WO2003087331A2 - Recepteur detectant les cations polyvalents dans du saumon atlantique - Google Patents

Recepteur detectant les cations polyvalents dans du saumon atlantique Download PDF

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WO2003087331A2
WO2003087331A2 PCT/US2003/011188 US0311188W WO03087331A2 WO 2003087331 A2 WO2003087331 A2 WO 2003087331A2 US 0311188 W US0311188 W US 0311188W WO 03087331 A2 WO03087331 A2 WO 03087331A2
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seq
salmokcar
nucleic acid
acid sequence
ofthe
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PCT/US2003/011188
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WO2003087331A3 (fr
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Jr. H. William Harris
Jacqueline Nearing
Marlies Betka
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Marical, Inc.
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Priority claimed from US10/125,778 external-priority patent/US6979558B2/en
Priority claimed from US10/125,792 external-priority patent/US6979559B2/en
Priority claimed from US10/125,772 external-priority patent/US6951739B2/en
Application filed by Marical, Inc. filed Critical Marical, Inc.
Priority to EP03746726.3A priority Critical patent/EP1572938A4/fr
Priority to CA2481827A priority patent/CA2481827C/fr
Priority to AU2003232002A priority patent/AU2003232002A1/en
Publication of WO2003087331A2 publication Critical patent/WO2003087331A2/fr
Publication of WO2003087331A3 publication Critical patent/WO2003087331A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • anadromous fish like salmon live most of their adulthood in seawater, but swim upstream to freshwater for the pmpose of breeding. As a result, anadromous fish hatch from their eggs and are born in freshwater. As these fish grow, they swim downstream and gradually adapt to the seawater.
  • the present invention relates to genes that allow fis.h to sense and adapt to ion concentrations in the sunounding environment. Modulating one or more of these genes allow anadromous fish like salmon to better adapt to seawater during smoltification, which in turn allows salmon to grow faster and stronger after transfer to seawater.
  • a gene called a PolyValent Cation-sensing Receptor (PVCR), has been isolated in several species offish, and in particular, in Atlantic Salmon.
  • PVCR PolyValent Cation-sensing Receptor
  • PVCR four forms ofthe PVCR have been isolated in Atlantic Salmon, and have been termed, "SalmoKCaR” genes and individually refened to as “SalmoKCaR#l”, “SalmoKCaR#2", “SafmoKCaR#3” and “SalmoKCaR#4".
  • PVCR and “SalmoKCaR” are used interchangeably when referring to Atlantic Salmon. These four genes work together to alter the salmon's sensitivity to the sunounding ion concentrations, as further described herein.
  • the invention embodies nucleic acid molecules (e.g., RNA, genomic DNA and cDNA) having nucleic acid sequences of SalmoKCaR#l (SEQ ID NO: 7), SalmoKCaR#2 (SEQ LD NO: 9), SalmoKCaR#3 (SEQ ID NO: 11), or
  • SalmoKCaR#4 (SEQ JD NO: 13).
  • the invention also embodies polypeptide molecules having amino acid sequences of SalmoKCaR#l (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), SafmoKCaR#3 (SEQ ID NO: 12), or SalmoKCaR#4 (SEQ ID NO: 14).
  • the present invention in particular, encompasses isolated nucleic acid molecules having nucleic acid sequences of SEQ ID NO: 7, 9, 11, or 13; the complementary strand thereof; the coding region of SEQ ID NO: 7, 9, 11, or 13; or the complementary strand thereof.
  • the present invention also embodies nucleic acid molecules that encode polypeptides having an amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.
  • the present invention in another embodiment, includes isolated polypeptide molecules having amino acid sequences that comprise SEQ ID NO: 8, 10, 12, or 14; or amino acid sequences encoded by the nucleic acid sequence of SEQ ID NO: 7, 9, 11, or 13.
  • the present invention pertains to isolated nucleic acid molecules that have a nucleic acid sequence with at least about 70% (e.g., 75%, 80%, 85%, 90%, or 95%) identity with SEQ ID NO: 7, 9, 11, or 13, or the coding region of SEQ ID NO: 7, 9, 11, or 13.
  • Such a nucleic acid sequence encodes a polj ⁇ eptide that allows for or assists in one or more ofthe following functions in Atlantic Salmon: sensing at least one SalmoKCaR modulator in serum or in the sunounding environment; adapting to at least one SalmoKCaR modulator present in the serum or surrounding environment; imprinting with an odorant; altering water intake; altering water abso ⁇ tion; or altering urine output.
  • the present invention further includes nucleic acid molecules that hybridize, preferably under high stringency conditions, with SalmoKCaR#l, SalmoKCaR#2, SahnoKCaR#3, or SalmoKCaR #4 but not to the Shark Kidney Calcium Receptor related protein (SKCaR) nucleic acid sequence (SEQ ID NO: 1, shown in Figure 1) and/or the Fugu pheromone receptor (described in Naito, T.
  • SSCaR Shark Kidney Calcium Receptor related protein
  • SKCaR is a PVCR isolated from dogfish shark.
  • the present invention relates to an isolated nucleic acid molecule that contains a nucleic acid sequence that hybridizes under high stringency conditions to SEQ ID NO: 7, 9, 11 , or 13; or the coding region of SEQ JD NO: 7, 9, 11, or 13; but excluding those that hybridize to SEQ ID NO: 1 or the nucleic acid sequence ofthe fugu pheromone receptor, as described herein, under the same conditions.
  • the present invention also includes probes, vectors, virases, plasmids, and host cells that contain the nucleic acid sequences, as described herein.
  • the present invention includes probes (e.g., nucleic acid probes or DNA probes) having a sequence from or hybridizes to SEQ JD NO: 7, 9, 11, or 13, but not SEQ ID NO: 1 or the nucleic acid sequence ofthe fugu pheromone receptor, as described herein.
  • the present invention encompasses nucleic acid or peptide molecules purified or obtained from clones deposited with American Type Culture Collection (ATCC), Accession No: PTA-4190, PTA-4191, PTA-4192, or (to be added).
  • the present invention includes isolated polypeptide molecules having at least about 70% (e.g., 75%, 80%, 85%, 90%, or 95%) identity with SEQ ID NO: 8, 10, 12, or 14; or an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 7, 9, 11, or 13.
  • polypeptide molecules e.g., SEQ ID NO: 8, 10, 12, or 14; or those encoded by the nucleic acid sequence of SEQ TD NO: 7, 9, 11, or 13
  • polypeptide molecules have one or more ofthe following functions in Atlantic Salmon: sensing at least one SalmoKCaR modulator in serum or in the surrounding environment; adapting to at least one SalmoKCaR modulator present in the serum or surrounding environment; imprinting with an odorant; altering water intake; altering water abso ⁇ tion; or altering urine output.
  • the present invention relates to antibodies that specifically bind to or are produced in reaction to polypeptide molecules described herein.
  • the invention further includes fusion proteins that contain one ofthe polj ⁇ eptide molecules described herein, and a portion of an immunoglobulin.
  • the present invention also pertains to assays for detennining the presence or absence of a SalmoKCaR in a sample by contacting the sample to be tested with an antibody specific to at least a portion ofthe SalmoKCaR polypeptide sufficiently to allow formation of a complex between SalmoKCaR and the antibody, and detecting the presence or absence ofthe complex- formation.
  • Another assay for determining the presence or absence of a nucleic acid molecule that encodes SalmoKCaR in a sample involves contacting the sample to be tested with a nucleic acid probe that hybridizes under high stringency conditions to a nucleic acid molecule having a sequence of SEQ ID NO: 7, 9, 11, or 13, sufficiently to allow hybridization between the sample and the probe; and detecting the SalmoKCaR nucleic acid molecule in the sample.
  • Such assay methods also include methods for determining whether a compound is a modulator of SalmoKCaR.
  • These methods include contacting a compound to be tested with a cell that contains SalmoKCaR.nucleic acid molecules and/or expresses SalmoKCaR proteins, and determining whether compounds are modulators by measuring the expression level or activity (e.g., phosphorylation, dimerization, proteolysis or intracellular signal transduction) of SalmoKCaR proteins. In one embodiment, one can measure changes that occur in one or more intracellular signal transduction systems that are altered by activation ofthe expressed proteins coded for by a single or combination of nucleic acids. Such methods can also encompass contacting a compound to be tested with a cell that comprises one or more of SalmoKCaR nucleic acid molecules; and determining the level of expression of said nucleic acid molecule.
  • the present invention relates to transgenic fish encoding a SalmoKCaR polj ⁇ eptide or having one or more nucleic acid molecules that contain the SalmoKCaR nucleic acid sequence, as described herein.
  • the present invention allows for a number of advantages, including the ability to more efficiently grow Atlantic Salmon, and in particular, transfer them to seawater with increased growth and reduce mortality.
  • the technology ofthe present invention also allows for assaying or testing these sahnon to determine if they are ready for transfer to seawater, so that they can be transfened at the best time.
  • the technology ofthe present invention provides for the imprinting of salmon with an odorant so that the salmon, once imprinted, can later more easily recognize and/or distinguish the odorant. For example, an attractant that has been used to imprint salmon can be added to feed so that the salmon will consume more feed and grow at a faster rate.
  • Figures 1 A-E show the annotated nucleotide sequence (SEQ ID NO: 1) and the deduced amino acids sequence (SEQ ID NO: 2) of SKCaR with the Open Reading Frame (ORF) starting at nucleotide (nt) 439 and ending at 3516.
  • ORF Open Reading Frame
  • Figure 2 is a graphical representation showing a normalized calcium response (%) against the amount of Calcium (mM) ofthe SKCaR-I protein when modulated by alternations in extracellular NaCl concentrations.
  • Figure 3 is a graphical representation showing a normalized calcium response (%) against the amount of magnesium(mM) ofthe SKCaR-I protein in increasing amounts of extracellular NaCl concentrations.
  • Figure 4 is a graphical representation showing the EC50 for calcium activation of shark CaR (mM) against the amount of sodium (mM) of he SKCaR ⁇ I protein in increasing amounts of extracellular NaCl concentrations.
  • Figure 5 is a graphical representation showing the EC50 for magnesium activation of shark CaR (mM) against the amount of sodium (mM) ofthe SKCaR-I protein in increasing amounts of extracellular NaCl concentrations.
  • Figure 6 is a graphical representation showing the EC50 for magnesium activation of shark CaR (mM) against the amount of sodium (mM) ofthe SKCaR-I protein in increasing amounts of extracellular NaCl concentrations and added amounts of calcium (3mM).
  • Figures 7A and 7B show an annotated partial nucleotide sequence (SEQ ID NO: 3) and the deduced amino acids sequence (SEQ ID NO: 4) of an Atlantic salmon polyvalent cation-sensing receptor protein.
  • Figures 8A-8C show a second annotated partial nucleotide sequence (SEQ ID NO: 5) and the deduced amino acids sequence (SEQ ID NO: 6) of an Atlantic salmon polyvalent cation-sensing receptor protein.
  • Figures 9 A-E show the nucleic acid (SEQ ID NO: 7) and amino acid (SEQ ID NO: 7)
  • Figures 10 A-E show the nucleic acid (SEQ JD NO: 9) and amino acid (SEQ ID NO: 10) sequences of a full length Atlantic Sahnon PVCR, SalmoKCaR#2 with the ORF starting at nt 270 and ending at 3095.
  • Figures 11 A-D show the nucleic acid (SEQ ID NO: 11) and amino acid (SEQ TD NO: 12) sequences of a full length Atiantic Salmon PVCR, SalmoKCaR#3 with the ORF starting at nt 181 and ending at 2733.
  • Figures 12A-E show the nucleic acid (SEQ JD NO: 13) and amino acid (SEQ ID NO : 14) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#4 with the ORF starting at nt 181 and ending at 3006.
  • Figures 13A-L are an alignment showing nucleic acid sequences of two partial Atlantic Salmon Clones (SEQ ID NO: 3 and 5), SalmoKCaR#l (SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), and SalmoKCaR#3 (SEQ ID NO: 11).
  • Figures 14A-C are an alignment showing amino acid sequences of two partial Atlantic Salmon Clones (SEQ ID NO: 4 and 6), SalmoKCaR#l (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), and SalmoKCaR#3 (SEQ LD NO: 12).
  • Figure 15A is photograph showing a Southern blot in which SalmoKCaR#l, 2, and 3 hybridize to nucleic acid derived from SKCaR.
  • Figure 15B is a photograph showing a Western blot protein produced by
  • HEK cells transiently transfected with SalmoKCaR#l and #3 constructs.
  • the left panel shows an immunoblotting analysis with SDD antiserum while the right panel shows immunoblotting analysis for Sail for the following: a standard, 5001 HEK (human) cells, HEK 293 mock transfected cells, SahnoKCaR#l HEK cells, SalmoKCaR#3 HEK cells, and Salmon SW Kidney.
  • Figures 16A-H are an alignment ofthe full length nucleic acid sequences of SalmoKCaR#l, 2, and 3 (SEQ LD NO: 7, 9, and 11, respectively). Alignment obtained using Clustal method with weighted residue weight table.
  • Figures 17A-L are an alignment showing nucleic acid sequences of SalmoKCaR#l (SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), SalmoKCaR#3 (SEQ JD NO: 11), and SalmoKCaR#4 (SEQ ID NO: 13). Alignment was obtained using Clustal method with weighted residue weight table.
  • Figures 18 A-C are an alignment showing amino acid sequences of SalmoKCaR#l (SEQ JD NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), SalmoKCaR#3 (SEQ JD NO: 12), and SalmoKCaR#4 (SEQ ID NO: 14).
  • Figures 19A-D are an alignment ofthe full length amino acid sequences of Human Parathyroid Calcium Receptor (HuPCaR) (SEQ JD NO: 30), SKCaR (SEQ ID NO: 2), SalmoKCaR#l (SEQ ID NO: 8), SalmoKCaR#2 (SEQ JD NO: 10), and SalmoKCaR#3 (SEQ JD NO: 12), and SalmoKCaR#4 (SEQ JD NO: 14). Alignment obtained using Clustal method with PAM250 residue weight table.
  • Figures 20A-F are graphical representations comparing six photographs of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis of freshwater ( Figures 20B, D and F) and seawater (Figur.es 20A, C and E) adapted Atlantic salmon tissues (gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and muscle) using either degenerate PVCR ( Figures 20A-D) or salmon actin PCR primers ( Figures 20E,F).
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction
  • Wells 1-14 for Figures 20A-F, top row, are designated as follows: ladder, gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and muscle, respectively.
  • Figures 20A, C, and E are designated as ladder, water, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively, and wells 1, 2, 3, 7, 9, and 12, bottom row, for Figures 20B,D, and F are designated as ladder, water, ovary, SalmoKCaR #1, SahnoKCaR#2 and SalmoKCaR#3, respectively.
  • Figure 21 A is photograph of a RT-PCR analysis using degenerate primers of steady state SalmoKCaR mRNA transcripts from kidney tissue of Atlantic Salmon adapted to freshwater, after 9 weeks of Process JJ treatment or 26 days after transfer to seawater. Process IT treatment is defined in the Exemplification.
  • Figure 2 IB is a photograph of a RT-PCR analysis showing increased steady state expression of SalmoKCaR transcripts in pyloric caeca of Process ⁇ treated and seawater fish as compared to freshwater Atlantic salmon smolt.
  • Figure 21C is a photograph of RT-PCR analysis showing expression of SalmoKCaR transcripts in various stages of Atlantic salmon embryo development.
  • degenerate SEQ JD Nos. 15 and 16
  • actin SEQ JD No 24 and 25
  • RNA obtained from samples of whole Atlantic salmon embryos at various stages of development were analyzed for expression of SalmoKCaRs using RT-PCR.
  • Ethidium bromide staining of samples from dechorionated embryos (Lane 1), 50% hatched (Lane 2), 100% hatched (Lane 3), 2 weeks post hatched (Lane 4) and 4 weeks post hatched (Lane 5) shows that SalmoKCaR transcripts are present in Lanes 1-4).
  • FIG. 22 is a photograph of a RNA blot containing 5 micrograms of poly A + RNA from kidney tissue dissected from either freshwater adapted (FW) or seawater adapted (SW) Atlantic salmon probed with full length SalmoKCaR #1 clone.
  • Figures 23A-F are graphical representations comparing six photographs showing RT-PCR analysis of freshwater ( Figures 23B, D and F) and seawater
  • Figures 23 A, C and E adapted Atlantic salmon tissues using either SalmoKCaR #3 specific PCR ( Figures 23 A-D) primers or salmon actin PCR primers ( Figures 23E,F).
  • Wells 1-14 for Figures 23A-F, top row, are designated as follows: ladder, gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and muscle, respectively.
  • Wells 1, 2, 8, 11, and 14, bottom row, for Figures 23 A, C, and E are designated as ladder, water, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively, and wells 1, 2, 3, 8, 11, and 14, bottom row, for Figures 23B,D, and F are designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and SahnoKCaR#3, respectively.
  • Figures 24A-F are graphical representations comparing six photographs showing RT-PCR analysis of freshwater ( Figures 24B, D and F) and seawater ( Figures 24A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR #1 specific PCR primers or salmon actin PCR primers.
  • Wells 1-14 for Figures 24A-F, top row, are designated as follows: ladder, gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and muscle, respectively.
  • Figures 24A, C, and E are designated as ladder, water, Kidney-RT, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively, and wells 1, 2, 3, 5, 6, and 7, bottom row, for Figures 24B, D, and F are designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.
  • Figures 25A-F are graphical representations comparing six photographs showing RT-PCR analysis of freshwater ( Figures 25B, D and F) and seawater ( Figures 25A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR #2 specific PCR primers ( Figures 25 A-D) or salmon actin PCR primers ( Figures 25 A-F).
  • Wells 1-14 for Figures 25A-F, top row, are designated as follows: ladder, gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and muscle, respectively. Wells 1, 2,3, 5, 6, and 7.
  • Figures 25 A, C, and E are designated as ladder, water, Kidney-RT, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively, and wells 1, 2, 3, 5, 6, and 7, bottom row, for Figures 25B, D, and F are designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.
  • FIG. 26 is a schematic diagram illustrating industry practice for salmon aquaculture production, prior to the discovery ofthe present invention.
  • the diagram depicts key steps in salmon production for SO (75 gram) and SI (100 gram) smolts. The wavy symbol indicates freshwater while the bubbles indicate seawater.
  • Figure 27A is a graphical representation comparing the weekly feed consumption on a per fish basis between Process I treated smolts weighing approximately 76.6 gm vs industry standard smolt weighing approximately 95.8 gm. These data are derived from individual netpens of fish containing about 10,000- 50,000 fish per pen. As shown, fish treated with Process I consumed approximately twice as much feed per fish during their first week after seawater transfer as compared to the large industry standard smolts weekly food consumption after 30 days. Process I treatment is defined in the Exemplification.
  • Figure 27B is a graphical representation illustrating length (cm) and weight (gm) of Process I Smolts 50 days after ocean netpen placement.
  • Process I smolts had an average weight of 76.6 gram, when placed in seawater and were sampled after 50 days.
  • Figure 28 is a graphical representation illustrating length (cm) and weight (gm) of representative Process I smolts prior to transfer to seawater.
  • Figure 29 is a graphical representation illustrating length (cm) and weight
  • Figure 30 is a three dimensional graph illustrating the survival over 5 days of Arctic Char in seawater after being maintained in freshwater, Process I for 14 days, and Process I for 30 days.
  • Figure 31 is a graphical representation illustrating the length (cm) and weight (gm) of St. John/St. John Process JJ smolts prior to seawater transfer. Process JJ is defined in the Exemplification Section.
  • Figures 32A and 32B are grapl ical representations illustrating weight (gm) and length (cm) of Process II smolt survivors and mortalities 5 days after transfer to seawater tanks (A), and 96 hours after transfer to ocean netpens (B).
  • Figures 33A-G are photographs of immunocytochemistry of epithelia ofthe proximal intestine of Atlantic Salmon illustrating SalmoKCaR localization and expression.
  • Figure 34 is a photograph of a Western Blot of intestinal tissue from salmon subjected to Process I for immune (lane marked CaR, e.g., a SalmoKCaR) and preimmune (lane marked preirmnune) illustrating SalmoKCaR expression.
  • CaR e.g., a SalmoKCaR
  • preimmune laminoKCaR
  • Figures 35 A-C are photographs of immunolocalization ofthe SalmoKCaR in the epidemiis of salmon illustrating SalmoKCaR localization and expression.
  • Figure 36 is a graphical representation quantifying the Enzyme-Linked ImmunoSorbent Assay (ELISA) protein (ng) for various tissue samples (e.g., gill, liver, heart, muscle, stomach, olfactory epithelium, kidney, urinary bladder, brain, pituitary gland, olfactory bulb, pyloric ceacae, proximal intestine, and distal intestine) from a single fish.
  • tissue samples e.g., gill, liver, heart, muscle, stomach, olfactory epithelium, kidney, urinary bladder, brain, pituitary gland, olfactory bulb, pyloric ceacae, proximal intestine, and distal intestine
  • Figure 37 is a photograph of a RT-PCR amplification of a partial SalmoKCaR mRNA transcript from various tissues (gill, nasal lamellae, urinary bladder, kidney, intestine, stomach, liver, and brain (Wells 1-8, respectively)) of Atlantic Salmon.
