CA2481827C - Polyvalent cation-sensing receptor in atlantic salmon - Google Patents

Abstract

The present invention encompasses four full length nucleic acid and amino.
acid sequences for PolyValent Cation-Sensing Receptors (PVCR) in Atlantic Salmon. These PVCR have been named SalmoKCaR#1, SalmoKCaR#2, SalmoKCaR#3, and SalmoKCaR#4. The present invention includes homologs thereof, antibodies thereto, and methods for assessing SalmoKCaR nucleic acid molecules and polypeptides. The present invention further includes plasmids, vectors, host cells containing the nucleic acid sequences of SalmoKCaR #1,2 3 and/or 4.

Description

POLYVALENT CATION-SENSING RECEPTOR IN ATLANTIC SALMON
.10 BACKGROUND OF THE INVENTION
In nature, anadromous fish like salmon live most of their adulthood in seawater, but swim upstream to freshwater for the purpose 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.
Currently, wild Atlantic salmon are classified as endangered species in multiple areas of their native habitats. Among the reasons for their decline has been man made alterations in freshwater conditions in their native streams that have produced multiple problems with their migration, spawning, smoltification and survival. One problem complicating the effective restoration of wild Atlantic salmon is the lack of a fundamental understanding of how these deleterious environmental conditions effect the salmon's ability to home to freshwater streams from the ocean, interchangeably adapt to freshwater and seawater as well as feed and grow in both salinity environments.

Despite the decline of wild populations, the global aquaculture industry has utilized Atlantic salmon as one of chief fish species for large-scale marine farming operations. At the present time, large scale breeding programs of Atlantic salmon provide for high quality fish used in production by selection of specific traits among them rapid growth, seawater adaptability, flesh quality and taste.
However, fish hatcheries have experienced some difficulty in raising salmon because the window of time in which the pre-adult salmon adapts to seawater (e.g., undergoes smoltification) is short-lived, and can be difficult to pinpoint. As a result, these hatcheries can experience significant morbidity and mortality when transferring salmon from freshwater to seawater. Additionally, many of the salmon that do survive the transfer from freshwater to seawater are stressed, and consequently, experience decreased feeding, and increased susceptibility to disease.
Therefore, salmon often do not grow-well after they-are transferred to seawater.
The aquaculture industry loses millions of dollars each year due to problems it encounters in transferring salmon from freshwater to seawater. Therefore, a need exists to gain a better understanding of the biological processes of salmon that are related to smoltification and adaptation to varying salinities, including seawater. In particular, a need exists to identify genes that play an important role in these areas.
SUMMARY OF THE INVENTION
The present invention relates to genes that allow fish to sense and adapt to ion concentrations in the surrounding 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 of fish, and in particular, in Atlantic Salmon. In fact, four foinis of the PVCR have been isolated in Atlantic Salmon, and have been termed, "SalmoKCall" genes and individually referred to as "SalmoKCaR#1", "SalmoKCaR#2", "SalmoKCaR#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 surrounding 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#1 (SEQ ED NO: 7), SalmoKCaR#2 (SEQ JD NO: 9), SalmoKCaR#3 (SEQ JD NO: 11), or SalmoKCaR#4 (SEQ ID NO: 13). The invention also embodies polypeptide molecules having amino acid sequences of SalmoKCaR#1 (SEQ II) NO: 8), SahnoKCaR#2 (SEQ ID NO: 10), SalmoKCaR#3 (SEQ ID NO: 12), or SalmoKCaR#4 (SEQ JD 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 JD NO: 7,9, 11, or 13.
In one embodiment, 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 ( e.g., SEQ ID
NO: 7, 9, 11, or 13) encodes a polypeptide that allows for or assists in one or more of the 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 absorption; or altering urine output.
The present invention further includes nucleic acid molecules that hybridize, preferably under high stringency conditions, with SalmoKCaR#1, SalmoKCaR#2, SalmoKCaR#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. et al., PNAS, 95:5178-5181 (1998), hereinafter "Naito" and deposited as GenBank Accession Nos:
AB008857 (Fugu CaR (01), SEQ ID NO: 31), A1B008858 (Fugu Ca02.1, SEQ ID
NO: 32), AB008859 (Fugu Ca09, SEQ JD NO: 33), AB008860 (Fugu Ca12, SEQ ID
NO: 34), AB008861 (Fugu Ca13, SEQ ID NO: 35), AB008862 (Fugu Ca15.1, SEQ
ID NO: 36), AB008863 (Fugu mGluR1, SEQ ID NO: 37), AB008864 (Fugu mGluR2, SEQ ID NO: 38), AB008865 (Fugu mGluR7, SEQ ID NO: 39), and AB008866 (Fugu mGluR8, SEQ ID NO: 40)). The sequences described in the Naito including those deposited under these GenBank Accession Numbers are incorporated by referenced in their entirety, and are referred to herein as the "fugu pheromone receptor" or "fugu receptor." SKCaR is a PVCR isolated from dogfish shark. Specifically, 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 ID NO:
7, 9, 11, or 13; but excluding those that hybridize to SEQ ID NO: 1 or the nucleic acid sequence of the fugu pheromone receptor, as described herein, under the same conditions.
The present invention also includes probes, vectors, viruses, plasmids, and _ host cells that contain the nucleic acid sequences, as described herein. In particular, the present invention includes probes (e.g., nucleic acid probes or DNA
probes) having a sequence from or hybridizes to SEQ ID NO: 7, 9, 11, or 13, but not SEQ ID
NO: 1 or the nucleic acid sequence of the fagu 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).
In another embodiment, 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. These polypeptide molecules (e.g., SEQ ID NO: 8, 10, 12, or 14; or those encoded by the nucleic acid sequence of SEQ
ID NO: 7, 9, 11, or 13) have one or more of the 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 absorption; or altering urine output.
Additionally, 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 of the polypeptide molecules described herein, and a portion of an immunoglobulin.
The present invention also pertains to assays for determining 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 of the SalmoKCaR polypeptide sufficiently to allow formation of a complex between SalmoKCaR and the antibody, and detecting the presence or absence of the 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 JD 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 detefinining 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 of the 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. An increase or decrease in the expression level, as compared to a control, indicates that the compound is a modulator.

Lastly, the present invention relates to transgenic fish encoding a SalmoKCaR polypeptide 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 of the present invention also allows for assaying or testing these salmon to determine if they are ready for transfer to seawater, so that they can be transferred at the best time. The technology of the 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. A number of additional advantages for the present invention exist and are apparent from the description provided herein.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A-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.
Figure 2 is a graphical representation showing a normalized calcium response (%) against the amount of Calcium (mM) of the SKCaR-I protein when modulated by altei-nations in extracellular NaC1 concentrations.
Figure 3 is a graphical representation showing a normalized calcium response (%) against the amount of magnesium(mM) of the SKCaR-I protein in increasing amounts of extracellular NaC1 concentrations.
Figure 4 is a graphical representation showing the EC50 for calcium activation of shark CaR (mM) against the amount of sodium (mM) of the SKCaR-I
protein in increasing amounts of extracellular NaC1 concentrations.
Figure 5 is a graphical representation showing the EC50 for magnesium activation of shark CaR (mM) against the amount of sodium (mM) of the SKCaR-I
protein in increasing amounts of extracellular NaC1 concentrations.
Figure 6 is a graphical representation showing the EC50 for magnesium activation of shark CaR (mM) against the amount of sodium (mM) of the SKCaR-I
protein in increasing amounts of extracellular NaC1 concentrations and added amounts of calcium (3mM).
Figures 7A and 7B show an annotated partial nucleotide sequence (SEQ JD
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
ED NO: 5) and the deduced amino acids sequence (SEQ ID NO: 6) of an Atlantic salmon polyvalent cation-sensing receptor protein.
Figures 9A-E show the nucleic acid (SEQ ID NO: 7) and amino acid (SEQ
ID NO: 8) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#1 with the ORF starting at nt 180 and ending at 3005.
Figures 10A-E show the nucleic acid (SEQ ID NO: 9) and amino acid (SEQ
ID NO: 10) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#2 with the ORF starting at nt 270 and ending at 3095.
Figures 11A-D show the nucleic acid (SEQ ID NO: 11) and amino acid (SEQ
ID NO: 12) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#3 with the ORF starting at nt 181 and ending at 2733.
Figures 12A-E show the nucleic acid (SEQ ID 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#1 (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#1 (SEQ ID NO:

8), SalmoKCaR#2 (SEQ ID NO: 10), and SalmoKCaR#3 (SEQ ID NO: 12).
Figure 15A is photograph showing a Southern blot in which SalmoKCaR#1, 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#1 and #3 constructs. In particular, the left panel shows an immunoblotting analysis with SDD antiserum while the right panel shows immunoblotting analysis for Sall for the following: a standard, 5001 HEK (human) cells, HEK 293 mock transfected cells, SalmoKCaR#1 HEK cells, SalmoKCaR#3 HEK cells, and Salmon SW Kidney.
Figures 16A-H are an alignment of the full length nucleic acid sequences of SalmoKCaR#1, 2, and 3 (SEQ ID 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#1 (SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), SalmoKCaR#3 (SEQ ID NO: 11), and SalmoKCaR#4 (SEQ ID NO: 13). Alignment was obtained using Clustal method with weighted residue weight table.
Figures 18A-C are an alignment showing amino acid sequences of SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), SalmoKCaR#3 (SEQ ID NO: 12), and SalmoKCaR#4 (SEQ ID NO: 14).
Figures 19A-D are an alignment of the full length amino acid sequences of Human Parathyroid Calcium Receptor (HuPCaR) (SEQ JD NO: 30), SKCaR (SEQ
ID NO: 2), SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), and SalmoKCaR#3 (SEQ ID NO: 12), and SalmoKCaR#4 (SEQ ID 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 (Figures 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). 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. Wells 1, 2, 7, 9, and 12, bottom row, for 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, SalmoKCaR#2 and SalmoKCaR#3, respectively.

Figure 21A 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 II treatment or 26 days after transfer to seawater. Process II treatment is defined in the Exemplification.
Figure 21B is a photograph of a RT-PCR analysis showing increased steady state expression of SalmoKCaR transcripts in pyloric caeca of Process II
treated and seawater fish as compared to freshwater Atlantic salmon smolt. Using degenerate (SEQ ID Nos 15 and 16) or actin (SEQ ID No 24 and 25) primers, samples of either freshwater (Panel A Lanes 3 and 6), Process II treated (Panel A Lanes 4 and 7) or seawater adapted (Panel A Lanes 5 and 8) Atlantic salmon smolt were analyzed by RT-PCR. To control for differences in sample loading, these identical samples were subjected to PCR analysis using actin specific primer (Panel A, Lanes 3-5).
Note that both ethidium bromide stained gel (Panel A) and its corresponding Southern blot (Panel C) show increased amounts of SalmoKCaR transcripts in pyloric caeca from Process II and seawater adapted fish as compared to freshwater. As a control, Panel B demonstrates that these degenerate primers amplify SalmoKCaR #1 (Lane 1), SalmoKCaR #2 (Lane 2) and SalmoKCaR #3 (Lane 3) transcripts.
Figure 21C is a photograph of RT-PCR analysis showing expression of SalmoKCaR transcripts in various stages of Atlantic salmon embryo development.
Using degenerate (SEQ ID Nos. 15 and 16) or actin (SEQ ID No 24 and 25) primers, RNA obtained from samples of whole Atlantic salmon embryos at various stages of develoiment 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 of the 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 of the 5 samples shows they are approximately equal (lanes 1-5).

Figure 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 23A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR
#3 specific PCR (Figures 23A-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 23A, 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 Sahnol(CaR#3, respectively.
Figures 24A-F are graphical representations comparing six photographs showing RT-PCR analysis of freshwater (Figures 248, 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. Wells 1, 2,3, 5, 6, and 7.
bottom row, for Figures 24A, C, and E are designated as ladder, water, Kidney-RT, SalmoKCaR #1, SalmoKCaR#2 and Sahnol(CaR#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 25A-D) or salmon actin PCR primers (Figures 25E,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. bottom row, for Figures 25A, 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.
Figure 26 is a schematic diagram illustrating industry practice for salmon aquaculture production, prior to the discovery of the 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 betvveen 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 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 (gm) of Process I smolts before transfer, and mortalities after transfer to ocean netp ens.
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 (gin) of St. John/St. John Process II smolts prior to seawater transfer.
Process II is defined in the Exemplification Section.
Figures 32A and 32B are graphical 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 of the 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 preinunune) illustrating SalmoKCaR expression.
Figures 35A-C are photographs of immunolocalization of the SalmoKCaR in the epidermis 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.
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 transferred 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 40A 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 immunocytochemistry 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 arrowhead).
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 corresponding 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 inununocytochemistry. 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). Note that anti-Sal ADD reactivity (denoted by arrows) is similar if not identical to that displayed by Sal-I antiserum. Corresponding 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 detemined 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).
Figure 44 shows the raw and integrated recordings from high resistance electrodes of freshwater adapted Atiantic Salmon when exposed to 500 M L-alanine, 1 mmol calcium, 50 p.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 with10 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 (finger 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 NaC1) 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 of the ratio from FURA-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').
Figure 49 is a graphical representation of the fractional Ca' response, as compared to the extracelluar Ca' (mM) for the PVCR in Ca' 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 53A-B show the nucleic acid sequence for a Fugu receptor, Fugu Ca12, (SEQ ID NO: 34) deposited under GenBank Accession Nos: AB008860.
Figures 54A-B show the nucleic acid sequence for a Fugu receptor, Fugu Ca13, (SEQ ID NO: 35) deposited under GenBank Accession Nos: AB008861.
Figures 55A-B show the nucleic acid sequence for a Fugu receptor, Fugu Ca15.1, (SEQ ID NO: 36) deposited under GenBank Accession Nos: A1B008862.
Figure 56 shows the nucleic acid sequence for a Fugu receptor, Fugu mGluR1, (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.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to at least four novel isolated sequences from PVCR genes, SalmoKCaR#1, SalmoKCaR#2, SalmoKCaR#3, and SalmoKCaR#4 in Atlantic Salmon. These genes encode four polypeptide sequences that are also the subject of the 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 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 absorption; or altering urine output.
Uses of the Present Invention One use of the 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 of the SalmoKCaR modulator in the serum. During this process, the serum level of the SalmoKCaR modulator significantly increases in the salmon, and causes modulated (e.g., increased and/or decreased) SalmoKCaR expression and/or altered SalmoKCaR sensitivity. This process prepares salmon for transfer to seawater, so that they can better adapt to seawater once they are transferred. The details of how to carry out this process is described in the Exemplification Section. In particular, the Exemplification describes two processes. Briefly, 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. 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 II 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 transferred to seawater.
Furthermore, experiments described in Example 6 demonstrate that modulated expression of one or more SahnoKCaR genes occurs in various tissues during Process I and Process II.
Process I and II, 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 II 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 (A13+, Zn21, Cu2+) 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' concentrations. Thus, treatment of wild strains of Atlantic salmon produced in restocking hatcheries with either Process I or Process 11 would render these treated smolt less susceptible to the effects of heavy metals since the smoltification process in these treated smolt was much further advanced that in untreated fish. Use of Process I or II to treat Atlantic salmon that would be released into rivers also have commercial utility in large-scale ocean ranching programs where large numbers of salmon smolt are released and captured for human consumption upon their return from 1-3 years in the ocean.
Similarly, since expression of the SalmoKCaR genes changes during Process - I and Process-II, assaying these genes allows one to determine if the salmon are ready for transfer to seawater. Examples of such assays are ELISAs, radioinununoassays (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 transferred 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. Using methods described herein, salmon can be assayed to determine if modulated levels of.the SalmoKCaR genes and/or polypeptides have occurred, 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 transferred to seawater. In one experiment, the increased expression of SalmoKCaR genes in the kidney of Atlantic Salmon subjected to Process II 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. When levels of SalmoKCaR genes and/or polypeptide encoded by these genes are similar to those levels seen in fish that have been transferred to seawater, then in the experiments described herein, the transfer of these salmon result in several benefits including increased survival and growth. Also, the optimal time periods for subjecting salmon to Process I or Process II are generally between 4-6 weeks, but vary depending on the strain of salmon or process used. Hence, the assays described herein can be used to deteiniine the optimal amount of time for subjecting the salmon to either Process I
or Process II before transferring to seawater.
Additionally, comparison of the SalmoKCaR #3 sequence with data generated from site directed mutagenesis studies of mammalian CaRs indicates that the Sahnol(CaR #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 terminal 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. In one experiment, it was found that increased expression of SalmoKCaR#3 was seen in tissues readily exposed to high concentrations of calcium and magnesium in the surrounding 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 of the 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.
Uses of nucleic acids of the present invention include one or more of the following: (1) producing receptor proteins which can be used, for example, for structure determination, to assay a molecule's activity, and to obtain antibodies binding to the receptor; (2) being sequenced to determine a receptor's nucleotide sequence which can be used, for example, as a basis for comparison with other receptors to determine one or more of the following: conserved sequences;
unique nucleotide sequences for noinial 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 as hybridization detection probes; and (5) for determining and isolating additional aquatic PVCR homologs in other species.
Another use for 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 of fish 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-broodstock enhancement program.
Cell lines expressing SahnoKCaR proteins, either individually or in various combinations, 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 SalmoKCaR #1-4 proteins either alone or in various combinations can be used in screening methods to identify both naturally occurring 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 detellnine 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 of the 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 is not limited to the uses described in this section.
Based on the data and information described herein, additional uses of the present invention may be readily appreciated by one of skill in the art.
The SalmoKCaR Polypeptides and its Function 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 of the full length SalmoKCaR amino acid sequence (SEQ ID 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 of the isolated full length SalmoKCaR nucleic acid sequences (SEQ ID NO: 7, 9, 11, or 13).
SalmoKCaR polypeptides referred 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 of the 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. "Analogous" or "homolgous"
amino acid sequences refer to amino acid sequences with sufficient identity of any one of the SalmoKCaR amino acid sequences so as to possess the biological activity of any one of the native SalmoKCaR polypeptides. For example, an analog polypeptide can be produced with "silent" changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues of any one of the SalmoKCaR protein, yet still possesses the function or biological activity of the SalmoKCaR. Examples of such differences include additions, deletions or substitutions of residues of the amino acid sequence of SalmoKCaR. Also encompassed by the present invention are analogous polypeptides that exhibit greater, or lesser, biological activity of any one of the SalmoKCaR proteins of the 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 of the isolated SalmoKCaR molecules described herein. Such mutations can be performed using methods described herein and those known in the art. In particular, 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. As used herein, "percent similarity" refers to the amount of similar or conservative amino acids between two amino acid sequences. Each of the SalmoKCaR polypeptides are homologous to one another.
The percent identity when comparing one SalmoKCaR amino acid sequence to another are as follows:
Percent Identity for Amino Acid Sequences*
Query SalmoKCaR#1 SalmoKCaR#2 SalrnoKCaR#3 SalmoKCaR#4 Sequence SalmoKCaR#1 N/A 99.9% 89.6% 100%
SalmoKCaR#2 99.9% N/A 89.5% 99.9%
SalmoKCaR#3 99.2% 99.1% N/A 99.2%
SalmoKCaR#4 100% 99.9% 99.2% N/A
* Note that the percentages are based on the number of aa's in the target sequence.
The 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 of the 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 surrounding environment; imprinting with an odorant; altering water intake;
altering water absorption; altering urine output. These and additional functions of the polypeptides are further described herein, and illustrated by the Exemplification.
The tem "sense" or "sensing" 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 performed at NCBI
against the GenBank, EMBL and SwissProt databases using, for example, the BLAST network service. Altschuler, S.F., et al., J Mol. Biol., 215:403 (1990), Altschuler, S.F., Nucleic Acids Res., 25:3389-3402 (1998). Computer analysis of nucleotide sequences can be performed using the MOTIFS and the FindPatterns subroutines of the Genetics Computing Group (GCG, version 8.0) software. Protein and/or nucleotide comparisons were performed according to Higgins and Sharp (Higgins, D.G. and Sharp, P.M., Gene, 73:237-244 (1988) e.g., using default parameters).
The SalmoKCaR proteins of the present invention also encompass biologically active or functional polypeptide fragments of the full length SalmoKCaR proteins. Such fragments can include the partial isolated amino acid sequences (SEQ ID NO: 3 and 5), or part of the full-length amino acid sequence (SEQ ID NO: 8, 10, 12, or 14), yet possess the function or biological activity of the full length sequence. For example, polypeptide fragments comprising deletion mutants of the 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.
In 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 of the SalmoKCaR modulator in the serum. This amount of NaC1 in the feed causes or induces the Atlantic Salmon to drink more freshwater. Since the freshwater contains = a SalmoKCaR modulator and the salmon ingest increased amounts of it, the serum level of the almoKCaR 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 of the 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 II 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)). Examples of Type IT
calcimimetics, which increase and/or decrease expression, and/or sensitivity of the SalmoKCaR genes, are, for example, NPS-R-467 and NPS-R-568 from NPS
Pharmaceutical Inc., (Salt Lake, Utah, Patent Nos. 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 p.M feed or water. See Nemeth, E.F. et al., PNAS 95:
4040-4045 (1998). Examples of inorganic polycations are divalent cations including calcium at a concentration 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 [tM. 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 [tM and 10 mM feed). See Brown, E.M. et al., Endocrinology 128: 3047-3054 (1991); Quinn, S.J. et al., Am. J. Physiol. 273:
C1315-1323 (1997). Additionally, 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. See Conigrave, A.D., et al., PNAS 97: 4814-4819 (2000). 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 NaC1, as described herein), so long as the combination modulates expression and/or sensitivity of one or more of the SalmoKCaR genes.
Another function of the 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 of the nerve impulse during sensing of an odorant. Generation of this nerve impulse occurs upon binding of the odorant to the olfactory lamellae in the-fish. The SalmoKCaR modulator alters the olfactory sensing of the salmon to the odorant. In some cases, 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. In other cases it can be heightened or increased.
By exposing the salmon in freshwater having a SalmoKCaR modulator to an odorant, the fish have an altered response which depending on the modulator would consist of either a decreased or heightened response to the odorant. Briefly, these imprinting methods involve adding at least one SalmoKCaR modulator (e.g., calcium and magnesium) to-the freshwater in .an amount sufficient to modulate expression and/or sensitivity of at least one SalmoKCaR genre; and adding feed for fish consumption to the freshwater. The feed contains at least one an attractant (e.g., alanine); an amount of NaC1 sufficient to contribute to a significantly increased level of the SalmoKCaR modulator in serum of the Atlantic Salmon; and optionally a SalmoKCaR modulator (e.g., tryptophan). The odorant can also be added to the water, instead of the 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 SahnoKCaR
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. 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 of the 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 of the attractant is modulated, at least in part, by the sensitivity and/or expression of the SalmoKCaR genes in the olfactory apparatus of the fish in response to a SalmoKCaR modulator. Examples of attractants 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 tnonophosphate), organic compounds (e.g., glycine-betaine and trimethylamine oxide), or a combination thereof. Similarly, a fish repellant is a compound that fish are repelled by, and the sensitivity of the fish to the repellant is altered through expression and/or sensitivity of a SafmoKCaR gene in the olfactory apparatus of the 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 of the 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.
Additionally, the function of SalmoKCaR polypeptides includes its ability to sense or adapt to ion concentrations in the surrounding 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 of the 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 absorption.
Responses also include changes in biological processes that affect its ability to excrete contaminants.
More specifically, 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 SalmoKCaR proteins present in cells involved in ion transport. For example, salinity tolerance of fish adapted (or acclimated) to freshwater can be increased by activating one or more of the SalmoKCaR
polypeptides, for example, by increasing the expression of one or more of SalmoKCaR genes, resulting in the secretion of ions and seawater adaption.
Alternatively, the salinity tolerance of fish adapted to seawater can be decreased by inhibiting one or more of the SalmoKCaR proteins, resulting in alterations in the absorption of ions and freshwater adaption.
"Salinity" refers to the concentration of various ions in a surrounding aquatic _ environment. In particular, salinity refers to the ionic concentration of calcium, magnesium and/or sodium (e.g., sodium chloride). "Normal salinity" levels refers to the range of ionic concentrations of typical water environment in which an aquatic species naturally lives. Normal salinity or normal seawater concentrations are about 10mM 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 of the PVCR allows fish to live in about four times and one-fiftieth, preferably, twice and one-tenth the normal The ability of 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. Both types of fish have to undergo similar physiological changes including alterations in their urine output, altering water intake and water absorption. Both types of fish utilize environments of either freshwater (Atlantic salmon) 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 of fish 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. Similarly, 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 of fish have PVCRs which respond to ion concentrations in a similar manner. Summer and Winter Flounder were adapted to live in 1/10th seawater (100 mOsm/kg) by reduction in salinity from 450 mM NaCl to 45 mM NaCl over an interval of 8 hrs. Summer and Winter Flounder can be maintained in 1/10 or twice the salinity for over a period of months. After a 10 day interval where the Summer and Winter Flounder were fed a normal diet, the distribution of the PVCR in their urinary bladder epithelial cells was examined using immunocytochemistry. PVCR immunostaining is reduced and localized primarily to the apical membrane of epithelial cells in the urinary bladder.
In contrast, the distribution of PVCR in epithelial cells lining the urinary bladders of control flounders continuously exposed to full strength seawater is more abundant and present in both the apical membranes as well ás in punctate sequences throughout the cell. These data are consistent with previous Northern data where more PVCR protein is present in the urinary bladders of seawater fish vs fish adapted to brackish water. These data show that PVCR protein is expressed in epithelial cells that line the urinary bladder where the PVCR protein comes into direct contact with the urine that is being foimed by the kidney. Due to its location in the cell membrane of these epithelial cells, the PVCR proteins can "sense"
changes in the urine's composition on a continuous basis. Depending on the specific ionic concentrations of the urine, the PVCR protein alters the transport of ions across the epithelium of the urinary bladder and, in this way, determines the final composition of the urine. This composition and the amount of water and NaCI

