tolerance in fish by modulating (e.g., alternating, activating and or expressing) the activity ofthe Aquatic PVCR protein present in epithelial cells involved in ion transport, as well as in endocrine and nervous tissue. For example, salinity tolerance of fish adapted (or acclimated) to fresh water can be increased by activating the Aquatic PVCR, for example, by increasing the expression of Aquatic PVCR in selected epithelial cells, resulting in the secretion of ions and seawater adaption. Specifically, this would involve regulatory events controlling the conversion of epithelial cells ofthe gill, intestine and kidney. In the kidney, PVCR activation facilitates excretion of divalent metal ions including Ca2+ and Mg2+ by renal tubules. In the gill, PVCR activation reduces reabsorption of ions by gill cells that occurs in fresh water and promote the net excretion of ions by gill epithelia that occurs in salt water. In the intestine,

PVCR activation will permit reabsorption of water and ions across the G.I. tract after their ingestion by fish.

Alternatively, the salinity tolerance offish adapted to seawater can be decreased by inhibiting the Aquatic PVCR, for example, by decreasing the expression of Aquatic PVCR in selected epithelial cells, resulting in alterations in the absorption of ions and freshwater adaption. Selected epithelial cells include, e.g., kidney, bladder, intestinal and gill cells.

The presence of Aquatic PVCR in brain reflects both its involvement in basic neurotransmitter release via synaptic vesicles (Brown, E.M. et al., New England J. of Med., 333:234-240 (1995)), as well as its activity to trigger various hormonal and behavioral changes that are necessary for adaptation to either fresh water or marine environments. For example, increases in water ingestion by fish upon exposure to salt water is mediated by PVCR activation in a manner similar to that described for humans where PVCR activation by hypercalcemia in the subfornical organ ofthe brain cause an increase in water drinking behavior (Brown, E.M. et al. , New England J. of Med., 333:234-240 (1995)). In fish, processes involving both alterations in serum hormonal levels and behavioral changes are mediated by the brain. These include the reproductive and spawning of euryhaline fish in fresh water after their migration from salt water as well as detection of salinity of their environment for purposes of feeding, nesting, migration and spawning. Data obtained recently from mammals now suggest that PVCR activation plays a pivotal role in coordinating these events. For example, alterations in plasma cortisol have been demonstrated to be critical for changes in ion transport necessary for adaptation of salmon smolts from fresh water to salt water (Veillette, P. A., et al, Gen. and Comp. Physiol, 97:250-258 (1995)). As demonstrated recently in humans, plasma Adrenocorticotrophic Hormone (ACTH) levels that regulate plasma cortisol levels are altered by PVCR activation.

The aquatic PVCR proteins in the present invention also encompasses biologically active polypeptide fragments ofthe aquatic PVCR proteins. Such fragments can include only a part of the full-length amino acid sequence of an aquatic PVCR yet possess osmoregulatory activity. For example, polypeptide fragments comprising deletion mutants ofthe aquatic PVCR proteins can be designed and expressed by well-known laboratory methods. Such polypeptide fragments can be evaluated for biological activity, as described herein. Aquatic PVCR protein are found in many tissues in fish including kidney, urinary bladder, intestine, gill, and brain. The aquatic PVCR proteins, described herein, can be isolated and characterized as to its physical characteristics (e.g., molecular weight, isoelectric point) using laboratory techniques common to protein purification, for example, salting out, immunoprecipation, column chromatography, high pressure liquid chromatography or electrophoresis. Aquatic PVCR proteins referred to herein as "isolated" are aquatic PVCR proteins separated away from other proteins and cellular material of their source of origin. Isolated aquatic PVCR proteins include essentially pure protein, proteins produced by chemical synthesis, by combinations of biological and chemical synthesis and by recombinant methods. The aquatic PVCR amino acid sequence deduced from the partial nucleotide sequence of cod (SEQ ID NO: 2), haddock (SEQ BD NO: 4), hake (SEQ ID NO: 6), halibut (SEQ ID NO: 8), mackeral (SEQ ID NO: 10), pollock (SEQ ID NO: 12), sea bass (SEQ ID NO: 14), swordfish (SEQ ID NO: 16), and winter flounder (SEQ ID NO: 20), tuna (SEQ ID NO: 24), catfish (SEQ ID NO: 26), atlantic salmon (SEQ ID NO: 32), chum sahnon (SEQ ID NO: 34), coho salmon (SEQ ID NO: 36), king salmon (SEQ ID NO: 38), pink salmon (SEQ ID NO: 40), sockeye salmon (SEQ ID NO: 42), rainbow trout (SEQ ID NO: 44), arctic char (SEQ ID NO: 46), seabream (SEQ ID NO: 48) and turbot (SEQ ID NO: 50) are disclosed (Figures 1-20 and 23- 24, respectively). h one embodiment, the present invention is directed to an isolated polypeptide molecule having at least 70% similarity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50; encoding a PVCR polypeptide in an aquatic species, wherein the PVCR allows fish to sense ion concentrations; alter water intake, water absorption or urine output; or modulate the percentage of fat, protein or moisture of muscle. The present invention is also directed toward an isolated polypeptide molecule that comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50; or encoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, and wherein the polypeptide allows fish to sense ion concentrations; alter water intake, water absorption or urine output; or modulate the percentage of fat, protein or moisture of muscle.

Antibodies can be raised to the aquatic PVCR proteins and analogs, using techniques known to those of skill in the art. Antibodies can be polyclonal, monoclonal, chimeric, or fragments thereof, and can be used to immunoaffinity purify or identify aquatic PVCR proteins contained in a mixture of proteins, using techniques well known to those of skill in the art. Additionally, antibodies, or antibody fragments, can also be used to detect the presence of aquatic PVCR proteins and homologs in other tissues using standard immunohistological methods. For example, immunohistochemical studies were performed using the 1169 antibody which was raised against a portion ofthe shark kidney calcium receptor demonstrating localized expression in the olfactory organ. Antibodies are absorbed to determine the PVCR protein levels. Antibodies could be used in a kit to monitor the PVCR protein level of fish in aquaculture. hi one embodiment, the present invention is directed toward a method for detecting the expression of an aquatic PVCR polypeptide comprising combining the sample to be tested with an antibody having specificity for PVCR, under suitable conditions for formation of a complex between said antibody and PVCR, and detecting or measuring the formation ofthe complex. As used herein, a "complex" refers to an association or binding between the antibody and the PVCR. Methods of detecting and/or measuring the presence or level of PVCR are described herein and well known to one of skill in the art. The present invention can optionally include a labeled primary antibody or secondary antibody. Labeled primary and secondary antibodies can be obtained commercially or prepared using methods know to one of skill in the art (see Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY).