  • RT-PCR reactions were separated by gel electrophoresis and either stained in ethidium bromide (EtBr) or transfened to a membrane and Southern Blotted (SB) using a 32P-labeled 653 basepair (bp) genomic DNA fragment from the Atlantic salmon SalmoKCaR gene.
  • Wells 9 and 10 are water (blank) and positive control, respectively.
  • Figure 38 is a series of photographs of immunocytochemistry showing the SalmoKCaR localization of Atlantic Salmon Olfactory Bulb Nerve and Lamellae using an anti-SalmoKCaR antibody.
  • Figure 39 is a schematic illustrating the effect of external and internal ionic concentrations on the olfactory lamellae in response to SalmoKCaR modulators.
  • Figure 40 A is a photograph of immunocytochemistry showing the SalmoKCaR protein expression in the developing nasal lamellae (Panel A) and olfactory bulb (Panel B) after hatching of Atlantic salmon using an anti-SalmoKCaR antibody.
  • Figure 40B is a photograph of iimnunocytochemistry of Atlantic salmon or trout larval fish using Sal-I antiserum shows abundant PVCR protein expression by selected cells. Specific binding of Sal-I antiserum denoting the presence of PVCR protein is shown by the dark reaction product. Staining of myosepta between various muscle bundles of larval fish is shown by asterisks (panel A). Panel B shows the head of a trout larvae in cross section where abundant PVCR protein is present in the skin (asterisks) and developing nasal lamellae (open anowhead). Panel C shows PVCR expression in the developing otolith as well as localized PVCR protein in epithelial cells immediately adjacent to it.
  • Panels D and E show high magnification views of myosepta shown in Panel A. Note the pattern of localized expression of PVCR protein where some cells contain large amounts of PVCR protein while those immediately adjacent to them have little or no expression.
  • Panel F shows a conesponding H+E section where myosepta (open arrowheads) can be clearly distinguished from intervening muscle bundles.
  • Figure 40C is a photograph showing localization of Sal ADD antiserum by immunocytochemistry.
  • Panel A shows the pattern of immunostaining of immune anti-Sal ADD serum as compared to lack of reactivity displayed by preimmune anti- Sal ADD serum when exposed to identical kidney tissue sections (Panel B).
  • anti-Sal ADD reactivity (denoted by arrows) is similar if not identical to that displayed by Sal-I antiserum.
  • Conesponding kidney tubules exposed to preimmune antiserum show no reactivity (denoted by asterisks).
  • Figure 41 is a photograph of immunocytochemistry showing the PVCR localization in nasal lamellae of dogfish shark using an anti-PVCR antibody.
  • Figure 42 is a photograph of a Southern blot of RT-PCR analyses of tissues from Atlantic Salmon showing the presence of SalmoKCaR mRNA in nasal lamellae of freshwater adapted fish.
  • Wells 1-10 are designated as follows: gill, nasal lamellae, urinary bladder, kidney, intestine, stomach, liver, brain, water (blank) and positive control, respectively.
  • Figure 43 is a histogram illustrating the amount of SalmoKCaR protein, as determined by an ELISA (ng) for various tissue samples (gill, liver, heart, muscle, stomach, olfactory epithelium, kidney, urinary bladder, brain, pituitary gland, olfactory bulb, pyloric ceacae, proximal intestine, and distal intestine).
  • tissue samples gill, liver, heart, muscle, stomach, olfactory epithelium, kidney, urinary bladder, brain, pituitary gland, olfactory bulb, pyloric ceacae, proximal intestine, and distal intestine.
  • Figure 44 shows the raw and integrated recordings from high resistance electrodes of freshwater adapted Atlantic Salmon when exposed to 500 ⁇ M L- alanine, 1 mmol calcium, 50 ⁇ M Gadolinium, and 250 mmol of NaCl.
  • the figures show the existence of an olfactory recording in response to L-alanine, calcium, gadolinium, and NaCl.
  • Figure 45 is a graph showing the response data for freshwater adapted Atlantic salmon nasal lamellae for calcium, magnesium, gadolinium, and sodium chloride normalized to the signal obtained with 10 mM Calcium.
  • Figure 46 shows raw recording from high resistance electrodes of olfactory nerve impulse in the presence of a repellant (finger rinse) and in the presence of a SalmoKCaR agonist (gadolinium) and a repellant (fmger rinse). The figure shows that the olfactory nerve impulse to the repellant is reversibly altered in the presence of a SalmoKCaR agonist.
  • Figure 47 shows the raw recordings from high resistance electrodes of freshwater adapted Atlantic Salmon in response to a series of repeated stimuli (L- alanine or NaCl) in 2 minute intervals. The figure shows that the olfactory nerve impulse to the attractant is reversibly altered in the presence of a SalmoKCaR agonist
  • Figure 48 is a graphical representation ofthe ratio from FTJRA-2 cells expressing a PVCR in the presence or absence of 10 mM L-Isoleucine in various concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 20.0 mM) of extracellular calcium (Ca 2+ ).
  • Figure 49 is a graphical representation ofthe fractional Ca 2+ response, as compared to the extracelluar Ca 2+ (mM) for the PVCR in Ca 2+ only, Phenylalanine, Isoleucine, or AA Mixture (a variety of L-isomers in various concentrations).
  • Figure 50 shows the nucleic acid sequence for a Fugu receptor, Fugu CaR (01), (SEQ ID NO: 31) deposited under GenBank Accession Nos: AB008857.
  • Figures 51A-B show the nucleic acid sequence for a Fugu receptor, Fugu Ca02.1, (SEQ ID NO: 32) deposited under GenBank Accession Nos: AB008858.
  • Figures 52A-B show the nucleic acid sequence for a Fugu receptor, Fugu Ca09, (SEQ ID NO: 33) deposited under GenBank Accession Nos: AB008859.
  • Figures 53 A-B show the nucleic acid sequence for a Fugu receptor, Fugu Cal2, (SEQ ID NO: 34) deposited under GenBank Accession Nos: AB00886O.
  • Figures 54A-B show the nucleic acid sequence for a Fugu receptor, Fugu Cal3, (SEQ ID NO: 35) deposited under GenBank Accession Nos: AB008861.
  • Figures 55 A-B show the nucleic acid sequence for a Fugu receptor, Fugu
  • Figure 56 shows the nucleic acid sequence for a Fugu receptor, Fugu mGluRl, (SEQ ID NO: 37) deposited under GenBank Accession Nos: AB008863.
  • Figure 57 shows the nucleic acid sequence for a Fugu receptor, Fugu mGluR2, (SEQ ID NO: 38) deposited under GenBank Accession Nos: AB008864.
  • Figure 58 shows the nucleic acid sequence for a Fugu receptor, Fugu mGluR7, (SEQ ID NO: 39) deposited under GenBank Accession Nos: AB008865.
  • Figure 59 shows the nucleic acid sequence for a Fugu receptor, Fugu mGluR8, (SEQ ID NO: 40) deposited under GenBank Accession Nos: AB008866.
  • the present invention relates to at least four novel isolated sequences from PVCR genes, SalmoKCaR#l, SalmoKCaR#2, SalmoKCaR#3, and SalmoKCaR#4 in Atlantic Salmon. These genes encode four polj ⁇ eptide sequences that are also the subject ofthe present invention. These polypeptide sequences allow for or assist in several functions in Atlantic Salmon including sensing at least one SalmoKCaR modulator in serum or in the sunounding environment; adapting to at least one SalmoKCaR modulator present in the serum or sunounding environment; imprinting with an odorant; altering water intake; altering water abso ⁇ tion; or altering urine output.
  • One use ofthe present invention relates to methods for improving the raising of salmon and/or methods for preparing salmon for transfer from freshwater to seawater. These methods involve adding one or more PVCR (e.g., SalmoKCaR) modulators to the freshwater (e.g., calcium and/or magnesium), and adding a specially made or modified feed to the freshwater for consumption by the fish.
  • the feed contains a sufficient amount of sodium chloride (NaCl) and/or a SalmoKCaR modulator (e.g., an amino acid like tryptophan) to significantly increase levels ofthe SalmoKCaR modulator in the serum.
  • Process I involves adding calcium and magnesium to the water, and providing feed containing NaCl; and Process II includes adding calcium and magnesium-to the water, and providing feed having both NaCl and tryptophan.
  • Example 7 Studies performed and described in Example 7 show that Atlantic Salmon maintained in freshwater and subjected to Process I had a survival rate of 91%, and those Atlantic Salmon subjected to Process JJ had a survival rate of 99%; as compared to control fish having a survival rate of only 67% after transfer to seawater. Similarly, in the same experiment, five days after transfer to seawater, Atlantic Salmon subjected to Process I had a survival rate of 90%, while Atlantic Salmon subjected to Process II had a survival rate of 99%. The control fish had a survival rate of only 50% after being transfened to seawater. Furthennore, experiments described in Example 6 demonstrate that modulated expression of one or more SalmoKCaR genes occurs in various tissues during Process I and Process II. Process I and JJ, as described herein, modulate the SalmoKCaR genes and allow for increased food consumption, growth and survival; and decreased morbidity and susceptibility to disease.
  • Process I and JJ likely have further utility in restoration of wild Atlantic salmon populations. Since a major cause of mortality of wild Atlantic salmon smolt is loss or capture by predators as they are adapting to seawater in river estuaries, treatment of wild Atlantic salmon produced in large numbers, as part of river restocking programs would boost the productivity and survival of fish produced in such programs. Moreover, several studies have shown that salmon smolt are also poisoned by exposure to heavy metals (Al 3+ , Zn 2+ , Cu 2+ ) that contaminate their native rivers in both the US and other countries such as Norway. These highly deleterious effects on salmon are manifested principally in rivers with low natural Ca 2+ concentrations.
  • heavy metals Al 3+ , Zn 2+ , Cu 2+
  • SalmoKCaR genes Similarly, since expression ofthe SalmoKCaR genes changes during Process - I and Process- ⁇ , assaying these genes allows one to determine if the salmon are ready for transfer to seawater. Examples of such assays are ELIS As, radioimmunoassays (RIAs), southern blots and RT-PCR assays, which are described herein in detail.
  • the salmon are subjected to either Process I or Process II for a period of time in freshwater before being transfened to seawater.
  • the SalmoKCaR genes, or polypeptides encoded by these genes can be assayed for determining the optimal time period for maintaining the salmon in the freshwater, before transfer to seawater.
  • salmon can be assayed to determine if modulated levels of .the SalmoKCaR genes and/or polypeptides have occvured, as compared to controls. For example, when fish that are maintained in freshwater and subjected to either Process I or Process II and changes in one or more of SalmoKCaR genes and/or polypeptide levels in at least one tissue are modulated such that they mimic changes in the same genes and/or polypeptide levels that would be seen in fish adapted to seawater, then this group of fish are ready to be transfened to seawater.
  • the increased expression of SalmoKCaR genes in the kidney of Atlantic Salmon subjected to Process IJ was similar to the increased expression in the same tissue for Atlantic Salmon already adapted to seawater, but dissimilar to expression to Atlantic Salmon adapted to freshwater (i.e., no increased expression in the kidney water fish was seen). See Example 6.
  • levels of SalmoKCaR genes and/or polypeptide encoded by these genes are similar to those levels seen in fish that have been transfened to seawater, then in the experiments described herein, the transfer of these sahnon result in several benefits including increased survival and growth.
  • the optimal time periods for subjecting salmon to Process I or Process U are generally between 4-6 weeks, but vary depending on the strain of salmon or process used.
  • the assays described herein can be used to determine the optimal amount of time for subjecting the salmon to either Process I or Process IJ before transfening to seawater.
  • SalmoKCaR #3 protein likely generates a dominant negative effect on the other SalmoKCaR #1, #2 and #4 proteins when they are expressed together in the same cell.
  • This dominant negative effect of SalmoKCaR #3 occurs since it lacks that necessary carboxyl tenninal domain to propagate signals generated by the binding of PVCR agonists. Interactions between the fully functional SalmoKCaR #1, #2 or #4 proteins and SalmoKCaR #3 would cause a marked reduction in the sensitivity of the SalmoKCaR #1, #2 or #4 proteins.
  • SalmoKCaR#3 increased expression of SalmoKCaR#3 was seen in tissues readily exposed to high concentrations of calcium and magnesium in the sunounding environment (e.g., gill and nasal lamellae) or tissues that excrete high concentrations of calcium and magnesium (e.g., urinary bladder and kidney). Therefore, such assays can be used to determine levels ofthe individual SalmoKCaR genes, and compare expression levels to one another, and to individual levels of these genes of seawater adapted salmon to determine whether the salmon being tested are ready for transfer to seawater.
  • nucleic acids ofthe present invention include one or more ofthe 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 one or more ofthe following: conserved sequences; unique nucleotide sequences for normal and altered receptors; and 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, as further described herein to determine the presence or level of SalmoKCaR in a sample for, e.g., assessing whether salmon are ready for transfer to seawater; (4) as PCR primers to generate particular nucleic acid sequence sequences, for example, to generate sequences to be used
  • nucleic acid sequences of SalmoKCaRs #1, #2, #3, or #4 is as probes for the screening of Atlantic salmon broodstock, eggs, sperm, embryos or larval and juvenile fish as part of breeding programs.
  • Use of SalmoKCaR probes would enable identification of desirable traits such as enhanced salinity responsiveness, homing, growth in seawater or freshwater or improve the feed utilization that were due to or associated with naturally occurring or induced mutations of SalmoKCaR genes.
  • Nucleic -acid sequences- of SalmoKCaRs #1 , #2, #3, or #4 can also be used as probes for screening of wild Atlantic salmon in various regions as a tool to identify specific strains offish from both sea run and land locked strains. Such strains could then be used to interbreed with existing commercial strains to produce further improvements in fish performance.
  • the structural-functional data generated via study of recombinant SalmoKCaRs after their expression in cells as functional proteins can be used to identify desirable alternations in the function of SalmoKCaR proteins that could then be screened for as part of genetic selection-bro ⁇ dstock enhancement program.
  • Cell lines expressing SahnoKCaR proteins would have utility and value as a means to assay various compounds, chemicals and water conditions that occur both in the natural and commercial environments.
  • Utilization of transfected cells expressing SahnoKCaR #1-4 proteins either alone or in various combinations can be used in screening methods to identify both naturally occuning and commercially synthesized compounds that would enhance the performance of wild or commercially produced Atlantic salmon including salinity adaption, feeding, growth and maturation, flesh quality, homing to areas of spawning, recognition of specific odorants as part of imprinting, utilization of nutrients with improved efficiency and altered behavior.
  • Such screening assay would be a vast improvement over existing assays where large numbers of fish are required and their end response (e.g., behavior, feeding, growth, survival or appearance is altered) to a given compound produce complicated assays that have many problems with data interpretation.
  • Transfected cells expressing SalmoKCaR #1-4 proteins either alone or in various combinations can also be used in screening methods to screen for specific water conditions including pH, ionic strength and composition of various compounds dissolved in the water to alter the function of SalmoKCaR proteins and thus lead to improved salinity responses in various life stages of Atlantic salmon.
  • Such assays would be designed to determine the interactions and effects of these conditions on SalmoKCaR proteins without having to test the effects of such compounds on either whole living fish or some tissue explants.
  • Fragments of recombinant SalmoKCaR proteins also provide a utility as modulators of PVCR function that could be added to water, applied to tissue surfaces such as gills or skin or injected into fish via standard techniques.
  • the present invention is also useful in immunization of any one ofthe various life stages of Atlantic salmon (eggs, embryo, larval or juvenile or adult fish) with either whole or fragments of recombinant SalmoKCaR proteins to create antibody responses that would, in turn, alter SalmoKCaR mediated functions of fish.
  • the present invention relates to isolated polypeptide molecules that have been isolated in Atlantic Salmon including four full length sequences.
  • the present invention includes polypeptide molecules that contain the sequence of any one ofthe full length SalmoKCaR amino acid sequence (SEQ JD NO: 8, 10, 12, or 14). See Figures 9, 10, 11, and 12.
  • the present invention also pertains polypeptide molecules that are encoded by nucleic acid molecules having the sequence of any one ofthe isolated full length SalmoKCaR nucleic acid sequences (SEQ ID NO: 7, 9, 11, or 13).
  • SalmoKCaR polypeptides refened to herein as "isolated” are polypeptides that separated away from other proteins and cellular material of their source of origin.
  • Isolated SalmoKCaR proteins include essentially pure protein, proteins produced by chemical synthesis, by combinations of biological and chemical synthesis and by recombinant methods.
  • the proteins ofthe present invention have been isolated and characterized as to its physical characteristics using laboratory techniques common to protein purification, for example, salting out, immunoprecipation, column chromatography, high pressure liquid chromatography or electrophoresis.
  • SalmoKCaR proteins are found in many tissues in fish including gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver, muscle, skin and brain.
  • the present invention also encompasses SalmoKCaR proteins and polypeptides having amino acid sequences analogous to the amino acid sequences of SalmoKCaR polypeptides.
  • Such polypeptides are defined herein as SalmoKCaR analogs (e.g., homologues), or mutants or derivatives.
  • Amalogous or “homolgous” amino acid sequences refer to amino acid sequences with sufficient identity of any one ofthe SalmoKCaR amino acid sequences so as to possess the biological activity of any one ofthe native SalmoKCaR polypeptides.
  • 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 of any one ofthe SalmoKCaR protein, yet still possesses the function or biological activity ofthe SalmoKCaR. Examples of such differences include additions, deletions or substitutions of residues ofthe amino acid sequence of SalmoKCaR. Also encompassed by the present invention are analogous polypeptides that exhibit greater, or lesser, biological activity of any one ofthe SalmoKCaR proteins ofthe present invention.
  • Such polypeptides can be made by mutating (e.g., substituting, deleting or adding) one or more amino acid or nucleic acid residues to any ofthe isolated SalmoKCaR molecules described herein. Such mutations can be performed using methods described herein and those known in the art.
  • the present invention relates to homologous polypeptide molecules having at least about 70%> (e.g., 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID NO: 8, 10, 12, or 14.
  • Percent “identity” refers to the amount of identical nucleotides or amino acids between two nucleotides or amino acid sequences, respectfully.
  • percent similarity refers to the amount of similar or conservative amino acids between two amino acid sequences.
  • Each ofthe SalmoKCaR polypeptides are homologous to one another.
  • polypeptides of the present invention including the full length sequences, the partial sequences, functional fragments and homologues, that allow for or assist in one or more ofthe following functions (e.g., in Atlantic Salmon): sensing at least one SalmoKCaR modulator in serum or in the surrounding environment; adapting to at least one SalmoKCaR modulator present in the serum or sunounding enviromnent; imprinting with an odorant; altering water intake; altering water abso ⁇ tion; altering urine output.
  • functions e.g., in Atlantic Salmon
  • sense refers to the SalmoKCaR' s ability to alter its expression and/or sensitivity in response to a SalmoKCaR modulator.
  • Homologous polypeptides can be determined using methods known to those of skill in the art. Initial homology searches can be perfomied at NCBI against the GenBank, EMBL and SwissProt databases using, for example, the BLAST network service. Altanner, S.F., et al, J. Mol Biol, 215:403 (1990), Altffler, S.F., Nucleic Acids Res., 25:3389-3402 (1998).
  • nucleotide sequences can be performed using the MOTIFS and the FindPattems subroutines of the Genetics Computing Group (GCG, version 8.0) software. Protein and/or nucleotide comparisons were performed according to Higgins and Sha ⁇ (Higgins, D.G. and Shaip, P.M., Gene, 75:237-244 (1988) e.g., using default parameters).
  • the SalmoKCaR proteins ofthe present invention also encompass biologically active or functional polypeptide fragments ofthe full length SalmoKCaR proteins.
  • Such fragments can include the partial isolated amino acid sequences (SEQ ID NO: 3 and 5), or part ofthe full-length amino acid sequence (SEQ JD NO: 8, 10, 12, or 14), yet possess the function or biological activity ofthe full length sequence.
  • polypeptide fragments comprising deletion mutants ofthe SalmoKCaR proteins can be designed and expressed by well-known laboratory methods. Fragments, homologues, or analogous polypeptides can be evaluated for biological activity, as described herein. hi one embodiment, the function or biological activity relates to preparing salmon for transfer to seawater.
  • the method for preparing Atlantic Salmon for transfer to seawater includes adding at least one SalmoKCaR modulator (e.g., PVCR modulator) to the freshwater, and adding a specially made or modified feed to the freshwater for consumption by the fish.
  • the feed contains a sufficient amount of sodium chloride (NaCl) (e.g., between about 1% and about 10% by weight, or about 10,000 mg/kg to about 100,000 mg/kg) to significantly increase levels ofthe SalmoKCaR modulator in the serum. This amount of NaCl in the feed causes or induces the Atlantic Salmon to drink more freshwater.
  • NaCl sodium chloride
  • the serum r level ofthe SalmoKCaR modulator significantly increases in the salmon, and causes modulated (e.g., increased and/or decreased) SalmoKCaR expression and/or altered SalmoKCaR sensitivity.
  • One function or activity ofthe SalmoKCaR genes is to sense SalmoKCaR modulators in the serum.
  • the SalmoKCaR expression is altered by the SalmoKCaR modulators in the serum, which provides the ability for the salmon to better adapt to seawater, undergo smoltification, survive, grow, consume food and/or to be less susceptible to disease.