absorbed from the urine are critical to salinity regulation in fish.
As urinary magnesium and calcium concentrations increase when fish are present in full strength sea water, activation of apical PVCR protein causes reduction in urinary bladder water transport. The invention provides methods to facilitate methods are now available to regulate salinity tolerance in fish by modulating (e.g., alternating, activating and or expressing) the activity of the PVCR protein present in epithelial cells involved in ion transport, as well as in endocrine and nervous tissue.
For example, salinity tolerance of fish adapted (or acclimated) to fresh water can be 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 of the gill, intestine and kidney. In the kidney, PVCR
activation facilitates excretion of divalent metal ions including calcium and ions by gill cells that occurs in fresh water and promote the net excretion of ions by gill epithelia that occurs in salt water. In the intestine, PVCR activation will permit reabsorption of water and ions across the G.I. tract after their ingestion by fish.
Alternatively, the salinity tolerance of fish adapted to seawater can be In another example, Winter and Summer Flounder were maintained in at 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 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. Preferably, the parasites and/or bacteria are reduced to a level that is safe for human consumption, raw or cooked. More preferably, the parasites and/or bacteria are reduced to having essentially no parasites and few bacteria. These fish must be maintained in a hypersalinity environment long enough to rid the fish of these parasites or bacteria, (e.g., for at least a few days or at least a few weeks).
The host range of many parasites is limited by exposure to water salinity.
For example, Diphyllobothrium species commonly known as fish tapeworms, is encountered in the flesh of fish, primarily fresh water or certain euryhaline species.
Foodbome Pathogenic Microorganisms and Natural Toxins Handbook. 1991. US
Food and Drug Administration Center for Food Safety and Applied Nutrition, the teachings of which are incorporated herein by reference in their entirety. In contrast, its presence in the flesh of completely marine species is much reduced or absent.
Since summer flounder can survive and thrive at salinity extremes as high as 58 ppt (1.8 times nonnal 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.
Data from Cole et al., (.1 Biol. Chem. 272:12008-12013 (1997)), show that winter flounder elaborate an antimicrobial peptide from their skin to prevent bacterial infections. Their data reveals that in the absence of pleurocidin, Escherichia coil are killed by high concentrations of NaCl. In contrast, low concentrations of NaC1 (<300mM NaC1) allow E. colt to grow and under these conditions pleurocidin presumably helps to kill them. These data provide evidence of NaC1 killing of E. coil, as well as highlight possible utility of bacterial elimination in fish.
Similarly, maintaining fish in a hyposalinity environment results in a fish with a reduced amount of contaminants (e.g., hydrocarbons, amines or antibiotics).
Preferably, the contaminants are reduced to a level that is safe for human consumption, raw or cooked fish. More preferable, the contaminants are reduced to having essentially very little contaminants left in the fish. These fish must be maintained in a hyposalinity environment long enough to rid the fish of these contaminants, (e.g., for at least a few days or a few weeks).
Organic amines, such as trimethylamine oxide (TMAO) produce a "fishy"
taste in seafood. They are excreted via the kidney in flounder. (Krogh, A., Osmotic Regulation in Aquatic Animals, Cambridge University Press, Cambridge, U.K. pgs 1-233 (1939), the teachings of which are incorporated herein by reference in their entirety). TMAO is synthesized by marine organisms consumed by fish that accumulate the TMAO in their tissues. Depending on the species of fish, the muscle content of TMAO and organic amines is either large accounting for the "strong"
taste of bluefish and herring or small such as in milder tasting flounder.
The presence of SalmoKCaR in brain reflects both its involvement in basic neurotransmitter release via synaptic vesicles (Brown, E.M. et al., New England J. of Med., 333:234-240 (1995)), as well as its activity to trigger various hormonal and behavioral changes that are necessary for adaptation to either fresh water or marine environments. For example, increases in water ingestion by fish upon exposure to salt water is mediated by SalmoKCaR activation in a manner similar to that described for humans where PVCR activation by hypercalcemia in the subfomical organ of the brain cause an increase in water drinking behavior (Brown, E.M.
et al., New England J. of Med., 333:234-240 (1995)). In fish, processes involving both alterations in serum hormonal levels and behavioral changes are mediated by the brain. These include the reproductive and spawning activities of euryhaline fish in fresh water after their migration from salt water as well as detection of salinity of their environment for purposes of feeding, nesting, migration and spawning.
The key events for successful reproduction in Atlantic salmon are to migrate to a specific streambed for spawning after 1-3 years of free-swimming existence on the open ocean. Successful achievement of this challenge depends on the combination of adult salmon being able to remember and navigate their way back to this original location as well as successful imprinting of larval and juvenile Atlantic salmon to =
odors present in freshwater in the freshwater streambed as well as the characteristics of the mouth of the river as the fish exit the river and enter the ocean.
Sensing of salinity by PVCR and its modulation of the 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. For example, alterations in plasma cortisol have been demonstrated to be critical for changes in ion transport necessary for adaptation of salmon smolts from fresh water to salt water (Veillette, P.A., et al., . Gen. and Comp. Physiol., 97:250-258 (1995)). As demonstrated recently in humans, plasma Adrenocorticotrophic Hormone (ACTH) levels that regulate plasma cortisol levels are altered by PVCR activation.
Additionally, the function or biological activity of the 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 proteins and their analogs are described in Brown, et al., New Eng.
J
Med., 333:243 (1995); Riccardi, etal., 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 of the SalmoKCaR to modulate signal transduction pathways in specific cells. Thus, depending on the distribution and nature of various signal transduction pathway proteins that are expressed in cells, biologically active SalmoKCaR proteins can modulate cellular functions in either an inhibitory or stimulatory manner.
Biologically active derivatives or analogs of the above described SalmoKCaR polypeptides, referred 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 Nos. 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 of the corresponding amino acid sequence. These peptide mimetics possess biological activity similar to the biological activity of the corresponding peptide compound, but possess a "biological advantage" over the corresponding 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 of the 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. Other forms of the SalmoKCaR polypeptides, encompassed by the present invention, include those which are "functionally equivalent." This term, as used herein, refers to any nucleic acid sequence and its encoded amino acid, which mimics the biological activity of the SalmoKCaR polypeptides and/or functional domains thereof.
SalmoKCaR Nucleic Acid Sequences, Plasmids, Vecfors and Host Cells The present invention, in one embodiment, includes an isolated full length nucleic acid molecule having a sequence of SalmoKCaR#1 (SEQ ID NO: 7), SalmoKCaR#2 (SEQ lD NO: 9), SalmoKCaR#3 (SEQ ID 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. As shown in these figures, the ORF
SalmoKCaR#1 begins at nt 180 and ends at nt 3005. For SalmoKCaR#2, it begins at nt 270 and ends at nt 3095, and for SalmoKCaR#3, the ORF=begins at nt 181 and ends at nt 2733. SahnoKCaR#4 has an OFR that begins at nt181 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 ID NO: 8, 10, 12, or 14. The SalmoKCaR full length nucleic acid sequences encode polypeptides that allow or assist in one or more of the 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 absorption; or altering urine output.

The present invention encompasses the SalmoKCaR full length nucleic acid sequences, SalmoKCaR#1 (SEQ IT) NO: 7), SalmoKCaR#2 (SEQ _________ NO: 9), SalmoKCaR43 (SEQ ID NO: 11), and SalmoKCaR #4 (SEQ ID 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), and PTA-4192 (March 29, 2002), (deposited by MariCal, LLC, 400 Commercial Street, Portland, Maine USA) for SalmoKCaR#1 (SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), and SalmoKCaR#3 (SEQ ID NO: 11) respectively. These clones are plasmid DNA which can be transformed into E.
Coli and cultured. The viability of the clones can be tested with ampicillin resistance.
The sequences of the present invention can be purified from these deposits using techniques known in the art.
As used herein, an "isolated" gene or nucleotide sequence which is not flanked by nucleotide sequences which normally (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). Thus, 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!! as used herein. Also, isolated nucleotide sequences include recombinant nucleic acid molecules and heterologous host cells, as well as partially or =
substantially or purified nucleic acid molecules in solution. in vivo and in vitro RNA transcripts of the present invention are also encompassed by "isolated"
nucleotide sequences. Such isolated nucleotide sequences are useful for the manufacture of the 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 of the present invention include homologues nucleic acid sequences. "Analogous" or "homologous" nucleic acid sequences refer to nucleic acid sequences with sufficient identity of any one of the SalmoKCaR nucleic acid sequences, such that once encoded into polypeptides, they possess the biological activity of any one of the native SalmoKCaR
polypeptides.
For example, 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 the 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. In particular, 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 of the SalmoKCaR genes are homologues to -one another.
The percent identity for the SalmoKCaR nucleic acid sequences are as follows:
Percent Identity for Nucleic Acid Sequences Query SalmoKCaR#1 SalmoKCaR#2 SalmoKCaR#3 SalmoKCaR#4 Sequence _ _ S almoKCaR#1 N/A 99.8% 95.8% 99%
SalmoKCaR#2 97.6% N/A 93.6% 98%
SalmoKCaR#3 98.7% 98.7% N/A 97%
SalmoKCaR#4 99% 98% 97% N/A
The nucleic acid molecules of the 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 of the 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 absorption; 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.
Also encompassed by the present invention are 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.
As defined herein, substantially complementary means that the nucleic acid need not reflect the exact sequence of the SalmoKCaR sequences, but must be sufficiently similar in sequence to permit hybridization with SalmoKCaR nucleic acid sequence under high stringency conditions. For example, 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 stlingency are described in e.g., Ausubel, F.M., et al.;
Current 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. For example, 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.
USA, 92:131-135 (1995), can be used. Positive clones can be isolated, subcloned and their sequences determined. Using the sequences of either a full length or several over-lapping partial cDNAs, the complete nucleotide sequence of the SalmoKCaR cDNA were obtained and the encoded amino acid sequence deduced.
The sequences of the SalmoKCaRs can be compared to each other and other aquatic PVCRs to determine differences and similarities. Methods for screening and identifying homologues genes as described in e.g., Ausubel, F.M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994).

SalmoKCaR genes were isolated by Polymerase Chain Reaction (PCR) of genomic DNA with degenerate primers (SEQ ID NOS: 15 and 16) specific to a highly conserved sequence of calcium receptors that does not contain introns.
For example, partial Atlantic Salmon clones were obtained by using degenerate primers that peiniit 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 ID NO: 3 with the addition of the sequence of the primers. The resulting amplified 653 bp fragment was ligated into a cloning vector and transformed into bacterial cells for growth, purification and sequencing. Additionally, SahnoKCaR genes can be isolated by Reverse Transciiptase-Polymerase Chain Reaction (RT-PCR) after isolation of poly A+
RNA
from aquatic species with the same or similar degenerate primers. Methods of PCR
and RT-PCR are well characterized in the art (See generally, PCR Technology:
Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila, et al., Nucleic Acids Res., 19:4967 (1991); Eckert, et al., PCR Methods and Applications, 1:17 (1991); PCR
(eds. McPherson, et al., 1RL Press, Oxford); and U.S. Patent 4,683,202. Poly A+
RNA can be isolated from any tissue which contains one or more of SalmoKCaR
polypeptides by standard methods as described. Preferred tissue for polyA+RNA
isolation can be determined using an antibody which is specific for the highly conserved sequence of calcium receptors, by standard methods. The partial genomic or cDNA sequences derived from a SalmoKCaR gene are unique and, thus, can be used as a unique probe to isolate the fall-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.
Also encompassed by the present invention are nucleic acid sequences, genomic DNA, cDNA, RNA or a combination thereof, which are substantially complementary to the DNA sequences encoding SalmoKCaR nucleic acid molecules and which 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. In another embodiment, the invention includes nucleic acid sequences that hybridize to the SalmoKCaR sequences, SEQ
ID 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. In one aspect, 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, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000).
Such molecules hybridize to one of the SalmoKCaR nucleic acid sequences (SEQ ID 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.
Typically 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. For example, a nucleic acid probe can be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the length of the SalmoKCaR nucleic acid sequence. The requirements of sufficient length and complementarity can be easily detefinined by one of skill in the art. Suitable hybridization conditions (e.g., high stringency conditions) are also described herein. Additionally, 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
preferred embodiment includes primers and probes which selectively hybridize to the nucleic acid constructs encoding any one of the SalmoKCaR proteins. For example, nucleic acid fragments which encode any one of the domains described herein are also implicated by the present invention.
Stringency conditions for hybridization refers to conditions of temperature and buffer composition which permit hybridization of a first nucleic acid sequence to a second nucleic acid sequence, wherein the conditions determine the degree of identity between those sequences which hybridize to each other. Therefore, "high stringency conditions" are those conditions wherein only nucleic acid sequences which are very similar to each other will hybridize. The sequences can be less similar to each other if they hybridize under moderate stringency conditions.
Still less similarity is needed for two sequences to hybridize under low stringency conditions. By varying the hybridization conditions from a stringency level at which no hybridization occurs:to a level at 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_suc_h as the length of the nucleic acid sequences, their base composition, the percent of mismatched base pairs between the two sequences, and the frequency of occurrence of subsets of the 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 (Tm) for any chosen SSC concentration.
Generally, a doubling of the concentration of SSC results in an increase in the Tm of about 17 C. Using these guidelines, the washing temperature can be detettnined empirically, depending on the level of mismatch sought. Hybridization and wash conditions are explained in Current Protocols in Molecular Biology (Ausubel, F.M.
et al., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) on pages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
High stringency conditions can employ hybridization at either (1) lx SSC

(10x SSC = 3 M NaC1, 0.3 M Na3-citrate=2H20 (88 g/liter), pH to 7.0 with 1 M
HC1), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA

at 65 C, (2) lx SSC, 50% fon-namide, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42 C, (3) 1% bovine serum albumin (fraction V), 1 m1VI Na2=EDTA, 0.5 M
NaHPO4 (pH 7.2) (1 M NaHPO4 = 134 g Na2HPO4.7H20, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 65 C, (4) 50%
foimamide, 5x SSC, 0.02 M Tris-HC1 (pH 7.6), lx Denh.ardt's solution (100x =
10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42 C, (5) 5x SSC, 5x Denhardt's solution, 1% SIDS, 100 g/ml denatured calf thymus DNA at 65 C, or (6) 5x SSC, 5x Denhardt's solution, 50%
formamide, 1% SDS, 100 pg/ml denatured calf thymus DNA at 42 C, with high stringency washes of either (1) 0.3 - 0.1x SSC, 0.1% SDS at 65 C, or (2) 1 mM
Na2EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS at 65 C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5 - 10 C below that of the calculated Tm of the hybrid, where Tm in C = (2 x the number of A and T bases) + (4 x the number of G and C
bases).
For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in C
(81.5 C + 16.6(log10M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where "M"
is the molarity of monovalent cations (e.g., Nat), and "L" is the length of the hybrid in base pairs.
Moderate stringency conditions can employ hybridization at either (1) 4x SSC, (10x SSC = 3 M NaC1, 0.3 M Na3-citrate=2H20 (88 g/liter), pH to 7.0 with HC1), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA
at 65 C, (2) 4x SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42 C, (3) 1% bovine serum albumin (fraction V), 1 mM Na2=EDTA, 0.5 M
NaHPO4 (pH 7.2) (1 M NaHPO4 -= 134 g Na2HPO4.7H20, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 65 C, (4) 50%
foiniamide, 5x SSC, 0.02 M Tris-HC1 (pH 7.6), lx Denhardt's solution (100x =
10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 42 C, (5) 5x SSC, 5x Denhardt's solution, 1% SDS, 100 p.g/m1 denatured calf thymus DNA at 65 C, or (6) 5x SSC, 5x Denhardt's solution, 50%
formamide, 1% SDS, 100 p.g/m1 denatured calf thymus DNA at 42 C, with moderate stringency washes of lx SSC, 0.1% SDS at 65 C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5 - 10 C below that of the calculated Tin of the hybrid, where Tin in C = (2 x the number of A and T bases) + (4 x the number of G and C
bases).
For hybrids believed to be about 18 to about 49 base pairs in length, the Tin in C --(81.5 C + 16.6(log10M) + 0.41(% G + C) - 0.61 (% fonnamide) - 500/L), where "M"
is the molarity of monovalent cations (e.g., Na), and "L" is the length of the hybrid in base pairs.
Low stringency conditions can employ hybridization-at either (1) 4x SSC, (10x SSC = 3 M NaCl, 0.3 M Na3-citrate2.H20 (88 g/liter), pH to 7.0 with 1 M
HC1), 1% SOS (sodium clodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA
at 50 C, (2) 6x SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 40 C, (3) 1% bovine serum albumin (fraction V), 1 mM Na2=EDTA, 0.5 M
NaHPO4 (pH 7.2) (1 M NaHPO4= 134 g Na,HPO4.7H20, 4m1 85% H3PO4 per liter), 7% SDS, 0.1 - 2 mg/nil denatured calf thymus DNA at 50 C, (4) 50%
foithamide, 5x SSC, 0.02 M Tris-HC1 (pH 7.6), lx Denhardt's solution (100x =
10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextra.n sulfate, 1% SDS, 0.1 - 2 mg/m1 denatured calf thymus DNA at 40 C, (5) 5x SSC, 5x Denhardt's solution, 1% SOS, 100 pz/m1 denatured calf thymus DNA at 50 C, or (6) 5x SSC, 5x Denhardt's solution, 50%
formamide, 1% SDS, 1001.1g/m1 denatured calf thymus DNA at 40 C, with low stringency washes of either 2x SSC, 0.1% SDS at 50 C, or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5 - 10 C below that of the calculated Tin of the hybrid, where Tin. in C = (2 x the number of A and T bases) + (4 x the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in C = (81.5 C + 16.6(log10M) + 0.41(% G + C) -0.61 (%
formamide) - 500/L), where "M" is the molarity of monovalent cations (e.g., Na), and "L" is the length of the hybrid in base pairs.
The SalmoKCaR nucleic acid sequence, or a fragment thereof, can also be used to isolate additional aquatic PVCR homologs. For example, 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).
In another embodiment, 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.
Methods foi- identifying a -nucleic acid by hybridization are routine in the art (see 'Current Protocols In Molecular Biology, Ausubel, F.M. et al., Eds., John Wiley &
Sons: New York, NY, (1997). The present method can optionally include a labeled SalmoKCaR probe.
The invention also provides vectors, plasmids or viruses containing one or _ more of the 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 Au-subel, F.M., et al:, Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., "Molecular Cloning: A Laboratory Manual,"

2nd ED. (1989).
Uses of plasmids, vectors or viruses containing the cloned SalmoKCaR
receptors or receptor fragments include one or more of the 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.
In one embodiment, 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-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 incorporated or inserted into the host cell by known methods. Examples of suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. "Transformation" or "transfection"
as used herein fefers to the acquisition of new or altered genetic features by incorporation of additional nucleic acids, e.g., DNA. "Expression" of the genetic information of a host cell is a term of art which refers to the directed transcription of DNA
to generate RNA which is translated into a polypeptide. Methods for preparing such recombinant host cells and incorporating nucleic acids are described in more detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition (1989) and Ausubel, et al. "Current Protocols in Molecular Biology," (1992), for example.
The host cell is then maintained under suitable conditions for expression and recovery of SalmoKCaR protein. Generally, the cells are maintained in a suitable buffer and/or growth medium or nutrient source for growth of the cells and expression of the 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, M199 and Grace's insect media. The growth media can contain a buffer, the selection of which is not critical to the invention.
The pH
of the 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.
Alternatively, 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.
Antibodies, Fusion Proteins and Methods of Assessment of the SalmoKCaR Nucleic Acid and Amino Acid Molecules The present invention includes methods of detecting the levels of the SalmoKCaR nucleic acid levels (mRNA levels) and/or polypeptide levels to deteindne whether fish are ready for transfer from freshwater to seawater. The present invention also includes methods for assaying compounds that modulate SalmoKCaR nucleic acid levels, expression levels or activity of SalmoKCaR
polypeptides. Activity of SalmoKCaR polypeptides includes, but is not limited to, phosphorylation of one or more of the SalmoKCaR polyp-eptides, dimerization of one of the SalmoKCaR polypeptides with a second SalmoKCaR polypeptide, proteolysis of one or more of the 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, radiohninunoassay, flow cytometry, immunocytochemistry, enzyme-linked immunosorbent assays (ELISA) and chemiluminescence assays.
Additionally, antibodies, or antibody fragments, can also be used to detect the r 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 of the 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 of fish in aquaculture. Any method known now or developed later can be used for measuring SalmoKCaR expression.
Antibodies reactive with any one of the SalmoKCaR or portions thereof can be used. In a preferred embodiment, the antibodies specifically bind with SalmoKCaR polypeptides or a portion thereof. The antibodies can be polyclonal or monoclonal, and the term antibody is intended to encompass polyclonal and monoclonal antibodies, and functional fragments thereof. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
In several of the preferred embodiments, immunological techniques detect SalmoKCaR levels by means of an anti-SalmoKCaR antibody (i.e., one or more antibodies). The term "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 SahnoKCaR, analogs or portion thereof (including synthetic molecules, such as synthetic peptides). In one embodiment, 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. In addition, 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:
511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S.
Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)).
Generally, fusing a suitable immortal or myeloma cell line, such as SP2/0, with antibody producing cells can produce a hybridoma. Animals immunized with the antigen of interest provide the antibody producing cell, preferably cells from the spleen or lymph nodes. Selective culture conditions isolate antibody producing hybridoma cells while limiting dilution techniques produce them. Researchers can use suitable assays such as ELISA to select antibody producing cells with the desired specificity.