The present invention also encompasses isolated nucleic acid sequences or the coding sequence of nucleic acid molecules encoding aquatic PVCR proteins described herein, and fragments of nucleic acid sequences encoding biologically active PVCR proteins, h one embodiment, the present invention is directed toward an isolated nucleic acid molecule encoding an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50. Fragments ofthe nucleic acid sequences, described herein, are useful as probes to detect the presence of marine species PVCRs. Specifically provided for in the present invention are DNA/RNA sequences encoding Aquatic PVCR proteins, the fully complementary strands of these sequences, and allelic variations thereof. Also encompassed by the present invention are nucleic acid sequences, genomic DNA, cDNA, RNA or a combination thereof, which are substantially complementary to the DNA sequences encoding Aquatic PVCR, and which specifically hybridize with the Aquatic PVCR DNA sequences under conditions of sufficient stringency to identify DNA sequences with substantial nucleic acid identity. In one embodiment, the present invention is directed toward an isolated nucleic acid molecule which hybridizes under high stringency conditions to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the coding sequence ofthe recited nucleic acid molecule; or the complementary strand ofthe recited nucleic acid; or a nucleic acid molecule that encodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50, and wherein the probe hybridizes to a nucleic acid molecule that encodes a polypeptide that allows fish to sense ion concentrations; alter water intake, water absoφtion or urine output; or modulate the percentage of fat, protein or moisture of muscle.

"Substantially complementary" as used herein refers to a sequence which is similar in identity of sequence to hybridize with aquatic PVCR DNA under high stringent conditions. For example, non-complementary bases can be interspersed in the sequence, or the sequences can be longer or shorter than aquatic PVCR DNA, provided that the sequence has a sufficient number of bases complementary to aquatic PVCR to hybridize therewith. Exemplary hybridization conditions are described herein (see also Brown, et al, Nature, 366:575 (1993).

The aquatic PVCR DNA sequence, or a fragment thereof, can be used as a probe to isolate additional aquatic PVCR homologs. For example, a cDNA or genomic DNA library from the appropriate organism can be screened with labeled aquatic PVCR DNA to identify homologous genes as described in e.g., Ausebel, et al. , Eds., Current Protocols In Molecular Biology, John Wiley & Sons, New York (1997). hi one embodiment, the present invention is directed toward an isolated nucleic acid probe wliich hybridizes under high stringency conditions to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the coding sequence ofthe recited nucleic acid molecule; or the complementary strand or the recited nucleic acid; or a nucleic acid molecule that encodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50, and wherein the probe hybridizes to a nucleic acid molecule that encodes a polypeptide that allows fish to sense ion concentrations; alter water intake, water absoφtion or urine output; or modulate the percentage of fat, protein or moisture of muscle.

In another embodiment, the present invention is directed toward a method of isolating an aquatic PVCR nucleic acid comprising contacting an isolated nucleic acid with an aquatic PVCR-specific hybridization probe and identifying an aquatic PVCR. Methods for identifying a nucleic acid by hybridization are routine in the art (see Current Protocols In Molecular Biology, Ausubel, F.M. et al, Eds., John Wiley & Sons: New York, NY, (1997). The present method can optionally include a labeled PVCR probe.

Additionally, the above probes could be used in a kit to identify aquatic PVCR homologs and their expression in various fish tissue. The present invention also encompasses the isolation of aquatic PVCR homologs and their expression in various fish tissues with a kit containing primers specific for conserved sequences of PVCR nucleic acids and proteins.

Typically the nucleic acid probe comprises a nucleic acid sequence (e.g. SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49) and is of sufficient length and complementarity to specifically hybridize to nucleic acid sequences which encode aquatic species PVCR. For example, a nucleic acid probe can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% the length ofthe PVCR. The requirements of sufficient length and complementarity can be easily determined by one of skill in the art. Suitable hybridization conditions are described herein and well know to one of skill in the art. Uses of nucleic acids encoding cloned receptors or receptor fragments include one or more the following: (1) producing receptor proteins which can be used, for example, for structure determination, to assay a molecule's activity, and to obtain antibodies binding to the receptor; (2) being sequenced to determine a receptor's nucleotide sequence which can be used, for example, as a basis for comparison with other receptors to determine conserved sequences, determine unique nucleotide sequences for normal and altered receptors, and to determine nucleotide sequences to be used as target sites for antisense nucleic acids, ribozymes, or PCR amplification primers; (3) as hybridization detection probes to detect the presence of a native receptor and/or a related receptor in a sample; and (4) as PCR primers to generate particular nucleic acid sequence sequences, for example to generate sequences to be screened with hybridization detection probes.

The aquatic PVCR proteins and/or nucleic acid sequences include fragment thereof. Preferably, the nucleic acid contains at least 14, at least 20, at least 27, at least 45, and at least 69, contiguous nucleic acids of a sequence provided in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. Advantages of longer-length nucleic acids include increased nucleic acid probe specificity under high stringent hybridization assay conditions; and higher specificity for related aquatic PVCR nucleic acid under lower stringency hybridization assay conditions and encoding longer-length protein fragments of an aquatic PVCR protein which can be used, for example, to produce antibodies.

Another aspect ofthe present invention features a purified nucleic acid encoding an aquatic PVCR protein or fragment thereof. The nucleic acid encodes at least 6 contiguous amino acids provided in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50. Due to the degeneracy ofthe genetic code, different combinations of nucleotides can code for the same polypeptide. Thus, numerous aquatic PVCR proteins and receptor fragments having the same amino acid sequences can be encoded for by difference nucleic acid sequences, hi preferred embodiments, the nucleic acid encodes at least 12, at least 18, at least 23, or at least 54 contiguous amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.

Another aspect ofthe present invention features a purified nucleic acid having a nucleic acid sequence sequence of at least 12 contiguous nucleotides substantially complementary to a sequence sequence in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49. Such nucleic acid sequences are particularly useful as hybridization probes for highly complementary nucleic acid sequences under stringent hybridization conditions. Preferably, such conditions prevent hybridization of nucleic acids having 4 mismatches out of 20 contiguous nucleotides, more preferably 2 mismatches out of 20 contiguous nucleotides, most preferably one mismatch out of 20 contiguous nucleotides. In preferred embodiments, the nucleic acid is substantially complementary to at least 20, at least 27, at least 45, or at least 69 contiguous nucleotides provided in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

Another aspect ofthe present invention features a purified polypeptide having at least 6 contiguous amino acids of an amino acid sequence provided in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50. By "purified" in reference to a polypeptide is meant that the polypeptide is in a form (i.e., its association with other molecules) distinct from naturally occurring polypeptide. Preferably, the polypeptide is provided as substantially purified preparation representing at least 75%, more preferably 85%, most preferably 95% or the total protein in the preparation. In preferred embodiments, the purified polypeptide has at least 12, 18, 23, or 54 contiguous amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.

Preferred receptor fragments include those having functional receptor activity, a binding site, or an epitope for antibody recognition (typically at least six amino acids). Such receptor fragments have various uses such as being used to obtain antibodies to a particular sequence, for example the 1169 antibody described in the related application 09/162,021 and being used to form chimeric receptors with fragments of other receptors to create a new receptor having unique properties or test receptor function.

The present invention is also encompasses plasmids, vectors or viruses that contain the nucleic acids described herein. In one embodiment, the present invention is directed toward a plasmid or vector containing a nucleic acid molecule comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; or the coding sequence ofthe recited nucleic acid molecule; or the complementary strand ofthe recited nucleic acid molecule. In another embodiment, the present invention is directed toward a plasmid or vector containing a nucleic acid molecule encoding the amino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38_? 40, 42, 44, 46, 48, or 50. Methods of preparing plasmids, vectors or viruses are well known to one of skill in the art (see Ausubel, F.M. et al, Eds., Current Protocols In Molecular Biology, John Wiley & Sons: New York, NY, (1997).