  • a “PVCR modulator” or “SalmoKCaR modulator” refers to a compound which modulates (e.g., increases and/or decreases) expression of SalmoKCaR, or alters the sensitivity or responsiveness of SalmoKCaR genes.
  • Such compounds include, but are not limited to, SalmoKCaR agonists (e.g., inorganic polycations, organic polycations and amino acids), Type JJ calcimimetics, and compounds that indirectly alter PVCR expression (e.g., 1,25 dihydroxyvitamin D in concentrations of about 3,000 -10,000 International Units /kg feed), cytokines such as Interleukin Beta, and Macrophage Chemotatic Peptide-1 (MCP-1)).
  • SalmoKCaR agonists e.g., inorganic polycations, organic polycations and amino acids
  • Type JJ calcimimetics e.g., 1,25 dihydroxyvitamin D in concentrations of about 3,000 -10,000 International
  • Type JJ calcimimetics which increase and/or decrease expression, and/or sensitivity ofthe SalmoKCaR genes, are, for example, NPS-R-467 and NPS-R-568 from NPS Pharmaceutical Inc., (Salt Lake, Utah, PatentNos. 5,962,314; 5,763,569; 5,858,684; 5,981,599; 6,001,884) which can be administered in concentrations of between about 0.1 ⁇ M and about 100 ⁇ M feed or water. See Nemeth, E.F. et al, PNAS 95: 4040-4045 (1998).
  • inorganic polycations are divalent cations including calcium at a concenfration between about 2.0 and about 10.0 mM and magnesium at a concentration between about 0.5 and about 10.0 mM; and trivalent cations including, but not limited to, gadolinium (Gd3+) at a concentration between about 1 and about 500 ⁇ M.
  • divalent cations including calcium at a concenfration between about 2.0 and about 10.0 mM and magnesium at a concentration between about 0.5 and about 10.0 mM
  • trivalent cations including, but not limited to, gadolinium (Gd3+) at a concentration between about 1 and about 500 ⁇ M.
  • Organic polycations include, but are not limited to, aminoglycosides such as neomycin or gentamicin in concentrations of between about 1 and about 8 gm/kg feed as well as organic polycations including polyamines (e.g., polyarginine, polylysine, polyhistidine, polyomithine, spermine, spennidine, cadaverine, putrescine, copolymers of poly arginine/histidine, poly lysine/arginine in concentrations of between about 10 ⁇ M and 10 mM feed).
  • polyamines e.g., polyarginine, polylysine, polyhistidine, polyomithine, spermine, spennidine, cadaverine, putrescine, copolymers of poly arginine/histidine, poly lysine/arginine in concentrations of between about 10 ⁇ M and 10 mM feed. See Brown, E.M. et ⁇ , End
  • SalmoKCaR agonists include amino acids such as L-Tryptophan L-Tyrosine, L-Phenylalanine, L- Alanine, L-Serine, L- Arginine, L-Histidine, L-Leucine, L-Isoleucine, L-Aspartic acid, L-Glutamic acid, L-Glycine, L-Lysine, L-Methionine, L- Asparagine, L-Proline, L-Glutamine, L-Threonine, L- Valine, and L-Cysteine at concentrations of between about 1 and about 10 gm/kg feed.
  • amino acids such as L-Tryptophan L-Tyrosine, L-Phenylalanine, L- Alanine, L-Serine, L- Arginine, L-Histidine, L-Leucine, L-Isoleucine, L-Aspartic acid, L-Glutamic acid, L-Glycine, L
  • Amino acids in one embodiment, are also defined as those amino acids that can be sensed by at least one SalmoKCaR in the presence of low levels of extracellular calcium (e.g., between about 1 mM and about 10 mM). In the presence of extracellular calcium, the SalmoKCaR in organs or tissues such as the intestine, pyloric caeca, or kidney can better sense amino acids.
  • the molar concentrations refer to free or ionized concentrations of the SalmoKCaR modulator in the freshwater, and do not include amounts of bound SalmoKCaR modulator (e.g., SahnoKCaR modulator bound to negatively charged particles including glass, proteins, or plastic surfaces). Any combination of these modulators can be added to the water or to the feed (in addition to the NaCl, as described herein), so long as the combination modulates expression and/or sensitivity of one or more of the SalmoKCaR genes.
  • bound SalmoKCaR modulator e.g., SahnoKCaR modulator bound to negatively charged particles including glass, proteins, or plastic surfaces.
  • SalmoKCaR polypeptides Another function ofthe SalmoKCaR polypeptides involves imprinting Atlantic Salmon with an odorant (e.g., an attractant or repellant). Atlantic Salmon can be imprinted with an odorant so that, when the fish are later exposed to the odorant, they can more easily distinguish the odorant or are sensitized to the odorant.
  • the SalmoKCaR polypeptides can work, for example, with one or more olfactory receptors to modify the generation ofthe nerve impulse during sensing of an odorant. Generation of this nerve impulse occurs upon binding of he odorant to the olfactory lamellae in the-fish.
  • the SalmoKCaR modulator alters the olfactory sensing ofthe salmon to the odorant.
  • the presence of a (e.g., at least one) SalmoKCaR modulator in freshwater reversibly reduces or ablates the fish' s ability to sense certain odorants. hi other cases it can be heightened or increased.
  • a SalmoKCaR modulator in freshwater reversibly reduces or ablates the fish' s ability to sense certain odorants. hi other cases it can be heightened or increased.
  • these imprinting methods involve adding at least one SalmoKCaR modulator (e.g., calcium and magnesium) to4he freshwater in .an amount sufficient to modulate expression and/or sensitivity of at least one SalmoKCaR gene; and adding feed for fish consumption to the freshwater.
  • the feed contains at least one an attractant (e.g., alanine); an amount of NaCl sufficient to contribute to a significantly increased level ofthe SalmoKCaR modulator in serum ofthe Atlantic Salmon; and optionally a SalmoKCaR modulator (e.g., tryptophan).
  • the odorant can also be added to the water, instead ofthe feed.
  • Salmon that has been imprinted with an attractant consume more feed having this attractant and, as a result, grow faster.
  • the imprinting process occurs during various developmental stages of salmon including the larval stage and the smoltification stage. Localizations of SalmoKCaR proteins and detection of SalmoKCaR expression using RT-PCR in various organs involved in the imprinting process including olfactory lamellae, olfactory bulb and brain is provided for both larval (Example 13) and smolt stages ( Figures 37 and 38).
  • the process of imprinting the salmon with an odorant refers to creating a lasting effect or impression on the fish so that the fish are sensitized to the odorant or can distinguish the odorant.
  • odorant Being sensitized to the odorant refers to the fish's ability to more easily recognize or recall the odorant. Distinguishing an odorant refers to the fish's ability to differentiate among one or more odorants, or have a preference for one odorant over another.
  • An odorant is a compound that binds to olfactory receptors and causes fish to sense odorants. Generation of an olfactory nerve impulse occurs upon binding ofthe odorant to the olfactory lamellae.
  • a fish odorant is either a fish attractant or fish repellant.
  • a fish attractant is a compound to which fish are attracted. The sensitivity ofthe attractant is modulated, at least in part, by the sensitivity and/or expression of the SalmoKCaR genes in the olfactory apparatus ofthe fish in response to a SalmoKCaR modulator.
  • Examples of at ractants in some fish include amino acids (e.g., L-Tryptophan L-Tyrosine, L-Phenylalanine, L- Alanine, L-Serine, L- Arginine, L-Histidine, L-Leucine, L-Isoleucine, L-Aspartic acid, L-Glutamic acid, L-Glycine, L-Lysine, L-Methionine, L- Asparagine, L-Proline, L-Glutamine, L-Threonine, L- Valine, and L-Cysteine), nucleotides (e.g., inosine monophosphate), organic compounds (e.g., glycine-betaine and trimethylamine oxide), or a combination thereof.
  • amino acids e.g., L-Tryptophan L-Tyrosine, L-Phenylalanine, L- Alanine, L-Serine, L- Arginine, L-H
  • a fish repellant is a compound that fish are repelled by, and the sensitivity ofthe fish to the repellant is altered through expression and/or sensitivity of a SalmoKCaR gene in the olfactory apparatus ofthe fish in the presence of a SalmoKCaR modulator.
  • An example of a repellant is a "finger rinse" which is a mixture of mammalian oils and fatty acids produced by the epidermal cells ofthe skin, and is left behind after human fingers are rinsed with an aqueous solution. Methods for performing a finger rinse is known in the art and is described in more detailed in the Exemplification Section.
  • SalmoKCaR polypeptides includes its ability to sense or adapt to ion concentrations in the sunounding environment.
  • the SalmoKCaR polypeptides sense various SalmoKCaR modulators including calcium, magnesium and/or sodium.
  • the SalmoKCaR polypeptides are modulated by varying ion concentrations. For instance, any one ofthe SalmoKCaR polypeptides can be modulated (e.g., increased or decreased) in response to a change in ion concentration (e.g., calcium, magnesium, or sodium).
  • Responses to changes in ion concentrations of Atlantic Salmon containing the SalmoKCaR polypeptides include the ability 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 abso ⁇ tion. Responses also include changes in biological processes that affect its ability to excrete contaminants.
  • methods are available to regulate salinity tolerance in fish by modulating (e.g., increasing, decreasing or maintaining the expression) the activity of one or more of the S almoKCaR proteins present in cells involved in ion transport.
  • salinity tolerance offish adapted (or acclimated) to freshwater can be increased by activating one or more ofthe SalmoKCaR polypeptides, for example, by increasing the expression of one or more of SalmoKCaR genes, resulting in the secretion of ions and seawater adaption.
  • the salinity tolerance offish adapted to seawater can be decreased by inhibiting one or more ofthe SalmoKCaR proteins, resulting in alterations in the abso ⁇ tion of ions and freshwater adaption.
  • “Salinity” refers to the concentration of various ions in a sunounding aquatic environment, hi 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. Nonnal 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.
  • anadromous fish (Atlantic salmon, trout and Arctic char) as well as euryhaline fish (flounders, alewives, eels) to traverse from freshwater to seawater environments and back again is of key importance to their lifecycles in the natural environment.
  • euryhaline fish (flounders, alewives, eels) to traverse from freshwater to seawater environments and back again is of key importance to their lifecycles in the natural environment.
  • Both types offish have to undergo similar physiological changes including alterations in their urine output, altering water intake and water abso ⁇ tion.
  • Both types of fish utilize environments of either freshwater (Atlantic sahnon) or partial salinity (flounders) to spawn and allow for the development of larval fish into juvenile forms that then undergo changes to migrate into full strength seawater.
  • Both types offish utilize PVCRs to sense when adult fish have arrived in a salinity environment suitable for spawning and to guide their return back to full strength seawater.
  • their resulting offspring utilize PVCRs to control various organs allowing for their normal development in fresh or brackish (partial strength seawater) water and subsequently to regulate the physiological changes that permit these fish to migrate into full strength seawater.
  • the following experiment was done in Summer and Winter Flounder, but is applicable to Atlantic Salmon because both species offish have PVCRs which respond to ion concentrations in a similar manner.
  • Summer and Winter Flounder were adapted to live in 1/lOth seawater (100 mOsm/kg) by reduction in salinity from 450 mM NaCl to 45 mM NaCl over an interval of 8 hrs. Summer and Winter Flounder can be maintained in 1/10 or twice the salinity for over a period of 6 months. After a 10 day interval where the Summer and Winter Flounder 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.
  • the PVCR protein alters the transport of ions across the epithelium ofthe urinary bladder and, in this way, determines the final composition ofthe urine.
  • This composition and the amount of water and NaCl absorbed from the urine are critical to salinity regulation in fish.
  • the invention provides methods to facilitate euryhalme adaptation of fish 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 PVCR protein present in epithelial cells involved in ion transport, as well as in endocrine and nervous tissue.
  • salinity tolerance of fish adapted (or acclimated) to fresh water can be increased by activating the PVCR, for example, by increasing the expression of 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.
  • PVCR activation facilitates excretion of divalent metal ions including calcium and magnesium by renal tubules, hi the gill, PVCR activation reduces reabso ⁇ tion 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, hi the intestine, PVCR activation will pennit reabsoiption of water and ions across the G.I. tract after their ingestion by fish.
  • the salinity tolerance offish adapted to seawater can be deceased by modulating one or more of the SalmoKCaR polypeptides, for example by decreasing the expression of one or more ofthe SalmoKCaR genes while others may be increased. The net result of these changes would be alterations in the abso ⁇ tion of ions that facilitate the adaption to freshwater conditions.
  • Winter and Summer Flounder were maintained in at least twice the normal salinity or 1/10 the normal salinity. See Exemplification. 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 nomial 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 fish in a hypersalinity environment also results in fish with a reduced number of parasites or bacteria.
  • 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).
  • 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 GI tract of summer flounder prior to their sale as product.
  • maintaining fish in a hyposalinity environment results in a fish with a reduced amount of contaminants (e.g., hydrocarbons, amines or antibiotics).
  • the contaminants are reduced to a level that is safe for human consumption, raw or cooked fish. More preferable, the contaminants are reduced to having essentially very little contaminants left in the fish.
  • 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).
  • TMAO trimethylamine oxide
  • SalmoKCaR The presence of SalmoKCaR in brain reflects both its involvement in basic neurofransmitter 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 SalmoKCaR activation in a manner similar to that described for humans where PVCR activation by hypercalcemia in the subfornical r organ ofthe brain cause an increase in water drinking behavior (Brown, E.M. et al, New England J. of Med., 333:234-240 (1995)).
  • Sensing of salinity by PVCR and its modulation ofthe odorant detection system of salmon for detecting various odorants is critical to the achievement of these processes.
  • Data obtained recently from mammals now suggest that PVCR activation plays a pivotal role in coordinating these events.
  • 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 (Neillette, P. A., et al, Gen. and Comp. Physiol, 97:250-258 (1995)).
  • plasma Adrenocorticotrophic Honnone (ACTH) levels that regulate plasma cortisol levels are altered by PVCR activation.
  • the function or biological activity ofthe SalmoKCaR " polypeptide or protein is defined, in one aspect, to mean the osmoregulatory activity of SalmoKCaR protein.
  • Assay techniques to evaluate the biological activity of SalmoKCaR protems and their analogs are described in Brown, et ⁇ l, New Eng. J. Med, 333:243 (1995); Riccardi, et ⁇ l, Proc. Nat. Acad. Sci USA, 92:131-135 (1995); and Sands, et al, J. Clinical Investigation 99:1399-1405 (1997).
  • the biological activity also includes the ability ofthe SalmoKCaR to modulate signal transduction pathways in specific cells.
  • biologically active SalmoKCaR proteins can modulate cellular functions in either an inhibitory or stimulatory mamier.
  • Biologically active derivatives or analogs ofthe above described SalmoKCaR polypeptides, refened to herein as peptide mimetics can be designed and produced by techniques known to those of skill in the art. (see e.g., U.S. Patent ⁇ os. 4,612,132; 5,643,873 and 5,654,276). These mimetics can be based, for example, on a specific SalmoKCaR amino acid sequence and maintain the relative position in space ofthe conesponding amino acid sequence.
  • peptide mimetics possess biological activity similar to the biological activity ofthe conesponding peptide compound, but possess a "biological advantage" over the conesponding SalmoKCaR amino acid sequence with respect to one, or more, of the following properties: solubility, stability and susceptibility to hydrolysis and proteolysis.
  • Methods for preparing peptide mimetics include modifying the N-terminal amino group, the C-terminal carboxyl group, and/or changing one or more ofthe amino linkages in the peptide to a non-amino linkage. Two or more such modifications can be coupled in one peptide mimetic molecule. Modifications of peptides to produce peptide mimetics are described in U.S. Patent Nos. 5,643,873 and 5,654,276.
  • SalmoKCaR polypeptides encompassed by the present invention, include those which are "functionally equivalent.”
  • This term refers to any nucleic acid sequence and its encoded amino acid, wliich mimics the biological activity of the SalmoKCaR polypeptides and/or functional domains thereof.
  • SalmoKCaR Nucleic Acid Sequences. Plasmids, Vectors and Host Cells
  • the present invention in one embodiment, includes an isolated full length nucleic acid molecule having a sequence of SahnoKCaR#l (SEQ JD NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), SalmoKCaR#3 (SEQ JD NO: 11), or
  • SalmoKCaR#4 (SEQ ID NO: 13). See Figures 9, 10, 11, and 12.
  • the present invention includes sequences to the full length SalmoKCaR nucleic acid sequences, as well as the coding regions thereof.
  • the ORF SalmoKCaR#l begins at nt 180 and ends at nt 3005.
  • the ORF SalmoKCaR#2 begins at nt 270 and ends at nt 3095
  • SalmoKCaR#3 the ORF-begins at nt 181 and ends at nt 2733.
  • SahnoKCaR#4 has an OFR that begins at ntl81 and ends at nt 3006.
  • the present invention also encompasses isolated nucleic acid sequences that encode SalmoKCaR polypeptides, and in particular, those which encode a polypeptide molecule having an amino acid sequence of SEQ JD NO: 8, 10, 12, or 14.
  • the SalmoKCaR full length nucleic acid sequences encode polypeptides that allow or assist in one or more ofthe following functions in Atlantic Salmon: sensing at least one SalmoKCaR modulator in serum or in the sunounding environment; adapting to at least one SalmoKCaR modulator present in the serum or surrounding environment; imprinting with an odorant; altering water intake; altering water abso ⁇ tion; or altering urine output.
  • the present invention encompasses the SalmoKCaR full length nucleic acid sequences, SalmoKCaR#l (SEQ JD NO: 7), SalmoKCaR#2 (SEQ JD NO: 9), SalmoKCaR#3 (SEQ JD NO: 11), and SalmoKCaR #4 (SEQ JD NO: 13), or polypeptides encoded by these sequences, which were deposited under the Budapest Treaty with the ATCC, 10801 University Boulevard, Manassas, VA 20110-2209, USA, under Accession Numbers PTA-4190 (March 29, 2002), PTA-4191 (March 29, 2002), PTA-4192 (March 29, 2002), (deposited by MariCal, LLC, 400 Commercial Street, Portland, Maine USA) and (Accession number to be added) (April 9, 2003) (deposited by MariCal, Inc,400 Commercial Street, Portland, Maine USA) respectively.
  • These clones are plasmid DNA which can be transformed into E. Coli and cultured. The viability of he clo
  • an "isolated" gene or nucleotide sequence which is not flanlced by nucleotide sequences which nonnally (e.g., in nature) flank the gene or nucleotide sequence (e.g., as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in a cDNA or RNA library).
  • an isolated gene or nucleotide sequence can include a gene or nucleotide sequence which is synthesized chemically or by recombinant means. Nucleic acid constructs contained in a vector are included in the definition of
  • isolated nucleotide sequences include recombinant r nucleic acid molecules and heterologous host cells, as well as partially or substantially or purified nucleic acid molecules in solution.
  • isolated RNA transcripts ofthe present invention are also encompassed by “isolated” nucleotide sequences.
  • Such isolated nucleotide sequences are useful for the manufacture ofthe encoded SalmoKCaR polypeptide, as probes for isolating homologues sequences (e.g., from other mammalian species or other organisms), for gene mapping (e.g., by in situ hybridization), or for detecting the presence (e.g., by Southern blot analysis) or expression (e.g., by Northern blot analysis) of related genes in cells or tissue.
  • the SalmoKCaR nucleic acid sequences ofthe present invention include homologues nucleic acid sequences.
  • “Analogous” or “homologous” nucleic acid sequences refer to nucleic acid sequences with sufficient identity of any one ofthe SahnoKCaR nucleic acid sequences, such that once encoded into polypeptides, they possess the biological activity of any one ofthe native SalmoKCaR polypeptides.
  • an analogous nucleic acid molecule can be produced with "silent" changes in the sequence wherein one, or more, nt differ from the nt of any one of the SalmoKCaR protein, yet, once encoded into a polypeptide, still possesses the function or biological activity of any one of he native SalmoKCaR. Examples of such differences include additions, deletions or substitutions. Also encompassed by the present invention are nucleic acid sequences that encode analogous polypeptides that exhibit greater, or lesser, biological activity of the SalmoKCaR proteins of the present invention.
  • the present invention is directed to nucleic acid molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity with SEQ ID NO: 7, 9, 11, or 13.
  • Each ofthe SalmoKCaR genes are homologues to - one another.
  • the percent identity for the SalmoKCaR nucleic acid sequences are as follows:
  • nucleic acid molecules ofthe present invention including the full length sequences, the partial sequences, functional fragments and homologues, once encoded into polypeptides, allow for or assist in one or more ofthe following functions in Atlantic Salmon: sensing at least one SalmoKCaR modulator in serum or in the surrounding environment; adapting to at least one SalmoKCaR modulator present in the serum or sunounding environment; imprinting with an odorant; altering water intake; altering water abso ⁇ tion; or altering urine output.
  • the homologous nucleic acid sequences can be determined using methods known to those of skill in the art, and by methods described herein including those described for determining homologous polypeptide sequences.
  • nucleic acid sequences DNA or RNA, which are substantially complementary to the DNA sequences encoding the SalmoKCaR polypeptides and which specifically hybridize with their DNA sequences under conditions of stringency known to those of skill in the art.
  • substantially complementary means that the nucleic acid need not reflect the exact sequence ofthe SalmoKCaR sequences, but must be sufficiently similar in sequence to permit hybridization with SalmoKCaR nucleic acid sequence under high stringency conditions.