Other suitable methods can produce or isolate antibodies of the 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 protein' s and thereby alter their function or specificity.
According to the method, an assay can determine the level of SalmoKCaR in a biological sample. In determining the amounts of SalmoKCaR, 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 formation of a complex. The sample can be obtained directly or indirectly, and can be prepared by a method suitable for the particular sample and assay format selected.
In particular, tissue samples, e.g., gill tissue samples, can be taken from fish after they are anaesthetized with MS-222. The tissue samples are fixed by immersion in 2% parafonnaldehyde in appropriate Ringers solution corresponding to the osmolality of the fish, washed in Ringers, then frozen in an embedding compound, e.g., 0.C.T.Tm (Miles, Inc., Elkahart, Indiana, USA) using methylbutane cooled with liquid nitrogen. After cutting 8-10 micron tissue sections with a cryostat, individual sections are subjected to various staining protocols. For example, sections are: 1) blocked with goat serum or serum obtained from the same species of fish, 2) incubated with rabbit anti-CaR or anti-SalmoKCaR
antiserum, and 3) washed and incubated with peroxidase-conjugated affinity-purified goat antirabbit antiserum. The locations of the 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 corresponding to amino acids numbers 214-236 localized in the extracellular domain of the RaKCaR
protein. The sequence of the 23-mer peptide is:
ADDDYGRPGIRKFREEABERDIC (SEQ ID NO.: 26) A small peptide with the sequence DDYGRPGIEKFREEABERDICI (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 chemiluminescent labels. They can also be indirectly detected using labels such as enzyme labels and other antigenic or specific binding paitners like biotin.
Examples of such labels include fluorescent labels such as fluorescein, rhodamine, chemiluminescent labels such as luciferase, radioisotope labels such as 32P, 125I, 131I, enzyme labels such as horseradish peroxidase, and alkaline phosphatase, f3-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).
Conventional methods-or other suitable methods can directly or indirectly label an antibody.
Labeled primary and secondary antibodies can be obtained commercially or prepared using methods know to one of skill in the art (see Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY).
Using the immunocytochemistry method, the levels of SalmoKCaR in various tissues can be detected and examined as to whether they change in comparison to control. Modulated levels or the presence of SalmoKCaR
expression in various tis-sues, as compared to a control, indicate that the fish or the population of fish from which a statistically significant amount of fish were tested, are ready for transfer to freshwater. A control refers to a level of SalmoKCaR, if any, from a fish that is not subjected to the steps of the present invention, e.g., not subjected to freshwater having a SalmoKCaR modulator and/or not fed a NaC1 diet. For example, 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 of the invention had SalmoKCaR levels that were easily detected.
In determining whether compounds are modulators, one can measure changes that occur in the expression levels of one or more the SalmoKCaR genes, or those that occur in one or more intracellular signal transduction systems or pathways. A
signal transduction pathway is a pathway involved in the sensing and/or processing of stimuli. In particular, such pathways are altered by activation of the expressed proteins coded for by a single or combination of nucleic acids of the present invention.
The SalmoKCaR polypeptides can be in the foul.' 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 comprising sequences which code for a biologically active portion of the 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. Upon expression, the fusion proteins can be isolated or purified from a cell by means of an affinity matrix. By measurement of the alternations in the functions of transfected cells occurring 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. There appear to be at least four distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb.
The SalmoKCaRs can also be assayed by hybridization, e.g., by hybridizing one of the SalmoKCaR sequences provided herein (e.g., SEQ ID NO: 7,9, 11, or 13) or an oligonucleotide derived from one of the sequences, to a DNA or RNA-containing tissue sample from a fish. Such a hybridization sequence can have a detectable label, e.g., radioactive, fluorescent, etc., attached to allow the detection of hybridization product. Methods for hybridization are well known, and such methods are provided in U.S. Pat. No. 5,837,490, by Jacobs et al., the entire teachings of which are herein incorporated by reference in their entirety. The design of the oligonucleotide probe should preferably follow these parameters: (a) it should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any, and (b) it should be designed to have a Tni 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 Sah-noKCaRs with PCR
Methods using primers disclosed or derived from sequences described herein.
For example, 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 talget sequence by repeated rounds of nucleic acid replication utilizing sequence-specific primers and a thermostable polymerase. PCR allows recovery of entire sequences between two ends of known sequence. Methods of PCR are described herein and are known in the art.
In particular, the 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. Methods of PCR and RT-PCR
are well characterized in the art (See generally, PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds.
McPherson et al., IRL Press, Oxford); Ausebel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience 1987, &
Supp.
49, 2000; and U.S. Patent 4,683,202). Briefly, mRNA is extracted from the tissue of interest and reverse transcribed. Subsequently, a PCR reaction is performed with SalmoKCaR-specific primers and the presence of the predicted SalmoKCaR product is deteiinined, for example, by agarose gel electrophoresis. Examples of SalmoKCaR-specific primers are SEQ ID NO: 18-23. The product of the RT-PCR

reaction that is performed with SalmoKCaR-specific primers is referred to herein as a RT-PCR product. The RT-PCR product can include nucleic acid molecules having part or all of the SalmoKCaR sequence. The RT-PCR product can optionally be radioactively labeled and the presence or amount of SalmoKCaR product can be determined using autoradiography. Two examples of commercially available fluorescent probes that can be used in such an assay are Molecular Beacons (Stratagene) and Taqman0 (Applied Biosystems). Alternative methods of labeling and quantifying the RT-PCR product are well known to one of skill in the art (see Ausebel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience 1987, & Supp. 49, 2000. Poly A+ 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. -Hence, the present invention includes 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 of SalmoKCaR.
Transgenic Fish Alterations in the expression or sensitivity of SalmoKCaRs could also be accomplished by introduction of a suitable transgene. 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 of the transgene in fish oocytes, embryos and adults are described in Chen, TT et al., Transgenic Fish, Trends in Biotechnology 8:209-215 (1990).
The present invention is further and more specifically illustrated by the following Examples, which are not intended to be limiting in any way.
EXEMPLIFICATION

The following examples refer to Process I and Process II throughout.
Process I is also referred 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 referred to as a "SUPERSMOLT
TM
I" or an "APS Process I" smolt. Likewise, Process ll is also referred to herein as "SUPERSMOLT TM II Process" or "Process II." A "Process II" fish or smolt refers to a fish or smolt that has undergone the steps of Process II. A Process II
smolt is also referred to as a "SUPERSMOLT TM II" 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 parr/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 II: Pre-adult anadromous fish (this includes both commercially produced SO, Si or S2 smolts as well as smaller parr/smolt fish) are exposed to or maintained in freshwater containing 2.0-10.0 mM Calcium and 0.5-10.0 mM
MagnesicUm 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) NaCI 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 32P-labeled full length rat kidney CaR (RaKCaR) cDNA
probe under intermediate stringency conditions (0.5X SSC, 0.1% SDS, 50 C.).
Individual positive plaques were identified by autoradiography, isolated and rescued using phagemid infections to transfer cDNA to KS Bluescript vector. The complete nucleotide sequence, Figure 1, (SEQ ID NO: 1) of the 4.1 kb shark kidney PVCR
related protein (SKCaR) clone was obtained using commercially available automated sequencing service that performs nucleotide sequencing using the dideoxy chain termination technique. The deduced amino acid sequence (SEQ ID
NO: 2) is shown in Figure 1. Northern analyses were perfotnied as described in Siner et. al. Am. J. Physiol. 270:C372-C381, 1996. The SKCAR-nucleotide -sequence was compared to others CaRs using commercially available nucleotide and protein database services including GENBANK and SWISS MR.
EXAMPLE 2. EXPRESSION/ACTIVATION STUDIES OF SKCaR Th HL1MAN
EMBRYONIC KIDNEY (HEK) CELLS
PVCRs serve as salinity sensors in 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 referred 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 NaC1, Ca2+ and Mg2+ in extracellular fluid bathing the HEK
cells. The ionic concentrations encompassed the range which includes the transition from freshwater to seawater. Expression of PVCR mRNA 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 lumpfish, by using nucleic acid amplification with degenerate primers.
In particular, the following was shown:
1. SKCaR encodes a functional ion receptor that is sensitive to both Mg2+ and Ca2+ as well as alterations in NaCl.
2. SKCaR's sensitivity to Ca2+, Mg2+ and NaC1 occur in the range that is found in marine environments and is consistent with SKCaRs role as a salinity sensor.
3. 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. These data can be used to design novel electrolyte solutions to maintain fish in salinities different from those present in their natural environment.
SKCaR cDNA was ligated into the mammalian expression vector PCDNA II
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 NaC1 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, 0. 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. I Biol. Chem., 32:19537-19545 (1996); and expressed as % normalized intracellular calcium response to receptor activation.
SKCaR is a functional extracellular Ca2+ sensror 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 NaC1 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 of the extracellular medium. This control experiment shows it is not alterations in cell osmolality effecting the changes observed.
The half maximal activation (EC50) by Ca2+ for SKCaR is reduced in increased concentrations of extracellular NaCl. See Figure 4. The EC50 for data shown on Figure 4 is displayed as a function of increasing extracellular NaC1 concentrations. Note the ECso for Ca2+ increases from less than 5 mM to approximately 18 mM as extracellular NaC1 concentrations increase from 50 mM
to 550 mM.
SKCaR is a functional extracellular Mg2+ sensor where its sensitivity is modulated by alterations in extracellular NaC1 concentrations. As shown in Figure 3, SKCaR is activated in the range of 5-40 mM extracellular Mg2+ and is modulated in a manner 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 (EC50) by Mg2+ for SKCaR is reduced in increased concentrations of extracellular NaCl. See Figure 5. The EC50 for data shown on Figure 5 is displayed as a function of increasing extracellular NaC1 concentrations. Note the ECso for Mg2+ increases from less than 20 mM to approximately 80 mM as extracellular NaC1 concentrations increase from 50mM to 550mM.
Addition of 3mM Ca2+ alters the sensitivity of SKCaR to Mg2+ and NaCl.
See Figure 6. The EC50 for Mg2+ of SKCaR is modulated by increasing concentrations of NaC1 as shown both in this Figure 6 and in Figure 5.
Addition of mM 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 NaC1 concentrations.
This method was also used to isolate partial genomic clones of PVCRs for Atlantic salmon and other species such as Arctic char and rainbow trout, as described herein. Figures 19A-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 of 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. Both anadromous fish (Atlantic salmon, trout and Arctic char) and euryhaline fish (flounders, alewives, eels) traverse from freshwater to seawater environments and back again as part of their lifecycles in the natural environment.
To successful accomplish this result; both types of fish have to undergo similar physiological changes including alterations in their urine output, altering water intake and water absorption. In some cases, naturally occurring mutations to PVCR
would provide for altered salinity adaptation capabilities that would have significant value for both commercial and environmental restoration uses. For example, identification of selective traits associated with PVCR mediated salinity responses might allow identification of new strains of fish for commercial aquaculture.
Similarly, 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. To permit the identification of individual fish possessing enhanced salinity responsive characteristics, assays must be designed that enable these fish to survive while others not possessing these characteristics will either die or perfoim poorly. As described below, such assays would take advantage _ _ of the ability of these anadromous and euryhaline fish to withstand a wide range of salinities. Fish that were identified using such assays would then be propagated in breeding-selection programs.
Winter and Summer Flounder can be grown and maintained in recycling water systems. Groups of both winter (Pleuronectes americanus) and summer (Paralichthus dentalus) flounder were maintained in multiple modular recycling water system units that are composed of a single 1 meter fish tank maintained by a 1 meter biofilter tank located directly above it. The upper tank of each unit contains 168 sq. ft. of biofilter surface area that will support a maximum of 31 lbs of flounder, while maintaining optimal water purity and oxygenation conditions.
Each unit is equipped with its own pump and temperature regulator apparatus. Both the temperature and photo-period of each unit can be independently regulated using black plastic curtains that partition each tank off from its neighbor. The inventors have a total of 12 independent modular units that permit 3 experiments each with 4 variables to be performed simultaneously. Using this experimental system, the following data have been obtained.
Salinity survival limits for winter and summer flounder with a constant ratio of divalent and monovalent ions were determined. The survival limit of both winter =
and summer flounder in waters of salinities greater than normal seawater (10 mM
Ca2+, 50 m_M Mg2+ and 450 mM NaC1) is water containing twice (20 mM Ca2+, 50 mM Mg2+ and 900 mM NaC1) the normal concentrations of ions present in noinial seawater. In contrast, the survival limit of both winter and summer flounder in waters of salinity less than nonnal seawater is 10% seawater (1 mM Ca2+, 5 mM
Mg2+ and 45 mM NaC1).
Use of a fully recycling water system patinits growth of flounder at vastly different salinities. Groups of flounder (n=10) were adapted over a 15 day interval and maintained at either low salinity (LS) (e.g., at 10% normal seawater), normal seawater (NS) or hypersalinity (HS) (e.g., 2X seawater) for intervals of 3 months, under otherwise identical conditions. Survival among the 3 groups were comparable (all greater than 80%) and there were no differences in the electrolyte content of their respective sera.
EXAMPLE 4: ISOLATION OF PARTIAL ATLANTIC SALMON PVCRs A partial PVCR gene of Atlantic Salmon was isolated as follows: sequences of shark kidney calcium receptor together with the nucleotide sequence of mammalian calcium receptors were used t6 design degenerate oligonucleotide primers, dSK-F3 (SEQ ID NO: 15) and dSK-R4 (SEQ ID 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).
Using these primers, 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. Using this methodology, 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 corresponds to the conserved transmembrane domain of the calcium receptors.
Atlantic salmon 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).
Primer sequences for PCR of PVCR clones:
dSK-F3 (SEQ ID NO:15) CKT GGA CGG AGC CCT TYG GRA TCG C-3' dSK-R4 (SEQ ID NO:16) 5f-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G-3' I=deoxyinosine, N=A+C+T+G, R=A+G, Y=C+T, M=A+C, K=T+G, S=C+G, W=A+T, H=A+T+C, B=T+C+G, D=A+T+G, V=A+C+G; Product from amplification = 653 nt 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 Jolla, CA) and screened using the Atlantic salmon PCR
product as a probe. A partial Atlantic salmon PVCR cDNA (SEQ ID NO: 5) is composed of 2021 nucleotides (nt) (Figure 8A) containing an open reading frame encoding 388 amino acids (SEQ ID NO: 6) (Figure 8B). The open reading frame encoded by SEQ ID NO: 5 begins at nucleotide position 87.
EXAMPLE 6: MOLECULAR CLONING OF 4 FULL LENGTH CDNA CLONES
FROM KIDNEY OF ATLANTIC SALMON (SALMO SALAR) AND
DETERMINATION OF THEIR TISSUE SPECIFIC EXPRESSION IN VARIOUS
SALMON TISSUES MODULATED BY WATER SALINITY.
In 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). Using sequence information derived from Examples 4 and 5, both nucleotide (nt) and antibody probes were designed to detect PVCRs in other fish species. Using degenerate primers whose sequence was derived from knowledge of the nt sequence of SKCaR, 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.
The data described in this Example show that the nt and putative protein sequences of 4 PVCR transcripts from Atlantic salmon kidney were isolated and characterized. Additionally, their tissue specific expression and modulation of tissue expression levels by alterations in water salinity were determined. This Example is divided into 2 parts: 1) isolation and sequence of 4 full length PVCR clones from salmon kidney (Salm oKCar#1 (SEQ 1D NO: 7), SalmoKCar#2 (SEQ ft) NO: 9), SalmoKCaR#3 (SEQ ID NO: 11), and SalmoKCaR#4 (SEQ ID NO: 13)) and 2) use of RT-PCR analysis with degenerate and clone specific SalmoKCaR PCR primers to determine the tissue specific expression of these 4 transcripts in seawater vs.
freshwater as well as the SuperSmoltTM process. Taken together, these data provide the framework for achieving a fundamental understanding of both PVCRs in salmonids as well as the their roles in the SuperSmoltTM process.
Part 1. Isolation and sequence of 4 full length PVCR clones from salmon kidney:
Materials and Methods: Total RNA was purified with Stat 60 reagent (Teltest B
Friendswood, TX) and poly A+ purified with the Micro FastTrack Kit (Invitrogen,Carlsbad, CA). cDNA was then synthesized and fractionated whereby selected fractions were ligated and packaged as AZAP libraries (Stratagene, La Jolla, CA). For SalmoKCaR #1,2, and 3, library phage were then plated and duplicate filter lifts performed that were screened under high stringency (0.1X SSC, 0.1% SDS
55 C) with a 32P-labeled (RadPrime Kit, Invitrogen, Carlsbad, CA) genomic fragment of Atlantic salmon PVCR (653 nt sequence) amplified using protocols and reagents described in Examples 1, 4 and 5. For SalmoKCaR #4, library phage were then plated and duplicate filter lifts performed that were screened under high stringency (0.1X SSC, 0.1% SDS @ 55 C) with a 3213-labeled (RadPrime Kit, Invitrogen, Carlsbad, CA) full length nucleic acid sequence of SalmoKCaR#2, SEQ
ID NO: 9, amplified using protocols and reagents described in Examples 1, 4 and 5.
Primary positive plaques were purified, excised and sequenced using commercial sequencing services (U. of Maine, Orono, ME) and their sequences compared with those of other PVCRs using BLAST. (National Library of Medicine, Bethesda, MD).
Results:
For isolation of SalmoKCaR#1,2, and 3:
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) #1,112 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.
For SalmoKCaR#4:
120,000 additional plaques were screened from the same cDNA library which was used to isolate SalMoKCaRs 1-3. Five positive plaques were picked, plated out and screened on secondary phage plates. Isolated plaques were picked, excised into bacterial plasmids, and transferred into bacterial cells as previously described. Plasmid DNA preparations of all 5 cDNAs showed strong hybridization to SalmoKCaR#3 by Southern blot analysis. Sequence analysis revealed two full length clones and three partials. One of the full length clones was a duplicate of a previously isolated SalmoKCaR, and the other was a new clone, SalmoKCaR4.
This clone was designated as SalmoKCaR#4, commensurate with the naming of the SalmoKCaR#1-3 clones, above. The aligned nt and putative protein sequence for SalmoKCaR #4 is shown in Figure 12.

Comparison of the different nt sequences of these 4 clones reveals the following similarities and differences:
- The SalmoKCaR 41 nucleic acid sequence (SEQ JD 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 9A-E.
The calculated molecular mass of this protein is 106,125 Daltons.
- The SalmoKCaR 42 nucleic acid sequence (SEQ ID 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 ID NO: 10). See Figures 10A-E. The calculated molecular mass of this protein is 106,180 Daltons.
- The SalmoKCaR '3 nucleic acid sequence (SEQ TD 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 11A-D. The calculated molecular mass of this protein is 96,538 Daltons.
- The SalmoKCaR#4 nucleic acid sequence (SEQ ID NO: 13) consists of 3988 nt of 5' and 3' regulatory elements together with full-length coding sequence for a 941 AA PVCR protein (SEQ ID NO: 13). See Figures 12A-E. The calculated molecular mass of this protein is 106,131 Daltons.
Figures 13A-L and 14A-C show an alignment of between the two partial SEQ ID NO: 12. Note that the amino acid sequence of SEQ ID 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 ID NO: 13. The second partial Atlantic Salmon clone, SEQ ID NO: 5, can also be found in the SalmoKCaR#4 nucleic acid sequence: between nt 1754 and 3774. Similarly, the amino acid sequence of SEQ
ID
NO: 4 is found between aa 601 and 797 of SEQ ID NO: 14. The amino acid sequence of the second Atlantic Salmon Clone, SEQ II) NO: 6, is found between aa 554 and 941 of SEQ ID NO: 14.
Additional differences between the partial Atlantic salmon PVCR (SEQ ID
NO: 5) and full length PVCR (SEQ ID NO: 7, 9, or 11) include: nt 1-112 do not align with any corresponding sequence in SEQ ID NO: 7, 9, or 11. There are also 4 single nt base pair substitutions that are present in SEQ ID NO: 5 that are different than corresponding nt in full length SEQ ID NO: 7, 9, or 11. These include:
_ nt 1893 change from G to A
nt 1970 change from G to A
nt 1973 change from G to A
nt 2001 change from G to A.
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.

However, in spite of the rather low overall % nt identity between the 4 SalmoKCaR
clones and SKCaR, all 4 full length SalmoKCaR clones hybridize to full length SKCaR clone under high stringency conditions (0.5XSSC, 0.1% SDS @ 65 C.) (See Figure 15A).