Uses of plasmids, vectors or viruses containing the cloned PVCR receptors or receptor fragments include one or more o the following; (1) generation of hybridization probes for detection and measuring level of PVCR in tissue or isolation of PVCR homologs; (2) generation of PVCR mRNA or protein in vitro or in vivo; and (3) generation of transgenic non-human animals or recombinant host cells. In one embodiment, the present invention is directed toward a host cell transformed with the plasmids, vectors or viruses described above. Host cells suitable in the present invention include a fish cell, a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell. The invention also features derivatives of full-length aquatic PVCR proteins and fragments thereof having the same, or substantially the same, activity as the full-length receptor or fragment. Such derivatives include amino acid addition(s), substitution(s), and deletion(s) to the receptor which do not prevent the derivative receptor from carrying out one or more ofthe activities ofthe parent receptor. Another aspect ofthe present invention features a recombinant cell or tissue containing a nucleic acid sequence encoding at least 6 contiguous amino acids provided in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 and a cell able to express the nucleic acid. Recombinant cells have various uses including acting as biological factories to produce polypeptides encoded for by the recombinant nucleic acid, and for producing cells containing a functioning aquatic PVCR protein. Cells containing a functioning aquatic PVCR can be used, for example, to screen to antagonists or agonists. Other uses will be well known to those skilled in the art.

As described herein, aquatic PVCR protein plays a role in the adaption of euryhaline fish to environmental salinity and their body composition. More specifically, methods are now available to regulate salinity tolerance in fish by modulating (e.g., increasing, decreasing or maintaining the expression) the activity ofthe aquatic PVCR protein present in cells involved in ion transport. For example, salinity tolerance of fish adapted (or acclimated) to freshwater can be increased by activating the aquatic PVCR, for example, by increasing the expression of aquatic PVCR in fish, resulting in the secretion of ions and seawater adaption. Alternatively, the salinity tolerance offish adapted to seawater can be decreased by inhibiting the aquatic PVCR, for example, by decreasing the expression of aquatic PVCR in fish, resulting in alterations in the absoφtion of ions and freshwater adaption. "Salinity" refers to the concentration of various ions in a surrounding aquatic environment. In particular, salinity refers to the ionic concentration of calcium, magnesium and/or sodium (e.g., sodium chloride). "Normal salinity" levels refers to the range of ionic concentrations of typical water environment in which an aquatic species naturally lives. For flounder and salmon, normal salinity or normal seawater concentrations are about lOmM Ca, about 40mM Mg, and about 450 mM NaCl. "Salinity tolerance" refers to the ability of a fish to live or survive in a salinity environment that is different than the salinity of its natural environment. Modulations ofthe PVCR allows fish to live in about four times and one-fiftieth, preferably, twice and one-tenth the normal salinity.

Alteration ofthe adaptability offish to environmental salinity has several advantages in aquaculture including alteration of body composition and enhancement of overall health of cultured fish. Body composition refers to various characteristics ofthe fish, including, but not limited to, weight, muscle, fat, protein, moisture, taste, or thickness. Alteration ofthe body composition means inducing a change in one of these characteristics. For example, culturing flounder in a hyposalinity enviromnent results in a fish that is twice as thick, 70% fatter and has a milder (less fishy) flavor than those maintained in a hypersalinity environment. Other benefits of altering the salinity ofthe environment for aquaculture include less parasites and less contaminants resulting in an increase in the overall health ofthe cultured fish.

Winter and summer flounder were maintained in at least twice the normal salinity or 1/10 the normal salinity. See Example 4. These fish can be maintained in these environments for long periods of time (e.g., over 3 months, over 6 months, or over 1 year). These limits were defined by decreasing or increasing the ionic concentrations of calcium, magnesium, and sodium, keeping a constant ratio between the ions. These salinity limits can be further defined by increasing and/or decreasing an individual ion concentration, thereby changing the ionic concentration ratio among the ions. Increasing and/or decreasing individual ion concentrations can increase and/or decrease salinity tolerance. "Hypersalinity" or "above normal salinity" levels refers to a level of at least one ion concentration that is above the level found in normal salinity. "Hyposalinity" or "below normal salinity" levels refers to a level of at least one ion concentration that is below the level found in normal salinity.

Maintaining winter and summer founder in this environment for about 3 months induced noticeable and significant changes occurred to the body composition ofthe flounder. These fish were slowly adapted to the hypersalinity or hyposalinity environments over a period of 15 days. Body composition refers to various characteristics ofthe fish, including, but not limited to, weight, muscle, fat, protein, moisture, taste, or thickness. Alteration ofthe body composition means inducing a change in one of these characteristics. Maintaining fish in 1/10 the normal salinity results in a fish that is twice as thick, 70% fatter, and "less fishy," (e.g., milder flavor) tasting fish than those fish maintained in hypersalinity environments. See Example 10. A fish maintained in low salinity or hyposalinity can increase its fat content by at least 10% or 20%, and preferably by at least 30%, 40%, or 50% than those fish maintained in normal salinity. Similarly, a fish maintained in low salinity or hyposalinity can increase its thickness by at least 30% or 40%, and preferably by at least 50%, 60%, or 70% than those fish maintained in normal salinity. A fish maintained in high salinity or hypersalinity can decrease its fat content by at least 10% or 20%, and preferably by at least 30%, 40%, or 50% than those fish maintained in normal salinity. Similarly, a fish maintained in high salinity or hypersalinity can decrease its thickness by at least 30% or 40%, and preferably by at least 50%), 60%, or 70% than those fish maintained in normal salinity.

Maintaining fish in a hypersalinity environment also results in fish with a reduced number of parasites or bacteria. Preferably, the parasites and/or bacteria are reduced to a level that is safe for human consumption, raw or cooked. More preferably, the parasites and/or bacteria are reduced to having essentially no parasites and few bacteria. These fish must be maintained in a hypersalinity environment long enough to rid the fish of these parasites or bacteria, (e.g., for at least a few days or at least a few weeks). The host range of many parasites is limited by exposure to water salinity.

For example, Diphyllobothrium species commonly known as fish tapeworms, is encountered in the flesh offish, primarily fresh water or euryhaline species including flounder of sahnon. Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. 1991. US Food and Drag Administration Center for Food Safety and Applied Nutrition, the teachings of which are incoφorated herein by reference in their entirety. In contrast, its presence in the flesh of completely marine species is much reduced or absent. Since summer flounder can survive and thrive at salinity extremes as high as 58 ppt (1.8 times normal seawater) for extended periods in recycling water, exposure of summer flounder to hypersalinity conditions might be used as a "biological" remediation process to ensure that no Diphyllobothrium species are present in the Gl tract of summer flounder prior to their sale as product. Recent data from Cole et al, (J. Biol. Chem. 272:12008-12013 (1997)), the teachings of which are incoφorated herein by reference in their entirety, show that winter flounder elaborate an antimicrobial peptide irom their skin to prevent bacterial infections. Their data reveals that in the absence of pleurocidin, E. coli are killed by high concentrations of NaCl. hi contrast, low concentrations of NaCl (<300mM NaCl) allow E. coli to grow and under these conditions pleurocidin presumably helps to kill them. These data provide evidence of NaCl killing of E. coli, as well as highlight possible utility of bacterial elimination in fish.