  • non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the SalmoKCaR nucleic acid sequence, provided that the sequence has a sufficient number of bases complementary to the SalmoKCaR sequence to allow hybridization therewith.
  • Conditions for stringency are described in e.g., Ausubel, F.M., et al.-, Cunent Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et al, Nature, 366:575 (1993); and further defined in conjunction with certain assays.
  • the SalmoKCaR sequence, or a fragment thereof, can be used as a probe to isolate additional homologues.
  • Nucleic acids encoding SalmoKCaR polypeptides were identified by screening a cDNA library with a SalmoKCaR-specific probe under conditions known to those of skill in the art to identify homologous receptor proteins.
  • the full length sequences were isolated by screening Atlantic Salmon intestinal and kidney cDNA libraries with a probe consisting of a 653 nt PCR amplified genomic sequence (SEQ ID NO: 3).
  • 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.
  • SalmoKCaR genes were isolated by Polymerase Chain Reaction (PCR) of genomic DNA with degenerate primers (SEQ JD NOS: 15 and 16) specific to a highly conserved sequence of calcium receptors that does not contain introns.
  • degenerate primers SEQ JD NOS: 15 and 16
  • partial Atlantic Salmon clones were obtained by using degenerate primers that permit selective amplification of a sequence (nucleotides 2279-2934 of SKCaR) that is highly conserved in both mammalian and shark kidney calcium receptors.
  • SalmoKCaR #4 was isolated using the full length SalmoKCaR#2, SEQ ID NO.:9 as a probe. See Exemplification.
  • the degenerate primers (SEQ ID NOS: 15 and 16) amplify a sequence of 653 base pairs that is present in the extracellular domain of calcium receptors.
  • This 653 nt sequence refers to SEQ JD NO: 3 with the addition ofthe sequence ofthe primers.
  • the resulting amplified 653 bp fragment was ligated into a cloning vector and transfonned into bacterial cells for growth, purification and sequencing.
  • SalfhoKCaR 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.
  • RNA can be isolated from any tissue wliich contains one or more of SalmoKCaR polypeptides by standard methods as described. Prefened 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 SalmoKCaR gene are unique and, thus, can be used as a unique probe to isolate the full-length cDNA from other species. Moreover, in one embodiment, this DNA fragment serves as a basis for specific assay kits for detection of SalmoKCaR expression in various tissues of Atlantic Salmon.
  • nucleic acid sequences genomic DNA, cDNA, RNA or a combination thereof, which are substantially complementary to the DNA sequences encoding SalmoKCaR nucleic acid molecules and wliich specifically hybridize with the SalmoKCaR nucleic acid sequences under conditions of sufficient stringency (e.g., high stringency) to identify DNA sequences with substantial nucleic acid identity.
  • the invention includes nucleic acid sequences that hybridize to the SalmoKCaR sequences, SEQ JD NO: 7, 9, 11, or 13, but not to SEQ ID NO: 1 (SkCaR) or 31-40 (Fugu) under the same conditions.
  • the present invention embodies nucleic acid molecules (e.g., probes or primers) that hybridize to SEQ ID NO: 7, 9, 11, or 13 under high stringency conditions, as defined herein.
  • the present invention includes molecules that hybridize to at least about 200 contiguous nucleotides or longer in length (e.g., 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2S00, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000).
  • Such molecules hybridize to one ofthe SalmoKCaR nucleic acid sequences (SEQ JD NO: 7, 9, 11, or 13) under high stringency conditions.
  • the present invention includes those molecules that hybridize with SalmoKCaR nucleic acid molecules and encode a polypeptide that has the functions or biological activity described herein.
  • the nucleic acid probe comprises a nucleic acid sequence (e.g. SEQ ID NO: 7, 9, 11, or 13) and is of sufficient length and complementarity to specifically hybridize to a nucleic acid sequence that encodes a SalmoKCaR polypeptide.
  • a nucleic acid probe can be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the length ofthe SalmoKCaR nucleic acid sequence.
  • Suitable hybridization conditions e.g., high stringency conditions are also described herein.
  • the present invention encompasses fragments that are biologically active SalmoKCaR polypeptides or nucleic acid sequences that encodes biologically active SalmoKCaR polypeptides, as described further herein. Such fragments are useful as probes for assays described herein, and as experimental tools, or in the case of nucleic acid fragments, as primers.
  • a prefened embodiment includes primers and probes which selectively hybridize to the nucleic acid constructs encoding any one ofthe SalmoKCaR proteins.
  • nucleic acid fragments which encode any one ofthe domains described herein are also implicated by the present invention.
  • Stringency conditions for hybridization refers to conditions of temperature and buffer composition which pennit 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 which 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 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 occunence 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.
  • T m melting temperature
  • 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.
  • the T m in °C (81.5°C + 16.6(log 10 M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where "M” is the molarity of monovalent cations (e.g., Na 4 ), and "L” is the length ofthe hybrid r in base pairs.
  • the T m in °C (81.5°C + 16.6(log ] 0 M) + 0.41(% G + C) - 0.61 (% fonnamide) - 500/L), where "M” is the molarity of monovalent cations (e.g., Na 4 ), and "L” is the length ofthe hybrid in base pairs.
  • the T m in °C (81.5°C + 16.6(log 10 M) + 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 SalmoKCaR nucleic acid sequence, or a fragment thereof, can also be used to isolate additional aquatic PVCR homologs.
  • a cDNA or genomic DNA library from the appropriate organism can be screened with labeled SalmoKCaR nucleic acid sequence to identify homologous genes as described in e.g. , Ausebel, et al. , Eds., Current Protocols In Molecular Biology, John Wiley & Sons, New York (1997).
  • the present invention pertains to a method of isolating a SalmoKCaR nucleic acid comprising contacting an isolated nucleic acid with a SalmoKCaR -specific hybridization probe and identifying an aquatic PVCR.
  • the present method can optionally include a labeled SalmoKCaR probe.
  • the invention also provides vectors, plasmids or virases containing one or _ more ofthe SalmoKCaR nucleic acid molecules.
  • Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F.M., et ah, Cunent Protocols in Molecular Biology, (Cunent r
  • Uses of plasmids, vectors or virases containing the cloned SalmoKCaR receptors or receptor fragments include one or more ofthe following; (1) generation of hybridization probes for detection and measuring level of SalmoKCaR in tissue or isolation of SalmoKCaR homologs; (2) generation of SalmoKCaR mRNA or protein in vitro or in vivo; and (3) generation of transgenic non-human animals or recombinant host cells.
  • the present invention encompasses host cells transformed with the plasmids, vectors or viruses described above. Nucleic acid molecules can be inserted into a construct which can, optionally, replicate and/or integrate into a recombinant host cell, by known methods.
  • the host cell can be a eukaryote or prokaryote and includes, for example, yeast (such as Pichia pastorius or Saccharomyces cerevisiae), bacteria (such as E. coli or Bacillus subtilis), animal cells or tissue, insect Sf9 cells (such as baculoviruses infected SF9 cells) or mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey COS-7 cells).
  • Host cells suitable in the present invention also include a fish cell, a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell.
  • the nucleic acid molecule can be inco ⁇ orated or inserted into the host cell by known methods. Examples of suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinj ection, infection, lipofection and direct uptake. "Transformation” or “transfection” as used herein refers to the acquisition of new or altered genetic features by inco ⁇ oration of additional nucleic acids, e.g., DNA. "Expression” ofthe genetic infonnation of a host cell is a tenn of art which refers to the directed transcription of DNA to generate RNA which is translated into a polypeptide.
  • the host cell is then maintained under suitable conditions for expression and recovery of SalmoKCaR protein.
  • the cells are maintained in a suitable buffer and/or growth medium or nutrient source for growth ofthe cells and expression ofthe gene product(s).
  • the growth media are not critical to the invention, are generally known in the art and include sources of carbon, nitrogen and sulfur. Examples include Luria broth, Superbroth, Dulbecco's Modified Eagles Media (DMEM), RPMI- 1640, Ml 99 and Grace's insect media.
  • the growth media can contain a buffer, the selection of which is not critical to the invention.
  • the pH ofthe buffered Media can be selected and is generally one tolerated by or optimal for growth for the host cell.
  • the host cell is maintained under a suitable temperature and atmosphere.
  • the host cell is aerobic and the host cell is maintained under atmospheric conditions or other suitable conditions for growth.
  • the temperature should also be selected so that the host cell tolerates the process and can be for example, between about 13°-40°C.
  • the present invention includes methods of detecting the levels ofthe SalmoKCaR nucleic acid levels (mRNA levels) and/or polypeptide levels to determine whether fish are ready for transfer from freshwater to seawater.
  • the present invention also includes methods for assaying compounds that modulate SahnoKCaR nucleic acid levels, expression levels or activity of SalmoKCaR polypeptides.
  • SalmoKCaR polypeptides includes, but is not limited to, phosphorylation of one or more ofthe SalmoKCaR polypeptides, dimerization of one ofthe SalmoKCaR polypeptides with a second SalmoKCaR polypeptide, proteolysis of one or more ofthe SalmoKCaR polypeptides, and/or increase or decrease in the intracellular signal transduction system or pathway of one or more of the SalmoKCaR polypeptides.
  • the present invention also includes assaying activities, as known in the art Methods that measure SahnoKCaR levels include several suitable assays.
  • Suitable assays encompass immunological methods, such as FACS analysis, radioimmunoassay, flow cytometry, immunocytochemistiy, enzyme-linked immunosorbent assays (ELISA) and chemiluminescence assays.
  • antibodies, or antibody fragments can also be used to detect the presence of SalmoKCaR 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 SalmoKCaR protein levels. Antibodies could be used in a kit to monitor the SalmoKCaR protein level offish in aquaculture.
  • Antibodies reactive with any one of the S almoKCaR or portions thereof can be used.
  • the antibodies specifically bind with SalmoKCaR polypeptides or a portion thereof.
  • the antibodies can be polyclonal or monoclonal, and the tenn antibody is intended to encompass polyclonal and monoclonal antibodies, and functional fragments thereof.
  • 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.
  • immunological techniques detect SalmoKCaR levels by means of an anti-SalmoKCaR antibody (i.e., one or more antibodies).
  • an anti-SalmoKCaR antibody i.e., one or more antibodies.
  • anti-SalmoKCaR antibody includes monoclonal and/or polyclonal antibodies, and mixtures thereof.
  • Anti-SalmoKCaR antibodies can be raised against appropriate immunogens, such as isolated and/or recombinant SalmoKCaR, analogs or portion thereof (including synthetic molecules, such as synthetic peptides).
  • antibodies are raised against an isolated and/or recombinant SalmoKCaR or portion thereof (e.g., a peptide) or against a host cell which expresses recombinant SalmoKCaR.
  • cells expressing recombinant SalmoKCaR, 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) and Eur. J. Immunol. 6:
  • 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. Such methods include immunization of various lifestages of Atlantic salmon to produce antibodies to native PVCR proteins and thereby alter their function or specificity.
  • an assay can determine the level of SalmoKCaR in a biological sample.
  • an assay includes combining the sample to be tested with an antibody having specificity for the SalmoKCaR, under conditions suitable for formation of a complex between antibody and the SalmoKCaR, and detecting or measuring (directly or indirectly) the fonnation 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.
  • tissue samples e.g., gill tissue samples
  • 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 conesponding to the osmolality ofthe fish, washed in Ringers, then frozen in an embedding compound, e.g., O.C.T.TM (Miles, Inc., Elkahart, Indiana, USA) using methylbutane cooled with liquid nitrogen.
  • O.C.T.TM Miles, Inc., Elkahart, Indiana, USA
  • sections are: 1) blocked with goat serum or serum obtained from the same species offish, 2) incubated with rabbit anti-CaR or anti-SalmoKCaR 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 SalmoKCaR protein is raised in rabbits using a 23-mer peptide conesponding 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. : 26)
  • a small peptide with the sequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO.: 27) or ARSRNSADGRSGDDLPC (SEQ ID NO.: 28) can also be used to make antisera containing antibodies to SalmoKCaRs.
  • Such antibodies can be monoclonal, polyclonal or chimeric.
  • Suitable labels can be detected directly, such as radioactive, fluorescent or 5 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 2 P, 125 I, L enzyme labels such as horseradish peroxidase, and alkaline phosphatase, 0 ⁇ -galactosidase, biotin, avidin, spin labels, magnetic beads 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).
  • a control refers to a level of SahnoKCaR, if any, from a fish that is not subjected to the steps ofthe present invention, e.g., not subjected to freshwater having a SalmoKCaR modulator and/or not fed a NaCl diet.
  • Figures 21 and 22 show that fish not subjected to the present invention had no detectable SalmoKCaR level, whereas fish that were subjected to the steps ofthe invention had SalmoKCaR levels that were easily detected.
  • a signal transduction pathway is a pathway involved in the sensing and/or processing of stimuli. In particular, such pathways are altered by activation ofthe expressed proteins coded for by a single or combination of nucleic acids ofthe present invention.
  • the SalmoKCaR polypeptides can be in the form of a conjugate or a fusion protein, which can be manufactured by known methods. Fusion proteins can be manufactured according to known methods of recombinant DNA technology. For example, fusion proteins can be expressed from a nucleic acid molecule compnsing sequences which code for a biologically active portion ofthe SalmoKCaR polypeptide and its fusion partner, for example a portion of an immunoglobulin molecule. For example, some embodiments can be produced by the intersection of a nucleic acid encoding immunoglobulin sequences into a suitable expression vector, phage vector, or other commercially available vectors. The resulting construct can be introduced into a suitable host cell for expression.
  • the fusion proteins can be isolated or purified from a cell by means of an affinity matrix.
  • an affinity matrix By measurement ofthe alternations in the functions of transfected cells occuning as a result of expression of recombinant SalmoKCaR proteins, either the cells themselves or SalmoKCaR proteins produced from the cells can be utilized in a variety of screening assays that all have a high degree of utility over screening methods involving tests on the same PVCR proteins in whole fish.
  • the SalmoKCaRs 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 PVCR expression is in gill tissue, followed by the kidney and the rectal gland.
  • the SalmoKCaRs can also be assayed by hybridization, e.g., by hybridizing one ofthe SalmoKCaR sequences provided herein (e.g., SEQ ID NO: 7,9, 11, or 13) or an oligonucleotide derived from one ofthe sequences, to a DNA or RNA- containing tissue sample from a fish.
  • a hybridization sequence can have a detectable label, e.g., radioactive, fluorescent, etc., attached to allow the detection of hybridization product.
  • 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). Additionally, the above probes could be used in a kit to identify SalmoKCaR homologs and their expression in various fish tissue.
  • the present invention also encompasses the isolation of SalmoKCaR homologs and their expression in various fish tissues with a kit containing primers specific for conserved sequences of SalmoKCaR nucleic acids and proteins.
  • the present invention encompasses detection of SalmoKCaRs with PCR methods using primers disclosed or derived from sequences described herein.
  • SalmoKCaRs can be detected by PCR using SEQ ID Nos: 15 and 16, as described in Example 6.
  • 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.
  • the levels of SalmoKCaR nucleic acid can be determined in various tissues by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) after isolation of poly A+ RNA from aquatic species.
  • RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
  • 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.
  • mRNA is extracted from the tissue of interest and reverse transcribed.
  • a PCR reaction is performed with SalmoKCaR-specific primers and the presence ofthe predicted SahnoKCaR product is determined, for example, by agarose gel electrophoresis.
  • Examples of SalmoKCaR-specific primers are SEQ ID NO: 18-23.
  • the product ofthe RT-PCR reaction that is perfonned with SalmoKCaR-specific primers is refened to herein as a RT-PCR product.
  • the RT-PCR product can include nucleic acid molecules having part or all ofthe SalmoKCaR sequence.
  • the RT-PCR product can optionally be radioactively labeled and the presence or amount of SahnoKCaR product can be determined using autoradiography.
  • RNA can be isolated from any tissue which contains at least one SalmoKCaR by standard methods. Such tissues include, for example, gill, nasal lamellae, urinary bladder, kidney, intestine, stomach, liver and brain.
  • kits for the detection of SalmoKCaR or the quantification of SalmoKCaR having either antibodies specific for SalmoKCaR or a portion thereof, or a nucleic acid sequence that can hybridize to the nucleic acid ofSalmoKCaR.
  • Suitable transgenes would include either the SalmoKCaR genes itself or modifier genes that would directly or indirectly influence SalmoKCaR 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).
  • Process I is also refened to herein as "SUPERSMOLT TM I Process” or "APS Process I.”
  • a "Process I” fish or smolt refers to a fish or smolt that has undergone the steps of Process I.
  • a Process I smolt is also refened to as a "SUPERSMOLT TM I” or an "APS Process I " smolt.
  • Process II is also refened to herein as "SUPERSMOLT TM JJ Process” or “Process II.”
  • a “Process II” fish or smolt refers to a fish or smolt that has undergone the steps of Process JJ.
  • a Process JJ smolt is also refened to as a "SUPERSMOLT TM H" or an "APS Process II " smolt.
  • Process I Pre-adult anadromous fish (this includes both commercially produced SO, SI or S2 smolts as well as smaller pan/smolt fish) are exposed to or maintained in freshwater containing either 2.0-10.0 mM Calcium and 0.5-10.0 mM Magnesium ions. This water is prepared by addition of calcium carbonate and/or chloride and magnesium chloride to the freshwater. Fish are fed with feed pellets containing 7% (weight/weight) NaCl. Fish are exposed to or maintained in this regimen of water mixture and feed for a total of 30-45 days, using standard hatchery care techniques. Water temperatures vary between 10-16°C. Fish are exposed to a constant photoperiod for the duration of Process I. A fluorescent light is used for the photoperiod.
  • Process IJ Pre-adult anadromous fish (this includes both commercially produced SO, SI or S2 smolts as well as smaller pan/smolt fish) are exposed to or maintained in freshwater containing 2.0-10.0 mM Calcium and 0.5-10.0 mM Magnesium ions. This water is prepared by addition of calcium carbonate and/or chloride and magnesium chloride to the freshwater. Fish are fed with feed pellets containing 7% (weight/weight) NaCl and either 2 gm or 4 gm of L-Tryptophan per kg of feed. Fish are exposed to or maintained in this regimen of water mixture and feed for a total of 30-45 days using standard hatchery care techniques. Water temperatures vary between 10-16°C Fish are exposed to a constant photoperiod for the duration of Process II. A fluorescent light is used for the photoperiod.
  • EXAMPLE 1 MOLECULAR CLONING OF SHARK KIDNEY CALCIUM RECEPTOR RELATED PROTEIN (SKCaR)
  • a shark ⁇ ZAP cDNA library was manufactured using standard commercially available reagents with cDNA synthesized from poly A+ RNA isolated from shark kidney tissue as described and published in Siner et al. Am. J. Physiol. 270:C372-C381, 1996.
  • the shark cDNA library was plated and resulting phage plaques screened using a 32 P-labeled full length rat kidney CaR (RaKCaR) cDNA probe under intermediate stringency conditions (0.5X SSC, 0.1% SDS, 50°C).
  • PVCRs serve as salinity sensors hi fish. These receptors are localized to the apical membranes of various cells within the fish's body (e.g., in the gills, intestine, kidney) that are known to be responsible for osmoregulation.
  • a full-length cation receptor (CaR, also refened to as "PVCR”) from the dogfish shark has been expressed in human HEK cells. This receptor was shown to respond to alterations in ionic compositions of NaCl, Ca2+ and Mg2+ in extracellular fluid bathing the HEK cells. The ionic concentrations encompassed the range which includes the transition from freshwater to seawater.
  • PVCR mRNA expression is also increased in fish after their transfer from freshwater to seawater, and is modulated by PVCR agonists.
  • Partial genomic clones of PVCRs have also been isolated from other fish species, including winter and summer flounder and lurnpfish, by using nucleic acid amplification with degenerate primers. hi particular, the following was shown:
  • SKCaR encodes a functional ion receptor that is sensitive to both g2+ and Ca2+ as well as alterations in NaCl.
  • SKCaR's sensitivity to Ca2+, Mg2+ and NaCl occur in the range that is found in marine environments and is consistent with SKCaRs role as a salinity sensor.
  • SKCaR' s sensitivity to Mg2+ is further modulated by Ca2+ such that SKCaR is capable to sensing various combinations of divalent and monovalent cations in seawater and freshwater.
  • SKCaR cDNA was ligated into the mammalian expression vector PCDNA JJ and transfected into HEK cells using standard techniques. The presence of SKCaR protein in transfeeted cells was verified by western blotting.
  • Activation of SKCaR by extracellular Ca2+, Mg2+ or NaCl was quantified using a well characterized FURA 2 based assay where increases in intracellular Ca2+ produced by SKCaR activation are detected using methodology published previously by Bai, M. , S . Quinn, S. Trvedi, O. Kifor, S.H.S. Pearce, M.R. Pollack, K. Krapcho, S.C. Hebert and E.M. Brown. Expression and characterization of inactivating and activating mutations in the human Ca2+-sensing receptor. J. Biol. Chem., 32:19537-19545 (1996); and expressed as % normalized intracellular calcium response to receptor activation.