The percentage identities between the aligned nucleotide sequences of the 4 full length SahnoKCaR clones (SEQ ID NO: 7, 9, 11, 13) include:
A total of 99.8% of the nt of SEQ ID NO: 7 are identical to those of corresponding SEQ ID NO: 9. A total of 97.6% of the nt of SEQ ID NO: 9 are identical to those corresponding nt of SEQ ID NO: 7.
A total of 93.6% of the nt of SEQ ID NO: 9 are identical to those corresponding nt of SEQ ID NO: 11. A total of 98.7% of the nt of SEQ ID
NO: 11 are identical to the corresponding nt present in SEQ ID NO: 9.
A total of 95.8% of the nt of SEQ ID NO: 7 are identical to the corresponding nt of SEQ ID NO: 11. A total of 98.7% of the nt of SEQ ID
NO: 11 are identical to those corresponding in SEQ ID NO: 7.
A total of 99% of the nt of SEQ ID NO: 7 are identical to corresponding nt of SEQ ID NO: 13. A total of 98% of the lit of SEQ ID NO: 9 are identical to corresponding nt of SEQ ID NO:13. A total of 97% of nt of SEQ ID NO: 13 are identical to corresponding nt of SEQ ID NO:11.
Table 1: Comparison of the % nucleotide (nt) identity of the complete nt sequence of SalmoKeaR clones #1, #2, #3, and #4 (including 5' and 3' regulatory elements) vs.
either the SKCaR done or the clone HuPCaR clone.
% NUCLEOTIDE IDENTITY
SalmoKCaR #1 SalmoKCaR 42 SalmoKCaR #3SalmoKCaR #4 SKCaR vs. 56.2 56.5 57.2 56 HuPCaR vs. 55.0 54.9 50.9 55 Table 2 compares both the overall and domain-specific percent amino acid (% AA) identity for each of the SalmoKCaR clones vs. shark kidney PVCR

(SKCaR-upper half) and human parathyroid CaR (HuPCaR-lower half). When compared to SKCaR, all 4 SalmoKCaR proteins possess approximately a 63-68%
overall AA identity to SKCaR.. However, their domain-specific identities show significant degrees of variation with the carboxyl terminal domain of the SalmoKCaR 3 being the most widely divergent. Not surprisingly, comparisons between the 4 SalmoKCaR proteins vs. HuPCaR reveal that the 7 transmembrane region possesses the highest degree of homology followed by the extracellular domain and finally the intracellular carboxy terminal domain.
The percentage identities between the aligned amino acid sequences of the 4 full length SalmoKCaR clones (SEQ ID NO: 8, 10, 12, or 14) include:
A total of 99.9% of the aa of SEQ ID NO: 8 are identical to those corresponding aas in SEQ D NO: 10. A total of 99.9% of the aa of SEQ ID
NO: 10 are identical to corresponding aa in SEQ ID NO: 8.
A total of 89.5% of the aa of SEQ JD NO: 10 are identical to those corresponding aas in SEQ ID NO: 12. A total of 99.1% of the aa of SEQ ID
NO: 12 are identical to those corresponding aa in SEQ ID NO: 10.
A total of 89.6% of the aa of SEQ JD NO: 8 are identical to those corresponding aas in SEQ ID NO: 12. A total of 99.2% of the aa of SEQ ID
NO: 12 are identical to those corresponding aa of SEQ ID NO: 8.
A total of 100% of the aa of SEQ ID NO: 8 are identical to those corresponding aa's in SEQ ID NO: 14. A total of 99.9% of the aa of SEQ ID NO:
10 are identical to those corresponding aa's in SEQ ID NO: 14. A total of 99.2% of the aa of SEQ ID NO: 12 are identical to those corresponding 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).
% AA Identity to SKCaR
SalmoKCaR SalmoKCaR SalmoKCaR SalmoKCaR

Overall Protein 68.4 68.3 63.3 68.4 N-terininal 70.0 69.8 70.0 70.0 Extracellular Ion Binding Domain 7 Transmembrane 87.2 87.2 86.4 87.2 Region Carboxyl Terminal 31.8 31.8 0.0 31.8 luta-Cellular Domain % AA Identity to HuPCaR
Overall Protein 66.3 66.3 61.4 66.3 N-terminal 71.9 - 71.9 72.1 71.9 Extracellular Ion Binding Domain 7 Transmembrane 89.2 89.2 88.4 89.2 Region Carboxyl Teaninal 24.1 24.1 0 24.1 Intra-Cellular Domain Figure 15A shows unique SalmoKCaR#1,2, 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:1, 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 normal agonist-mediated CaR activation. SalmoKCaR#1-4 possess Cys at AAs corresponding to HuPCaR
AA#101 and AA#236 and presumably functions as dimers in a manner similar to mammalian CaRs.
Nucleotide Sequence Differences in the 5' and 3' untranslated regions or UTRs of SalmoKCaRs 41, 42 43 and #4:
Figure 16 displays the aligned nucleotide sequences of SalmoKCaR clones #1, #2, and #3, and Figure 17 displays the aligned nucleotide sequences of SalmoKCaR #1-4. As compared to SalmoKCaR #1 and '3, 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 ORE of SalnioKCaR# 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 of the 4 SahnoKCaRs 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 nt 1039 A to G; nt 3837 A to G; nt 3862 A to G
SalmoKCaR 43: 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 44: nt 3634 A to G; nt 3645 A to G; nt 3661 A to G, nt 3726 G to A, nt 3740 A to G, nt 3746 A to G, nt 3773 A to G
Although the functional significance of each of these nt differences in the 5' or 3' UTRs is unknown at the present time, each nt difference either individually or in combinations could represent a means for controlling either the stability or processing of the RNA transcript or its translation into each of the 4 SalmoKCaR proteins.
Sequence Differences in the Coding Regions of SalmoKCaRs 41, 42, 43 and #4:
Figure 19 displays the aligned AA sequences of SalmoKCaRs #1, #2, #3, and #4 as well as the Shark SKCaR protein and HuPCaR
proteins. As compared to SalmoKCaR 41 and #4, SalmoKCaR
possesses 2 different AA's present at AA#257 and AA#941 of its AA
sequence. In contrast to SalmoKCaR 41 and #4 that possesses an Asp in AA#257, SalmoKCaR 42 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.
At AA #443, SalmoKCaR 41, 42 and #4 both possess a Leu whereas SalmoKCaR 43 contains a Phe. The conserved hydrophobic nature of the AA at this position appears to be important since Fugu PVCR also contains a Leu whereas SKCaR contains an Ile. As compared to SahnoKCaRs 41, 2, or #4 SalmoKCaR 43 possesses a truncated carboxyl teiminus as described below.
Sequence Differences in the Coding Regions of SahnoKCaRs 41, 42 43 and #4 as Compared to Mammalian CaRs.
The putative AA sequences of SalmoKCaR 41, 42, 43 and #4 proteins possess multiple differences in AAs at various positions throughout their extracellular, 7 transmembrane and carboxyl terminal domains when compared to mammalian CaRs such as HuPCaR (see aligned differences with HuPCaR in Figure 19). While many of the 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 structure-functional studies of mammalian CaRs. The actual functional consequences of these AA differences in SalmoKCaR proteins are described herein in the following expression study.
Expression _of SalmoKCaR #1 and #3 in Human Embryonic Kidney (HEK) Cells This express study demonstrates that SahnoKCaR #1 and #3 clones can be expressed in HEK cells to yield corresponding 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 of the use of SDD and_Sal-1 antisera, respectively, as reagents to detect either both SalmoKCaRs #1 and #3, or selectively detest SalmoKCaR #1.
Methods for the Transfection and HEK Expression of SalmoKCaRs:
Full length SalmoKCaR 1 and SalmoKCaR 3 cDNAs were directionally cloned into the Kpnl and XbaI sites of the mammalian expression vector pcDNA3. 1/Hyg- (Invitrogen) as described for the transfection and transient expression of SKCaR PVCR. DNA was prepared using the Wizard Midiprep Kit (Promega) and 15 jig of sterilized Salm.oKCaR 1 or SalmoKCaR 3 DNA was diluted in 1 ml Opti-Mem I reduced serum media (Gibco) and combined with 45111 Lipofectamine 2000 (Gibco). 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% CO2 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% CO2. At 24 hours post transfection, the media was replaced with 20 ml full growth media. Flasks were kept at 37 C, 5% CO2 for 24-48 hrs to optimize expression of protein.
Methods for Immunoblotting Analysis of HEK Expression of SahnoKCaR #1 or #2 Using SDD and Sal-1.2 Antisera:
The transfected cells were then rinsed with PBS and treated with trypsin-EDTA (Gibco) for 2 minutes at 37 C, 5% CO2 to detach cells from the surface of the 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 (20 mM Tris, 250 mM sucrose, at pH 7.4 plus protease inhibitors, including 1 g/m1pepstatin, .5 g/m1 leupeptin, and 1mM PMSF). The cells were homogenized in 14 ml (Falcon) tubes using a polytron 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 I 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). For the gel shown in Figure 15B, 20 jig of protein was loaded into each lane except the 5001 HEK of which 15 g was loaded. Novex 10% Tris-Glycine pre-cast gels (Invitrogen) were used to separate protein bands in each sample by gel electrophoresis. The protein bands were subsequently electro-transferred to a Hybond-P PVDF
nitrocellulose membrane (Amersham-Pharmacia). The membranes were blocked overnight at 4 C in a 5% block solution (Amersham-Pharmacia).
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-Phannacia) and exposed to high performance chemiluminescence film (Amersham-Phanuacia).
Figure 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. In 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
#3 and salmon SW kidney. Thus, HEK cells expressing HuPCaR
(Positive Control) display a prominent -115kDa band, as well as higher molecular mass bands which represent oligomers of HuPCaR. In contrast, mock transfected HEK cells (Negative Control) show minimal inunuoreactivity similar to that present when lanes are exposed to preimmune SDD antiserum. However, SDD antiserum displays intense reactivity to HEK cells transfect with SalmoKCaR #1 (SalmoKCaR #1) or SahnoKCaR #3 (SalmoKCaR #3). Note 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 corresponding clones. Note also the presence of larger immunoreactive bands present in SalmoKCaR #3 lane that corresponds to oligomeric complexes of SalmoKCaR protein. The ability of SalmoKCaR #3 to farm oligomeric complexes could result in formation of it exerting a dominant negative effect on the activity of other PVCRs expressed in the same cell. The lane designated Salmon 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 salmon.
Figure 15B, right panel, shows immunoblotting analysis with SAL1 antiserum. SAL1 antiserum recognizes an aa sequence in the carboxyl terminal of SalmoKCaR #1 that is not present in either HuPCaR or SalmoKCaR #3. As a result, minimal immunoreactivity is displayed in either immune or preimmune lanes except for SalmoKCaR #1 and Salmon SW Kidney lanes. Note the presence of higher molecular wight immunoreactive SalmoKCaR proteins in both SalmoKCaR #1 and Salmon SW Kidney. The absence of immunoreactivity of SalmoKCaR
#3 lane is expected since nucleotide and sequence analyses show that SalmoKCaR #3 is missing the aa sequence that is recognized by Sal-1 antiserum.
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 location. In contrast, 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.
- Another notable difference between SalmoKCaRs vs. mammalian CaRs and SKCaR is differences in AA #227 where mutagenesis studies have identified the presence of the positively charged Arg as important in CaR
sensitivity since its alteration in HuPCaR to a Leu results in over a 2 fold reduction in EC50 Ca from 4.0mM to 9.3mM but not Gd' sensitivity. In contrast to mammalian CaRs and SKCaR, all 4 SalmoKCaRs possess a negatively charged Glu at AA#227. Fugu PVCR also exhibits the same Glu at AA#227. interestingly, the 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.
- Lastly, all 4 SalmoKCaR clones as well as Fugu possess an in frame deletion of a single AA at position #757 (between TM4 and 5) as compared to either mammalian CaRs or SKCaR.
- SalmoKCaR #3 possesses a truncated carboxyl terminal domain as compared to either SalmoKCaRs #1, #2 or #4. The number of AA that comprise the carboxyl terminal domains of the 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.
Studies from multiple site directed mutagenesis studies of HuPCaR reveal that alterations to the structure of the carboxyl terminal domain of PVCRs have profound effects on their function and sensitivity to ligands such as Ca" and Mg".
Various truncations of the carboxyl terminal domain of HuPCaR have highlighted the importance of HuPCaR AAs #860-910. Truncation of the 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' as well as a decrease in the apparent cooperativity of Ca" dependent activation.
While the exact functional characteristics of SalmoKCaR #3 remain to be determined using similar HEK transfection studies, these data derived from HuPCaR mutagenesis studies suggest that SahnoKCaR #3 protein is either inactive or exhibits a greatly reduced functional affinity for Ca'. 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.
These dominant negative effects could occur where SalmoKCaR#3 reduces the overall sensitivity of cells to Ca' via combinations between SalmoKCaR #3 and SalmoKCaR #1/#2/#4 to reduce the sensitivity of the latter PVCRs via cooperative interactions (dimers and higher oligomers) with them. -Certain mutagenesis studies also highlight the importance of the Threonine AA at AA#888 in mediation of HuPCaR's sensitivity to Ca2+ and normal signal transduction. Figure 19 shows that 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.
Of interest is also the presence of consensus sites for receptor kinase phosphorylation (Ser-Ser-Ser) that are present at AA#907-909 in HuPCaR, RaKCaR, SKCaR BoPCaR and SalmoKCaR #1,- #2 and #4. In contrast, Fugu PVCR
possesses an Asn at AA#908 that would render its site nonrecongizable to protein kinases. A similar protein kinase site also appears in the region of AA#918-where HuPCaR, RaKCaR and BoPCaR possess a Ser-Ser-Ser motif. In contrast, SKCaR possesses an inactive site due to its sequence of Ala-Ser-Ser. Fugu PVCR
and SalmoKCaR #1, #2 and #4 also have intact Ser-Ser-Ser motifs at position AA

#918-920 or #919-921. The exact functional significance of these Ser-Ser-Ser sites possessed by SalmoKCaR #1, #2 and #4 await expression studies.
The presence of multiple differences in the nucleotide and putative protein sequences of SahnoKCaR clones #1-#4 strongly suggest the presence of multiag PVCR genes within Atlantic salmon:

Recent studies in rainbow trout provide direct evidence of the existence of multiple genes encoding two different forms of a specific type of protein, each of which are differentially expressed in specific tissues of trout. These proteins are aryl hydrocarbon receptor Type 2 (AhRs). Detailed studies on AhRs have shown the presence of 2 functional genes that produce different closely related AhR
proteins, "Two forms of aryl hydrocarbon receptor type 2 in rainbow trout (Oncorhynchus mykiss)," by Abnet, C. C., et al., J. of Biological Chemistry 274: 15159-15166, (1999). These two proteins are differentially expressed in various tissues where they perform closely related but distinct functions.
The presence of single nucleotide substitutions together with specific large scale alterations in the sequence of SalmoKCaR clones #1-4 including the gapping of large numbers of nucleotides and alterations in reading frame of the resulting _ SalmoKCaR transcript are not readily explainable on the basis of differential splicing of RNA transcripts derived from a single gene, or perhaps some complex process where different alleles of a single gene are present in salmon.
Alternatively, these data suggest that there are multiple PVCR genes present in Atlantic salmon that work in concert to enable Atlantic salmon and likely other salmonids to carry out their lifecycle stages that include hatching as well as development of larval and juvenile phases in freshwater followed by smoltification and migration into seawater with a subsequent return to freshwater for spawning.
Detailed studies in mammals including mice and humans show the presence of a single functionarPVCR gene. However, multiple published reports provide support for the possibility that multiple PVCR genes exist in fish, while only a single functional PVCR gene exists in mammals including humans. Support for multiple PVCR genes is provided by detailed studies of well characterized genes that have demonstrated that teleost fish including salmonids possess multiple sets of duplicated genes as compared to mammals. These duplicated genes have arisen as a result of either genomic duplication events occurring early in the evolutionary history of fishes with subsequent gene drop out or via more recent selective duplication of genes or some combination of both. Moreover, it is widely aclmowledged that salmonids are polyploid with respect to other teleost fish and have undergone an additional genome duplication. This additional genomic duplication further heightens the possibility that multiple functional PVCR
genes exist in sahnonids particularly Atlantic salmon.
If the products of a duplicated gene are not important in the development, growth or maintenance of an organism, the nonfunctional gene accumulates natural mutations and is either inactivated becoming a pseudogene or lost from the genome altogether. However, multiple authors have provided evidence that preservation of duplicated genes likely involves changes in the developmental or tissue specific expression pattern of the duplicated vs. original gene or formation of a new functional gene protein product that would interact with the original gene product in novel ways. (See AhR data above). These data provide support for the possible roles of SalmoKCaR transcripts #1-4 as either differentially expressed in various tissues of Atlantic salmon as well as SalmoKCaR #3 exerting a dominant negative effect on the remaining functional SalmoKCaR proteins. As discussed below, such -interactions amongst SalmoKCaR transcripts would provide Atlantic salmon and perhaps all salmonids with the ability to exploit a wide variety of freshwater and seawater environments.
Part 2: Use of RT-PCR and northern analysis to deteimine the expression of SalmoKCaR clones #1, #2 and 43 in various tissues of Atlantic salmon:
Background:
SalmoKCaR clones #1, #2 and #3 were originally isolated from a Atlantic _ salmon kidney cDNA library. To determine the pattern of tissue specific expression of these various SalmoKCaR clones, both degenerate (to amplify all Salmo PVCRs species) and SalmoKCaR primers that will specifically amplify either SalrnoKCaR
41 or 42 or 43 were utilized. As shown in "Materials and Methods" Section below, these primers amplify DNA products of different sizes that can be distinguished by agarose gel electrophoresis. 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 41 and #2 span introns and therefore DNAase treatment is not required to interpret 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 41, #2 versus #4.
Materials and Methods:
Primers:
Degenerate Primers DSK-F3 and DSK-R4 primers are shown in Example 3.
SalmoKCaR #1 Specific Primers SalmoKCaR #1 nts AS1-F17 5'¨ CAA GCA TTA TCA AGA TCA AG 3' nt 47-(SEQ ID NO:18) AS2-R14 5' ¨ CTC AGA GTG GCC
TTG GC ¨3' nt 2800-(SEQ ID NO:19) Product from amplification 2754 nt. The.SalmoKCaR #1 primer pair consists of a forward primer (AS1-F17) spanning the 5' UTR insertion in SalmoKCaR #2, and a reverse primer (AS2-R14) within the 158 bp deleted from SalmoKCaR #3.
SalmoKCaR #2 Specific Primers SalmoKCaR #2 nts AS2-F13 5'¨ CAG TTC TCT CTT TAA TGG AC 3' nt 109-128 (SEQ ID NO:20) AS2-R14 5' ¨ CTC AGA GTG GCC TTG GC ¨3' nt 2890-2874 (SEQ ID NO:21) Product from amplification = 2782 nt. The 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 AS5-F'11 5' ¨ AGT CTA CAT CAT CCA TCA GCC ¨3' nt 2700-2720 (SEQ ID NO:22) AS5-R12 5' ¨ GAT TTT ATT GTC ATT GGA TGC ¨3' nt 3810-3790 (SEQ ID NO:23) Product from amplification = 1111 nt. The SalmoKCaR #3 primer pair consists of a forward primer (AS5-F11) which spans the 158 bp deletion, and a reverse primer (AS5-R12) located in the 36 bp insertion at the 3' end of the SalmoKCaR #3 clone.
Salmon Actin Primers SA-Fl 5' ¨ TGG AAG ATG AAA TCG CCG C ¨3' nt 2-20 (SEQ ID NO:24) SA-R2 5' ¨ GTG GTG GTG AAA CTG TAA CCG C ¨3' nt 608-587 (SEQ ID NO:25) Product from amplification = 607 nt. This primer set is used to amplify salmon actin mRNA that serves as a control to quantify differences in mRNA content.
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 (Invitrogen,Carlsbad, CA). The cDNA was amplified (30 cycles of lmin @ 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). Aliquots of PCR reactions were subjected to gel electrophoresis and ethidium bromide (EtBr) staining or blotted onto Magnagraph membranes (Osmonics, Westboro, MA) and probed with a 32P-atlantic salmon genomic PCR product (653 bp sequence identical to that shown in SEQ ED NO: 3 with added nt sequences, washed (0.1x SSC, 0.1% SDS @ 55 C) and autoradiographed. Selected amplified PCR products from Atlantic salmon tissues were sequenced as described above. The following conditions were utilized for each of the SalmoKCaR specific primers and corresponding blots:
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 (0.1x 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 (0..lx SSC, 0.1% -SDS @ 55 C) and autoradiographed for 168 hr.
SalmoKCaR #3 amplification conditions and primer set: PCR: lmin @ 94 C, linin @ 52 C, 3min @ 72 C, 35 cycles. Amplification products attached to membrane were probed with full length SalmoKCaR #3 clone and washed (0.1x SSC, 0.1%
SDS @ 55 C) and autoradiographed for 72 hr.
Results:
Analysis of Atlantic salmon tissues from freshwater vs. seawater adapted fish using degenerate primers:
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. In contrast, samples from nasal lamellae, urinary bladder, olfactory bulb and pituitary gland were all pooled samples from all 10 fish.
Note that the amplification products from these degenerate primers do not distinguish between SalmoKCaR #1, #2, #3 or #4 since their nt sequences in the region amplified by the primers are all identical (lanes 7, 9 and 12 Lower gel ¨
Panels A, B, C and D). Moreover, these degenerate primers also possess the capacity to amplify additional PVCRs (if any are present) in salmon tissues that could be distinct from either SalmoKCaR #1-4. Thus, amplified RT-PCR products are referred to as PVCR products since use of these degenerate primers do not distinguish between various PVCR species.
Analysis of panels A-D of Figure 20 shows that the PVCR degenerate primers yield PCR products in various tissues of both seawater and freshwater adapted fish. These various bands are more visible in Southern blots (Panels C, D) of corresponding ethidium bromide gels (Panels A and B) because detection of PVCR amplified products via hybridization of a 3213-PVCR probe is more sensitive as compared to ethidium bromide staining. Prominent ethidium bromide stained bands are visible in urinary bladder (lane 4), kidney (lane 5) and muscle (lane 14) in seawater adapted fish (Panel A) while either faint or no bands are seen in other tissues. In contrast, ethidium bromide bands are also visible in nasal lamellae (lane 3), urinary bladder (lane 4) and kidney (lane 5) as well as olfactory bulb (lane 12) in freshwater fish (Panel B). In summary, these data show differential tissue expression of PVCRs 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. (Panels A and B): Ethidium bromide stained agarose gel. DNA markers in lane 1 of both Panels A and B were used to indicate size of amplification products. (Panels C and D) Southern blot of gel in Top Panel using 3213-labeled Atlantic salmon genomic fragment. (Panels E
and F) Ethidium bromide stained gels 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.