Similarly, maintaining fish in a hyposalinity environment results in a fish with a reduced amount of contaminants (e.g., hydrocarbons, amines or antibiotics). Preferably, the contaminants are reduced to a level that is safe for human consumption, raw or cooked, and produces a milder, "less fishy" tasting fish. More preferable, the contaminants are reduced to having essentially very little contaminants left in the fish. These fish must be maintained in a hyposalinity environment long enough to rid the fish of these contaminants, (e.g., for at least a few days or a few weeks).

Organic amines, such as trimethylamine oxide (TMAO) produce a "fishy" taste in seafood. They are excreted via the kidney in flounder. (Krogh, A., Osmotic Regulation in Aquatic Animals, Cambridge University Press, Cambridge, U.K. pgs 1-233 (1939), the teachings of which are incoφorated herein by reference in their entirety). TMAO is synthesized by marine organisms consumed by fish that accumulate the TMAO in their tissues. Depending on the species offish, the muscle content of TMAO and organic amines is either large accounting for the "strong" taste of bluefish and herring or small such as in milder tasting flounder.

Methods of Assessment ofthe PVCR The present invention includes methods of detecting the level ofthe PVCR to determine whether fish are ready for transfer from freshwater to seawater. Methods that measure PVCR levels include several suitable assays. Suitable assays encompass immunological methods, such as FACS analysis, radioimmunoassay, flow cytometry, immunocytochemistry, enzyme-linked immunosorbent assays (ELISA) and chemiluminescence assays. Any method known now or developed later can be used for measuring PVCR expression.

Antibodies reactive with the PVCR or portions thereof can be used. In a preferred embodiment, the antibodies specifically bind with the PVCR or a portion thereof. The antibodies can be polyclonal or monoclonal, and the term antibody is intended to encompass polyclonal and monoclonal antibodies, and functional fragments thereof. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.

In several ofthe preferred embodiments, immunological techniques detect PVCR levels by means of an anti-PVCR antibody (i.e., one or more antibodies). The term "anti-PVCR" antibody includes monoclonal and/or polyclonal antibodies, and mixtures thereof.

Anti-PVCR antibodies can be raised against appropriate immunogens, such as isolated and/or recombinant PVCR or portion thereof (including synthetic molecules, such as synthetic peptides). hi one embodiment, antibodies are raised against an isolated and/or recombinant PVCR or portion thereof (e.g., a peptide) or against a host cell which expresses recombinant PVCR. hi addition, cells expressing recombinant PVCR, such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor. Any suitable technique can prepare the immunizing antigen and produce polyclonal or monoclonal antibodies. The art contains a variety of these methods (see e.g., Kohler et al, Nature, 256: 495-497 (1975) andEwr. J. Immunol. 6: 511-519 (1976); Milstein et al, Nature, 266: 550-552 (1977); Koprowski et al, U.S. Patent No. 4,172,124; Harlow, Ε. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.M. et al, Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, fusing a suitable immortal or myeloma cell line, such as SP2/0, with antibody producing cells can produce a hybridoma. Animals immunized with the antigen of interest provide the antibody producing cell, preferably cells from the spleen or lymph nodes. Selective culture conditions isolate antibody producing hybridoma cells while limiting dilution techniques produce them. Researchers can use suitable assays such as ELISA to select antibody producing cells with the desired specificity.

Other suitable methods can produce or isolate antibodies ofthe requisite specificity. Examples of other methods include selecting recombinant antibody from a library or relying upon immunization of transgenic animals such as mice.

According to the method, an assay can determine the level of PVCR in a biological sample. In determining the amounts of PVCR, an assay includes combining the sample to be tested with an antibody having specificity for the PVCR, under conditions suitable for formation of a complex between antibody and the PVCR, and detecting or measuring (directly or indirectly) the formation of a complex. The sample can be obtained directly or indirectly, and can be prepared by a method suitable for the particular sample and assay format selected.

In particular, tissue samples, e.g., gill tissue samples, can be taken from fish after they are anaesthetized with MS-222. The tissue samples are fixed by immersion in 2% paraformaldehyde in appropriate Ringers solution corresponding to the osmolality ofthe fish, washed in Ringers, then frozen in an embedding compound, e.g., O.C.T.™ (Miles, Inc., Elkahart, Indiana, USA) using methylbutane cooled with liquid nitrogen. After cutting 8-10μ tissue sections with a cryostat, individual sections are subjected to various staining protocols. For example, sections are: 1) blocked with goat serum or serum obtained from the same species of fish, 2) incubated with rabbit anti-CaR or anti-PVCR antiserum, and 3) washed and incubated with peroxidase-conjugated affinity-purified goat antirabbit antiserum. The locations ofthe bound peroxidase-conjugated goat antirabbit antiserum are then visualized by development of a rose-colored aminoethylcarbazole reaction product. Individual sections are mounted, viewed and photographed by standard light microscopy techniques. The anti-CaR antiserum used to detect fish PVCR protein is raised in rabbits using a 23-mer peptide corresponding to amino acids numbers 214-236 localized in the extracellular domain ofthe RaKCaR protein. The sequence ofthe 23-mer peptide is: ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO: 51) A small peptide with the sequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO: 52) or ARSRNSADGRSGDDLPC (SEQ ID NO: 53) can also be used to make antisera containing antibodies to PVCRs. Such antibodies can be monoclonal, polyclonal or chimeric.

Suitable labels can be detected directly, such as radioactive, fluorescent or chemiluminescent labels. They can also be indirectly detected using labels such as enzyme labels and other antigenic or specific binding partners like biotin. Examples of such labels include fluorescent labels such as fluorescein, rhodamine, chemiluminescent labels such as luciferase, radioisotope labels such as ³²P, ¹²⁵1, ¹³¹I, enzyme labels such as horseradish peroxidase, and alkaline phosphatase, β-galactosidase, biotin, avidin, spin labels and the like. The detection of antibodies in a complex can also be done immunologically with a second antibody which is then detected (e.g. , by means of a label). Conventional methods or other suitable methods can directly or indirectly label an antibody. hi performing the method, the levels of PVCR in various tissues change in comparison to control. Modulated levels or the presence of PVCR expression in various tissues, as compared to a control, indicate that the fish or the population of fish from which a statistically significant amount offish were tested, are ready for transfer to freshwater. A control refers to a level of PVCR, if any, from a fish that is not subjected to the steps ofthe present invention, e.g., not subjected to freshwater having a PVCR modulator and/or not fed a NaCl diet. For example, Figures 13 and 18 of related U.S. Patent Application No.: 09/162,021 show that fish not subjected to the present invention had no detectable PVCR level, whereas fish that were subjected to the steps ofthe invention had PVCR levels that were easily detected. The PVCRs can also be assayed by Northern blot analysis of mRNA from tissue samples. Northern blot analysis from various shark tissues has revealed that the highest degree of PVCRs expression is in gill tissue, followed by the kidney and the rectal gland. There appear to be at least three distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb. The PVCRs can also be assayed by hybridization, e.g. , by hybridizing one of the PVCR sequences provided herein (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49) or an oligonucleotide derived from one ofthe sequences, to a DNA-containing tissue sample from a fish. Such a hybridization sequence can have a detectable label, e.g., radioactive, fluorescent, etc., attached to allow the detection of hybridization product. Methods for hybridization are well known, and such methods are provided in U.S. Pat. No. 5,837,490, by Jacobs et al, the entire teachings of which are herein incoφorated by reference in their entirety. The design ofthe oligonucleotide probe should preferably follow these parameters: (a) it should be. designed to an area ofthe sequence which has the fewest ambiguous bases ("N's"), if any, and (b) it should be designed to have a T_m of approx. 80°C (assuming 2°C for each A or T and 4 degrees for each G or C).