  • SKCaR is a functional extracellular Ca2+ sensor where its sensitivity is modulated by alterations in extracellular NaCl concentrations. As shown in Figure 2, SKCaR is activated by increasing concentrations of extracellular Ca2+ where half maximal activation of SKCaR ranges between 1-15 mM depending on the extracellular concentration of NaCl. These are the exact ranges of Ca2+ (1-10 mM present in marine estuarian areas). Note that increasing concentrations of NaCl reduce the sensitivity of SKCaR to Ca2+. This alteration in SKCaR sensitivity to Ca2+ was not observed after addition of an amount of sucrose sufficient to alter the osmolality ofthe extracellular medium. This control experiment shows it is not alterations in cell osmolality effecting the changes observed.
  • the half maximal activation (EC 50 ) by Ca2+ for SKCaR is reduced in increased concentrations of extracellular NaCl. See Figure 4.
  • the EC 50 for data shown on Figure 4 is displayed as a function of increasing extracellular NaCl concentrations. Note the EC 50 for Ca2+ increases from less than 5 mM to approximately 18 mM as extracellular NaCl concentrations increase from 50 mM to 550 mM.
  • SKCaR is a functional extracellular Mg2+ sensor where its sensitivity is modulated by alterations in extracellular NaCl concentrations. As shown in Figure 3, SKCaR is activated in the range of 5-40 mM extracellular Mg2+ and is modulated in a mamier similar to that shown in Figures 2 and 4 by increasing concentrations of extracellular NaCl. Similarly, this alteration in SKCaR sensitivity to Ca2+ was not observed after addition of an amount of sucrose sufficient to alter the osmolality of the extracellular medium.
  • the half maximal activation (EC 50 ) by Mg2+ for SKCaR is reduced in increased concentrations of extracellular NaCl. See Figure 5.
  • the EC 50 for data shown on Figure 5 is displayed as a function of increasing extracellular NaCl concentrations. Note the EC 50 for Mg2+ increases from less than 20 mM to approximately 80 mM as extracellular NaCl concentrations increase from 50mM to 550mM.
  • 3mM Ca2+ to the extracellular solution alters the sensitivity characteristics of SKCaR as shown. Note the 3mM Ca2+ increases the sensitivity of SKCaR to Mg2+ as a function of extracellular NaCl concentrations. This method was also used to isolate partial genomic clones of PVCRs for
  • FIGS 19 A-D show the amino acid sequences and alignment for the PVCRs from four full length Atlantic salmon clones (SalmKCar #1, #2, #3, and #4) relative to the PVCR from the kidney o the dogfish shark (Squalus acanthias) (SKCaR) and human parathyroid calcium receptor (HuPCaR).
  • EXAMPLE 3 DEFINING SALINITY LIMITS AS AN ASSAY TO IDENTIFY FISH WITH ENHANCED SALINITY RESPONSIVE AND ALTERED PVCR FUNCTION.
  • Anadromous fish Atlantic salmon, trout and Arctic char
  • euryhaline fish flounders, alewives, eels
  • both types of fish have to undergo similar physiological changes including alterations in their urine output, altering water intake and water abso ⁇ tion.
  • naturally occurring mutations to PVCR would provide for altered salinity adaptation capabilities that would have significant value for both commercial and environmental restoration uses.
  • identification of selective traits associated with PVCR mediated salinity responses might allow identification of new strains offish for commercial aquaculture.
  • identification of selected environmental parameters from a host of natural and man made variables that are the most important to improve the survival and successful restocking and/or ocean ranching of either wild Atlantic salmon or winter flounder would also be of great utility.
  • assays must be designed that enable these fish to survive while others not possessing these characteristics will either die or perform poorly. As described below, such assays would take advantage ofthe ability of these anadromous and euryhaline fish to withstand a wide range of salimties. Fish that were identified using such assays would then be propagated in breeding-selection programs.
  • 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 M 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.
  • 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).
  • LS low salinity
  • NS nonnal seawater
  • HS hypersalinity
  • a partial PVCR gene of Atlantic Salmon was isolated as follows: sequences of shark kidney calcium re ⁇ eptor together with the nucleotide sequence of mammalian calcium receptors were used to design degenerate oligonucleotide primers, dSK-F3 (SEQ JD NO: 15) and dSK-R4 (SEQ JD NO: 16), to highly conserved regions in the transmembrane 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, pages 303-312, 1993).
  • genomic DNA from the above species was amplified using standard PCR methodology.
  • the PCR product (653 nt) was then purified by agarose gel electrophoresis and ligated into appropriate plasmid vector that was then 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.
  • a total of 8 nucleotide sequences from 8 fish species including Atlantic Salmon were amplified. Each clone is 594 nt (with-out primer sequences) and encodes a 197 amino acid sequence which conesponds to the conserved transmembrane domain ofthe calcium receptors.
  • Atlantic sahnon partial PVCR nucleic acid sequence (SEQ ID NO: 3) is composed of 594 nucleotides (nt) containing an open reading frame encoding 197 amino acids (SEQ ID NO: 4) ( Figure 7).
  • EXAMPLE 5 MOLECULAR CLONING OF A SECOND PARTIAL ATLANTIC SALMON PVCR A second Atlantic salmon partial PVCR was isolated, as described herein.
  • An Atlantic salmon ⁇ ZAP cDNA library was manufactured using standard commercially available reagents with cDNA synthesized from poly A+ RNA isolated from Atlantic salmon intestine tissue according to manufacturers instructions (Stratagene, La JoUa, CA) and screened using the Atlantic salmon PCR product as a probe.
  • a partial Atlantic sahnon PVCR cDNA (SEQ JD NO: 5) is composed of 2021 nucleotides (nt) ( Figure 8 A) containing an open reading frame encoding 388 amino acids (SEQ ID NO: 6) ( Figure 8B). The open reading frame encoded by SEQ JD NO: 5 begins at nucleotide position 87.
  • EXAMPLE 6 MOLECULAR CLONING OF 4 FULL LENGTH CDNA CLONES FROM KIDNEY OF ATLANTIC SALMON (S ALMO S ALAR) AND
  • Example 5 a homology based approach was used to screen cDNA libraries under moderate stringency conditions to obtain a full length shark kidney PVCR clone (SKCaR).
  • SKCaR shark kidney PVCR clone
  • nt nucleotide
  • antibody probes were designed to detect PVCRs in other fish species.
  • PCR was utilized to amplify a series of genomic and cDNA (RT-PCR) sequences that contain partial nt and putative protein sequences of PVCRs from multiple fish including Atlantic salmon. See Examples 1, 4, and 5.
  • SalmoKCaR#1.2 A total of seven cDNA clones containing PVCR sequence were identified and purified from Atlantic Salmon kidney and intestine libraries. A total of three of the seven contain full length coding sequences for PVCR proteins together with 5 ' and 3' regulatory elements. For convenience, these clones are designated Salmo salar Kidney PVCRs (SalmoKCaRs) # 1, # 2 and 3 and their aligned nt and putative protein sequences are shown in Figures 9-11, respectively. The remaining 4 positive clones were partial PVCR clones very nearly identical to these 3 full-length SalmoKCaR clones.
  • SalrnoKCaR#4 This clone was designated as SalrnoKCaR#4, commensurate with the naming ofthe SalmoKCaR#l-3 clones, above.
  • the aligned nt and putative protein sequence for SalmoKCaR #4 is shown in Figure 12. Comparison ofthe different nt sequences of these 4 clones reveals the following similarities and differences:
  • SalmoKCaR H nucleic acid sequence (SEQ ID NO: 7) consists of 3941 nts of 5' and 3' regulatory elements together with full-length coding sequence for a 941 AA PVCR protein (SEQ ID NO: 8). See Figures 9 A-E.
  • the calculated molecular mass of this protein is 106,125 Daltons.
  • the SalmoKCaR # 2 nucleic acid sequence (SEQ TD NO: 9) consists of 4031 nts of 5' and 3' regulatory elements together with full-length coding sequence for a 941 AA PVCR protein (SEQ JD NO: 10). See Figures 10A- E. The calculated molecular mass of this protein is 106, 180 Daltons.
  • SalmoKCaR # 3 nucleic acid sequence (SEQ JD NO: 11) consists of 3824 nts of 5' and 3' regulatory elements together with full-length coding sequence for a 850 AA PVCR protein (SEQ ID NO: 12). See Figures 11 A-
  • the calculated molecular mass of this protein is 96,538 Daltons.
  • the SalmoKCaR#4 nucleic acid sequence (SEQ ID NO: 13) consists of
  • Figures 13A-L and 14A-C show an alignment of between the two partial sequences of Atlantic Salmon PVCRs isolated and 3 full length clones, SalmoKCaR #1-3, for both the nucleic acid and amino acid sequences, respectively.
  • One partial nucleic acid sequence of an Atlantic Salmon PVCR, SEQ ID NO: 3 can be found in all three SalmoKCaR nucleic acid sequences between nt 1979 and 2572; nt 2069 and 2662; and nt 1980 and 2573 of SEQ ID NO: 7, 9, and 11, respectively.
  • the second partial Atlantic Salmon clone, SEQ JD NO: 5 can also be found in all three
  • SalmoKCaR nucleic acid sequences between nt 1753 and 3773; 1843 and 3863, and 1754 and 3616 of SEQ JD NO: 7, 9, and 11, respectively.
  • amino acid sequence of SEQ ID NO: 4 is found between aa 601 and 797 of each of SEQ ID NO: 8, 10, and 12.
  • the amino acid sequence ofthe second Atlantic Salmon Clone, SEQ ID NO: 6, is found in each ofthe polypeptides: between aa 554 and 941 of SEQ ID NO: 8; between aa 554 and 941 of SEQ ID NO: 10; and between aa 554 and 850 of SEQ ID NO: 12.
  • amino acid sequence of SEQ JD NO: 6 extends 91 aa past the end of SEQ ID NO: 12.
  • Figures 17 and 18 show an alignment of between the two partial sequences of Atlantic Salmon PVCRs isolated and all 4 full length clones, SalmoKCaR #1-4, for both the nucleic acid and amino acid sequences, respectively.
  • One partial nucleic acid sequence of an Atlantic Salmon PVCR, SEQ ID NO: 3, can be found from 1980 to 2573 of SalmoKCaR#4, SEQ JD NO: 13.
  • the second partial Atlantic Salmon clone, SEQ JD NO: 5 can also be found in the SalmoKCaR#4 nucleic acid sequence: between nt 1754 and 3774.
  • the amino acid sequence of SEQ ID NO: 4 is found between aa 601 and 797 of SEQ JD NO: 14.
  • the amino acid sequence ofthe second Atlantic Salmon Clone, SEQ JD NO: 6, is found between aa 554 and 941 of SEQ JD NO: 14.
  • nt 1-112 do not align with any conesponding sequence in SEQ JD NO: 7, 9, or 11.
  • Table 1 compares the overall % identity of nucleotides (nt) between cDNA clones that contain the SalmoKCaRs #1,2, 3, and 4 vs. shark kidney calcium receptor (SKCaR containing 4079 nts) or human parathyroid CaR (HuPCaR containing 3783 nts). Note that all 4 SalmoKCaR clones possess approximately a 56-57% nt identity to SKCaR and an approximately 50-55%> nt identity to HuPCaR.
  • a total of 99.8% ofthe nt of SEQ JD NO: 7 are identical to those of conesponding SEQ JD NO: 9.
  • a total of 97.6% ofthe nt of SEQ JD NO: 9 are identical to those conesponding nt of SEQ ID NO: 7.
  • a total of 93.6% ofthe nt of SEQ JD NO: 9 are identical to those conesponding nt of SEQ JD NO: 11.
  • a total of 98.7% ofthe nt of SEQ ID NO: 11 are identical to the conesponding nt present in SEQ ID NO: 9.
  • a total of 95.8% ofthe nt of SEQ JD NO: 7 are identical to the conesponding nt of SEQ JD NO: 11.
  • a total of 98.7% ofthe nt of SEQ JD NO: 11 are identical to those conesponding in SEQ ID NO: 7.
  • a total of 99% ofthe nt of SEQ ID NO: 7 are identical to conesponding nt of SEQ ID NO: 13.
  • a total of 98% ofthe nt of SEQ ID NO: 9 are identical to conesponding nt of SEQ ID NO: 13.
  • a total of 97% of nt of SEQ ED NO: 13 are identical to conesponding nt of SEQ ID NO: 11.
  • Table 1 Comparison ofthe % nucleotide (nt) identity ofthe complete nt sequence of SalmoKCaR clones #1, #2, #3, and #4 (including 5' and 3' regulatory elements) vs. r either the SKCaR clone or the clone HuPCaR clone.
  • Table 2 compares both the overall and domain-specific percent amino acid (% AA) identity for each ofthe SalmoKCaR clones vs. shark kidney PVCR (SKCaR-upper half) and human parathyroid CaR (HuPCaR-lower half).
  • % AA percent amino acid identity
  • the percentage identities between the aligned amino acid sequences ofthe 4 full length SalmoKCaR clones include:
  • a total of 99.9% ofthe aa of SEQ ID NO: 8 are identical to those conesponding aas in SEQ ID NO: 10.
  • a total of 99.9% ofthe aa of SEQ ID NO: 10 are identical to conesponding aa in SEQ ID NO: 8.
  • a total of 89.5% ofthe aa of SEQ JD NO: 10 are identical to those conesponding aas in SEQ ID NO: 12.
  • a total of 99.1% ofthe aa of SEQ ID NO: 12 are identical to those conesponding aas in SEQ ID NO: 12.
  • a total of 89.6% ofthe aa of SEQ JD NO: 8 are identical to those conesponding aas in SEQ JD NO: 12.
  • a total of 99.2% ofthe aa of SEQ JD NO: 12 are identical to those conesponding aa of SEQ JD NO: 8.
  • a total of 100% ofthe aa of SEQ JD NO: 8 are identical to those conesponding aa's in SEQ TD NO: 14.
  • a total of 99.9% ofthe aa of SEQ ID NO: 10 are identical to those conesponding aa's in SEQ ID NO: 14.
  • a total of 99.2% of the aa of SEQ ID NO: 12 are identical to those conesponding aa's in SEQ ID NO: 14.
  • Table 2 Comparison of % amino acid (AA) identities of 4 SalmoKCaR proteins vs. AA sequence of shark kidney CaR (SKCaR-Upper Half) and human parathyroid CaR (HuPCaR -Lower Half).
  • Figure 15A shows unique SalmoKCaR#l,2, and 3 clones hybridize to full length shark kidney CaR (SKCaR) under high stringency conditions (0.5XSSC, 0.1% SDS @ 65° C). Representative autoradiogram of Southern blot was exposed for 30 min. Since the SalmoKCaR nt sequence, SEQ ID NO:l, is 97-99% identical to SalmoKCaR's #1, 2, and 3, and so it also would be expected to hybridize under these conditions. Site directed mutagenesis studies of mammalian CaRs, notably HuPCaR, have identified AAs that are particularly important in the various functions of CaRs.
  • Cysteine AAs at AA#101 and AA#236 mediate dimerization of HuPCaR.
  • HuPCaR and native CaRs in rat kidney exist primarily as dimers within the cell membrane where disulfide bond-mediated dimerization is required for nonnal agonist-mediated CaR activation.
  • SalmoKCaR#l-4 possess Cys at AAs conesponding to HuPCaR AA#101 and AA#236 and presumably functions as dimers in a manner similar to mammalian CaRs.
  • FIG. 17 displays the aligned nucleotide sequences of SalmoKCaR #1-4.
  • SalmoKCaR *'2 possesses an 89 nt insert in its 5' UTR.
  • SalmoKCaR#4 does not contain the 89 nt insert in the 5' UTR that SalmoKCaR# 2 contains. It does not share the 158 nt deletion in the ORF of SalmoKCaR# 3. However, it does share a 39 nt insertion in the 3' UTR just prior to the poly A tail with SalmoKCaR# 3. This insertion is likely a regulatory element.
  • SalmoKCaR #4 looks as though it would have the same structure and function as SalmoKCaR #1, but would be co-regulated with SalmoKCaR 3. Differences between the 3' UTRs ofthe 4 SalmoKCaRs include a 36 nt insert just prior to the poly A tail in SalmoKCaR # 3 as well as other single nt differences listed below where each difference is compared to the 3 other SalmoKCaR clones:
  • SalmoKCaR *1 nt 3660 A to G; nt 3739 A to G; nt 3745 A to G SalmoKCaR # 2: nt 1039 A to G; nt 3837 A to G; nt 3862 A to G SalmoKCaR # 3: nt 1462 C to T; nt 3472 A to G; nt 3487 A to G; nt 3564 A to G; nt 3568 G to A; nt 3603 A to G; nt 3786 A to C.
  • SalmoKCaR H nt 3634 A to G; nt 3645 A to G; nt 3661 A to G, nt
  • each nt difference either individually or in combinations could represent a means for controlling either the stability or processing ofthe RNA transcript or its translation into each of the 4 SalmoKCaR proteins.
  • FIG 19 displays the aligned AA sequences of SalmoKCaRs #1, #2, #3, and #4 as well as the Shark SKCaR protein and HuPCaR proteins.
  • SalmoKCaR # 2 Compared to SalmoKCaR # 1 and #4, SalmoKCaR # 2 possesses 2 different AA's present at AA#257 and AA#941 of its AA sequence.
  • SalmoKCaR # 1 and #4 that possesses an Asp in AA#257, SalmoKCaR *2 possesses a Gly.
  • the negative charge in this location may be important since both SKCaR and Fugu PVCR possess Asp at #257 while the mammalian CaRs, HuPCaR and RaKCaR possess a Glu.
  • SalmoKCaR #3 also contains a Asp at AA#257.
  • SalmoKCaR # 1, 2 and #4 both possess a Leu whereas SalmoKCaR # 3 contains a Phe.
  • the conserved hydrophobic nature ofthe AA at this position appears to be important since Fugu PVCR also contains a Leu whereas SKCaR contains an He.
  • SalmoKCaRs # 1, 2, or #4 SalmoKCaR # 3 possesses a truncated carboxyl terminus as described below.
  • SalmoKCaR species While many ofthe differences between SalmoKCaR species and HuPCaR are conserved substitutions that preserve the overall net charge or hydrophobicity characteristics at that specific position in the PVCR protein, other substitutions may have functional consequences as based on previous structu ⁇ -e-functional studies of mammalian CaRs. The actual functional consequences of these AA differences in SalmoKCaR proteins are described herein in the following expression study.
  • SalmoKCaR #1 and #3 clones can be expressed in HEK cells to yield conesponding PVCR proteins of predicted molecular mass. It also is a demonstration of creation of HEK cells expressing SalmoKCaRs #1 and #3 as tools for assay systems to measure PVCR interactions and sensitivity to PVCR modulators for another aquatic PVCR, SKCaR. Furthermore, it is a demonstration ofthe use of SDD and_Sal-l antisera, respectively, as reagents to detect either both SalmoKCaRs #1 and #3, or selectively detect SahnoKCaR #1.
  • the DNA/Lipofectamine mixture was diluted with 8 ml Optio-Mem I and added to the flask. Flasks were incubated for 5 hours at 37°C, 5% C0 2 to allow transfer of DNA into the cells. 10 ml of Opti-Mem I media supplemented with 20% FBS was added to each flask, for a final concentration of 10%> serum. Flasks were incubated overnight at 37°C, 5% C0 2 . At 24 hours post transfection, the media was replaced with 20 ml full growth media. Flasks were kept at 37°C, 5% C0 2 for 24-48 hrs to optimize expression of protein.
  • the transfected cells were then rinsed with PBS and treated with trypsin-EDTA (Gibco) for 2 minutes at 37°C, 5% C0 2 to detach cells from the surface ofthe plate.
  • the detached cells were added to 10 ml of full growth media (serum deactivates trypsin) and pelleted at 250 x g for 10 minutes.
  • the cells were resuspended in 3 ml homogenization buffer
  • the cells were homogenized in 14 ml (Falcon) tubes using apolytron motorized homogenizer. The homogenate was centrifuged at 3, 000 x g for 15 minutes at 4°C twice, re-suspending pellet in 3 ml of homogenization buffer between spins and pooling supernatant from both spins into high speed centrifuge tubes. Mitochondrial material was then sedimented from pooled supernatant at 22, 000 x g for 20 minutes at 4°C.
  • the supernatant was subsequently sedimented at 37, 000 x g for 30 minutes at 4°C to pellet the plasma membranes, and the resulting pellet was solubilized with 100 ⁇ l homogenization buffer.
  • a fresh sample of salmon kidney from a fish 7 weeks post transfer into salt water was prepared using the same homogenization protocol, along with the mock transfected HEK 293 cells and a flask of HEK cells stably transfected with human calcium receptor (5001 HEK) to be used as positive and negative controls on the western blots. Protein was quantified from the plasma membrane preparations using the Bio-Rad Protein Assay (Bio- Rad Laboratories).
  • the membranes were washed in TBS-T and incubated for 1 hour at room temperature in primary antibodies of SalmoSDD or Sal 1 or the preimmunie serum to those antibodies, at 1 :4,000 dilutions.
  • the secondary antibody used was an antirabbit horseradish peroxidase (Amersham-Pharmacia) at 1:50,000 dilution, incubated for 1 hour at room temperature.
  • the protein bands shown in Figure 15B were detected with an Enhanced Chemiluminescence system (Amersham-Pharmacia) and exposed to high performance chemiluminescence film (Amersham- Pharmacia).