Southern blots (Panels C and D) of the corresponding gels shown in Panels A
and B reveal that amplified PVCR products are present in additional tissues not shown by simple ethidium bromide staining as described above. As shown in Panel C, 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. In contrast, 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 perfolured on identical aliquots of the RT-PCR
reactions serve to quantify any differences in pools of cDNA from the individual RT
reactions in each sample. Isolation and subcloning of the ethidium bromide stained bands from olfactory lamellae and urinary bladder show that nucleotide sequences of multiple subclones from these bands all are identical to the nucleotide sequence present in SalmoKCaR clones #1-3.
Close examination of the differences in Panel C (seawater adapted) vs. Panel D (freshwater adapted) reveal differences in the apparent abundance of PVCR
mRNA in specific tissues. Apparent increases in tissue PVCR mRNA abundance in seawater-adapted salmon vs. freshwater-adapted salmon are present in gill, kidney, stomach, pyloric caeca, distal intestine, and muscle. The increased expression of PVCRs in Atlantic salmon exposed to seawater is consistent with other data that an increase in PVCR expression in at least one tissue occurs upon transfer of Atlantic salmon from freshwater to seawater. In contrast, the abundance of PVCR mRNA
species in olfactory bulb tissue of seawater adapted salmon appears to be reduced as compared to olfactory bulbs of freshwater adapted counterparts (Lane 12 in Panels C
vs. D). In other tissues such as nasal lamellae (Lane 3 in Panel C vs. D) there is little or no apparent change in the steady state PVCR mRNA content. In summary, these data demonstrate tissue specific changes in the steady state expression of PVCR
mRNA species in seawater adapted vs. freshwater adapted Atlantic salmon.
Depending on the tissue, steady state PVCR mRNA content is either increased, decreased or remains unchanged when freshwater adapted fish are compared to seawater adapted counterparts. Since these analyses shown in Figure 20 use PVCR
degenerate primers, it is not possible to determine from these experiments whether the alterations in steady state PVCR mRNA content are the result of changes in individual SalmoKCaRs #1-4.
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 21A 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 of the SuperSmoltml process in freshwater (lane 2) or transferred to seawater and maintained for 26 days.
Figure 21B shows RT-PCR analysis of a single representative experiment using pyloric caeca from the same fish shown in Figure 21A. Note the significant increase in amplified PVCR product present in kidney (Figure 21A) and pyloric caeca (Figure 21B) 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). The increased expression of PVCRs in these 2 tissues of Atlantic salmon exposed to the SuperSmoltTM process where this increased PVCR
expression mimics that produced after seawater transfer is consistent with earlier data that an increase in PVCR expression in at least one tissue occurs upon either treatment with the SuperSmoltTM process or transfer of Atlantic salmon to seawater.
Figure 21C shows RT-PCR analysis using the same degenerate primers to detect expression of SalmoKCaR transcripts in various stages of Atlantic salmon embryo development. Using degenerate (SEQ ID Nos 15 and 16) or actin (SEQ ID
No 24 and 25) primers, 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 of the 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 weeks after hatching. Panel B shows the series of controls where PCR
amplification of actin content of each of the 5 samples shows they are approximately equal.
Northern blotting of kidney poly .A+ RNA with SalmoKCaR 41 reveals an increase in PVCR expression in seawater-adapted vs. freshwater-adapted Atlantic salmon.
To both confirm the size of SalmoKCaR transcripts and test for changes in SalmoKCaR expression in fish exposed to different salinities, poly A+ RNA from kidney of either freshwater adapted (FW) or seawater adapted (SW) Atlantic salmon were probed with SalmoKCaR 41. As shown in Figure 22, kidney RNA contains a 4.2kb band that corresponds to the 3.9-4.0 kb sizes of SalmoKCaR 41-4 as determined by nucleotide sequence analysis. Because of the high degree of nucleotide identities between SalmoKCaR #1-4, the 4.2kb band is actually derived from the combination of all 4 SalmoKCaR species and any additional PVCR
species in salmon kidney due to crosshybridization of SalmoKCaR #1. However, these data show an increase in the intensity of the 4.2kb SalmoKCaR band in SW adapted fish as compared to their FW adapted counterparts.
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.
Use of RT-PCR with SalmoKCaR43 specific primers demonstrates that tissue specific alterations in the steady state tissue content of SalmoKCaR 43 mRNA
in freshwater vs. seawater adapted Atlantic salmon.
To determine whether specific SalmoKCaRs 43 are modulated by exposure to different salinities, 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 43 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 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. (Panels C and D) Southern blot of gel in Top Panel using 32P-labeled Atlantic salmon genomic fragment. (Panels 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. The specificity of these - SalmoKCaR#3 primers is demonstrated in the bottom half of Panels A and B
of Figure 23. The specific SalmoKCaR #3 primers only amplify product from SalmoKCaR #3 clone (lane 14) and not SalmoKCaR #1 (lane 8) or SalmoKCaR #2 (lane 11) or SalmoKCaR #4. Note that in the tissue sample lanes, ethidium bromide stained bands are present in the kidney of seawater adapted salmon (lane 5 upper gel- Panel A) and only very faintly in urinary bladder of freshwater adapted salmon (lane 4 upper gel- Panel B). The corresponding Southern blots of freshwater adapted tissue samples (Panel D) reveal detectable SalmoKCaR #3 product only in urinary bladder (lane 4) and a small amount in kidney (lane 5). In contrast, in seawater-adapted salmon (Panel C) there are detectable increases in SalmoKCaR #3 product in both urinary bladder (lane 4) and kidney (lane 5) as well as the presence of SalmoKCaR #3 amplified product in gill (lane 2), nasal lamellae (lane 3), pyloric caeca (lane 7) and muscle (lane 14) of seawater adapted fish.
As described above, the increase in tissue expression of SalmoKCaR #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. In contrast to freshwater where the ambient water concentrations of both Ca2+ and Me are low and require a high degree of sensitivity from SalmoKCaRs to sense changes in concentration, the concentrations of Ca2+
and Mg2+ 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. It is of interest that many of these specific tissues exhibiting significant SalmoKCaR
#3 expression are either exposed directly to the high Ca' and Mg" content of seawater (gill, nasal lamellae) or experience high Ca' and Mg' concentrations as the result of the excretion of these divalent cations (urinary bladder, kidney).
Use of RT-PCR with SalmoKCaR 41 specific primers demonstrates tissue specific alterations in the steady state tissue content of SalmoKCaR 41 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 of fish. As controls to demonstrate primer specificity, selected reactions were subjected to primer amplification of portions of individual SalmoKCaR clones or water alone (Panels A
and B): Ethidium bromide stained agarose gel. DNA markers in lane 1 of both Panels A and B were used to indicate size of amplification products. (Panels C
and D) Southern blot of gel in Top Panel using 3213-labeled Atlantic salmon genomic 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.
As shown in lower halves of Panels A and B of Figure 24, 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 of the 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.
In the various tissue samples, 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). The exact nature of the 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. These data show tissue specific expression of SalmoKCaR #1 in both freshwater and seawater adapted salmon.
Use of RT-PCR with SalmoKCaR #2 specific primers demonstrates tissue specific alterations in the steady state tissue content of SalmoKCaR #2 mRNA in freshwater vs. seawater adapted Atlantic salmon.
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. As controls to demonstrate primer specificity, selected reactions were subjected to primer amplification with samples of portions of individual SalmoKCaR clones or water alone (Panels A and B): Ethidium bromide stained agarose gel. 6NA markers in lane 1 Panels A, B, E and F were used to indicate size of amplification products. (Panels C and D) Southern blot of gel in Top Panel using 32P-labeled Atlantic salmon genomic 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. 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. Corresponding 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 Sahnol(CaR #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 current salmon farming 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 of the anadrornous 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. As shown herein, one utility for the present invention is its use in conjunction with transferring _ Atlantic Salmon from freshwater to seawater. For standard 70-110 gram smolt, application of the invention eliminates the phenomenon known as "smolt window"
and permits fish to be maintained and transferred into ocean water at 15 C or higher.
Use of these methods in 15 gram or larger smolt permits greater utilization of freshwater hatchery capacities followed by successful seawater transfer to ocean _ netpens. In both cases, 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 foun the key features of current 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 fingerlings and parr. Faster growing parr are able to undergo smoltification and placement in ocean netpens as SO smolt (70 gram) during year 01. In contrast, slower growing parr are smoltified in year 02 and placed in netpens as Si 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 transferred smolt. This is particularly true for 51 smolt since freshwater hatcheries are often located at significant distances from ocean netpen growout sites and their water temperatures rise rapidly during early summer. Thus, the combination of rising water temperatures and the tendency of smolt to revert or die when held for prolonged intervals in freshwater produces a need to transfer smolt into seawater during the smolt window.
Standard molts that are newly placed in ocean netpens are not able to grow optimally during their first 40-60 day interval in seawater because of the presence of osmotic stress that delays their feeding. This interval of osmotic adaptation prevents the smolts from taking advantage of the large number of degree days present immediately after either spring or fall placement. The combination of the presence of the 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 occurrence of grilsing in maturing fish that are exposed to reductions in ambient photoperiod.
Methods The smolt were subjected to the steps of Process I and II, as described herein.
RESULTS AND DISCUSSION:
SECTION I: Demonstration of the Benefits of the Process I For Atlantic Salmon Demonstration of the Benefits of the Process I For Atlantic Salmon:
Process I increases the survival of small Atlantic Salmon Sllike 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 NaC1 diet. In contrast, administration of calcium and magnesium either via the food only Or without NaC1 dietary supplementation does not produce results equivalent to Process I.
Table 3 shows data obtained from Atlantic salmon S2 like smolts less than 1 year old weighing approximately 25 gm. This single group of fish 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 of the 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 transferred to seawater. After transfer to seawater, this group also experienced high mortality where only one half of these smolts survived.
Inclusion of calcium (10 mM) and magnesium (5 mM) within the feed offered to smolt (Ca2+/Mg2+diet) reduced survival as compared to controls both in freshwater (51% vs 67%) as well after seawater transfer (1% vs 50%). In contrast, inclusion of mM Ca2+ and 5 mM Mg2+ in the freshwater (Process I Water Only) improved smolt survival in Process I water as well as after transfer of smolt to seawater.
10 However, optimal results were obtained (99% survival in both the Process I water mixture as well as after seawater transfer) when smolt were maintained in Process I
water mixture and fed a diet supplemented with 7% sodium chloride.

Table 3: Comparison of the Survival of Atlantic Salmon S2 like Smolts After Various Treatments Parameter Control Ca2+/Mg2+ Process I Process I
Sampled Freshwater Diet Water Only Water + NaC1 Diet Starting # of 66 70 74 130 fish # of fish 44 36 67 129 % of fish 67% 51% 91% 99%
surviving after 45 days in freshwater or Process mixture # of fish 22 2 60 128 % of fish 50% 6% 90% 99%
surviving 5 days after transfer to seawater 'Survival percentages expressed as rounded whole numbers Application of the Process Ito the Placement of 70-100 gm smolts in seawater.
These data show that use of the Process I eliminates the "smolt window" and provides for immediate smolt feeding and significant improvement in smolt growth rates.

Experimental Protocol:
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 transferred with standard commercially available smolt trucks and transfer personnel. Si 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 transferred 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. After receiving the Process I treatment, 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 of the netpens were fed by hand or feed blower to satiation twice per day using camera visualization of feeding. Mort dives were performed on a regular basis and each netpen received identical standard care practices established on this salmon farm. Sampling of fish _ for growth analyses was performed at either 42 days (Process l) or 120 days or greater (Control) fish. In both cases, fish were removed from the netpens and multiple analyses performed as described below.
All calculations to obtain feed conversion ratio (FCR) Or specific growth rate (SGR) and growth factor (GF3) were perfatined using standard accepted formulae (Willoughby, S. Manual of Salmonid Farming Blackwell Scientific, Oxford UK
1999) and established measurements of degree days for the Blue Hill Bay site as provided in Table 4 below. A degree day is calculated by multiplying the number of days in a month by the mean daily temperature in degrees Celsius.

Table 4: Degree days for Blue Hill Bay Salmon Aquaculture Site Month Degree Days Jan 60 Feb 30 Mar 15 April 120 May 210 June 300 July 390 Aug 450 Sept 420 Oct 360 Nov 240 Dec 180 Table 5 displays data obtained after seawater transfer of Control Si smolt.
Smolt ranging from 75-125 gm were placed into 3 independent netpens and subjected to normal farm practices and demonstrated characteristics typical for present rday salmon aquaculture in Maine. Significant mortalities (average 3.3%) were experienced after transfer into cool (10 C) seawater and full feeding was achieved only after a significant interval (-56 days) in ocean netpens. As a result, the average SGR. and GF3 values for these 3 netpens were 1.09 and 1.76 respectively for the 105-121 day interval measured.
In contrast to the immediate transfer of Control Si smolt as described above to ocean netpens (Table 5), a total of 10,600 Si smolt possessing an average size of 63.6 grams were transported on 11 May 2000 from the Deblois freshwater hatchery to the research facility. While being maintained in standard circular tanks, these fish were held for a total of 45 days at an average water temperature of 11 C and were subjected to Process I. During this interval, smolt mortality was only 64 fish (0.6%).

As a matched control for the Process I fish, a smaller group of control fish (n=220) were held under identical conditions but did not receive the Process I
treatment. The mortalities of these control fish were minimized by the holding temperature of and were equivalent to treated smolts prior to transfer to seawater.

Table 6: Characteristics of St. John Si smolt subjected to immediate placement in ocean netpens after transport form the freshwater hatchery without Process I
or Process II technology (the Control fish) Netpen Number #17 #18 #10 Total Fish 51,363 43,644 55,570 Mean Date of 5/1/00 5/5/00 5/14/00 Seawater Transfer Average Size at (117.6) Transfer (grams) 100-125 75-100 75-100 Mortalities after 30 1,785;_3.5% 728; 1.7% 2503; 4.5%
days (# and % total) Time to achieve full 68 days 48 days 50 days feeding after transfer Interval between 121 120 105 _ netpen placement and analysis Average size at Analysis Weight (gram) 376.8 174 305.80164 298.90137.40 Length (cm) 33.411.9 28.3019.0 30.4011.17 Condition Factor 1.02 1.34 1.06 (k) SGR 0.96 1.10 1.17 during initial 120 days During the 45 day interval when Si smolts were receiving Process I, fish grew an average of 10 grams and thus possessed an average weight of 76.6 gm when transferred to an ocean netpen. The actual smolt transfer to seawater occurring on 26 June 2000 was notable for the unusual vigor of the smolt that would have normally been problematic since this time is well past the normal window for ocean placement of smolt. The ocean temperature at the time of Process I smolt netpen placement was 15.1 C. In contrast to the counterpart S1 smolts subjected to standard industry practices described above, Process I smolts fed vigorously within 48 hours of ocean placement and continued to increase their consumption of food during the immediate post-transfer period. 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 incurred during the transfer process itself.
In contrast, corresponding 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 (1.5mX1.5m*1.5m) 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.
Both Process I and control smolts were fed on a daily basis in a manner identical to that experienced by the Industry Standard Fish shown on Table 6. Process I
fish were sampled 51 days after their seawater placement and compared to the Industry Standard smolts shown on Table 6. As shown in Table 7, comparison of their characteristics reveals dramatic differences between Industry Standard smolts vs Process I.

Table 7: Comparison of the characteristics of St. John Si Process I Smolts subjected to Process I treatment and then placed in ocean netpens vs corresponding industry standard smolts.
Process I Smolts Averaged Industry Standard Data from Table 6 in this Example Total Fish 10,600 150,577 Mean Date of Seawater 6/26/00 5/7/00 Transfer Average Size at Transfer 76.6 95.8 (grams) Mortalities after 30 days 648; 6.1% 5,016; 3.3%
(# and %) Time to achieve full 2 days 56 days Feeding after transfer Interval between netpen 51 115 placement and analysis Average size at Analysis Weight (gram) 175.48 50 327.2 97 Length (cm) 26.2 32 30.7 Condition Factor (k) 0.95 0.9 1.14 SGR 1.80 1.09 In summary, notable differences between Process I, Control smolt and Industry Standard smolt include:
1. The mortalities observed after ocean netpen placement were low in Process I

(6.1%) vs Control (63%) despite the that fact these fish were transferred 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 of the accepted Industry Standard smolt (3-10%) transferred 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" currently used in industry practice.
2. The Process I fish were in peak condition during and immediately after seawater transfer. Unlike industry standard smolt that required 56 days to reach fall 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 (Stradmeyer, L. Is feeding nonstarters a waste of time.
Fish Fanner 3:12-13, 1991; Usher, ML, C Talbot and FB Eddy. Effects of transfer to seawater on growth and feeding in Atlantic salmon smolts (Salmo salar L.) Aquaculture 94:309-326, 1991) for SGR's range between 0.2-0.8). In fact, the growth rates of Process I smolts are significantly larger as compared to Industry standard smolts placed into seawater on the same site despite that industry standard smolt were both larger at the time of seawater placement as well as that their growth was measured 120 days after seawater placement. These data provide evidence that the Process I smolts were not subjected to significant osrnoregulatory stress which would prevent them from feeding immediately.
3. The rapid growth of Process I smolts immediately upon ocean netpen placement provides for compounding increases in the size of salmon as seawater growout proceeds. Thus, it is anticipated that if Industry Standard Smolts weighing 112.5 gram '(gm) were subjected to Process I treatment, placed in ocean netpens and examined at 120 days after ocean netpen placement their size would be average gram instead of 377 gram as observed. This provides for more than a doubling in size of fish in the early stages of growout. Such fish would reach market size more rapidly as compared to industry standard fish.
In contrast to the counterpart Si smolts subjected to standard industry practices, smolt treated with Process I fed vigorously within 48 hours of ocean placement and continued to increase their consumption of food during the immediate post-transfer period. By comparison, the industry standard smolts consumed little or no feed within the first week after transfer, Figure 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 278 provides data on the characteristics of Process I smolts after seawater transfer. These experiments were carried out for over 185 days.
Application of the Process Ito Atlantic Salmon pre-adult Fish that are Smaller than the Industry Standard "Critical Size" Smolt.
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 Hatchery, 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 truck 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. The Process I were then vaccinated with the Lipogen Forte product (Aquahealth LTD.) and transported to ocean netpens by conventional truck 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. Alternatively, 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 grain 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 SO-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 John strain Process I smolts possessing a range of body sizes that were transferred to seawater either in ocean netpens or corresponding 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.
Experiments with these data were carried out for 84 days after the transfer of fish to seawater tanks, and the data from these experiment's are described in co-pending application No: 09/975,553, Attorney Docket No: 2213.1004-001.

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.
Laboratory Tank Ocean Netpen Total Fish 140 1,069 Date of Seawater 9/5/00 (40); 9/12/00 9/12/00 Transfer (100) Average Size at Transfer 26.6 26.6 (gram) Total mortalities after 4 1; 0.7% 143;13.4%
days (# and % total) % mortality of fish 0; 0.0% 4; 0.4%
weighing 25 gm and above Time to achieve feeding 48 hrs 72 lirs 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 froth 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.
Length and weight measurements for all mortalities collected from the bottom of the ocean netpen were compared to the distribution of Process I smolt body characteristics obtained from analysis of a representative sample prior shown in Figure 29. The data show that the mortalities occurred selectively amongst Process I
smolts possessing small body sizes such that the mean body weight of mortalities was 54% of the mean body weight of the total transfer population (14.7/27 gram or 54%). Thus, the actual mortality rates of Process I smolts weighing 25-30 gram is 0.4% (4/1069) and those weighing 18-30 gram is 2.9% (31/1069).

Application of Process I to Trout pre-adult Fish that are Smaller than the Industry Standard "Critical Size" Smolt.
Table 9 displays data on the use of the 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 of the full strength seawater present in standard ocean netpens.
After a prolonged interval of osmotic adaptation, trout are then transferred to more standard ocean netpen sites to complete their growout cycle. In general, trout are transferred to these ocean sites for growout at body weights of approximately 70-90 or 90-gram.

Table 9: Comparison of the Survival of Rainbow Trout (3-5 gram) in Seawater After Various Treatments.
Percent Survival of Fish' Hours Post Control Constant 14 Constant 14 Constant 23 Seawater Freshwater day day day Transfer Photoperiod Photoperiod Photoperiod +
Process I Process I

Number of Fish Per 10 20 30 80 Experiment 1Survival percentages expressed as rounded whole numbers 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. When control freshwater rainbow trout are transferred directly into seawater, there is 100% mortality within 24 hr (Control Freshwater). Exposure of the trout to a constant photoperiod for 14 days results in a slight improvement in survival after their transfer to seawater. In contrast, 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% of the fish respectively have survived after a 5 day interval in seawater. These data demonstrate that application of the Process I increases in the survival of pre-adult trout that are less than 7% of the size of standard "critical size" trout produced by present day industry standard techniques.
Application of the Process Ito Arctic Char pre-adult Fish that are Smaller than the Industry Standard "Critical Size" Smolt.
Although arctic char are salmonids and anadromous fish, their tolerance to seawater transfer is far less as compared to either salmon or trout. 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% of the 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.
SECTION The Use of the Process If to Peanit Successful Transfer of 10-30 gram Smolt into Seawater Netpens and Tanks.
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 salmon smolt transfers.