Stringency conditions for hybridization refers to conditions of temperature and buffer composition which permit hybridization of a first nucleic acid sequence to a second nucleic acid sequence, wherein the conditions determine the degree of identity between those sequences which hybridize to each other. Therefore, "high stringency conditions" are those conditions wherein only nucleic acid sequences which are very similar to each other will hybridize. The sequences can be less similar to each other if they hybridize under moderate stringency conditions. Still less similarity is needed for two sequences to hybridize under low stringency conditions. By varying the hybridization conditions from a stringency level at which no hybridization occurs, to a level at wliich hybridization is first observed, conditions can be determined at which a given sequence will hybridize to those sequences that are most similar to it. The precise conditions determining the stringency of a particular hybridization include not only the ionic strength, temperature, and the concentration of destabilizing agents such as formamide, but also on factors such as the length ofthe nucleic acid sequences, their base composition, the percent of mismatched base pairs between the two sequences, and the frequency of occurrence of subsets ofthe sequences (e.g., small stretches of repeats) within other non-identical sequences. Washing is the step in which conditions are set so as to determine a minimum level of similarity between the sequences hybridizing with each other. Generally, from the lowest temperature at which only homologous hybridization occurs, a 1% mismatch between two sequences results in a 1°C decrease in the melting temperature (T_m) for any chosen SSC concentration. Generally, a doubling ofthe concentration of SSC results in an increase in the T_m of about 17°C. Using these guidelines, the washing temperature can be determined empirically, depending on the level of mismatch sought. Hybridization and wash conditions are explained in Current Protocols in Molecular Biology (Ausubel, F.M. et al, eds., John Wiley & Sons, Inc., 1995, with supplemental updates) on pages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6. High stringency conditions can employ hybridization at either (1) lx SSC

(lOx SSC = 3 M NaCl, 0.3 M Na₃-citrate-2H₂O (88 g/liter), pH to 7.0 with 1 M HCI), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA at 65°C, (2) lx SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42°C, (3) 1% bovine serum albumin (fraction V), 1 mM Na^EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄ = 134 g Na₂HPO₄-7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 65°C, (4) 50% formamide, 5x SSC, 0.02 M Tris-HCl (pH 7.6), lx Denhardt's solution (lOOx = 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42°C, (5) 5x SSC, 5x Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 65°C, or (6) 5x SSC, 5x Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 42°C, with high stringency washes of either (1) 0.3 - O.lx SSC, 0.1% SDS at 65°C, or (2) 1 mM Na^DTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS at 65°C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5 - 10°C below that ofthe calculated T_m ofthe hybrid, where T_m in °C = (2 x the number of A and T bases) + (4 x the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the T_m in °C = (81.5°C + 16.6(log₁₀M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where "M" is the molarity of monovalent cations (e.g., Na ), and "L" is the length ofthe hybrid in base pairs.

Moderate stringency conditions can employ hybridization at either (1) 4x SSC, (lOx SSC = 3 M NaCl, 0.3 M Na₃-citrate-2H₂O (88 g/liter), pH to 7.0 with 1 M HCI), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA at 65°C, (2) 4x SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42°C, (3) 1% bovine serum albumin (fraction V), 1 mM Na^EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄ = 134 g Na₂HPO₄-7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 65°C, (4) 50% formamide, 5x SSC, 0.02 M Tris-HCl (pH 7.6), lx Denhardt's solution (lOOx = 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42°C, (5) 5x SSC, 5x Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 65°C, or (6) 5x SSC, 5x Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 42°C, with moderate stringency washes of lx SSC, 0.1 % SDS at 65°C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5 - 10°C below that ofthe calculated T_m ofthe hybrid, where T_m in °C = (2 x the number of A and T bases) + (4 x the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the T_m in °C = (81.5°C + 16.6(log₁₀M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where "M" is the molarity of monovalent cations (e.g., Na ), and "L" is the length ofthe hybrid in base pairs.

Low stringency conditions can employ hybridization at either (1) 4x SSC, (lOx SSC = 3 M NaCl, 0.3 M Na₃-citrate-2H₂O (88 g/liter), pH to 7.0 with 1 M HCI), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA at 50°C, (2) 6x SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 40°C, (3) 1% bovine serum albumin (fraction V), 1 mM Na^EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄ = 134 g Na^PO^H_jO, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 50°C, (4) 50% formamide, 5x SSC, 0.02 M Tris-HCl (pH 7.6), lx Denhardt's solution (lOOx - 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 40°C, (5) 5x SSC, 5x Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 50°C, or (6) 5x SSC, 5x Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 40°C, with low stringency washes of either 2x SSC, 0.1% SDS at 50°C, or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na_jEDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5 - 10°C below that ofthe calculated T_m ofthe hybrid, where T_m in °C = (2 x the number of A and T bases) + (4 x the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the T_m in °C = (81.5°C + 16.6(log₁₀M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where "M" is the molarity of monovalent cations (e.g., Na ), and "L" is the length ofthe hybrid in base pairs.

The present invention encompasses detection of PVCRs with PCR methods using primers disclosed or derived from sequences described herein. For example, PVCRs can be detected by PCR using SEQ ID NOs: 21 and 22, as described in Example 1. PCR is the selective amplification of a target sequence by repeated rounds of nucleic acid replication utilizing sequence-specific primers and a thermostable polymerase. PCR allows recovery of entire sequences between two ends of known sequence. Methods of PCR are described herein and are known in the art.