  • FIG. 15B shows a Western blot analysis of PVCR proteins produced by HEK cells transiently transfected with SalmoKCaR #1 and #3 constructs. Both Right and Left Panels each containing 10 lanes were loaded and electrophoresed with 20 micrograms of protein except for HEK-5001 (HuPCaR control) that contained 15 micrograms.
  • Figure 15B left panel, shows an immunoblotting analysis with SDD antiserum. SDD antiserum recognizes an aa sequence in the extracellular domain of PVCRs that is present in both HUPCaR, SalmoKCaR #1 , SalmoKCaR
  • HEK cells expressing HuPCaR display a prominent ⁇ 115kDa band, as well as higher molecular mass bands which represent oligomers of HuPCaR.
  • HuPCaR Positive Control
  • mock transfected HEK cells show minimal innnuoreactivity similar to that present when lanes are exposed to preimmune SDD antiserum.
  • SDD antiserum displays intense reactivity to HEK cells transfect with SalmoKCaR #1 (SalmoKCaR #1) or SahnoKCaR #3 (SalmoKCaR #3).
  • the immunoreactive band produced by SalmoKCaR #1 is slightly larger (higher molecular mass) when compared to SalmoKCaR #3 as predicted by their nucleotide and amino acid sequence analysis of conesponding clones. Note also the presence of larger immunoreactive bands present in SalmoKCaR #3 lane that conesponds to oligomeric complexes of SalmoKCaR protein. The ability of SalmoKCaR #3 to form oligomeric complexes could result in formation of it exerting a dominant negative effect on the activity of other PVCRs expressed in the same cell.
  • SW kidney is a positive control showing that several bands in both SalmoKCaR #1 and #3 lanes are an identical molecular mass to PVCR proteins present in kidney tissue of Atlantic sahnon.
  • FIG 15B shows immunoblotting analysis with SAL1 antiserum.
  • SALl antiserum recognizes an aa sequence in the carboxyl terminal of SalmoKCaR #1 that is not present in either HuPCaR or SalmoKCaR #3.
  • minimal immunoreactivity is displayed in either immune or preimmune lanes except for SalmoKCaR #1 and Salmon SW Kidney lanes.
  • SalmoKCaR proteins Differences between SalmoKCaR proteins vs. mammalian and other fish PVCRs include:
  • SalmoKCaR #1 , #2, #3 and #4 possess a deletion of 15 AA' s beginning at AA #369 as compared to either HuPCaR or RaKCaR. Fugu PVCR also exhibits a 19 AA deletion at the same al ⁇
  • SKCaR does not exhibit any deletion in this area and thus is more similar to mammalian CaRs as compared to either SalmoKCaR or Fugu in this regard.
  • SalmoKCaRs vs. mammalian CaRs and SKCaR Another notable difference between SalmoKCaRs vs. mammalian CaRs and SKCaR is differences in AA #227 where mutagenesis studies have identified the presence ofthe positively charged Arg as important in CaR sensitivity since its alteration in HuPCaR to a Leu results in over a 2 fold reduction in EC 50 Ca 2+ from 4.0mM to 9.3mM but not Gd 3+ sensitivity.
  • all 4 SalmoKCaRs possess a negatively charged Glu at AA#227. Fugu PVCR also exhibits the same Glu at AA#227.
  • AA sequence immediately following AA#227 is Glu-Glu-Ala in the mammalian HuPCaR and elasmobranch SKCaR whereas it is Lys-Glu-Met in all 4 SalmoKCaRs and Fugu.
  • SalmoKCaR 3 possesses a truncated carboxyl tenninal domain as compared to either SalmoKCaRs #1, #2 or #4.
  • the number of AA that comprise the carboxyl terminal domains ofthe 4 SalmoKCaRs are different and include: SalmoKCaR #1 - 96 AA; SalmoKCaR #2 - 97 AA, SalmoKCaR #3 - 5 AA, and SalmKCaR #4 - 96 AA. Reduction in the 91- 92AA's in SalmoKCaR #3 vs. SalmoKCaRs #1, #2 or #4 would reduce its estimated molecular mass by 9,600 Daltons.
  • HuPCaR Studies from multiple site directed mutagenesis studies of HuPCaR reveal that alterations to the structure ofthe carboxyl terminal domain of PVCRs have profound effects on their function and sensitivity to ligands such as Ca 2+ and Mg 2"1" .
  • Various truncations ofthe carboxyl terminal domain of HuPCaR have highlighted the importance of HuPCaR AAs #860-910.
  • Truncation ofthe carboxyl terminal domain of HuPCaR to AAs less than AA#870 produced either an inactive receptor or a modified HuPCaR with a marked decrease in its affinity for extracellular Ca 2+ as well as a decrease in the apparent cooperativity of Ca 2+ dependent activation.
  • SalmoKCaR #3 protein is either inactive or exhibits a greatly reduced functional affinity for Ca 2+ .
  • Significant expression of SalmoKCaR #3 together with other SalmoKCaRs #1, #2 or #4 could result in an overall reduction in • the response to extracellular Ca2+ due to so called dominant negative effects.
  • SalmoKCaR#3 reduces the overall sensitivity of cells to Ca 2+ via combinations between SalmoKCaR #3 and SalmoKCaR #l/#2/#4 to reduce the sensitivity ofthe latter PVCRs via cooperative interactions (dimers and higher oligomers) with them.
  • AA #888 is a Thr in all wild type CaR and PVCR proteins including HuPCaR, RaKCaR, SKCaR, BoPCaR and SalmoKCaR #1 , #2, and #4.
  • SalmoKCaR #3 is missing Thr #888 because of its truncated tail.
  • Ser-Ser-Ser consensus sites for receptor kinase phosphorylation
  • PCR on specific cDNA clones confirms that these primer pairs function exclusively on the clones for which they have been designed. Note that both the degenerate and SalmoKCaR #3 specific primers do not span an intron and therefore RNA was treated with DNAse to ensure that there was not amplification of contaminating genomic DNA in the results shown. Primers specific for SahnoKCaR #1 and #2 span introns and therefore DNAase treatment is not required to inte ⁇ ret these results. As a control, the amounts of mRNA added to each RT-PCR reaction was determined by separate amplification of actin using primers designed from the published sequence of Atlantic salmon actin (Genbank Accession #AF012125 Salmo salar beta actin mRNA). SalmoKCaR #4 could be distinguished from SalmoKCaRs #1 or #2 by use of appropriate restriction enzymes that would identify single nt differences between SalmoKCaR's #1, #2 versus #4.
  • SalmoKCaR #1 Specific Primers SalmoKCaR #1 nts
  • The.SalmoKCaR #1 primer pair consists of a forward primer (AS1-F17) spanning the 5' UTR insertion m SalmoKCaR #2, and a reverse primer (AS2-R14) within the 158 bp deleted from SalmoKCaR #3.
  • SalmoKCaR #2 primer pair is a forward primer (AS2-F13) in the 5' UTR insertion in SalmoKCaR #2 clone, and the same reverse primer as SalmoKCaR #1 primer (AS2-R14).
  • SalmoKCaR #3 Specific Primers SalmoKCaR #3 nts
  • the SalmoKCaR #3 primer pair consists of a forward primer (AS5-F11) which spans the 1 8 bp deletion, and a reverse primer (AS5-R12) located in the 36 bp insertion at the 3' end ofthe SalmoKCaR #3 clone.
  • RNA blotting analysis and RT-PCR of Atlantic salmon and elasmobranch tissues Total RNA was purified with Stat 60 reagent (Teltest B Friendswood, TX) DNAse (Introgen, Carlsbad, CA) treated and used for RT-PCR after cDNA production with cDNA Cycle Kit (lnvitrogen,Carlsbad, CA). The cDNA was amplified (30 cycles of Imin @ 94°C, lmin @ 57°C, 3' @72°C) using degenerate primers [forward primer dSK-F3 (SKCaR nts 2279-2306) and reverse primer dSK- R4 (SKCaR nts 2904-2934 ).
  • SalmoKCaR #1 amplification conditions and primer set PCR: lmin @ 94°C, lmin @ 50°C, 3min @ 72°C, 35 cycles. Amplification products attached to membrane were probed with full length SalmoKCaR #1 clone and washed (O.lx SSC, 0.1% SDS @ 55°C) and autoradiographed for 48 hr.
  • SalmoKCaR #2 amplification conditions and primer set PCR: lmin @ 94°C, lmin @ 50°C, 3min @ 72 °C, 35 cycles. Amplification products attached to membrane were probed with full length SalmoKCaR #2 clone and washed (O.lx SSC, 0.1% - SDS @ 55°C) and autoradiographed for 168 hr.
  • SalmoKCaR #3 amplification conditions and primer set PCR: lmin @ 94°C, lmin @ 52°C, 3min @ 72°C, 35 cycles. Amplification products attached to membrane were probed with full length SalmoKCaR #3 clone and washed (O.lx SSC, 0.1 % SDS @ 55°C) and autoradiographed for 72 hr.
  • Figure 20 shows data obtained from 14 tissues of freshwater or seawater adapted Atlantic salmon using the degenerate primers described above. Samples were obtained from a single representative seawater adapted salmon (866gm and 41cm in length) from a group of 10 fish of average weight of 678gm. Samples from nasal lamellae, urinary bladder, olfactory bulb and pituitary gland were all pooled samples from all 10 fish. The samples were from a representative single freshwater adapted fish (112gm and 21.5cm) selected from a group of 10 fish with an average weight of 142.8gm.
  • Figure 20 shows a RT-PCR analysis of freshwater (Panels B, D and F) and seawater (Panels A, C and E) adapted Atlantic salmon tissues using either degenerate PVCR or salmon actin PCR primers.
  • Total RNA from 13 (seawater adapted) and 14 (freshwater adapted) tissues of Atlantic salmon was first treated with DNAase to remove any genomic DNA contamination then used to synthesize cDNA that was amplified using degenerate primers.
  • PVCRs are present in tissues of seawater-adapted salmon including gill (lane 2), nasal lamellae (lane 3), urinary bladder (lane 4), kidney (lane 5), stomach (lane 6), pyloric caeca (lane 7), proximal (lane 8) and distal (lane 9) intestine, pituitary gland (11) and muscle (lane 14). Ovary tissue was not tested in seawater-adapted fish.
  • freshwater-adapted salmon possess amplified PVCR products in gill (lane 2), nasal lamellae (lane 3), urinary bladder (lane 4), kidney (lane 5), proximal intestine (lane 8), brain (lane 10), pituitary (lane 11), olfactory bulb (lane 12), liver (lane 13), muscle (lane 14) and ovary (lower lane 3).
  • the intensity of individual actin bands shown in Panels E and F performed on identical aliquots ofthe RT-PCR reactions serve to quantify any differences in pools of cDNA from the individual RT reactions in each sample.
  • RT-PCR analysis using degenerate primers shows that steady state content of kidney PVCRs is increased by the SuperSmoltTM process similar to that produced by transfer of Atlantic salmon to seawater.
  • Figure 21 A shows RT-PCR analysis of a single representative experiment where kidney tissue was harvested from Atlantic salmon that had either been freshwater adapted (lane 1), exposed to 9 weeks ofthe SuperSmoltTM process in freshwater (lane 2) or transfened to seawater and maintained for 26 days.
  • Figure 2 IB shows RT-PCR analysis of a single representative experiment using pyloric caeca from the same fish shown in Figure 21 A. Note the significant increase in amplified PVCR product present in kidney ( Figure 21 A) and pyloric caeca (Figure 2 IB) for both SuperSmoltTM (lanes 2 and 7, respectively) and seawater adapted (lanes 3 and 8, respectively) fish as compared to freshwater (lanes 1 and 6, respectively).
  • Figure 21 C shows RT-PCR analysis using the same degenerate primers to detect expression of SalmoKCaR transcripts in various stages of Atlantic salmon embryo development.
  • degenerate SEQ ID Nos 15 and 16
  • actin SEQ JD No 24 and 25
  • RNA obtained from samples of whole Atlantic salmon embryos at various stages of development were analyzed for expression of SalmoKCaRs using RT-PCR.
  • Ethidium bromide staining of samples from dechorionated embryos (Lane 1), 50% hatched (Lane 2), 100% hatched (Lane 3), 2 weeks post hatched (Lane 4) and 4 weeks post hatched (Lane 5) shows that
  • SalmoKCaR transcripts are present in Lanes 1-4. Southern blotting ofthe same gel (Panel C) confirms expression of SalmoKCaRs in embryos from very early stages up to 2 weeks after hatching. No expression of SalmoKCaR was observed in embryos 4 weeks after hatching. Panel B shows the series of controls where PCR amplification of actin content of each ofthe 5 samples shows they are approximately equal.
  • kidney RNA contains a 4.2kb band that conesponds to the 3.9-4.0 kb sizes of SalmoKCaR #1-4 as determined by nucleotide sequence analysis.
  • Figure 22 shows a RNA blot containing 5 micrograms of poly A + RNA from kidney tissue dissected from either freshwater adapted (FW) or seawater adapted (SW) Atlantic salmon probed with full length SalmoKCaR #1 clone. Autoradiogram exposure after 7 days. r
  • RT-PCR Use of RT-PCR with SalmoKCaR *3 specific primers demonstrates that tissue specific alterations in the steady state tissue content of SalmoKCaR # 3 mRNA in freshwater vs. seawater adapted Atlantic salmon.
  • nucleotide primer sets that allows for the specific amplification of SalmoKCaR transcripts were designed.
  • Figure 23 shows RT-PCR analysis of freshwater (Panels B, D and F) and seawater (Panels A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR #3 specific PCR primers or salmon actin PCR primers.
  • RNA from 13 (seawater adapted) and 14 (freshwater adapted) tissues of Atlantic salmon identical to those shown in Figure 20 were first treated with DNAase to remove any genomic DNA contamination, then used to synthesize cDNA that was amplified using SalmoKCaR #3 primers. All RNA samples were prepared from a single fish with the exception of olfactory bulb, pituitary, urinary bladder and nasal lamellae that are composed of RNA from pooled samples of fish. Selected reactions were subjected to primer amplification using SalmoKCaR#3 specific primers. DNA markers in lane 1 of both Panels A and B were used to indicate size of amplification products.
  • the increase in tissue expression of SahnoKCaR #3 serves to provide for a possible means to reduce the overall tissue sensitivity to PVCR-mediated sensing via an action where SalmoKCaR #3 would act as a dominant negative effector.
  • the concentrations of Ca + and Mg 2+ in seawater are 10 fold and 50 fold higher and thus may require reduction of the high sensitivity of SalmoKCaRs #1, #2 and #4 by SalmoKCaR #3.
  • RT-PCR with SalmoKCaR 1 specific primers demonstrates tissue specific alterations in the steady state tissue content of SalmoKCaR # 1 mRNA in freshwater vs. seawater adapted Atlantic salmon.
  • Figure 24 shows RT-PCR analysis of freshwater (Panels B, D and F) and seawater (Panels A, C and E) adapted Atlantic salmon tissues using either
  • SalmoKCaR #1 specific PCR primers or salmon actin PCR primers Total RNA from 13 (seawater adapted) and 14 (freshwater adapted) tissues of Atlantic salmon identical to those shown in Figures 20 and 23 were used to synthesize cDNA that was amplified using SalmoKCaR #1 primers. All RNA samples were prepared from a single fish with the exception of olfactory bulb, pituitary, urinary bladder and nasal lamellae that are composed of RNA from pooled samples offish. As controls to demonstrate primer specificity, selected reactions were subjected to primer amplification of portions of individual SalmoKCaR clones or water alone (Panels A andB): Ethidium bromide stained agarose gel.
  • DNA markers in lane 1 of both Panels A and B were used to indicate size ⁇ f amplification products.
  • (Panels C and D) Southern blot of gel in Top Panel using 32 P-labeled Atlantic salmon geri mic fragment.
  • (Panes E and F) Ethidium bromide stained gel of RT-PCR amplification products using Atlantic salmon beta actin primers as described above. These reactions serve as controls to ensure that samples contain equal amounts of RNA.
  • PCR amplification with these primers yields an ethidium bromide staining band (lane5) when SalmoKCaR #1 clone is used as a template but not either SalmoKCaR #2 (lane 6) or SalmoKCaR #3 (lane 7). Similar analysis could be performed on SalmoKCaR #4. Southern blotting analysis ofthe gels shown in Panels A and B reveals that the amplification product of the SalmoKCaR #3 is highly positive (lanes 5) - - Panels C and D.
  • SalmoKCaR #1 product is amplified in selected tissues including urinary bladder (lane 4) and pyloric caeca (lane 7) in seawater-adapted salmon (Panel C) as compared to urinary bladder (lane 4) and kidney (lane 5) in freshwater-adapted salmon (Panel D).
  • urinary bladder lane 4
  • pyloric caeca lane 7
  • seawater-adapted salmon Panel C
  • kidney lane 5
  • freshwater-adapted salmon Pulpyloric caeca
  • the exact nature ofthe smaller and larger than expected PCR amplification products present in gill (lane 2 -Panels C, D) and nasal lamellae (lane 3 - Panel D) are not known at present.
  • Figure 25 shows RT-PCR analysis of freshwater (Panels B, D and F) and seawater (Panels A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR #2 specific PCR primers or salmon actin PCR primers.
  • Total RNA from 13 (seawater adapted) and 14 (freshwater adapted) tissues of Atlantic salmon was used to synthesize cDNA that was amplified using SalmoKCaR #2 primers. All RNA samples were prepared from a single fish with the exception of olfactory bulb, pituitary, urinary bladder and nasal lamellae that are composed of RNA from pooled samples of fish.
  • Figure 25 shows data obtained using SalmoKCaR #2 specific primers and the identical tissue RT and plasmid samples as shown in Figures 20, 23, and 24.
  • Conesponding Southern blots shown in Panels C and D reveal the presence of SalmoKCaR #2 PCR amplification product in urinary bladder of seawater-adapted salmon (lane 4) as well as urinary bladder (lane 4) and kidney (lane 5) of freshwater-adapted salmon.
  • These data provide evidence of the tissue specific expression of SahnoKCaR #1 in both freshwater and seawater adapted salmon.
  • EXAMPLE 7 SURVIVAL AND GROWTH OF PRE-ADULT ANADROMOUS FISH BY MODULATING PVCRS An important feature of cunent salmon fanning is the placement of smolt from freshwater hatcheries to ocean netpens. Present day methods use smolt that have attained a critical size of approximately 70- 110 grams body weight. The methods described herein to modulate one or more PVCRs ofthe anadromous fish including Atlantic Salmon, can either be utilized both to improve the ocean netpen transfer of standard 70-110 grams smolt as well as permit the successful ocean netpen transfer of smaller smolts weighing, for example, only 15 grams.
  • one utility for the present invention is its use in conjunction with transferring Atlantic Sahnon from freshwater to seawater.
  • application ofthe invention eliminates the phenomenon known as "smolt window" and permits fish to be maintained and transfened into ocean water at 15°C or higher.
  • Use of these methods in 15 gram or larger smolt pennits greater utilization of freshwater hatchery capacities followed by successful seawater transfer to ocean netpens.
  • fish that undergo the steps described herein feed vigorously within a short interval of time after transfer to ocean netpens and thus exhibit rapid growth rates upon transfer to seawater.
  • Figure 26 shows in schematic form the key features of cunent aquaculture of Atlantic salmon in ocean temperatures present in Europe and Chile. Eggs are hatched in inland freshwater hatcheries and the resulting fry grow into f ⁇ ngerlings and parr. Faster growing pan are able to undergo smoltification and placement in ocean netpens as SO smolt (70 gram) during year 01. hi contrast, slower growing pan are smoltified in year 02 and placed in netpens as S 1 smolt (100 gram). In both SO and SI transfers to seawater, the presence of cooler ocean and freshwater temperatures are desired to minimize the stress of osmotic shock to newly transfened smolt.
  • SO smolt 70 gram
  • S 1 smolt 100 gram
  • Standard smolts that are newly placed in ocean netpens are not able to grow optimally during their first 40-60 day interval in seawater because ofthe presence of osmotic stress that delays their feeding.
  • This interval of osmotic adaptation prevents the smolts from taking advantage ofthe large number of degree days present immediately after either spring or fall placement.
  • the combination ofthe presence ofthe smolt window together with delays in achieving optimal smolt growth prolong the growout interval to obtain market size fish. This is particularly problematic for SO's since the timing of their harvest is sometimes complicated by the occunence of grilsing in maturing fish that are exposed to reductions in ambient photoperiod.
  • Process I increases the survival of small Atlantic Salmon S2flike smolt after their transfer to seawater when compared to matched freshwater controls. Optimal survival is achieved by using the complete process consisting of both the magnesium and calcium water mixture as well as NaCl diet. In contrast, administration of calcium and magnesium either via the food only or without NaCl dietary supplementation does not produce results equivalent to Process I.
  • Table 3 shows data obtained from Atlantic sahnon S2 like smolts less than 1 year old weighing approximately 25 gm. This single group offish was apportioned into 4 specific groups as indicated below and each were maintained under identical laboratory conditions except for the variables tested. All fish were maintained at a water temperature of 9-13°C and a continuous photoperiod for the duration ofthe experiment. The control freshwater group that remained in freshwater for the initial 45 day interval experienced a 33% mortality rate under these conditions such that only 67% were able to be transfened to seawater. After transfer to seawater, this group also experienced high mortality where only one half of these smolts survived.