In summary, Process II reduces the "critical size" for successful seawater transfer to less than one fifth the size of the present day industry standard SO smolt.
Application of Process II to Atlantic Salmon Fingerlings:
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. Kennebec Hatchery, Kennebec Maine on 27 April 2000. These fish were transported to the treatment facility using standard conventional truck transport. After their arrival, these parr were first grown in conventional flow through freshwater growout conditions that included a water temperature of 9.6 C and a standard freshwater parr diet (Moore-Clark Feeds).
On 17 July 2000, fingerlings were begun on Process II 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 II
as well as immediately prior to transfer to seawater. St John/St John Process If smolts were transported to ocean netpens by conventional truck 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 II smolts possessing a range of body sizes were transferred to seawater either in ocean netpens or corresponding laboratory seawater tanks. Note that these Process II 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 II smolts. The average measurements of these St. John/St. John Process II
smolts included a body weight of 11.50+/-5.6 gram, length of 9.6+1-1.5 cm and condition factor of 1.19+1-0.09. The data displayed in Table 10 shows the outcomes for two groups of Process II smolts derived from a single production pool of fish after their seawater transfer into either laboratory tanks or ocean netpens.
Although important variables such as the water temperatures and transportation of fish to the site of seawater transfer were identical, these 2 groups of Process II smolts experienced differential post seawater transfer mortalities after 5 days into laboratory tanks (10% mortality) and ocean netpens (37.7% mortality).
The probable explanation for this discrepancy in mortalities between seawater laboratory tanks (10% mortality) and ocean netpens (37.7% mortality) is exposure of these fish to different photoperiod regimens after seawater placement.
Exposure of juvenile Atlantic salmon to a constant photoperiod after seawater placement reduced their post-seawater transfer mortality from approximately 34% to 6%. Fish transferred to ocean netpens experienced natural photoperiod that was not continuous and thus suffered an approximate 4-fold increase in mortality. As shown in Table 10, a separate seawater transfer of St John/St John juvenile Atlantic salmon possessing an average weight of 21 gins exhibited only 0.2% mortality after a six week treatment with Process II and underwater lights. These fish were exposed to a continuous photoperiod by underwater halogen lights for an interval of 30 days.
Table 10: Characterization and survival of St. John/St. John Process II fish after their placement into seawater in ocean netpens containing underwater lights.
Total Fish 15,000 Seawater Transfer Date 8/9/01 Water Temperature (oC) 12.6 Size at Transfer (gram) 21+/-4.5 Total Mortalities after 30 days (# and % total) 250 1.7%
% Mortalities weighing15 grams or greater 30 0.2%
Time to achieve feeding after transfer 48 hr Table 12: Characteristics and survival of St. John/St. John Process II fish after their placement into seawater in either a laboratory tank or ocean netpen.
Laboratory Tank Ocean Netpen Total Fish 100 1,316 Seawater Transfer Date 8/31/00 9/5/00 Water Temperature ( C) 15.6 15.6 Size at Transfer (gram) 11.5 11.5 Total Mortalities after 5 10; 10% 496; 37.7%
days (# and % total) % mortalities weighing 0; 0% 1; 0.08%
_ 13 grams or-greater Time to achieve feeding 48 hrs 48 hrs after transfer No apparent problems were observed with the smaller (10-30 gram) Process II
smolts negotiating the conditions that exist within the confines of their ocean netpen. This included the lack of apparent problems including the ability to school freely as well as the ability to swim normally against the significant ocean currents that are continuously present in the commercial Blue Hill Bay salmon aquaculture site. While these observations are (-still ongoing, these data do not suggest that the placement and subsequent growth of Process II smolts in ocean netpens will be comprised because of lack of ability of these pre-adult anadromous fish to swim against existing ocean currents and therefore be unable to feed or develop properly.
Figure 32 compares characteristics of survivors and mortalities of Process II
smolts after seawater transfer to either laboratory tanks (Figure 32A) or ocean netpens (Figure 32B) . Figure 32A data are derived from analyses of 100 Process II
smolts transferred to seawater tank where all fish were killed and analyzed on Day 5.
In contrast, 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 of the average body size of those Process II smolts that survived seawater transfer vs. those Process II smolts that died shows that unsuccessful Process II smolts possessed significantly smaller body weights as compared to average body size of whole Process II smolt transfer group. Thus, the average weight of mortalities in laboratory tank (5.10+/-2.2 gram) and ocean netpen (6.46+1-1.5 gram) are 44% and 56% respectively the value of the average body weight possessed by the entire transfer cohort (11.5 gram). In contrast, the mortalities of Process 1I smolts with body weights greater than 13 gram is 0/100 in the laboratory tank and 1/1316 or 0.076% for ocean netpens. Together, these data demonstrate that Process II is able to redefine the "critical size" of Atlantic salmon smolts from 70-100 gram to approximately 13 gram.
Quantitation of Feeding and Growth of Process I and II smolts after Seawater Transfer:
Landcatch/St John Process I smolts were offered food beginning 48 hr after their seawater transfer to either laboratory tanks or ocean netpens. While these Process I
smolts that were transferred to laboratory tanks began to feed after 48 hr, those fish transferred to ocean netpens were not observed to feed substantially until 7 days. To validate these observations, the inventors performed direct visual inspection of the 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.
The St John/St John Process II smolts fed vigorously when first offered food hrs after their seawater transfer regardless of whether they were housed in laboratory tanks or ocean netpens. An identical direct analysis of Process II smolts gut contents performed as described above revealed that 61/83 or 73.5% of fish were feeding 4 days after transfer to seawater. The vigorous feeding activity of Process II
smolts in an ocean netpen as well as laboratory tanks occurred. Taken together, these data suggest that Process I and II smolts do not suffer from a prolonged (20-40 day) interval of poor feeding after seawater transfer as is notable for the much larger industry standard Atlantic salmon smolts not treated with the process.

The growth rates of identical fish treated with either Process I or II within laboratory seawater tanks has been quantified. As shown in Table 12, both Atlantic salmon treated with Process I or II grow rapidly during the initial interval (21 days) after transfer to seawater. In contrast to industry standard smolt weighing 70-grams that eat poorly and thus have little or no growth during their first 20-30 days after transfer to seawater, pre-adult Atlantic salmon receiving Process I or II both exhibited substantial weight gains and growth despite the fact that they are only 27-38% (Process I) and 12-16% (Process II) of the critical size of industry standard smolts. Data that relates to mortalities, SGR, temperature corrected SGR
(GF3), FCR, body weights, lengths and condition factors for these same fish were obtained a total of 4 additional intervals during an interval that now extends for 157 days.

Table 12: Comparison of Growth Rates of Pre-adult Atlantic Salmon Exposed to either Process I or Process II and Placed in Laboratory Tanks During Initial Interval After Seawater Transfer Process I Process II
Number of Fish 140 437 Weight at Placement into 26.6 11.50 Seawater Days in Seawater 22 21 Placement Weight 26.6* 13.15*
Corrected for Mortalities _ Weight after Interval in 30.3 15.2 Seawater Weight Gained in 3.75 2.05 Seawater SGR (% body 0.60 0.68 weight/day) FCR 1.27 2.04 * Weight gain corrected for selective mortalities amongst smaller fish (4/140 or 2.9%' Process I; 103/437 or 23.6% Process II) EXAMPLE 8. EXPOSURE OF SALMON SMOLTS TO CA2+ AND MG2+
INCREASES EXPRESSION OF PVCR IN CERTAIN TISSUES.
In smolts that were exposed to 10 mM Ca2+ and 5.2 mM me+, the expression of PVCR was found to increase in a manner similar to that in smolts that are untreated, but are transferred directly to seawater.
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% parafomialdehyde, washing in Ringers then frozen in an embedding compound, e.g., 0.C.T.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. Briefly, sections mounted on glass slides were: 1) blocked with goat serum or serum obtained from the same species of fish, 2) incubated with rabbit anti-CaR antiserum, and 3) washed and incubated with peroxidase-conjugated affinity-purified goat antirabbit antiserum. The locations of the 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.
The results are shown in Figs. 33A - 33G, which are a set of seven photomicrographs showing immunocytochemistry of epithelia of the proximal intestine of Atlantic salmon smolts using anti-PVCR antiserum, and in Fig. 34, -which is a Western blot of intestine of a salmon smolt exposed to Ca2+- and Mg2+-treated freshwater, then transferred to seawater. The antiserum was prepared by immunization of rabbits with a 16-mer peptide containing the protein sequence encoded by the carboxyl terminal domain of the dogfish shark PVCR ("SKCaR") (Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A). Specific binding of the anti-PVCR antibody is indicated by aminoethylcarbazole (AEC) reaction product.
Figs. 33A and 33B show stained intestinal epithelia from smolts that were maintained in freshwater then transferred to seawater and held for an interval of 3 -days. Abundant PVCR immunostaining is apparent in cells that line the luminal surface of the intestine. The higher magnification (1440X) shown in Fig. 33B
displays PVCR protein localized to the apical (luminal-facing) membrane of intestinal epithelial cells. The pattern of PVCR staining is localized to the apical membrane of epithelial cells (small arrowheads) as well as membranes in globular round cells (arrows). 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 33A and 33B including apical membrane staining (small arrowheads) as well as larger globular round cells (arrows). 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 mI\4 Ca2+
and 5.2 mM Mg2+ for 50 days and fed 1% NaC1 in the diet. Again, small arrowhead and arrows denote PVCR staining of the apical membrane and globular cells respectively. In contrast to the prominent PVCR staining shown in Figures 33A-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. Both 13E and 13F display a marked lack of significant PVCR staining. Fig. 33G (1440X) shows the lack of any apparent PVCR staining upon the substitution of preimmune serum on a section corresponding to that shown in Figure 33A where anti-PVCR antiserum identified the PVCR protein. The lack of any PVCR staining with preinunune antiserum is a control to demonstrate the specificity of the anti-PVCR antiserum under these-imimmocytochemistry 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. In contrast, the PVCR protein content of the corresponding intestinal epithelial cells was significantly increased upon the transfer of these smolts to seawater (Figs. 33A and 33B). Importantly, 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 of the anti-PVCk antiserum, since substitution of the immune antiserum by the preimmune eliminated all reaction product from intestinal epithelial cell sections (Fig. 33G).
Disclosure of localization of PVCR protein(s) in additional areas of osmoregulatory organs of Atlantic Salmon using paraffin sections. Demonstration that PVCR
proteins are localized to both the apical and basolateral membranes of intestinal epithelial cells.
Using the methods described herein, immunolocalization data from paraffin sections of various osmoregulatory organs of seawater-adapted juvenile Atlantic salmon smolt were obtained. 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 of the skin, nasal lamellae and gill each of which are bathed by the water surrounding the fish. The PVCR was also seen in various portions of the G.I.
tract including stomach, pyloric caeca, proximal intestine and distal intestine that process seawater ingested by fish. These tissues were analyzed after treatment with Processes I and II, and after their transfer from freshwater to seawater. In addition, it is believed that the PVCR protein can also act as a nutrient receptor for various amino acids that are reported to be present in stomach, proximal intestine, pyloric caeca.
In particular, higher magnification views of PVCR immunolocalizations in selected cells of the stomach, proximal intestine and pyloric caeca were obtained.
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 of the crypts of the stomach, but also is present in neuroendocrine cells that are located in the submucosal area of the stomach. From its location on neuroendocrine cells of the 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 hoiniones (e.g., 5-hydroxytryptamine (5-HT), serotonin, or colecystokinin (CCK)). Importantly, it is believed that the constituents of Process II effect G.I. 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.
In a similar manner, 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 luminal and blood contents of divalent cations, NaC1 and specific amino acids and thereby integrate the multiple nutrient and ion absorptive-secretory functions of the intestinal epithelial cells.
Epithelial cells of pyloric caeca also possess abundant apical PVCR protein.
To further demonstrate the specificity of the anti-CaR antiserum to recognize salmon smolt PVCRs, Fig. 34 shows a Western blot of intestinal protein from salmon smolt maintained in 10 mM Ca2+, 5 mM Mg2+ and fed 1% NaC1 in the diet.
Portions of the proximal and distal intestine were homogenized and dissolved in SDS-containing buffer, subjected to SDS-PAGE using standard techniques, transferred to nitrocellulose, and equal amounts of homogenate proteins as determined by both protein assay (Pierce Chem. Co, Rocford, IL) as well as Coomassie Blue staining were probed for presence of PVCR using standard western blotting techniques. The results are shown in the left lane, labeled "CaR", and shows a broad band of about 140-160 kDa and several higher molecular weight complexes.
The pattern of PVCR bands is similar to that previously reported for shark.kidney (Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A) and rat kidney inner medullary collecting duct (Sands, J.M. et al., 1997, J. Clin. Invest. 99:1399-1405). The lane on the right was treated with the preimmune anti-PVCR serum used in Fig. 33G, and shows a complete lack of bands. Taken together with immunocytochemistry data shown in Figure 33, this immtmoblot demonstrates that the antisenun used is specific for detecting the PVCR protein in salmon.
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.
Thus, the presence of PVCR proteins in selected cells of the fish's epidermal layer would be able to "sense" the salinity of the surrounding water as it flowed past and provide for the opportunity for continuous remodeling of the salmonid's skin based on the composition of the water where it is located.
Methods: Samples of the skin from juvenile Atlantic Salmon resident in seawater for over 12 days were fixed in 3% parafonnaldehyde dissolved in buffer (0.1M NaPO4, 0.15M NaC1, 0.3M sucrose pH 7.4), manually descaled, rinsed in buffer and frozen at -80 C for cryosectioning. Ten micron sections were either utilized for immunolocalization of PVCR using anti-shark PVCR antiserum or stained directly with 1% Alcian Blue dye to localize cells containing acidic glycoprotein components of mucous.
Results and Discussion: Figure 35A shows that salmon epidennis contains multiple Alcian Blue staining cells present in the various skin layers. Note that only a portion of some larger cells (that containing acidic mucins) stains with Alcian Blue (denoted by the open arrowheads). For purposes of orientation, note that scales have been removed so asterisks denote surface that was previously bathed in seawater.
Figure 35B shows immunolocalization of salmon skin PVCR protein that is localized to multiple cells (indicated by arrowheads) within the epidermal layers of the skin. Note that anti-PVCR staining shows the whole cell body, which is larger than its corresponding apical portion that stains with Alcian Blue as shown in Figure 35A. The presence of bound anti-CaR antibody was indicated by the rose color reaction product. Although formal quantitation has not yet been performed on these sections, it appears that the number of PVCR cells is less than the total number of Alcian Blue positive cells. These data indicate that only a subset of Alcian Blue positive cells contain abundant PVCR protein. Figure 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.
These data demonstrate the presence of PVCR protein in discrete epithelial cells (probably mucocytes) localized in the epidermis of juvenile Atlantic salmon.
From this location, the PVCR protein could "sense" the salinity of the surrounding 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 of the present invention. This treatment of Atlantic salmon smolts by the process of the 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 IN 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
96 well plate assay system. The data, described herein, demonstrate the utility of a 96 well ELISA assay to quantify the tissue content of PVCR protein using a rabbit polyclonal anti-PVCR antibody utilized to perfouu immunocytochemistry and western blotting. These data form the basis for development of commercial assay kits that would monitor the expression levels of PVCR proteins in various tissues of juvenile anadromous fish undergoing the processes of the present invention, as described herein. The sensitivity of this ELISA is demonstrated by measurement of the relative PV-CR content of 14 tissues from a single juvenile Atlantic salmon, as shown in Figure 36.
Description of Experimental Protocol:
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 KC1, 1mM Phenylmethylsulfonyl fluoride (PMSF), 0.5 dithiothreitol (DTT) and linM benzamidine pH 8.8) and using a standard glass Potter-Elvenhiem homogenizer with a rotary pestle. After centrifugation at 2,550Xg forr 20 min. at 4 C to remove larger debris, the supernatant was either used directly or frozen at -80 C until further use. 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 transferred 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 KC1 pH 8.0), primary antiserum (either rabbit anti-PVCR
immune or corresponding rabbit pre-irnmune antiserum) at a 1:1500 dilution was added.
After a lhr incubation, individual wells were rinsed 3 times with 500 microliters of TBS, an 1:3000 horseradish peroxidase conjugated goat anti-rabbit (Gibco-BRL ) were added and allowed to incubate for 1 hr. Individual wells were then rinsed and bound complex of primary-secondary antibody detected with Sigma A3219 2,2' Azino-bis(3-ethylbenzthiazidine-6-sulfonic acid) color reagent after 15 min of incubation using a Molecular Devices 96 well plate reader (Molecular Devices, VMAX) at 405nm. Relative amounts of tissue PVCR content were determined after corrections for minimal background and nonspecific antibody binding as measured by binding of preimmune antiserum.
Results and Data Interpretation:
Figure 36 shows the data obtained from a representative single ELISA
determination of PVCR protein content of 14 tissues of a single juvenile 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. Immunocytochemistry 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. In contrast, other organs-including liver, , heart and muscle contain minimal PVCR protein. Note that the highest PVCR
content of any tissue tested is the olfactory lamellae where salmon possess the ability to "smell" alterations in calcium concentration in water. The olfactory bulb containing neurons that innervate the olfactory lamellae also possess abundant PVCR. Taken together, these data demonstrate the utility of ELISA kits to measure tissue content of PVCR proteins and form the basis for development of commercial assay kits that would monitor the expression levels of PVCR proteins in various tissues of juvenile anadromous fish undergoing the processes of the present invention. Alterations in PVCR tissue content measured in either relative changes in tissue PVCR content or absolute quantity of PVCR per tissue mass could, in turn, be utilized as correlative assays to determine the readiness ofjuvenile anadromous fish for sea water transfer or initiation of feeding. These data demonstrate the ability to perform such assays on individual juvenile Atlantic salmon in the range of body sizes that would be utilized to transfer fish from fresh to seawater after treatment with the methods of the present invention.
EXAMPLE 11: ANTIBODMS MADE FROM THE CARBOXYL TERMINAL
PORTION OF AN ATLANTIC SALMON PVCR PROTEIN ARE EFFECTIVE IN
IMMLTNOCYTOCHEMISTRY AND IIVIMUNOBLOTTING 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.
To obtain more cDNA sequence from anadromous fish PVCRs, in particular the putative amino acid sequence of the carboxyl terminal domain of PVCRs that are targets for generation of specific peptides and, as a result, specific anti-Atlantic Salmon PVCR antisera, an unamplified cDNA library from Atlantic salmon intestine was constructed. Phage plaques originating from this cDNA library were screened under high stringency usineP-labeled 653 bp genomic Atlantic Salmon PCR
product. From-this cDNA library screening effort, a 2,021 bp cDNA clone was isolated and contained a single open reading fi-aine for a putative amino acid sequence corresponding to approximately one half of a complete cDNA sequence from an intestinal PVCR protein. This putative amino acid sequence corresponds exactly to the sequence encoded by the corresponding genomic probe as well as the putative amino acid sequence corresponding to the carboxyl terminal domain of the PVCR.
On the basis of the knowledge of this putative amino acid sequence, a peptide, shown below, was synthesized and corresponded to a separate region of the putative carboxyl terminal PVCR amino acid sequence:
The peptide sequence for antibody production is as follows:

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 immunoblotting and immunocytochemistry techniques to determine whether the antibody bound to protein bands corresponding to PVCR proteins or yielded staining patterns similar to those produced using other anti-PVCR antiserum. A
photograph of an immun.oblot 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). As expected, 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). Immunostaining of juvenile Atlantic salmon kidney sections with 3 different anti-PVCR antisera (anti-Sail, anti-4641, and anti-SKCaR) 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 corresponding to extracellular domain _ of mammalian PVCRs that is very similar to SKCaR (SEQ ID NO: 2). These PVCR
protein patterns stained identically to that produced by SAL-1 antiserum. Anti-Sal-1 antisenun also exhibits a similar staining pattern for the distribution of intestinal PVCR protein, as compared to anti-SKCaR. Thus, 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 of fish, 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 salmon 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 significant commercial importance since they are one of the principal determinants of texture of smoked Atlantic salmon fillets.
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: CSDDEYGRPGTFKFEKEM (SEQ ID NO: 29).
This peptide was synthesized, derivatized in a manner 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 immonostaining on sections of juvenile Atlantic salmon that is identical to that exhibited by Sal L (See Figure 40C). Since both SalmoKCaR #1, #2 and #4, but not SalmoKCaR #3 possess the carboxyl terminal 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.
In contrast, the Salmo ADD antibody binds to a peptide sequence present in the extracellular domain of all 4 SalmoKCaR proteins. Thus, 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
In 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 of the genomic DNA sequences of PVCRs from various anadromous fish. These same primers can also be used to amplify a portion of corresponding 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:
1. 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.
2. 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.
3. 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.
RT-PCR Method:
Total RNA was purified from selected tissues using Teltest B reagent (Friendswood, TX) and accompanying standard protocol. A total of 5 micrograms of total-RNA was reversc transcribed with oligo dT primers using Invitrogen's cDNA Cycle Kit (Invitrogen Inc, Madisbn, WI). The resulting cDNA product was denatured and a second round of purification was perfouned. Two microliters of the resulting reaction mixture was amplified in a PCR reaction (30 cycles of 1 min. @
94 C, 2 min. @ 57 C, 3 min. @72 C) using degenerate primers dSK-F3 (SEQ ED
NO: 15) and dSK-R4 (SEQ ID NO: 16). The resulting products were electrophoresed on a 2% (w/v) agarose gel using TAE buffer containing ethidium bromide for detection of amplified cDNA products. Gels were photographed using standard laboratory methods.
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 Amersham's MicroSpin S-400 FIR 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 MacVector (GCG) and LaserGene (DNA STAR) sequence analysis software.
Detection of Amplified RT-PCR cDNA products by Southern Blotting:
Alternatively, the presence of amplified PVCR products was detected by Southern blotting analyses of gel fractionated RT-PCR products using a '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). After crosslinking of the DNA, blots were prehybridized and then probed overnight (68 C
_ in 6X SSC, 5X Denhardt's Reagent, 0.5% SDS, 10Oug/m1 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 0.1x 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 transferred to a membrane and Southern blotted using a 32P-labeled 653 bp genomic DNA fragment from the Atlantic salmon PVCR gene. Figure 37 shows the detection of the PVCR in several tissue types of Atlantic Salmon 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 IN NASAL
LAMELLAE AND OLFACTORY BULB AS WELL AS GI TRACT OF FISH.
The data described herein described the roles of PVCR proteins in the olfactory organs (nasal lamellae and olfactory bulb) of fish as it relates to the ability of fish to sense or "smell" both alterations in the water salinity and/or ionic composition as well as specific amino acids. These data are particularly applicable to anadromous fish (salmon, trout and char) that are either transferred from freshwater directly to seawater or exposed to Process I or Process II in freshwater and then transferred to seawater.
These data described herein were derived from a combination of sources including immunocytochemistry using anti-PVCR antisera, RT-PCR amplification of PVCRs from nasal lamellae tissue, studies of the function of recombinant aquatic PVCR proteins expressed in cultured cells-where these proteins "sense"
specific ions or amino acids as well as electrophysiological recordings of nerve cell electrical activity from olfactory nerves or bulb of freshwater salmon.
The combination of immunocytochemistry and RT-PCR data, described herein, reveal the presence of PVCR proteins in both major families of fish (elasmobranch-shark; teleost-salmon) in both larval, juvenile and adult life stages.
Immunocytochemistry analyses reveal that one or more PVCR proteins are present both on portions of olfactory receptor cells located in the nasal lamellae of fish (where they are bathed in water from the surrounding environmpt) as well as on nerve cells that compose olfactory glomeruli present in the olfactory bulb of fish brain (where these cells are exposed to the internal ionic environment of the fish's body). Thus, from these locations fish are able to compare the ionic composition of the surrounding water with reference to their own internal ionic composition.
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 control of the 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.
One feature of this biological system is alteration in the sensitivity of the PVCR protein for divalent cations such as Ca 2+ and Mg2+ by changes in the NaC1 concentration of the water. Thus, PVCRs in fish olfactory organs have different apparent sensitivity to Ca2+ in either the presence or absence of NaCl. These data presented here are the first direct evidence for these functions via PVCR
proteins present in the olfactory apparatus of fish.
Another feature of PVCR protein function in the olfactory apparatus of fish 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:
1. responded to PVCR agonists in a concentration-dependent manner similar to that shown previously for other fish tissues including that shown for winter flounder urinary bladder. These data provide the functional evidence of the presence of a PVCR protein; and 2. that the presence of a PVCR agonist reduces or ablates the signal resulting from odorants including both attractants or repellants. Thus, PVCRs in the orfactory 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 surrounding environment. Using the full-length recombinant 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 spemndine) 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 of fish including G.I. tract and endocrine organs of fish also function to sense specific concentrations of amino acids providing 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.
Description of Experimental Results and Data Interpretation:
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 of the PVCR
protein. _ Thus, antiserum anti-4641 (recognizing an extracellular domain PVCR region) and antiserum anti-SKCaR (recognizing an intracellular domain PVCR region) exhibit similar staining patterns that include various glomeruli on serial sections of olfactory bulb. Using anti-SKCaR antiserum, specific staining of PVCR proteins is observed in discrete regions of the olfactory nerve as well as epithelial cells in the nasal lamellae that are exposed to the external ionic environment.
The presence of 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 of the salmon. For this purpose, cells containing internally-exposed PVCRs are connected to externally-exposed PVCRs via electrical connections within the nervous system. As shown schematically in Figure 39, these data suggest that externally and internally-exposed PVCRs function together to provide for the ability to sense the ionic concentrations of the surrounding ionic environment using as a reference the ionic concentration of the salmon's body fluids. Changes in the expression and/or sensitivity of the external set of PVCRs vs internal PVCRs would then provide a long term "memory"
of the adaptational state of the fish as it travels through ionic environments of different composition. 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 salmon 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 of the salmon. Myosepta are also of significant commercial importance since they are one of the principal determinants of texture for smoked Atlantic salmon fillets. Sah-nol(CaR proteins are also present in the otolith which is a calcified structure located in the inner ear of the salmon 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.
Data obtained from using anti-SKCaR antiserum from other fish species including elasmobranchs display similar staining of PVCR protein in cells (marked A) their nasal lamellae (Figure 41). Use of other methodology including RT-PCR