In particular, the level of aquatic PVCR can be determined in various tissues by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) after isolation of poly A+ RNA from aquatic species. Methods of PCR and RT-PCR are well characterized in the art (See generally, PCR Technology: Principles and Applications for DNA Amplification (H.A. Erlich, Ed., Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (h nis, et al, Eds., Academic Press, San Diego, CA, 1990); Manila et al, Nucleic Acids Res., 19:4967 (1991); Eckert et al, PCR Methods and Applications, 1:17 (1991); PCR

(McPherson et al, Eds., IRL Press, Oxford); Ausebel, F. M. et al, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-friterscience 1987, & Supp. 49, 2000; and U.S. Patent 4,683,202). Briefly, mRNA is extracted from the tissue of interest and reverse transcribed. Subsequently, a PCR reaction is performed with PVCR-specific primers and the presence of the predicted PVCR product is determined, for example, by agarose gel electrophoresis. Examples of PVCR-specific primers are SEQ K> NO: 21 and/or SEQ ID NO: 22. The product of the RT-PCR reaction that is performed with PVCR-specific primers is referred to herein as a RT-PCR product. The RT-PCR product can include nucleic acid molecules having part or all ofthe PVCR sequence. The RT-PCR product can optionally be radioactively labeled and the presence or amount of PVCR product can be determined using autoradiography. Two examples of commercially available fluorescent probes that can be used in such an assay are Molecular Beacons (Stratagene) and Taqman® (Applied Biosystems). Alternative methods of labeling and quantifying the RT-PCR product are well known to one of skill in the art (see Ausebel, F. M. et al, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-hiterscience 1987, & Supp. 49, 2000). Poly A+ RNA can be isolated from any tissue which contains at least one PVCR by standard methods. Such tissues include, for example, gill, nasal lamellae, urinary bladder, kidney, intestine, stomach, liver and brain. Hence, the present invention includes kits for the detection ofthe PVCR or the quantification ofthe PVCR having either antibodies specific for the PVCR or a portion thereof, or a nucleic acid sequence that can hybridize to the nucleic acid of the PVCR. Alterations in the expression or sensitivity of PVCRs could also be accomplished by introduction of a suitable transgene. Suitable transgenes would include either the PVCR gene itself or modifier genes that would directly or indirectly influence PVCR gene expression. Methods for successful introduction, selection and expression ofthe transgene in fish oocytes, embryos and adults are described in Chen, TT, et al., Transgenic Fish, Trends in Biotechnology 8:209-215 (1990).

The present invention encompasses the identification and characterization of polyvalent cation-sensing receptor (PVCR) proteins in aquatic species. Aquatic PVCR proteins play a critical role in the adaptation of fish to marine and freshwater environments and influences the growth and development of fish. The present invention can be used in methods of aquaculture to regulate the adaptation to environmental salinity, manipulate the body composition, and increase the overall health of cultured fish.

The present invention will be illustrated in the following examples, which is not meant to be limiting in any way.

EXAMPLE 1: PVCRs ISOLATED IN VARIOUS AQUATIC SPECIES

Aquatic PVCR are provided in the present invention including cod, haddock, hake, halibut, mackeral, pollock, sea bass, swordfish, tilapia tuna, catfish, seabream, turbot and sahnonid fish such as atlantic salmon, chum salmon, coho salmon, king salmon, pink salmon, sockeye salmon, rainbow trout, and arctic char, by a protocol described in the Patent Application No. 09/162,021, whose teaching are incoφorated herein.

The above partial aquatic PVCR genes were isolated as follows: sequences of shark kidney calcium receptor (disclosed in Patent Application No. 09/162,021) together with the nucleotide sequence of mammalian calcium receptors were used to design degenerate oligonucleotide primers, dSK-F3 (SEQ ID NO: 21), dSK-R4 (SEQ ID NO: 22), DUFL-F1(SEQ ID NO: 27) and DUFL-R1(SEQ ID NO: 29) to highly conserved sequences in the extracellular domain of polyvalent cation receptor proteins using standard methodologies (See GM Preston, "Polymerase chain reaction with degenerate oligonucleotide primers to clone gene family members," Methods in Mol. Biol. Vol. 58, Edited by A. Harwood, Humana Press, pp. 303-312 (1993)). Genomic DNA isolated from muscle (or other PVCR containing-tissue) from the above species were amplified using pairs of degenerate primers (dSK-F3/dSK-R4 or DUFL-Fl/DUFL-Rl) using standard PCR methodology. The PCR product (653 nt) is then purified by agarose gel electrophoresis and ligated into appropriate plasmid vector that is transformed into a bacterial strain. After growth in liquid media, vectors and inserts are purified using standard techniques, analyzed by restriction enzyme analysis and sequenced. Using this methodology, a total of 21 nucleotide sequences from 21 fish species were amplified. Each clone is 594 nt (with-out primer sequences) and encodes 197 amino acid sequence which corresponds to the conserved extracellular domain ofthe calcium receptors (except tilapia). The open reading frame encoded by these sequences begins at nucleotide position 2 (except tilapia).

Cod PVCR (SEQ ID NO: 1) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 2) (Figure 1). Haddock PVCR (SEQ ID NO: 3) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 4) (Figure

2).

Hake PVCR (SEQ ID NO: 5) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 6) (Figure 3).

Halibut PVCR (SEQ ID NO: 7) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 8) (Figure

4).

Mackeral PVCR (SEQ ID NO: 9) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 10) (Figure 5). Pollock PVCR (SEQ ID NO: 11) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 12) (Figure 6).

Sea bass PVCR (SEQ ID NO: 13) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 14) (Figure 7).

Swordfish PVCR (SEQ ID NO: 15) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 16) (Figure 8). Tuna PVCR (SEQ ID NO: 23) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 24) (Figure 11).

Catfish PVCR (SEQ ID NO: 25) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 26) (Figure 12).

The tilapia PVCR was isolated using the DUFL-F1(SEQ ID NO: 27) and DUFL-R1(SEQ ID NO: 29) degenerate primers as described above. Tilapia PVCR (SEQ ID NO: 17) is composed of 1915 nucleotides (Figure 9).

Chum sahnon PVCR (SEQ ID NO: 33) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 34) (Figure 14).

Coho salmon PVCR (SEQ ID NO: 34) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 35) (Figure 15). King salmon PVCR (SEQ ID NO: 37) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 38) (Figure 16).

Pink salmon PVCR (SEQ ID NO: 39) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 40) (Figure 17). ' Sockeye salmon PVCR (SEQ ID NO: 41) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 42) (Figure 18).

Rainbow trout PVCR (SEQ ID NO: 43) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO:44) (Figure 19).

Arctic char PVCR (SEQ ID NO: 45) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 46) (Figure 20). Seabream PVCR (SEQ ID NO: 47) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 48) (Figure 23).

Turbot PVCR (SEQ ID NO: 49) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 50) (Figure 24).

Primer sequences for PCR of PVCR clones: dSK-F3 (SEQ ID NO:21) 5'-TGT CKT GGA CGG AGC CCT TYG GRA TCG C-3' dSK-R4 (SEQ ID NO:22) 5'-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G3' DUFL-F1(SEQ ID NO:27) 5'-YCA RRG AGT TYT GGG ARG ARA CHT TYA AYT G-3'

DUFL-R1 (SEQ ID NO:29) 5'-GCA DSC CRW AGC TGG MBG CCA GRA TGG C-3'

I=deoxyinosine, N=A+C+T+G, R=A+G, Y=C+T, M=A+C, K=T+G, S=C+G, W=A+T, H=A+T+C, B=T+C+G, D=A+T+G, V=A+C+G

EXAMPLE 2: MOLECULAR CLONING OF PARTIAL ATLANTIC SALMON PVCR

An atlantic salmon PVCR was isolated by PCR using degenerate primers dSK-F3 (SEQ ID NO: 21) and dSK-R4 (SEQ ID NO: 22), as described in Example 1.