  • Smolts derived from the St. John strain of Atlantic salmon produced by the Connors Brothers Deblois Hatchery located in Cherryfield, Maine, USA were utilized for this large scale test. Smolts were produced using standard practices at this hatchery and were derived from a January 1999 egg hatching. All smolts were transfened with standard commercially available smolt trucks and transfer personnel. S 1 smolt were purchased during Maine's year 2000 smolt window and smolt deliveries were taken between the dates of 29 April 2000 - 15 May 2000. Smolts were either transfened directly to Polar Circle netpens (24m diameter) located in Blue Hill Bay Maine (Controls) or delivered to the treatment facility where they were treated with Process I for a total of 45 days.
  • the smolt were then transported to the identical Blue Hill Bay netpen site and placed in an adjacent rectangular steel cage (15mX15mX5m) for growout. Both groups of fish received an identical mixture of moist (38% moisture) and dry (10%> moisture) salmonid feed (Connors Bros). Each ofthe netpens were fed by hand or feed blower to satiation twice per day using camera visualization of feeding. Mort dives were perfomied on a regular basis and each netpen received identical standard care practices established on this salmon fann. Sampling of fish for growth analyses was performed at either 42 days (Process I) or 120 days or greater (Control) fish. In both cases, fish were removed from the netpens and multiple analyses perfonned as described below,
  • Table 5 displays data obtained after seawater transfer of Control S 1 smolt.
  • Table 6 Characteristics of St. John SI smolt subjected to immediate placement in ocean netpens after transport fonn the freshwater hatchery without Process I or Process II technology (the Control fish)
  • Process I smolts The mortality of Process I smolts was comparable to that of smolts placed earlier in the summer (6.1%) during initial 50 days after ocean netpen placement and two thirds of those mortalities were directly attributable to scale loss and other physical damage inclined during the transfer process itself.
  • conesponding control fish (held under identical conditions without Process I treatment) did not fare well during transfer to the netpen (17% transfer mortality) and did not feed vigorously at any time during the first 20 days after ocean netpen placement.
  • This smaller number of control fish (176) were held in a smaller (1.5mX1.5mX1.5m) netpen floating within the larger netpen containing Process I smolts. Their mortality post-ocean netpen placement was very high at 63% within the 51 day interval.
  • Standard smolt include:
  • Process I The mortalities observed after ocean netpen placement were low in Process I (6.1%) vs Control (63%) despite the that fact these fish were transfened to seawater 1.5 months after the smolt window and into a very high (15.1°C) ocean water temperature.
  • the mortality of Process I was comparable to that ofthe accepted Industry Standard smolt (3-10%) transfened to cooler (10°C) seawater during the smolt window.
  • This characteristic of Process I provides for a greater flexibility in freshwater hatchery operations since placement of Process I smolts are not rigidly confined the conventional "smolt window" cunently used in industry practice.
  • Process I fish were in peak condition during and immediately after seawater transfer. Unlike industry standard smolt that required 56 days to reach full feeding, the Process I smolts fed vigorously within 2 days. Moreover, the initial growth rate (SGR 1.8) demonstrated by Process I smolts are significantly greater than published data for standard smolt during their initial 50 days after seawater placement (published values (Sfradmeyer, L. Is feeding nonstarters a waste of time. Fish Farmer 3: 12-13, 1991; Usher, ML, C Talbot and FB Eddy.
  • FIG. 27A compares the weekly feed consumption on a per fish basis between Process I treated smolts and industry standard smolts. As shown, Process I treated smolts consumed approximately twice as much feed per fish during their FIRST WEEK as compared to the industry standard smolts after 30 days. Since smolts treated with Process I fed significantly more as compared to Industry standard smolts, the Process I treated smolts grew faster.
  • Figure 27B provides data on the characteristics of Process I smolts after seawater transfer. These experiments were can ⁇ ed out for over 185 days.
  • a total of 1,400 Landcatch/St John strain fingerlings possessing an average weight of 20.5 gram were purchased from Atlantic Salmon of Maine Inc., Quossic Hatcheiy, Quossic, Maine, USA on 1 August 2000. These fingerlings were derived from an egg hatching in January 2000 and considered rapidly growing fish. They were transported to the treatment facility using standard conventional track transport. After their arrival, these fingerlings were first placed in typical freshwater growout conditions for 14 days. These fingerlings were then subjected to Process I for a total of 29 days while being exposed to a continuous photoperiod.
  • Process I were then vaccinated with the Lipogen Forte product (Aquahealth LTD.) and transported to ocean netpens by conventional track transport and placed into seawater (15.6°C) in either a research ocean netpen possessing both a predator net as well as net openings small enough (0.25 inch) to prevent loss of these smaller Process I smolts.
  • Process I smolts were placed in circular tanks within the laboratory. Forty eight hours after sea water transfer, Process I smolts were begun on standard moist (38% moisture) smolt feed (Connors Bros.) that had been re-pelletized due to the necessity to provide for smaller size feed for smaller Process I smolts, as compared to normal industry salmon. In a manner identical to that described for 70 gram smolts above, the mortality, feed consumption, growth and overall health of these 30 gram Process I smolts were monitored closely.
  • Figure 28 displays the characteristics of a representative sample of a larger group of 1,209 Process I smolts immediately prior to their transfer to seawater. These parameters included an average weight of 26.6+8.6 gram, length of 13.1+1.54 cm and condition factor of 1.12+0.06. After seawater transfer, Process I smolts exhibited a low initial mortality despite the fact that their average body weight is 26-38% of industry standard 70-100 gram S0-S1 smolts. As shown in Table 8, Process I smolts mortality within the initial 72 hr after seawater placement was 1/140 or 0.07% for the laboratory tank. Ocean netpen mortalities after placement of Process I smolts were 143/1069 or 13.4%.
  • Figure 28 shows representative Landcatch/St Jolm strain Process I smolts possessing a range of body sizes that were transfened to seawater either in ocean netpens or conesponding laboratory seawater tanks.
  • Process I smolts possess a wide range of sizes (e.g., from about 5.6 grams to about 46.8 grams body weight) with an average body weight of 26.6 gram.
  • Table 8 Characteristics and survival of Landcatch/St. John Process I fish after their placement into seawater in either a laboratory tank or ocean netpen.
  • Figure 29 shows a comparison of the distributions of body characteristics for total group of Landcatch/St John Process I smolts vs. mortalities 72 hr after seawater ocean netpen placement. Length and body weight data obtained from the 143 mortalities ocpurring after seawater placement of 1,069 Process I smolts were plotted on data obtained from a 100 fish sampling as shown previously in Figure 28. Note that the mortalities are exclusively distributed among the smaller fish within the larger Process I netpen population.
  • Table 9 displays data on the use of he Process I on small (3-5 gram) rainbow trout. Juvenile trout are much less tolerant of abrupt transfers from freshwater to seawater as compared to juvenile Atlantic salmon. As a result, many commercial seawater trout producers transfer their fish to brackish water sites located in estuaries or fresh water lenses or construct "drinking water” systems to provide fresh water for trout instead ofthe full strength seawater present in standard ocean netpens. After a prolonged interval of osmotic adaptation, trout are then transfened to more standard ocean netpen sites to complete their growout cycle, hi general, trout are transfened to these ocean sites for growout at body weights of approximately 70-90 or 90-120 gram.
  • a total of 140 trout from a single pool of fish less than 1 year old were divided into groups and maintained at a water temperature of 9-13°C and pH 7.8-8.3 for the duration of the experiment described below.
  • Control Freshwater Exposure ofthe trout to a constant photoperiod for 14 days results in a slight improvement in survival after their transfer to seawater.
  • exposure of trout to Process I for either 14 days or 23 days results in significant reductions in mortalties after transfer to seawater such that 30% and 46%» ofthe fish respectively have survived after a 5 day interval in seawater.
  • Figure 30 shows the results of exposure of smaller char (3-5 gram) to the Process I for a total of 14 and 30 days. All fish shown in Figure 30 were exposed to a continuous photoperiod. Transfer of char to seawater directly from freshwater results in the death of all fish within 24 hr. In contrast, treatment of char with the Process I for 14 and 30 days produces an increase in survival such that 33% (3/9) or 73% (22/30) respectively are still alive after a 3 day exposure. These data demonstrate that the enhancement of survival of arctic char that are less than 10% ofthe critical size as defined by industry standard methods after their exposure to the Process I followed by transfer to seawater.
  • Figure 30 shows a comparison of survival of arctic char after various treatments.
  • a single group of arctic char (3-5 gram were obtained from Pierce hatcheries (Buxton, ME) and either maintained in freshwater or treated with the Process I prior to transfer to seawater.
  • the Process II protocol is utilized to treat pre-adult anadromous fish for placement into seawater at an average size of 25-30 gram or less. This method differs from the Process I protocol by the inclusion of L-tryptophan in the diet of pre-adult anadromous fish prior to their transfer to seawater. Process II further improves the osmoregulatory capabilities of pre-adult anadromous fish and provides for still further reductions in the "critical size" for Atlantic sahnon smolt transfers. In summary, Process II reduces the "critical size" for successful seawater transfer to less than one fifth the size ofthe present day industry standard SO smolt.
  • St John/St John strain pre-adult fingerlings derived from a January 2000 egg hatching and possessing an average weight of 0.8 gram were purchased from Atlantic Salmon of Maine Inc. Kemiebec Hatchery, Kennebec Maine on 27 April 2000. These fish were transported to the treatment facility using standard conventional truck transport. After their arrival, these pan were first grown in conventional flow through freshwater growout conditions that included a water temperature of 9.6°C and a standard fi-eshwater pan diet (Moore-Clark Feeds). On 17 July 2000, fingerlings were begun on Process U for a total of 49 days while being exposed to a continuous photoperiod.
  • Process II smolts were then vaccinated with the Lipogen Forte product (Aquahealth LTD.) on Day 28 (14 August 2000) of Process II treatment.
  • Process II smolts were size graded prior to initiating Process JJ as well as immediately prior to transfer to seawater.
  • St John/St John Process II smolts were transported to ocean netpens by conventional track transport and placed into seawater (15.2°C) in either a single ocean netpen identical to that described for placement of Process I smolts or into laboratory tanks (15.6°C) within the research facility.
  • Figure 31 shows representative St. John/St John strain Process JJ smolts possessing a range of body sizes were transfened to seawater either in ocean netpens or conesponding laboratory seawater tanks.
  • Process JJ smolts possess a wide range of body weights (3.95-28 gram) that comprised an average body weight of 11.5 gram.
  • Figure 31 shows the characteristics of St. John/St John Process JJ smolts. The average measurements of these St. John/St. John Process JJ smolts included a body weight of 11.50+/-5.6 gram, length of 9.6+/- 1.5 cm and condition factor of 1.19+/-0.09.
  • the data displayed in Table 10 shows the outcomes for two groups of Process ⁇ smolts derived from a single production pool of fish after their seawater transfer into either laboratory tanks or ocean netpens.
  • Table 10 Characterization and survival of St. John/St. John Process JJ fish after their placement into seawater in ocean netpens containing underwater lights.
  • Table 12 Characteristics and survival of St. John/St. John Process JJ fish after their placement into seawater in either a laboratory tank or ocean netpen.
  • Figure 32 compares characteristics of survivors and mortalities of Process JJ smolts after seawater transfer to either laboratory tanks ( Figure 32A) or ocean netpens (Figure 32B) .
  • Figure 32 A data are derived from analyses of 100 Process JJ smolts transfened to seawater tank where all fish were killed and analyzed on Day 5.
  • Figure 32B displays only mortality data from ocean netpen. In both cases, only smaller Process II smolts experienced mortality. Note differences in Y axis scales of Figures 32A-B. Comparison ofthe average body size of those Process IJ smolts that survived seawater transfer vs.
  • Landcatch/St Jolm Process I smolts were offered food beginning 48 hr after their seawater transfer to either laboratory tanlcs or ocean netpens. While these Process I smolts that were transfened to laboratory tanks began to feed after 48 hr, those fish transfened to ocean netpens were not observed to feed substantially until 7 days. To validate these observations, the inventors performed direct visual inspection ofthe gut contents from a representative sample of 49 Process I smolts 4 days after their seawater transfer to laboratory tanks. A total of 21/49 or 42.9% possessed food within their gut contents at that time.
  • Table 12 Comparison of Growth Rates of Pre-adult Atlantic Salmon Exposed to either Process I or Process II and Placed in Laboratory Tanlcs During Initial Interval After Seawater Transfer
  • EXAMPLE 8 EXPOSURE OF SALMON SMOLTS TO CA2+ AND MG2+ INCREASES EXPRESSION OF PVCR FN CERTAIN TISSUES.
  • Tissues were taken from either Atlantic salmon or rainbow trout, after anesthesitizing the animal with MS-222. Samples of tissues were then obtained by dissection, fixed by immersion in 3% paraformaldehyde, washing in Ringers then frozen in an embedding compound, e.g., OCT.TM (Miles, Inc., Elkahart, Indiana, USA) using methylbutane cooled on dry ice. After cutting 8 micron thick tissue sections with a cryostat, individual sections were subjected to various staining protocols.
  • OCT.TM Miles, Inc., Elkahart, Indiana, USA
  • sections mounted on glass slides were: 1) blocked with goat serum or serum obtained from the same species offish, 2) incubated with rabbit anti-CaR antiserum, and 3) washed and incubated with peroxidase-conjugated affinity-purified goat antirabbit antiserum.
  • the locations ofthe bound peroxidase-conjugated goat anti-rabbit antiserum were visualized by development of a rose-colored aminoethylcarbazole reaction product.
  • Individual sections were mounted, viewed and photographed by standard light microscopy techniques. The methods used to produce anti-PVCR antiserum are described below.
  • Figs. 33A - 33G are a set of seven photomicrographs showing immunocytochemistry of epithelia of he proximal intestine of Atlantic salmon smolts using anti-PVCR antiserum
  • Fig. 34 - which is a Western blot of intestine of a salmon smolt exposed to Ca2+- and Mg2+-treated freshwater, then transfened to seawater.
  • the antiserum was prepared by immunization of rabbits with a 16-mer peptide containing the protein sequence encoded by the carboxyl tenninal domain ofthe dogfish shark PVCR ("SKCaR”) (Nearing, J. et al., 1997, J. Am.
  • Figs. 33A and 33B show stained intestinal epithelia from smolts that were maintained in freshwater then transfened to seawater and held for an interval of 3 - days. Abundant PVCR immunostaining is apparent in cells that line the luminal surface ofthe intestine. The higher magnification (1440X) shown in Fig. 33B displays PVCR protein localized to the apical (luminal-facing) membrane of intestinal epithelial cells.
  • Fig. 33C shows stained intestinal epithelia from a representative smolt that was exposed Process I and maintained in freshwater containing 10 mM Ca2+ and 5.2 mM Mg2+ for 50 days. Note that the pattern of PVCR staining resembles the pattern exhibited by epithelial cells displayed in Figures 33 A and 33B including apical membrane staining (small anowheads) as well as larger globular round cells (anows). Fig.
  • FIG. 33D shows a 1900X magnification of PVCR-stained intestinal epithelia from another representative fish that was exposed to the Process I and maintained in freshwater containing 10 mM Ca2+ and 5.2 mM Mg2+ for 50 days and fed 1% NaCl in the diet.
  • small anowhead and arrows denote PVCR staining ofthe apical membrane and globular cells respectively, hi contrast to the prominent PVCR staining shown in Figures 33 A-D
  • Figs. 33E (1440X) and 13F (1900X) show staining of intestinal epithelia from two representative smolt that were maintained in freshwater alone without supplementation of Ca2+ and Mg2+ or dietary NaCl.
  • Fig. 33G (1440X) shows the lack of any apparent PVCR staining upon the substitution of preimmune serum on a section conesponding to that shown in Figure 33A where anti-PVCR antiserum identified the PVCR protein.
  • the lack of any PVCR staining with preimmune antiserum is a control to demonstrate the specificity ofthe anti-PVCR antiserum under these immunocytochemistry conditions.
  • the relative amount of PVCR protein present in intestinal epithelial cells of freshwater smolts (Figs. 33E and 33F) was negligible as shown by the faint staining of selected intestinal epithelial cells.
  • the PVCR protein content ofthe conesponding intestinal epithelial cells was significantly increased upon the transfer of these smolts to seawater (Figs. 33 A and 33B).
  • the PVCR protein content was also significantly increased in the intestinal epithelial cells of smolts maintained in freshwater supplemented with Ca2+ and Mg2+ (Fig. 33C and 33D).
  • the AEC staining was specific for the presence ofthe anti-PVCR antiserum, since substitution ofthe immune antiserum by the preimmune eliminated all reaction product from intestinal epithelial cell sections (Fig. 33G).
  • PVCR proteins as determined by the binding of a specific anti-PVCR antibody, were present in the following organs. These organs are important in various osmoregulatory functions. These organs include specific kidney tubules and urinary bladder responsible for processing of urine, and selected cells ofthe skin, nasal lamellae and gill each of which are bathed by the water sunounding the fish.
  • the PVCR was also seen in various portions ofthe G.I. tract including stomach, pyloric caeca, proximal intestine and distal intestine that process seawater ingested by fish.
  • the PVCR protein is not only present on both the apical (luminally facing) and basolateral (blood-facing) membranes of stomach epithelial cells localized at the base ofthe crypts ofthe stomach, but also is present in neuroendocrine cells that are located in the submucosal area ofthe stomach. From its location on neuroendocrine cells ofthe G.I. tract, the PVCR protein is able to sense the local environment immediately adjacent to intestinal epithelial cells and modulate the secretion and .synthesis of important G.I. tract hormones (e.g., 5-hydroxytryptamine (5-HT), serotonin, or cnolecystokinin (CCK)). Importantly, it is believed that the constituents of Process U effect G.I.
  • 5-hydroxytryptamine (5-HT), serotonin, or cnolecystokinin (CCK) e.g., 5-hydroxytryptamine (5-HT), serotonin, or cnolecy
  • neuroendocrine cells by at least two means.
  • the first way that constituents of Process II remodel the G.I endocrine system is through alterations in the expression and/or sensitivity of PVCRs expressed by these cells.
  • the second way is to supply large quantities of precursor compounds, for example, tryptophan that is converted into 5-HT and serotonin by G.I. metabolic enzymes.
  • PVCR protein is localized to both the apical and basolateral membranes of epithelial cells lining the proximal intestine. From their respective locations, PVCR proteins can sense both the luniinal and blood contents of divalent cations, NaCl and specific amino acids and thereby integrate the multiple nutrient and ion abso ⁇ tive-secretory functions ofthe intestinal epithelial cells. Epithelial cells of pyloric caeca also possess abundant apical PVCR protein.
  • Fig. 34 shows a Western blot of intestinal protein from salmon smolt maintained in 10 mM Ca2+, 5 mM Mg2+ and fed 1% NaCl in the diet. Portions ofthe proximal and distal intestine were homogenized and dissolved in SDS-containing buffer, subjected to SDS-PAGE using standard techniques, transfened to nitrocellulose, and equal amounts of homogenate proteins as determined by both protein assay (Pierce Chem. Co, Rocford, FL) as well as Coomassie Blue staining were probed for presence of PVCR using standard western blotting techniques.
  • EXAMPLE 9 IMMUNOLOCALIZATION OF POLYVALENT CATION RECEPTOR (PVCR) IN MUCOUS CELLS OF EPIDERMIS OF SALMON.
  • the skin surface of salmonids is extremely important as a barrier to prevent water gain or loss depending whether the fish is located in fresh or seawater.
  • the presence of PVCR proteins in selected cells ofthe fish's epidermal layer would be able to "sense" the salinity ofthe sunounding water as it flowed past and provide for the opportunity for continuous remodeling ofthe salmonid's skin based on the composition ofthe water where it is located.
  • FIG. 35B shows immunolocalization of salmon skin PVCR protein that is localized to multiple cells (indicated by anowheads) within the epidennal layers of the skin.
  • anti-PVCR staining shows the whole cell body, which is larger than its conesponding apical portion that stains with Alcian Blue as shown in Figure 35 A. The presence of bound anti-CaR antibody was indicated by the rose color reaction product.
  • FIG. 35C shows the Control Preimmune section where the primary anti-PVCR antiserum was omitted from the staining reaction. Note the absence of rose colored reaction product in the absence of primary antibody.
  • PVCR protein in discrete epithelial cells (probably mucocytes) localized in the epidennis of juvenile Atlantic salmon. From this location, the PVCR protein could "sense" the salinity ofthe sunounding water and modulate mucous production via changes in the secretion of mucous or proliferation of mucous cells within the skin itself.
  • the PVCR agonists (Ca2+, Mg2+) present in the surrounding water activate these epidermal PVCR proteins during the interval when smolts are being exposed to the process ofthe present invention. This treatment of Atlantic salmon smolts by the process ofthe present invention is important to increased survival of smolts after their transfer to seawater.
  • EXAMPLE 10 DEMONSTRATION OF THE USE OF SOLID PHASE ENZYME- LINKED ASSAY FOR DETECTION OF PVCRS TN VARIOUS TISSUES OF INDIVIDUAL ATLANTIC SALMON USING ANTI-PVCR POLYCLONAL ANTISERUM.