_ using specific degenerate primers (Figure 42) and ELISA methods (Figure 43) detects the presence of PVCR proteins and mRNA in nasal lamellae of fish.
While neither of these 2 techniques provide quantitative measurements as described, both sets of data are consistent and show abundant PVCR protein present in this tissue.
Measurement of extracellular electrical potentials (EEG's) from olfactory nerve from freshwater adapted Atlantic salmon reveals the presence of functional PVCR
proteins:
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 Sparus Aurata, Exp. Biol. 203:3821-3829 (2000). 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 stream of distilled water via a tube held in position in the inhalant olfactory opening. The olfactory nerves of the 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.
As shown in Figure 44, irrigation of salmon olfactory epithelium with distilled water produces minimal generation of large signals in olfactory nerve. The data in Figure 44 are displayed as both raw recordings (left column) and the corresponding integrated signals for each raw recording shown in the right column.
Exposure of the olfactory epithelium to 500 micromolar L-alanine ( a well known amino acid attractant for fish) produces large increases in both the firing frequency and amplitude in the olfactory nerve lasting approximately 2 seconds in duration.
Similarly, application of either 1mM Ca" or 250 inM NaC1 also produce responses in EEG activity. To test for the presence of furictional PVCR protein, the olfactory epithelium was exposed to 50 micromolar gadolinium (Ge-a PVCR agonist) and also obtained a response. Figure 45 shows dose response data from multiple fish to various PVCR agonists or modulators where the relative magnitudes of individual olfactory nerve response were noimalized relative to the response produced by the exposure of the olfactory epithelium to 10 mM Ca'. As shown in Figure 45, the olfactory epithelium of freshwater adapted juvenile salmon is very sensitive to Ca' where the half maximal excitatory response (EC50) is approximately 1-10 micromolar. Similarly, exposure of olfactory epithelium to the PVCR agonist Geproduces responses of a similar magnitude to those evoked by Ca" in a concentration range of 1-10 micromolar. In contrast, olfactory epithelium responses to Mg" do not occur until 10-100 micromolar solutions are applied. These dose response curves (EC50 Ge<Ca2<Mg2+) are similar to those obtained for PVCR
modulated responses in other fish epithelium (flounder urinary bladder NaC1-mediated water transport-see SKCaR application).
In contrast, analysis of the olfactory epithelium responses to NaC1 exposure shows that it is unresponsive until a concentration of 250 millimolar NaC1 is applied.
Since NaCl does not directly activate PVCRs in a manner such as Gd' Ca' or Mg' but rather reduces the sensitivity of PVCRs to these agonists, these data are also consistent with the presence of an olfactory epithelium PVCR. The response evoked by exposure of the epithelium to significant concentrations of NaC1 likely occurs via other PVCR independent mechanisms.
These data suggest that PVCR proteins present in olfactory epithelium are capable of sensing and generating corresponding olfactory nerve signals in response to PVCR agonists at appropriate concentrations in distilled water.
Addition of PVCR agonists such as Ca2+ or Gd3+ to distilled water containing well known salmon repellants reversibly ablates the response of the olfactory epithelium to these stimuli:
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 of the PVCR agonists 5mM Ca' or 50 micromolar Gd3+ reversibly ablated the response by the olfactory epithelium to mammalian finger rinse. These data show that PVCR agonists modulated the response of the olfactory epithelium to an odorant such as mam_malian 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.
Addition of PVCR agonists such as Ca2+ or Gd3+ but not NaC1 to distilled water containing the well known salmon attractant L-alanine reversibly ablates the response of the olfactory epithelium to these stimuli:
Figure 47 shows a time series of stimuli (2 mm 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.
Note that the addition of either 5 inM Ca2+ (recording #2) or 50 micromolar (recording #7) to 500 micromolar L-alanine resulted in the complete loss of the corresponding response from the olfactory nerve after injection of this mixture. In both cases, this was not due to a permanent alteration of the olfactory epithelium by either of these PVCR agonists because a subsequent identical stimulus without the PVCR agonist (recordings #3 and #8) caused a return of the signal. It is noteworthy that in the case of Ge addition, the magnitude of the subsequent L-alanine signal was decreased as compared to control (compare recordings #6 vs #8) indicating that the olfactory epithelium prefers an interval of recovery from its exposure to this potent PVCR agonist. However, the alteration of response to the L-Alanine stimulus is not permanent or nonspecific since combining the same dose of L-Alanine with _ 250 mM NaC1 resulted initially in a similar response (recordings #4 and #9) followed by an enhanced response to L-Alanine alone (recordings #5 and #10).
In summary, the data displayed in Flores 46 and 47 show that inclusion of a PVCR agonist in solutions containing either a repellant (finger rinse) or attractant (L-alanine) causes a dramatic reduction in the response of the olfactory epithelium to those odorants. For both repellants and attractants, some form of complex interactions occur within olfactory epithelial cells since mixing of PVCR
agonists and odorants renders the epithelia temporary unresponsive to either stimulus.
While the nature of such interactions are not known at the present time, such interactions do not occur at the level of the PVCR molecule itself as shown by data from experiments using recombinant PVCR protein SKCaR. As further described herein, inclusion of amino acids in the presence of Ca' enhances the response of SKCaR
to ambient Ca"- concentrations. Regardless of their nature, these negative modulatory effects of PVCR agonists including Ca' is likely to produce major effects on how freshwater salmon smell objects in their environment after transfer from a low calcium to a high calcium environment. Use of this assay system would permit the identification and analyses of both specific classes of PVCR agonists and antagonists as well as the specific effects of each PVCR modulator on specific odorants including both repellants and attractants.
Recombinant PVCR protein SKCaR possesses the capability to sense concentrations of amino acids after its expression in human embryonic kidney (HEK) cells:
Full length recombinant dogfish (Squalus acanthias) 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

NaC1, 4rnM KC1, 0.5 mM CaC12, 0.5 MgC12, 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' concentration was 0.5 mM. Aliquots of Ca' were added to produce final extracellular concentrations of 2.5 mM, 5mM, 7.5mM, 10mM and 20 mM Ca' with changes in the fluorescence recorded. Note that increases in cell fluorescence were greater in the presence of = 20 10mM Phe for extracellular Ca" concentrations less than 10 mM.
Figure 49 shows data plotted from multiple experiments as described in Figure 48 where the effects of 10 in_M Phe, 10mM Ile 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, Arg, 75 Ile, 150 Leu. Note that both 10mM Phe and 10 mM Ile as well as the mixture of amino acids increase SKCaR's response to a given Ca' concentration.

Thus, these data show that presence of amino acids either alone or in combination increase the apparent sensitivity to Ca'pennitting SKCaR to "sense"amino acids in the presence of physiological concentrations of Ca". These data obtained for SKCaR are comparable to those obtained for the human CaR.

The significance of these data for aquatic organisms stand in marked contrast to the roles of human CaRs amino acid sensing capabilities. Figure 48 shows that SKCaR's maximal capability to sense amino acids is confined to a range of Ca'that is present both in aquatic external environments as well as the body fluids of various fish. The following physiological processes occur: 1) Sensing of amino acids in the proximal intestine and pyloric caeca of fish: The PVCR present on the apical surface of intestinal epithelial cells is capable of responding to amino acids such as tryptophan as part of the Process II. Inclusion of tryptophan in the feed of fish interacts with the intestinal PVCR to improve the development of juvenile anadromous fish to tolerate seawater transfer. 2) In both adult, juvenile and larval fish, PVCR localized to the apical membrane of stomach and intestinal epithelial cells could "sense" the presence of amino acids produced by the proteolysis of proteins into amino acids. This mechanism could be used to infoun both epithelial and neuroendocrine cells of the intestine of the presence of nutrients (proteins) and trigger a multitude of responses including growth and differentiation of intestinal epithelia as well as their accompanying transport proteins, secretion or reabsorption of ions such as gastric acid. The apical PVCR also regulates the secretion of intestinal hormones such as cholecystokin (CCK) and others. 3) PVCR proteins _ present in cells of the nasal lamellae of fish "smell" both water salinity (via Ca', Mg' and NaC1) and amino acids which is an example of an attractant. At the present time, it is unclear whether the amino acid sensing capabilities of PVCRs are utilized by the olfactory epithelium to enable fish to smell various amino acid attractants.
These data show that PVCR sensing of amino acids occurs in a range of extracellular calcium that is present in various concentrations of seawater present in estuaries and fish migration routes as well as various compartments of a fish's body including serum and body cavities including intestine, pyloric caeca and kidney where transepithelial amino acid absorption occurs. These data constitute the first report showing the amino acid sensitivity of a PVCR in fish.
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09/975, 553, entitled "Methods for Raising Pre-adult Anadromous Fish," filed on October 11, 2001; International PCT Application No. PCT/US01/31562, entitled, "Methods for Raising Pre-adult Anadromous Fish," filed on October 11, 2001; Provisional Patent Application No. 60/382, 464, "Methods for Growing and Imprinting Fish Using an Odorant," filed October 11, 2001; and Patent Application No.
10/268,051, "Methods for Growing and Imprinting Fish Using an Odorant," filed October 8, 2002.
Additionally, Patent No 6, 334, 391, issued on January 8, 2002, International PCT application No. PCT/US97/05031, filed on March 27, 1997, and Application No. 08/622,738 filed March 27, 1996, all entitled, " Polycation Sensing Receptor in Aquatic Species and Methods of Use Thereof'. Additionally, EPO Application No.: 97937017.8, filed July 24, 1997, which describes the SkCaR receptor.