An atlantic salmon ΛZAP cDNA library was manufactured using standard commercially available reagents with cDNA synthesized from poly A+ RNA isolated from atlantic salmon intestine tissue according to manufactures instructions

(Stratagene, La Jolla, CA) and screened using the atlantic salmon PCR product as a probe. A partial atlantic salmon PVCR cDNA (SEQ ID NO: 31) is composed of 2021 nucleotides (nt) (Figure 13 A) containing an open reading frame encoding 388 amino acids (SEQ ID NO: 32) (Figure 13B). The open reading frame encoded by SEQ ID NO: 31 begins at nucleotide position 87.

EXAMPLE 3: MOLECULAR CLONING OF PARTIAL WINTER FLOUNDER PVCR

A winter flounder λZAP cDNA library was manufactured using standard commercially available reagents with cDNA synthesized from poly A+ RNA isolated from winter, flounder urinary bladder tissue as described and published in Siner, et al, Am. J. Physiol. 270:C372-C381 (1996). The winter flounder urinary bladder cDNA library was plated and resulting phage plaques screened using a 32p-i_abeled shark kidney calcium receptor cDNA probe under intermediate stringency conditions (0.5X SSC, 0.1% SDS, 50°C). Individual positive plaques were identified by autoradiography, isolated and rescued using phagemid infections to transfer cDNA to KS Bluescript vector. The nucleotide (nt) sequence, Figure 10, (SEQ ID NO: 19) of the winter flounder PVCR clone was obtained using commercially available automated sequencing service that performs nucleotide sequencing using the dideoxy chain termination technique. The open reading frame encoded by SEQ ID NO: 19 begins at nucleotide position 1. The deduced amino acid sequence (SEQ ID NO: 20) is shown in Figure 10. The winter flounder PVCR nucleotide sequence was compared to others aquatic PVCR using commercially available nucleotide and protein database services including GENBANK and SWISSPROT.

EXAMPLE 4: ALTERING THE BODY COMPOSITION OF FISH AND DEFINING SALINITY LIMITS

Winter and Summer Flounder can be grown and maintained in recycling water systems. Groups of both winter (Pleuronectes americanus) and summer (Paralichthus dentalus) flounder were maintained in multiple modular recycling water system units that are composed of a single 1 meter fish tank maintained by a 1 meter biofilter tank located directly above it. The upper tank of each unit contains 168 sq. ft. of biofilter surface area that will support a maximum of 31 lbs of flounder, while maintaining optimal water purity and oxygenation conditions. Each unit is equipped with its own pump and temperature regulator apparatus. Both the temperature and photo-period of each unit can be independently regulated using black plastic curtains that partition each tank off from its neighbor. The inventors have a total of 12 independent modular units that permit 3 experiments each with 4 variables to be performed simultaneously. Using this experimental system, the following data have been obtained.

Salinity survival limits for winter and summer flounder with a constant ratio of divalent and monovalent ions were determined. The survival limit of both winter and summer flounder in waters of salinities greater than normal seawater (10 mM Ca2+, 50 mM Mg2+ and 450 mM NaCl) is water containing twice (20 mM Ca2+, 50 mM Mg2+ and 900 mM NaCl) the normal concentrations of ions present in normal seawater. In contrast, the survival limit of both winter and summer flounder in waters of salinity less than normal seawater is 10% seawater (1 mM Ca2+, 5 mM Mg2+ and 45 mM NaCl).

Flounder grown and/or maintained in low and hypersalinities possess different fat contents and taste as compared to flounder maintained in normal sea water. Use of a fully recycling water system permits growth of flounder at vastly different salinities. Groups of flounder (n=10) were adapted over a 15 day interval and maintained at either low salinity (LS) (e.g., at 10% normal seawater), normal seawater (NS) or hypersalinity (HS) (e.g., 2X seawater) for intervals of 3 months, under otherwise identical conditions. Survival among the 3 groups were comparable (all greater than 80%o) and there were no differences in the electrolyte content of their respective sera. Analyses of fillet muscle from summer flounder for total fat, protein and moisture content are shown on Table I. TABLE I: Comparison of Total Fat, Protein and Moisture Content of Muscle from Flounders Grown at Differing Water Salinities for 3 months. All values an average of 4 individual fish.

*Values significantly different from each other (p<0.05).

Muscle from low salinity flounder contains approximately 30% higher fat content as compared to flounder maintained in normal seawater and approximately 70% greater fat content when compared to flounder maintained in 2X seawater (e.g., the fat of a flounder maintained in normal salinity is 40% greater than flounder maintained in twice seawater). These differences appear selective because no significant differences were observed in either muscle protein or moisture content. Furthermore, fillets were sampled in a blinded protocol where tasters (n=6) were offered either raw or cooked fillets without knowledge of salinity conditions. Tasters could distinguish little difference between the taste of fillets of individual fish from each specific salinity group. However, when asked to compare fillets from flounder grown at differing salinities, a majority (5/6) clearly distinguished a taste difference between fillets from fish maintained at 10% salinity describing them as "sweet and buttery tasting with a soft consistency" as compared to fillets from fish maintained at either normal seawater or 2X seawater that were described as "wild and fishy tasting with a firmer consistency. These data provides evidence that "finishing" growth of winter flounder at different water salinities can be used to alter the taste and fat content ofthe resulting fillets in summer and winter flounder.

Groups of tagged hatchery raised summer flounder obtained from identical broodstock were exposed to either 10% seawater or 2X seawater for an interval of 3 months under conditions identical to that described above. There were no significant differences in either length or width in fish maintained 10% seawater or 2X seawater. However, there was a significant difference in the weights ofthe respective fish where 10% seawater fish weighted 80+14% (n=10) more than summer flounder maintained in 2X seawater. Moreover, the summer flounder maintained in 10% seawater were nearly twice (2.1+0.4 times n=6) as thick as compared to fish maintained in 2X seawater. These data show that flounder maintained at different water salinities exhibit significant differences in the thickness of their fillets. Thus, flounder could be "finished" using water of differing compositions to alter the thickness of their fillets.

The teachings of all patents, patent applications and all other publications and websites cited herein are incoφorated by reference in their entirety. Also, companion Patent Application No. not yet assigned (Attorney Docket No. 2213.2004-001), entitled "Methods for Growing and Imprinting Fish Using Odotant," filed October 11, 2001; Patent Application No. not yet assigned (Attorney Docket No. 2213.1004- 001), entitled "Methods for Raising Pre-adult Anadromous Fish," filed October 11, 2001 ; International Patent Docket No. not yet assigned (Attorney Docket No. 2213.1003-003), entitled "Growing Marine Fish in Freshwater," filed October 11, 2001. Also, Patent Application No. 09/687,372, entitled "Methods for Raising Pre- adult Anadromous Fish," filed on October 12, 2000; Patent Application No. 09/687,476, entitled "Methods for Raising Pre-adult Anadromous Fish," filed on October 12, 2000; Patent Application No. 09/687,477, entitled "Methods for Raising Marine Fish," filed on October 12, 2000; Provisional Patent Application No. 60/240,392, entitled "Polyvalent Cation Sensing Receptor Proteins in Aquatic Species," filed on October 12, 2000; Provisional Patent Application No. 60/240,003, entitled "Polyvalent Cation Sensing Receptor Proteins in Aquatic Species," filed on October 12, 2000, are all hereby incoφorated by reference in their entirety.