  • the PVCR content of various tissues of fish can be quantified using an ELISA
  • Homogenates were prepared by placing various tissues of juvenile Atlantic salmon (St. John St. John strain average weight 15-20 gm) into a buffer (10 mM HEPES, 1.5 mM MgC12, 10 mM KCl, ImM Phenylmethylsulfonyl fluoride (PMSF), 0.5 dithiothreitol (DTT) and ImM benzamidine pH 8.8) and using a standard glass Potter-Elvenhiem homogenizer with a rotary pestle. After centrifugation at 2,550Xg r for 20 mm. at 4°C to remove larger debris, the supernatant was either used directly or frozen at -80°C until further use.
  • a buffer 10 mM HEPES, 1.5 mM MgC12, 10 mM KCl, ImM Phenylmethylsulfonyl fluoride (PMSF), 0.5 dithiothreitol (DTT) and ImM benzamidine pH 8.8
  • Homogenate protein concentrations were determined using the BCA assay kit (Pierce Chem. Co.). Aliquots of individual tissue homogenates were diluted into a constant aliquot size of 100 microliters and each was transfened to a 96 well plate (Costar Plastic Plates) and allowed to dry in room air for 15hr. After blocking of nonspecific binding with a solution of 5% nonfat milk powder + 0.5% Tween 20 in TBS (25 mM Tris 137 mM sodium chloride, 2.7 mM KCl pH 8.0), primary antiserum (either rabbit anti-PVCR immune or conesponding rabbit pre-immune antiserum) at a 1:1500 dilution was added.
  • TBS 25 mM Tris 137 mM sodium chloride, 2.7 mM KCl pH 8.0
  • Figure 36 shows the data obtained from a representative single ELISA determination of PVCR protein content of 14 tissues of a single j uvenile Atlantic salmon. Under the conditions specified in the Experimental Protocol as outlined above, nonspecific binding of both primary and secondary antibodies were - minimized. While these quantitative values are measured relative to each other and not in absolute amounts, they provide data that parallels extensive immunocytochemistry examination of each of the tissues. Note that the PVCR content of various organs reflects their importance in osmoregulation of Atlantic salmon, hnmunocytochemistry data described herein shows that tissues such as intestine (proximal and distal segments), gill, urinary bladder and kidney contained PVCR protein. In each case, epithelial cells that contact fluids that bathe the surfaces of these tissues express PVCR.
  • EXAMPLE 11 ANTIBODIES MADE FROM THE CARBOXYL TERMINAL PORTION OF AN ATLANTIC SALMON PVCR PROTEIN ARE EFFECTIVE IN IMMUNOCYTOCHEMISTRY AND MUNOBLOTTING ASSAYS TO DETERMINE THE PRESENCE, ABSENCE OR AMOUNT OF THE PVCR PROTEIN Degenerate primers, dSK-F3 (SEQ ID NO: 15) and dSK-R4 (SEQ ID NO: 16), described herein were constructed specifically from the SKCaR DNA sequence. These primers have proved to be useful reagents for amplification of portions of PVCR sequences from both genomic DNA as well as cDNA.
  • Peptide #1 Ac-CTNDNDSPSGQQRIHK-amide (SEQ ID NO. : 17) producing rabbit antiserum SAL-1
  • the peptide was derivatized to carrier proteins and utilized to raise peptide specific antiserum in two rabbits using methods for making a polyclonal antibody.
  • the resulting peptide specific antiserum was then tested using both immunob lotting and immunocytochemistry techniques to determine whether the antibody bound to protein bands conesponding to PVCR proteins or yielded staining patterns similar to those produced using other anti-PVCR antiserum.
  • a photograph of an immunob lot was taken showing protein bands that were recognized by antisera raised against peptides containing either SAL-1 (SEQ JD NO.: 17) or SKCaR (SEQ ID NO:2).
  • antiserum raised to the peptide identified protein bands that co-electrophorese with PVCR proteins that are recognized by antisera raised to SKCaR (SEQ ID NO:2).
  • Immuno staining of juvenile Atlantic salmon kidney sections with 3 different anti-PVCR antisera produces similar localizations of PVCR protein within the tubules of salmon kidney. Staining produced by anti-SKCaR antiserum is identical to that produced by anti-4641 antiserum, an anti-peptide antisera conesponding to extracellular domain of mammalian PVCRs that is very similar to SKCaR (SEQ ID NO: 2).
  • Anti-Sal- 1 antiserum also exhibits a similar staining pattern for the distribution of intestinal
  • This new antiserum is specific for a PVCR in Atlantic Salmon tissues.
  • This antiserum can be used to determine the presence, absence or amount of PVCR in various tissues offish, using the methods described herein.
  • the Sal I antiserum is also useful in localization of SalmoKCaR proteins in larval Atlantic salmon (See Figure 40B).
  • the Sal I antiserum localizes SalmoKCaR proteins in the developing nasal lamellae of anadromous fish, including Atlantic sahnon and trout, skin, myosepta, otolith and sensory epithelium.
  • the myoseptae are collagenous sheets that separate the various muscle bundles in the fish. Myosepta are important in both the development of muscle in larval fish as well as its function for muscle force generation in adult fish. Myosepta are also of -i n ⁇
  • the otolith is also of considerable importance to Atlantic salmon. It is a calcified structure located in the inner ear of salmon where it is closely associated with epithelial cells responsible for sensing sound and direction. It is likely that the SalmoKCaRs associated with the otolith participate in the calcification of the otolith structure that consists of proteins and calcium precipitate.
  • a second peptide sequence was used for antibody production:
  • Peptide #2 CSDDEYGRPGIEKFEKEM (SEQ ID NO: 29). This peptide was synthesized, derivatized in a mamier identical to that described for Peptide #1 and antiserum was raised in rabbits as described above. As expected, this antiserum (Salmo ADD) produced a pattern of i monostaining on sections of juvenile Atlantic salmon that is identical to that exhibited by Sal I. (See Figure 40C).
  • SalmoKCaR #1, #2 and #4, but not SalmoKCaR #3 possess the carboxyl tenninal sequence recognized by the Sal I antibody the antibody-staining pattern displayed by Sal I show the distribution of SalmoKCaR proteins #1, #2 and #4 but not #3 within the kidney of Atlantic salmon.
  • Salmo ADD antibody binds to a peptide sequence present in the extracellular domain of all 4 SalmoKCaR proteins.
  • any cells that possess no staining of Sal I but staining with Salmo ADD likely express either SalmoKCaR #3 or some similar SalmoKCaR protein.
  • EXAMPLE 12 USE OF REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) TO DETECT EXPRESSION OF PVCRS IN VARIOUS TISSUES
  • Example 4 2 degenerate primers, dSK-F3 (SEQ ID NO: 15) and dSK-R4
  • SEQ ID NO: 16 are disclosed. These two primers were used to amplify genomic DNA and obtain the sequence of a portion ofthe genomic DNA sequences of PVCRs from various anadromous fish. These same primers can also be used to amplify a portion of conesponding PVCR mRNA transcripts in various tissues. DNA sequence analyses of amplified cDNAs from specific Atlantic salmon tissues (olfactory lamellae, kidney, urinary bladder) verifies these are all identical to certain genomic PVCR sequences described herein. These data show that:
  • PVCR mRNA transcripts are actually expressed in specific tissues of anadromous fish. These data reinforce the data regarding PVCR protein expression as detected by anti-PVCR antisera.
  • RT-PCR methods can be used to detect and quantify the degree of PVCR expression in various tissues, as a means to predict the readiness of anadromous fish for transfer to seawater.
  • cDNA probes can be generated from specific tissues of anadromous -fish for use as- specific DNA probes to either detect PVCR expression using solution or solid phase DNA-DNA or DNA-RNA nucleic acid hybridization or obtain putative PVCR protein sequences used for generation of specific anti-PVCR antisera.
  • DNA sequencing of RT-PCR products were performed as follows: A total of 15 microliters of Atlantic Salmon urinary bladder, kidney and nasal lamellae RT-PCR reactions were diluted in 40 microliters of water and purified by size exclusion on Amershan s MicroSpin S-400 HR spin columns (Amersham Inc, Piscataway, NJ). Purified DNA was sequenced using degenerate PVCR primers (SEQ ID NO.: 15 and 16) as sequencing primers. Automated sequencing was performed using an Applied Biosystems Inc. Model 373A Automated DNA Sequencer (University of Maine, Orono, Maine). The resulting DNA sequences were aligned using Mac Vector (GCG) and LaserGene (DNA STAR) sequence analysis software.
  • GCG Mac Vector
  • LaserGene DNA STAR
  • the presence of amplified PVCR products was detected by Southern blotting analyses of gel fractionated RT-PCR products using a 32 P-labeled 653 bp Atlantic salmon amplified genomic PCR product. A total of 10 microliters of each PCR reaction was electrophoresed on a 2% agarose gel using TAE buffer then blotted onto Magnagraph membrane (Osmonics, Westboro, MA).
  • blots were prehybridized and then probed overnight (68°C in 6X SSC, 5X Denhardt's Reagent, 0.5% SDS, lOOug/ml calf thymus DNA) with the 653 bp Atlantic salmon PCR product (labeled with RadPrime DNA Labeling System, Gibco Life Sciences). Blots were then washed with O.lx SSC, 0.1% SDS @ 55°C and subjected to autoradiography under standard conditions.
  • Figure 37 shows the results of RT-PCR amplification of a partial PVCR mRNA transcript from various tissues of juvenile Atlantic salmon. RT-PCR reactions were separated by gel electrophoresis and either stained in ethidium bromide(EtBr) or transfened to a membrane and Southern blotted using a 32 P -labeled 653 bp genomic DNA fragment from the Atlantic salmon PVCR gene.
  • Figure 37 shows the detection ofthe PVCR in several tissue types of Atlantic Sahnon using the RT-PCR method, as described herein. The types of tissue are gill, nasal lamellae, urinary bladder, kidney, intestine, stomach, liver, and brain.
  • EXAMPLE 13 PRESENCE AND FUNCTION OF PVCR PROTEIN TN NASAL LAMELLAE AND OLFACTORY BULB AS WELL AS GI TRACT OF FISH.
  • PVCR proteins Alterations in the expression and/or sensitivity of PVCR proteins provides the means to enable fish to determine on a continuous basis whether the water composition they encounter is different from that they have been adapted to or exposed to previously. This system is likely to be integral to both the confrol ofthe homing of salmon from freshwater to seawater as smolt and their return to freshwater from seawater as adults. Thus, fish have the ability to "smell" changes in water salinity directly via PVCR proteins and respond appropriately to regulate remain in environments that are best for their survival in nature.
  • PVCR protein for divalent cations such as Ca 2+ and Mg 2"1" by changes in the NaCl concentration ofthe water.
  • PVCRs in fish olfactory organs have different apparent sensitivity to Ca 24 in either the presence or absence of NaCl.
  • PVCR protein function in the olfactory apparatus offish is to modulate responses of olfactory cells to specific odorants (attractants or repellants). Transduction of cellular signals resulting from the binding of specific odorants to olfactory cells occurs via changes in standing ionic gradients across the plasma membranes of these cells. The binding of specific odorants to olfactory cells results in-electrical nerve conduction signals.that can be recorded using standardized electrophysiological electrodes and equipment. Using this apparatus, the olfactory apparatus of freshwater adapted salmon:
  • PVCRs in the olfactory apparatus of salmon possess the capacity of modulating responses to various odorants.
  • Another feature of PVCR proteins is their ability to "sense" specific amino acids present in sunounding enviromnent.
  • SKCaR cDNA functional SKCaR protein was expressed in HEK cells and shown to respond in a concentration-dependent manner to both single and mixtures of L- amino acids. Since PVCR agonists including amino acids as well as polyamines (putrescine, spermine and spermidine) are attractants to marine organisms including fish and crustaceans, these data provide for another means by which PVCR proteins would serve not only as modulators of olfaction in fish but also as sensors of amino acids and polyamines themselves. PVCR proteins in other organs offish including G.I.
  • amino acids provide for integration of a wide variety of cellular processes in epithelial cells (amino acid transport, growth, ion transport, motility and growth) with digestion and utilization of nutrients in fish.
  • PVCR protein and mRNA are localized to the olfactory lamellae, olfactory nerve and olfactory bulb of freshwater adapted larval, juvenile and adult Atlantic salmon as well as the olfactory lamellae of dogfish shark:
  • Figure 38 show representative immunocytochemistry photographs of PVCR protein localization in olfactory bulb and nerve as well as olfactory lamellae in juvenile Atlantic salmon.
  • the specificity of staining for PVCR protein is verified by the use of 2 distinct antisera each directed to a different region ofthe PVCR protein.
  • antiserum anti-4641 recognizing an extracellular domain PVCR region
  • antiserum anti-SKCaR recognizing an intracellular domain PVCR region
  • PVCR protein in both nasal lamellae cells as well as olfactory bulb and nerve shows that these respective PVCR proteins would be able to sense both the internal and external ionic environments ofthe salmon.
  • cells containing internally-exposed PVCRs are connected to externally- exposed PVCRs via electrical connections within the nervous system.
  • these data suggest that externally and internally-exposed PVCRs function together to provide for the ability to sense the ionic concentrations ofthe sunounding ionic environment using as a reference the ionic concentration of the salmon's body fluids.
  • Figure 40 shows immunocytochemistry using anti-SKCaR antiserum that reveals the presence of PVCR protein in both the developing nasal lamellae cells and olfactory bulb of larval Atlantic salmon only days after hatching (yolk sac stage). As described herein, imprinting of sahnon early in development as well as during smoltification have been shown to be key intervals in the successful return of wild salmon to their natal stream.
  • the Sal I antiserum also localizes SalmoKCaR proteins in a variety of tissues in larval Atlantic salmon (Figure 40B). These tissues include the developing nasal lamellae of salmon and trout, their skin, myosepta, otolith and sensory epithelium. Myosepta are important in both the development of muscle in larval fish since they separate and define the muscle bundles ofthe salmon. Myosepta are also of significant commercial importance since they are one ofthe principal detenninants of texture for smoked Atlantic salmon fillets.
  • SalmoKCaR proteins are also present in the otolith which is a calcified structure located in the inner ear of he sahnon where it is closely associated with epithelial cells responsible for sensing sound and direction.
  • the presence of PVCR proteins at these developmental stages of salmon lifecycle indicate that PVCRs participate in this process.
  • EEG extracellular electrical potentials
  • Figure 44 displays representative recordings obtained from 6 freshwater adapted juvenile Atlantic salmon (approximately 300-400gm) using methods similar to those described in Bodznick, D. J. Calcium ion: an odorant for natural water discriminations and the migratory behavior of sockeye salmon, Comp. Physiol A 127:157-166 (1975), and Hubbard, PC, et al, Olfactory sensitivity to changes in environmental Ca2+ in the marine teleost Spams Aurata, J. Exp. Biol 203:3821- 3829 (2000).
  • V-clamp apparatus After anaesthetizing the fish, it was placed in V-clamp apparatus where its gills were irrigated continuously with aerated seawater and its nasal lamellae bathed continuously by a sfream of distilled water via a tube held in position in the inhalant olfactory opening.
  • the olfactory nerves ofthe fish were exposed by removal of overlying bony structures.
  • Stimuli were delivered as boluses to the olfactory epithelium via a 3 way valve where 1 cc of water containing the stimulus was rapidly injected into the tube containing a continuously stream of distilled water.
  • Extracellular recordings were obtained using high resistance tungsten electrodes where the resultant amplified analog signals (Grass Amplifier Apparatus) were digitized, displayed and analyzed by computer using MacScope software. Using this experimental approach, stable and reproducible recordings could be obtained for up to 6 hr after the initial surgery on the fish.
  • FIG. 45 shows dose response data from multiple fish to various PVCR agonists or modulators where the relative magnitudes of individual olfactory nerve response were normalized relative to the response produced by the exposure ofthe olfactory epithelium to 10 mM Ca 2+ .
  • the olfactory epithelium of freshwater adapted juvenile sahnon is very sensitive to Ca 2+ where the half maximal excitatory response (EC J0 ) is approximately 1-10 micromolar.
  • PVCR proteins present in olfactory epithelium are capable of sensing and generating conesponding olfactory nerve signals in response to PVCR agonists at appropriate concentrations in distilled water.
  • Figure 46 shows representative data obtained from a single continuous recording where the olfactory epithelium was first exposed to a well-known repellant, mammalian finger rinse.
  • Finger rinse is obtained by simply rinsing human fingers of adherent oils and fatty acids using distilled water and has been shown previously to be a powerful repellant stimulus both in EEG recordings as well as behavioral avoidance assays (Royce-Malmgren and W.H Watson J. Chem. Ecology 13:533-546 (1987)). Note however that inclusion ofthe PVCR agonists 5mM Ca 2+ or 50 micromolar Gd 3+ reversibly ablated the response by the olfactory epithelium to mammalian finger rinse.
  • PVCR agonists modulated the response ofthe olfactory epithelium to an odorant such as mammalian finger rinse.
  • the ablation of responses to both the PVCR agonists as shown in Figure 45 as well as mammalian finger rinse indicate that there are some complex interactions between PVCR proteins and other odorant receptors. It is also extremely unlikely that inclusion of PVCR agonists removed all the stimulatory components of mammalian finger rinse from solution such that they were not able to stimulate the epithelium.
  • Figure 47 shows a time series of stimuli (2 min between each stimulus in a single fish) similar to that displayed on Figure 47 except that 500 micromolar L- Alanine (a salmon attractant) was used to produce a signal in the olfactory nerve.
  • Recombinant PVCR protein SKCaR possesses the capability to sense concentrations of amino acids after its expression in human embryonic kidney (HEK) cells:
  • SKCaR Full length recombinant dogfish (Squ ⁇ lus ⁇ c ⁇ nthi ⁇ s) shark kidney calcium receptor (SKCaR) was expressed in human embryonic kidney cells using methods described herein. The ability of SKCaR to respond to individual amino acids as well as various mixtures was quantified using FURA-2 ratio imaging fluorescence.
  • Figure 48 shows a comparison of fluorescence tracings of FURA2-loaded cells stably expressing SKCaR that were bathed in physiological saline (125 mM NaCl, 4mM KCl, 0.5 mM CaCl 2 , 0.5 MgCl 2 , 20 mM HEPES (NaOH), 0.1% D- glucose pH 7.4) in the presence or absence of 10 mM L-Isoleucine (L-Ile) before being placed into the fluorimeter. Baseline extracellular Ca 2+ concentration was 0.5 mM.
  • Figure 49 shows data plotted from multiple experiments as described in Figure 48 where the effects of 10 mM Phe, lOmM He or an amino acid mixture (AA Mixture) containing all L-isomers in the following concentrations in micromoles/liter: 50 Phe, 50 Trp, 80 His, 60 Tyr, 30 Cys, 300 Ala, 200 Thr, 50 Asn, 600 Gin, 125 Ser, 30 Glu, 250 Gly, 180 Pro, 250 Val, 30 Met, 10 Asp, 200 Lys, 100 Arg, 75 He, 150 Leu. Note that both 10 mM Phe and 10 M He as well as the mixture of amino acids increase SKCaR's response to a given Ca 2+ concentration.
  • AA Mixture amino acid mixture
  • This mechanism could be used to inform both epithelial and neuroendocrine cells ofthe intestine ofthe presence of nutrients (proteins) and trigger a multitude of responses including growth and differentiation of intestinal epithelia as well as their accompanying fransport proteins, secretion or reabso ⁇ tion of ions such as gastric acid.
  • the apical PVCR also regulates the secretion of intestinal honnones such as cholecystokin (CCK) and others.
  • CCK cholecystokin

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Abstract

L'invention concerne quatre séquences d'acides nucléiques et d'aminoacides pleine longueur destinées à des récepteurs détectant les cations polyvalents (PVCR) dans le saumon atlantique. Ces PVCR ont été nommés SalmoKCaR n°1, SalmoKCaR n°2, SalmoKCaR n°3, et SalmoKCaR n°4. L'invention concerne des homologues de ceux-ci, des anticorps dirigés contre ceux-ci, et des procédés permettant d'évaluer des molécules d'acides nucléiques et des polypeptides des récepteurs SalmoKCaR. L'invention concerne en outre des plasmides, des vecteurs, et des cellules hôtes contenant les séquences d'acides nucléiques des récepteurs SalmoKCaR n°1, 2, 3 et/ou 4.
PCT/US2003/011188 2000-10-12 2003-04-09 Recepteur detectant les cations polyvalents dans du saumon atlantique WO2003087331A2 (fr)

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EP03746726.3A EP1572938A4 (fr) 2002-04-11 2003-04-09 Recepteur detectant les cations polyvalents dans du saumon atlantique
CA2481827A CA2481827C (fr) 2002-04-11 2003-04-09 Recepteur detectant les cations polyvalents dans du saumon atlantique
AU2003232002A AU2003232002A1 (en) 2002-04-11 2003-04-09 Polyvalent cation-sensing receptor in atlantic salmon

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US24000300P 2000-10-12 2000-10-12
US24039200P 2000-10-12 2000-10-12
US12144102A 2002-04-11 2002-04-11
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US10/125,778 2002-04-18
US10/125,792 2002-04-18
US10/125,778 US6979558B2 (en) 2000-10-12 2002-04-18 Polyvalent cation-sensing receptor in Atlantic salmon
US10/125,792 US6979559B2 (en) 2000-10-12 2002-04-18 Polyvalent cation-sensing receptor in atlantic salmon
US10/125,772 2002-04-18
US10/125,772 US6951739B2 (en) 2000-10-12 2002-04-18 Polyvalent cation-sensing receptor in atlantic salmon

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