Additionally, Application No. 09/162,021, filed on September 28, 1998, International PCT application No. PCT/US97/05031, filed on March 27, 1997, and Application No. 08/622,738 filed March 27, 1996, all entitled, "Polycation Sensing Receptor in Aquatic Species and Methods of Use Thereof are all hereby incoφorated by reference in their entirety. All references, patent applications, and patents cited herein are included by reference in their entirety.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes can be made therein without departing from the scope of the invention encompassed by the appended claims.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule llbis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page (_s) _rtf, lines-20-23; pq 13 , lines 3-12 ; pg 49, lines Q-22

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [ ]

Name of depositary institution

AMERICAN TYPE CULTURE COLLECTION

Address of depositary institution (including ostal code and country)

American Type Culture Collection 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America

Date of deposit Accession NumberC s ⁾

05 October 2000 PTA2538; PTA2549

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet [X]

In respect of those designations for which a European patent is sought, the Applicant hereby informs the International Bureau that the Applicant wishes that, until the publication of the mention ofthe grant of a European patent or for 20 years from the date of filing if the application is refused or withdrawn or deemed to be withdrawn, the biological material deposited with the American Type Culture Collection under Accession No. [*ε*f shall be made available as provided in Rule 28(3) EPC only by the issue of a sample to an expert nominated by the requester (Rule 28(4) EPC).

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature ofthe indications e.g., "Accession Number of Deposit")

For International Bureau use only D This sheet was received by the International Bureau on:

Authorized officer

Based on PCT/RO/134 (My 1998) INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICIAL MATERIAL

(Additional Sheet)

C. ADDITIONAL INDICATIONS (Continued)

In respect ofthe designation of Australia in the subject PCT application, and in accordance with Regulation 3.25(3) ofthe Australian Patents Regulations, the Applicant hereby gives notice that the furnishing of a sample ofthe biological material deposited with the American Type Culture Collection under Accession No. f &**] shall only be effected prior to the grant of a patent, or prior to the lapsing, refusal or withdrawal ofthe application, to a person who is a skilled addressee without an interest in the invention and who is nominated in a request for the furnishing of a sample.

In respect ofthe designation of Canada in the subject PCT application, the Applicant hereby informs the International Bureau that the Applicant wishes that, until either a Canadian patent has been issued on the basis of an application or the application has been refused, or is abandoned and no longer subject to reinstatement, or is withdrawn, the Commissioner of Patents only authorizes the furnishing of a sample ofthe biological material deposited with the American Type Culture Collection under Accession No. [*&**] and referred to in the application to an independent expert nominated by the Commissioner.

Claims

CLAIMSWhat is claimed is:

1. An isolated nucleic acid molecule having at least 70% identity with SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

2. An isolated nucleic acid molecule having at least 70% identity with a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; encoding a PolyNalent Cation-sensing Receptor (PNCR) polypeptide, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

3. An isolated PNCR polypeptide having at least 70% similarity with SEQ ID ΝOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50; wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

4. An isolated nucleic acid molecule comprising: a) SEQ ID ΝOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; b) a coding sequence of SEQ ED ΝOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; and c) a complementary strand of SEQ ID ΝOS: 1, 3, 5, 1, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

5. An isolated RNA molecule encoded by a nucleic acid molecule of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49.

6. An isolated RNA molecule encoded by a nucleic acid molecule of a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35,

37, 39, 41, 43, 45, 47, or 49.

7. An isolated nucleic acid molecule encoding a complementary strand of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, wherein the nucleic acid molecules is RNA.

8. An isolated nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50, wherein the nucleic acid molecule is RNA.

9. An isolated nucleic acid molecule encoding an amino acid sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24,

26, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50, wherein the nucleic acid molecule is RNA.

10. An isolated nucleic acid molecule that hybridizes under high stringency conditions to a nucleic acid sequence selected from the group consisting of: a) SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33,

35, 37, 39, 41, 43, 45, 47, and 49; b) a coding sequence of SEQ ID NOS: 1, 3, 5, 1, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; c) a complementary strand of SEQ ID NOS : 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; and d) a nucleic acid encoding an amino acid sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,

16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.

11. An isolated nucleic acid molecule that hybridizes under high stringency conditions to a nucleic acid sequence selected from the group consisting of: a) SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; b) a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,

17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; c) a complementary strand of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,

15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49; and d) a nucleic acid encoding an amino acid sequence selected from the group consisting of: SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50, wherein the probe hybridizes to a nucleic acid molecule that senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

12. A plasmid or vector containing a nucleic acid molecule comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

13. A plasmid or vector containing a nucleic acid molecule comprising a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

14. A plasmid or vector containing a nucleic acid molecule encoding an amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50.

15. A host cell transformed with a nucleic acid molecule selected from the group consisting of: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33,

35, 37, 39, 41, 43, 45, 47 and 49.

16. A host cell transformed with a nucleic acid molecule selected from the group consisting of: a coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49.

17. A host cell transformed with a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.

18. A host cell expressing a polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.

19. The host cell of Claim 18, wherein the polypeptide is expressed in a cell selected from the group consisting of: a fish cell, a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell.

20. A cDNA from an ATCC Nos.: PTA-2538; PTA-2539; PTA-2541 ; PTA-2542; PTA-2543; PTA-2544; PTA-2545; PTA-2546; PTA-2547; PTA-2548; or PTA-2549.

21. An isolated polypeptide molecule comprising SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

22. An isolated polypeptide molecule comprising a polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

23. A composition comprising a polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49; and a pharmaceutically-acceptable carrier.

24. A composition comprising a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50; and a pharmaceutically-acceptable carrier.

25. An antibody specific for a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 26, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

26. An antibody specific for a polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, wherein the polypeptide senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.

27. A method for detecting an aquatic PNCR polypeptide in a sample comprising the steps of: a) combimng a sample to be tested with a PNCR-specific antibody, under suitable conditions for formation of a complex between the antibody and PNCR; b) detecting the formation ofthe complex.

26. The method of Claim 27, wherein the antibody is labeled.

27. The method of Claim 26, wherein the labeled antibody is radioactively labeled.

28. The method of Claim 27, further including detecting the formation ofthe complex with a second antibody, wherein the second antibody has a detectable label.

29. A method for detecting an aquatic PNCR nucleic acid molecule in a sample comprising the steps of: a) combining a sample to be tested with a PNCR-specific hybridization probe, under suitable conditions for specific hybridization ofthe PNCR-specific probe and the sample; b) detecting the PNCR nucleic acid molecule in the sample, and wherein the PNCR nucleic acid detected in the sample encodes a polypeptide that senses ion concentrations; alters water intake; alters water absorption; alters urine output; or modulates the percentage of fat, protein or moisture of muscle in an aquatic species.