SEQUENCE LISTING
<110> MariCal, Inc.
<120> Polyvalent Cation-Sensing Receptor in Atlantic Salmon <130> 08901536CA
<140> 2,481,827 <141> 2003-04-09 <150> 10/125,778 <151> 2002-04-18 <150> 10/125,772 <151> 2002-04-18 <150> 10/125,792 <151> 2002-04-18 <150> 10/121,441 <151> 2002-04-11 <150> 60/240,392 <151> 2000-10-12 <150> 60/240,003 <151> 2000-10-12 <160> 40 <170> PatentIn version 3.2 <210> 1 <211> 4134 <212> DNA
<213> Squalus acanthias <400> 1 aattccgttg ctgtcggttc agtccaagtc tcctccagtg caaaatgaga aatggtggtc 60 gccattacag gaacatgcac tacatctgtg ttaatgaaat attgtcagtt atctgaaggt 120 tattaaaatg tttctgcaag gatggcttca cgagaaatca attctgcacg ttttcccatt 180 gtcattgtat gaataactga ccaaagggat gtaacaaaat ggaacaaagc tgaggaccac 240 gttcaccctt tcttggagca tacgatcaac cctgaaggag atggaagact tgaggaggaa 300 atggggattg atcttccagg agttctgctg taaagcgatc cctcaccatt acaaagataa 360 gcagaaatcc tccaggcatc ctctgtaaac gggctggcgt agtgtggctt ggtcaaggaa 420 cagagacagg gctgcacaat ggctcagctt cactgccaac tcttattctt gggatttaca 480 ctcctacagt cgtacaatgt ctcagggtat ggtccaaacc aaagggccca gaagaaagga 540 gacatcatac tgggaggtot etteccaata cactttggag tagccgccaa ggatcaggac 600 ttaaaatcga gaccggaggc gacaaaatgt attcggtaca attttcgagg cttccgatgg 660 ctccaggcga tgatattcgc aattgaagag attaacaaca gtatgacttt cctgcccaat 720 otn oqooqqfreob loaevoq336 ofoovoogoo Beoovoopoq vaeepobErb ofIcz olgoz6.61D5 135155E33y yoatembq5 EqopTeobqo ogolvo6.4.6.4 opzEoqqaft.
insz ozeo.66.4113 a65oorepa6 opqp663q.6.4 op.eb6lovE6 fto33bv.63B Bolvoqqoqe 09tz pqa6p4o6vo oloqlo.6.43.6 lozeoqoppq oqqoqobqa6 loovqooqbq 4.6.e.666paep ootz oproo65pp.6 q6oqroopqp eopt66P3qg BveolvD113 q66.6.65qa6.4 Boqloogoop ovEg ogebqopTeo 65.64peq6oo BolqpTeopv 5qoqoBoTeb .6.6oqqp3o6E E6ovE,E46o1 oEizz BqoaegftED qvBeaftpoo B3qya6.4.601 Bovoy3opv6 v.6.4.2.e.63q6.6 loqqlvEzey inn Boopbqftee ovlBz606.4.6 vra6qpbove veflgyfafto 6S666 .6-ea6-4.6qva6 091z 5qya6zey6q .4.43B.43,61.3o voopEm65.65 5y6oqvoqp6 B.6.6-eva6op vobbBoof45 00-Ez lbqop6v5pq 6eD6qp-epop qoqq4Daqq.6 .64.6-eeepow qloa6.4.6p6.6 wwozeppv oton BReeefoevo qeoze3g3e6 33s6q6e333 Ereewbovw 0.63PEopqae va6.6.64.66p5 0861 weooqq.6.446 15.6ozes6we BBebroBool oqaftaBBlo tvoqvqqp= poeqope5.66 061 epr3qope6q 6.6.ev3le63e .614q3p6446 6pa5e6q.666 weofmoveop vq1q6Ev5qo 0981 Te35q363q voovrowoq 6.6vo6.61.33.6 Bpbqqaepv.e. PvqzeweBeo 6464.4Di:e56 0081 3pppo6qqqo qva6.6Boro6 B000vvea61 qpwroolro pftpobqoop Bopola6.41E.
polovw46o 365q.60.ezeq Blevaeqopq ozefiftbloo vaeovzeiqv .66qopeqop3 0891 povE.E.66.46o Broovolpov pbefeefiBE5 govobwoop voobovqoop Bvoy6Baebe 0g9T ooq3ve66.6.6 355 rooq36 6ovE6E6pro qa66a6boor .66DpoBowo ofiqbeepool 0991 ze6me.64a6 ea6pe6qpoo eftebebooe oqqaeqa6qo Peoll.porft 6.6e66bqoqq 0091 5.efeepoz6.4 qq6Bblppot. 65oqopl.65 oaeopoovoo 1.5.6.ebbpp5.4 ooqqaepovp owc oqq5.6.6popo leq6368.660 666powqp6 olloMpleD peof6a66pq 651.63pooqq 08u 3eq5efee3p6 eepoblqwq pEoqqoqoBe 3a6.66T43.65 sEoBeopfaq obbqozefLo 0E1zefopeoqvp evteeMolq .6-eweft.66-e3 oweo.43533.6 s65wov5oD 336.61Ev3ol 091 35553 Teolfiftwo 6e BleoDqvoqfio vfoobozeoq 1.6R66T45-eo oogi ftebropoto vqaelbeoqo 4pqrbze6s6 1.6Poqlovbq leq.61.olvae 65.6-ebp.e.446 01top56p6.6.96.6 Booqqbevov 5.4.4.2056vo3 o5oo.66Tew p6qt,Bor63o Ema6.6qopae 0801 te66q5E643 vy6.6.4.6vo3l. loeaftBowe owebe633E6 1e338.63e3p BEe3ep3bs6 (not TeBzepoppo Te33RE6p.61 poqqopEase 3vq5e.6.4ey.E. Evovp36-23.4 365 35 096 Boqooqoobi vqaftolb&E, oppoqqvp-eq qqqvqqr5.66 lzeqolvvoo .66461D66or 006 opwlevWe pqMbopeep .6.6.653.46645 voftweoup ozeopoTeze pos6wwbq 0178 ovv.46.401.4.6 RE.Te&eqqae P6loboqoe.E. ozerProve6 poopb646qq.
qoaeoqopor 08L vofiBr5ela6 o65evoo.4.6q Boovopp.46.4 6ovoR6111r wea6olele5 664poovoqp !
ctggtcttcc tctgcatcct ggtgcaaatc gtcacctgca tcatctggct ctacaccgcg 2700 cctccctcca gctacaggaa ccatgagctg gaggacgagg tcatcttcat cacctgcgac 2760 gagggctcgc tcatggcgct gggcttcctc atcggctaca cctgcctcct cgccgccatc 2820 tgcttcttct tcgccttcaa gtcccgtaag ctgccggaga acttcaacga ggctaagttc 2880 atcaccttca gcatgttgat cttcttcatc gtctggatct ccttcatccc cgcctatgtc 2940 agcacctacg gcaagtttgt gtcggccgtg gaggtgattg ccatcctggc ctccagcttc 3000 gggctgctgg gctgcattta cttcaacaag tgttacatca tcctgttcaa gccgtgccgt 3060 aacaccatcg aggaggtgcg ctgcagcacg gcggcccacg ccttcaaggt ggcggcccgg 3120 gccaccctcc ggcgcagcgc cgcgtctcgc aagcgctcca gcagcctgtg cggctccacc 3180 atctcctcgc ccgcctcgtc cacctgcggg ccgggcctca ccatggagat gcagcgctgc 3240 agcacgcaga aggtcagctt cggcagcggc accgtcaccc tgtcgctcag cttcgaggag 3300 acaggccgat acgccaccct cagccgcacg gcccgcagca ggaactcggc ggatggccgc 3360 agcggcgacg acctgccatc tagacaccac gaccagggcc cgcctcagaa atgcgagccc 3420 cagcccgcca acgatgcccg atacaaggcg gcgccgacca agggcaccct agagtcgccg 3480 ggcggcagca aggagcgccc cacaactatg gaggaaacct aatccaactc ctccatcaac 3540 cccaagaaca tcctccacgg cagcaccgtc gacaactgac atcaactcct aaccggtggc 3600 tgcccaacct ctcccctctc cggcactttg cgttttgctg aagattgcag catctgcagt 3660 tccttttatc cctgattttc tgacttggat atttactagt gtgcgatgga atatcacaac 3720 ataatgagtt gcacaattag gtgagcagag ttgtgtcaaa gtatctgaac tatctgaagt 3780 atctgaacta ctttattctc tcgaattgta ttacaaacat ttgaagtatt tttagtgaca 3840 ttatgttcta acattgtcaa gataatttgt tacaacatat aaggtaccac ctgaagcagt 3900 gactgagatt gccactgtga tgacagaact gttttataac atttatcatt gaaacctgga 3960 ttgcaacagg aatataatga ctgtaacaaa aaaattgttg attatcttaa aaatgcaaat 4020 tgtaatcaga tgtgtaaaat tggtaattac ttctgtacat taaatgcata tttcttgata 4080 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcgg cccgacagca acgg 4134 <210> 2 <211> 1027 <212> PRT
<213> Squalus acanthias <400> 2 Met Ala Gin Leu His Cys Gin Leu Leu Phe Leu Gly Phe Thr Leu Leu Gin Ser Tyr Asn Val Ser Gly Tyr Gly Pro Asn Gin Arg Ala Gin Lys Lys Gly Asp Ile Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp Gin Asp Leu Lys Ser Arg Pro Glu Ala Thr Lys Cys Ile Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser Met Thr Phe Leu Pro Asn Ile Thr Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Ser Asp His Ile Pro Ser Thr Ile Ala Val Val Gly Ala Thr Gly Ser Gly Ile Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gin Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Glu Tyr Lys Ala Phe Leu Arg Thr Ile Pro Asn Asp Glu Gin Gin Ala Thr Ala Met Ala Glu Ile Ile Glu His Phe Gin Trp Asn Trp Val Gly Thr Leu Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Asp Lys Phe Arg Glu Glu Ala Val Lys Arg Asp Ile Cys Ile Asp Phe Ser Glu Met Ile Ser Gin Tyr Tyr Thr Gin Lys Gin Leu Glu Phe Ile Ala Asp Val Ile Gin Asn Ser Ser Ala Lys Val Ile Val Val Phe Ser Asn Gly Pro Asp Leu Glu Pro Leu Ile Gin Glu Ile Val Arg Arg Asn Ile Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu Ile Ala Lys Pro Glu Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe Ala Leu Arg Ala Gly Arg Ile Pro Gly Phe Asn Lys Phe Leu Lys Glu Val His Pro Ser Arg Ser Ser Asp Asn Gly Phe Val Lys Glu Phe Trp Glu Glu Thr Phe Asn Cys Tyr Phe Thr Glu Lys Thr Leu Thr Gin Leu Lys Asn Ser Lys Val Pro Ser His Gly Pro Ala Ala Gin Gly Asp Gly Ser Lys Ala Gly Asn Ser Arg Arg Thr Ala Leu Arg His Pro Cys Thr Gly Glu Glu Asn Ile Thr Ser Val Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val Tyr Val Ala Val Tyr Ser Ile Ala His Ala Leu Gin Asp Ile His Ser Cys Lys Pro Gly Thr Gly Ile Phe Ala Asn Gly Ser Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gin Val Leu Asn His Leu Leu His Leu Lys Phe Thr Asn Ser Met Gly Glu Gin Val Asp Phe Asp Asp Gin Gly Asp Leu Lys Gly Asn Tyr Thr Ile Ile Asn Trp Gin Leu Ser Ala Glu Asp Glu Ser Val Leu Phe His Glu Val Gly Asn Tyr Asn Ala Tyr Ala Lys Pro Ser Asp Arg Leu Asn Ile Asn Glu Lys Lys Ile Leu Trp Ser Gly Phe Ser Lys Val Val Pro Phe Ser Asn Cys Ser Arg Asp Cys Val Pro Gly Thr Arg Lys Gly Ile Ile Glu Gly Glu Pro Thr Cys Cys Phe Glu Cys Met Ala Cys Ala Glu Gly Glu Phe Ser Asp Glu Asn Asp Ala Ser Ala Cys Thr Lys Cys Pro Asn Asp Phe Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala Lys Glu Ile Glu Tyr Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Thr Ile Phe Ala Val Leu Gly Ile Leu Ile Thr Ser Phe Val Leu Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu Pro Arg Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Val Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Cys Ile Leu Val Gin Ile Val Thr Cys Ile Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg Asn His Glu Leu Glu Asp Glu Val Ile Phe Ile Thr Cys Asp Glu Gly Ser Leu Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Val Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu Leu Gly Cys Ile Tyr Phe Asn Lys Cys Tyr Ile Ile Leu Phe Lys Pro Cys Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val Ala Ala Arg Ala Thr Leu Arg Arg Ser Ala Ala Ser Arg Lys Arg Ser Ser Ser Leu Cys Gly Ser Thr Ile Ser Ser Pro Ala Ser Ser Thr Cys Gly Pro Gly Leu Thr Met Glu Met Gin Arg Cys Ser Thr Gin Lys Val Ser Phe Gly Ser Gly Thr Val Thr Leu Ser Leu Ser Phe Glu Glu Thr Gly Arg Tyr Ala Thr Leu Ser Arg Thr Ala Arg Ser Arg Asn Ser Ala Asp Gly Arg Ser Gly Asp Asp Leu Pro Ser Arg His His Asp Gin Gly Pro Pro Gin Lys Cys Glu Pro Gin Pro Ala Asn Asp Ala Arg Tyr Lys Ala Ala Pro Thr Lys Gly Thr Leu Glu Ser Pro Gly Gly Ser Lys Glu Arg Pro Thr Thr Met Glu Glu Thr <210> 3 <211> 594 <212> DNA
<213> Salmo salar <400> 3 cttggcatta tgctctgtgc tgggggtatt cttgacagca ttcgtgatgg gagtgtttat 60 caaatttcgc aacaccccaa ttgttaaggc cacaaacaga gagctatcct acctcctcct 120 gttctcactc atctgctgtt tctccagttc cctcatcttc attggtgaac cccaggactg 180 gacatgccgt ctacgccagc ctgcattcgg gataagtttt gttctctgca tctcctgcat 240 cctggtaaaa actaaccgag tacttctagt gttcgaagcc aagatcccca ccagtctcca 300 tcgtaagtgg tgggggctaa acttgcagtt cctgttagtg ttcctgttca catttgtgca 360 agtgatgata tgtgtggtct ggctttacaa tgctcctccg gcgagctaca ggaaccatga 420 cattgatgag ataattttca ttacatgcaa tgagggctct atgatggcgc ttggcttcct 480 aattgggtac acatgcctgc tggcagccat atrcttcttc tttgcattta aatcacgaaa 540 actgccagag aactttactg aggctaagtt catcaccttc agcatgctca tctt 594 <210> 4 <211> 197 <212> PRT
<213> Salmo salar <220>
<221> misc_feature <222> (171)..(171) <223> Xaa = Any Amino Acid <400> 4 Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Xaa Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile <210> 5 <211> 2021 <212> DNA
<213> Salmo salar <400> 5 gtgatcacaa aggtaagaaa gacagtgaaa aatctgaact accccattat ataatctgtt 60 gctatttcat atgtttctat caataataca aacactactt ctctattcct gcagatgcca 120 gtgtttgtac caagtgtccc aatgactcat ggtctaatga gaaccacaca tcttgtttcc 180 tgaaggagat agagtttctg tcttggacag agccctttgg gatcgccttg gcattatgct 240 ctgtgctggg ggtattcttg acagcattcg tgatgggagt gtttatcaaa tttcgcaaca 300 ccccaattgt taaggccaca aacagagagc tatcctacct cctcctgttc tcactcatct 360 gctgtttctc cagttccctc atcttcattg gtgaacccca ggactggaca tgccgtctac 420 gccagcctgc attcgggata agttttgttc tctgcatctc ctgcatcctg gtaaaaacta 480 accgagtact tctagtgttc gaagccaaga tccccaccag tctccatcgt aagtggtggg 540 ggctaaactt gcagttcctg ttagtgttcc tgttcacatt tgtgcaagtg atgatatgtg 600 tggtctggct ttacaatgct cctccggcga gctacaggaa ccatgacatt gatgagataa 660 ttttcattac atgcaatgag ggctctatga tggcgcttgg cttcctaatt gggtacacat 720 gcctgctggc agccatatgc ttcttctttg catttaaatc acgaaaactg ccagagaact 780 ttactgaggc taagttcatc accttcagca tgctcatctt cttcatcgtc tggatctctt 840 tcatccctgc ctacttcagc acttacggaa agtttgtgtc ggctgtggag gtcatcgcca 900 tactagcctc cagctttggc ctgctggcct gtattttctt caataaagtc tacatcatcc 960 tcttcaaacc gtccaggaac actatagagg aggttcgctg tagcactgcg gcccattctt 1020 tcaaagtggc agccaaggcc actctgagac acagctcagc ctccaggaag aggtccagca 1080 gtgtgggggg atcctgtgcc tcaactccct cctcatccat cagcctcaag accaatgaca 1140 atgactcccc atcaggtcag cagagaatcc ataagccaag agtaagcttt ggaagtggaa 1200 cagttactct gtccttgagc tttgaggagt ccagaaagaa ttctatgaag tagggaagtg 1260 tcttttggtg ggccgagagc cttgtcaaaa cctgagttgg tgttgcattc tttgttggct 1320 gggtagttgg agcagaaatt atgatattaa aagctttgat gtattcagaa tggtgacaca 1380 gcataggtgg ccaagattcc attatattac aataatctgt gttgttcatt atgaggacat 1440 ttcaaaatgc tgaaaatcat caaatacata atttactgag ttttcttgat aatcttgaga 1500 atagaatagc ctattcaagt catcgttgag cagacattaa ttaacaatga tgtaatactt 1560 tccataccta ttttctttaa caatagattc acattgttaa agttcaacta tgacctgtaa 1620 aatacatgag gtataacagg agacaataaa actatgcata tcctagcttc tgggcctgag 1680 tagcaggcag tttactctgg gcacgctttt catccaaact tccgaatgct gcccccaatc 1740 ctagtgaggt taaaggccca gtgcagtcat atcttttctc taggcacgct tttcatccaa 1800 acttccgaat gcggctatat cagtctcttt cctactgtct ttttcattag gccagtgttt 1860 aacaaccctg gtccttaagt acacacaaca gaacacattt ttgttgtagc cctggacaat 1920 cactcctcac tcagctcatt gagggcctga tgattagttg acaagttgaa tcaggtgtgc 1980 ttgtccaggg ttacaataca aatgtgtact gttgggggta c 2021 <210> 6 <211> 388 <212> PRT
<213> Salmo salar <400> 6 Tyr Lys His Tyr Phe Ser Ile Pro Ala Asp Ala Ser Val Cys Thr Lys Cys Pro Asn Asp Ser Trp Ser Asn Glu Asn His Thr Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ser Phe Lys Val Ala Ala Lys Ala Thr Leu Arg His Ser Ser Ala Ser Arg Lys Arg Ser Ser Ser Val Gly Gly Ser Cys Ala Ser Thr Pro Ser Ser Ser Ile Ser Leu Lys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gin Gin Arg Ile His Lys Pro Arg Val Ser Phe Gly Ser Gly Thr Val Thr Leu Ser Leu Ser Phe Glu Glu Ser Arg Lys Asn Ser Met Lys <210> 7 <211> 3941 <212> DNA
<213> Salmo salar <400> 7 ttccaacagc atatttttgt tgtatttgct ttggtttgtc tgaaatcaag cattatcaag 60 atcaagaaca gcatgagtca gaaacaaggc gacagccaga gtcactggag gggacaagac 120 tgaggttaac tctgaagtct aatgtgctga gaggacaagg ccctcctgag agctgaacga 180 tgagatttta cctgtattac ctggtgcttt tgggcttcag ttctgtcatc tccacctatg 240 ggcctcatca gagagcacag aagactgggg atattctgct gggcgggctg tttccaatgc 300 actttggtgt tacctccaaa gaccaagacc tggcagcgcg gccagaatcc acagagtgtg 360 ttaggtacaa tttccgggga ttccgttggc ttcaggccat gatttttgca atagaggaga 420 tcaacaacag cagtactctc ctgcccaaca tcacactggg ctacaggatc tttgacacct 480 gcaacaccgt gtccaaggcc ctggaggcta ccctcagttt cgtagcacag aataagattg 540 actctctgaa cttggatgaa ttctgtaact gcactgatca catcccatcg actatagcag 600 tggtgggggc ttctgggtca gcggtctcca ctgctgttgc caatctgttg ggccttttct 660 acatcccaca gatcagctat gcctcttcca gtcgcctact aagcaacaag aaccagttca 720 aatccttcat gaggaccatt cccacagatg agcaccaggc cactgccatg gcagatatca 780 tcgactactt ccaatggaat tgggtcattg cagttgcgtc tgatgatgag tatggacgtc 840 cggggattga aaaatttgag aaagagatgg aagaacgaga catttgtatc catctgagtg 900 , , agctgatctc tcagtacttt gaggagtggc agatccaagg attggttgac cgtattgaga actcctcagc taaagttata gtcgttttcg ccagtgggcc tgacattgag cctcttatta 1020 aagagatggt cagacggaac atcaccgacc gcatctggtt ggccagcgag gcttgggcaa 1080 ccacctccct catcgccaaa ccagagtacc ttgatgttgt agttgggacc attggctttg 1140 ctctcagagc aggcgaaata cctggcttca aggacttctt acaagaggtc acaccaaaga 1200 aatccagcca caatgaattt gtcagggagt tttgggagga gacttttaac tgctatctgg 1260 aagacagcca gagactgaga gacagtgaga atgggagcac cagtttcaga ccattgtgta 1320 ctggcgagga ggacattatg ggtgcagaga ccccatatct ggattacact catcttcgta 1380 tttcctataa tgtgtatgtt gcagttcact ccattgcaca ggccctacag gacattctca 1440 cctgcattcc tggacggggt cttttttcca acaactcatg tgcagatata aagaaaatag 1500 aagcatggca ggttctcaag cagctcagac atttaaactt ctcaaacagt atgggagaaa 1560 aggtacattt tgatgagaat gctgatccgt caggaaacta caccattatc aattggcacc 1620 ggtctcctga ggatggttct gttgtgtttg aagaggtcgg tttctacaac atgcgagcta 1680 agagaggagt acaacttttc attgataaca caaagattct atggaatgga tataatactg 1740 aggttccatt ctctaactgt agtgaagatt gtgaaccagg caccagaaag gggatcatag 1800 aaagcatgcc aacgtgttgc tttgaatgta cagaatgctc agaaggagag tatagtgatc 1860 acaaagatgc cagtgtttgt accaagtgtc ccaatgactc atggtctaat gagaaccaca 1920 catcttgttt cctgaaggag atagagtttc tgtcttggac agagcccttt gggatcgcct 1980 tggcattatg ctctgtgctg ggggtattct tgacagcatt cgtgatggga gtgtttatca 2040 aatttcgcaa caccccaatt gttaaggcca caaacagaga gctatcctac ctcctcctgt 2100 tctcactcat ctgctgtttc tccagttccc tcatcttcat tggtgaaccc caggactgga 2160 catgccgtct acgccagcct gcattcggga taagttttgt tctctgcatc tcctgcatcc 2220 tggtaaaaac taaccgagta cttctagtgt tcgaagccaa gatccccacc agtctccatc 2280 gtaagtggtg ggggctaaac ttgcagttcc tgttagtgtt cctgttcaca tttgtgcaag 2340 tgatgatatg tgtggtctgg ctttacaatg ctcctccggc gagctacagg aaccatgaca 2400 ttgatgagat aattttcatt acatgcaatg agggctctat gatggcgctt ggcttcctaa 2460 ttgggtacac atgcctgctg gcagccatat gcttcttctt tgcatttaaa tcacgaaaac 2520 tgccagagaa ctttactgag gctaagttca tcaccttcag catgctcatc ttcttcatcg 2580 tctggatctc tttcatccct gcctacttca gcacttacgg aaagtttgtg tcggctgtgg 2640 aggtcatcgc catactagcc tccagctttg gcctgctggc ctgtattttc ttcaataaag 2700 tctacatcat cctcttcaaa ccgtccagga acactataga ggaggttcgc tgtagcactg 2760 cggcccattc tttcaaagtg gcagccaagg ccactctgag acacagctca gcctccagga 2820 agaggtccag cagtgtgggg ggatcctgtg cctcaactcc ctcctcatcc atcagcctca 2880 agaccaatga caatgactcc ccatcaggtc agcagagaat ccataagcca agagtaagct 2940 ttggaagtgg aacagttact ctgtccttga gctttgagga gtccagaaag aattctatga 3000 agtagggaag tgtcttttgg tgggccgaga gccttgtcaa aacctgagtt ggtgttgcat 3060 tctttgttgg ctgggtagtt ggagcagaaa ttatgatatt aaaagctttg atgtattcag 3120 aatggtgaca cagcataggt ggccaagatt ccattatatt acaataatct gtgttgttca 3180 ttatgaggac atttcaaaat gctgaaaatc atcaaataca taatttactg agttttcttg 3240 ataatcttga gaatagaata gcctattcaa gtcatcgttg agcagacatt aattaacaat 3300 gatgtaatac tttccatacc tattttcttt aacaatagat tcacattgtt aaagttcaac 3360 tatgacctgt aaaatacatg aggtataaca ggagacaata aaactatgca tatcctagct 3420 tctgggcctg agtagcaggc agtttactct gggcacgctt ttcatccaaa cttccgaatg 3480 ctgcccccaa tcctagtgag gttaaaggcc cagtgcagtc atatcttttc tctaggcacg 3540 cttttcatcc aaacttccga atgcggctat atcagtctct ttcctactgt ctttttcatt 3600 aggccagtgt ttaacaaccc tggtccttaa gtacacacaa cagagcacat ttttgttgtg 3660 gccctggaca atcactcctc actcagctca ttgagggcct gatgattagt tgacaagttg 3720 agtcgggtgt gcttgtccgg ggttgcaata cagatgtgta ctgttggggg tactcgagga 3780 ccaggattgg gaaacattac attaggacta ctgtaggttc ttcaatatgg tgtcatacgg 3840 tcatatggtg tcatatggtg tctggttgtt ttctgcatat gtgtatttca ccaagttact 3900 gcacatgtta gacctataca ctggaataaa catttttttt c 3941 <210> 8 <211> 941 <212> PRT
<213> Salmo salar <400> 8 Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gln Lys Thr Gly Asp Ile Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp Gin Asp Leu Ala Ala Arg Pro Glu Ser Thr Glu Cys Val Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser Ser Thr Leu Leu Pro Asn Ile Thr Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Thr Asp His Ile Pro Ser Thr Ile Ala Val Val Gly Ala Ser Gly Ser Ala Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gin Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gin Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His Gin Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gin Trp Asn Trp Val Ile Ala Val Ala Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu Lys Phe Glu Lys Glu Met Glu Glu Arg Asp Ile Cys Ile His Leu Ser Glu Leu Ile Ser Gin Tyr Phe Glu Glu Trp Gin Ile Gin Gly Leu Val Asp Arg Ile Glu Asn Ser Ser Ala Lys Val Ile Val Val Phe Ala Ser Gly Pro Asp Ile Glu Pro Leu Ile Lys Glu Met Val Arg Arg Asn Ile Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp Ala Thr Thr Ser Leu Ile Ala Lys Pro Glu Tyr Leu Asp Val Val Val Gly Thr Ile Gly Phe Ala Leu Arg Ala Gly Glu Ile Pro Gly Phe Lys Asp Phe Leu Gin Glu Val Thr Pro Lys Lys Ser Ser His Asn Glu Phe Val Arg Glu Phe Trp Glu Glu Thr Phe Asn Cys Tyr Leu Glu Asp Ser Gin Arg Leu Arg Asp Ser Glu Asn Gly Ser Thr Ser Phe Arg Pro Leu Cys Thr Gly Glu Glu Asp Ile Met Gly Ala Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val Tyr Val Ala Val His Ser Ile Ala Gin Ala Leu Gin Asp Ile Leu Thr Cys Ile Pro Gly Arg Gly Leu Phe Ser Asn Asn Ser Cys Ala Asp Ile Lys Lys Ile Glu Ala Trp Gin Val Leu Lys Gin Leu Arg His Leu Asn Phe Ser Asn Ser Met Gly Glu Lys Val His Phe Asp Glu Asn Ala Asp Pro Ser Gly Asn Tyr Thr Ile Ile Asn Trp His Arg Ser Pro Glu Asp Gly Ser Val Val Phe Glu Glu Val Gly Phe Tyr Asn Met Arg Ala Lys Arg Gly Val Gin Leu Phe Ile Asp Asn Thr Lys Ile Leu Trp Asn Gly Tyr Asn Thr Glu Val Pro Phe Ser Asn Cys Ser Glu Asp Cys Glu Pro Gly Thr Arg Lys Gly Ile Ile Glu Ser Met Pro Thr Cys Cys Phe Glu Cys Thr Glu Cys Ser Glu Gly Glu Tyr Ser Asp His Lys Asp Ala Ser Val Cys Thr Lys Cys Pro Asn Asp Ser Trp Ser Asn Glu Asn His Thr Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ser Phe Lys Val Ala Ala Lys Ala Thr Leu Arg His Ser Ser Ala Ser Arg Lys Arg Ser Ser Ser Val Gly Gly Ser Cys Ala Ser Thr Pro Ser Ser Ser Ile Ser Leu Lys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gin Gin Arg Ile His Lys Pro Arg Val Ser Phe Gly Ser Gly Thr Val Thr Leu Ser Leu Ser Phe Glu Glu Ser Arg Lys Asn Ser Met Lys <210> 9 <211> 4031 <212> DNA
<213> Salmo salar <400> 9 gttccaacag catatttttg ttgtatttgc tttggtttgt ctgaaatcaa gcattatcaa 60 ggattgagca agacaactga gttgtcagac taagaatata cacatttcca gttctctctt 120 taatggactt ctcacactga tgttcttcag atcaagaaca gcatgagtca gaaacaaggc 180 gacagccaga gtcactggag gggacaagac tgaggttaac tctgaagtct aatgtgctga 240 gaggacaagg ccctcctgag agctgaacga tgagatttta cctgtattac ctggtgcttt 300 tgggcttcag ttctgtcatc tccacctatg ggcctcatca gagagcacag aagactgggg 360 atattctgct gggcgggctg tttccaatgc actttggtgt tacctccaaa gaccaagacc 420 tggcagcgcg gccagaatcc acagagtgtg ttaggtacaa tttccgggga ttccgttggc 480 ttcaggccat gatttttgca atagaggaga tcaacaacag cagtactctc ctgcccaaca 540 tcacactggg ctacaggatc tttgacacct gcaacaccgt gtccaaggcc ctggaggcta 600 ccctcagttt cgtagcacag aataagattg actctctgaa cttggatgaa ttctgtaact 660 gcactgatca catcccatcg actatagcag tggtgggggc ttctgggtca gcggtctcca 720 ctgctgttgc caatctgttg ggccttttct acatcccaca gatcagctat gcctcttcca 780 gtcgcctact aagcaacaag aaccagttca aatccttcat gaggaccatt cccacagatg 840 agcaccaggc cactgccatg gcagatatca tcgactactt ccaatggaat tgggtcattg 900 cagttgcgtc tgatgatgag tatggacgtc cggggattga aaaatttgag aaagagatgg 960 aagaacgaga catttgtatc catctgagtg agctgatctc tcagtacttt gaggagtggc 1020 agatccaagg attggttggc cgtattgaga actcctcagc taaagttata gtcgttttcg 1080 ccagtgggcc tgacattgag cctcttatta aagagatggt cagacggaac atcaccgacc 1140 gcatctggtt ggccagcgag gcttgggcaa ccacctccct catcgccaaa ccagagtacc 1200 ttgatgttgt agttgggacc attggctttg ctctcagagc aggcgaaata cctggcttca 1260 aggacttctt acaagaggtc acaccaaaga aatccagcca caatgaattt gtcagggagt 1320 tttgggagga gacttttaac tgctatctgg aagacagcca gagactgaga gacagtgaga 1380 atgggagcac cagtttcaga ccattgtgta ctggcgagga ggacattatg ggtgcagaga 1440 ccccatatct ggattacact catcttcgta tttcctataa tgtgtatgtt gcagttcact 1500 ccattgcaca ggccctacag gacattctca cctgcattcc tggacggggt cttttttcca 1560 acaactcatg tgcagatata aagaaaatag aagcatggca ggttctcaag cagctcagac 1620 atttaaactt ctcaaacagt atgggagaaa aggtacattt tgatgagaat gctgatccgt 1680 caggaaacta caccattatc aattggcacc ggtctcctga ggatggttct gttgtgtttg 1740 aagaggtcgg tttctacaac atgcgagcta agagaggagt acaacttttc attgataaca 1800 caaagattct atggaatgga tataatactg aggttccatt ctctaactgt agtgaagatt 1860 gtgaaccagg caccagaaag gggatcatag aaagcatgcc aacgtgttgc tttgaatgta 1920 cagaatgctc agaaggagag tatagtgatc acaaagatgc cagtgtttgt accaagtgtc 1980 ccaatgactc atggtctaat gagaaccaca catcttgttt cctgaaggag atagagtttc 2040 tgtcttggac agagcccttt gggatcgcct tggcattatg ctctgtgctg ggggtattct 2100 tgacagcatt cgtgatggga gtgtttatca aatttcgcaa caccccaatt gttaaggcca 2160 caaacagaga gctatcctac ctcctcctgt tctcactcat ctgctgtttc tccagttccc 2220 tcatcttcat tggtgaaccc caggactgga catgccgtct acgccagcct gcattcggga 2280 taagttttgt tctctgcatc tcctgcatcc tggtaaaaac taaccgagta cttctagtgt 2340 tcgaagccaa gatccccacc agtctccatc gtaagtggtg ggggctaaac ttgcagttcc 2400 tgttagtgtt cctgttcaca tttgtgcaag tgatgatatg tgtggtctgg ctttacaatg 2460 ctcctccggc gagctacagg aaccatgaca ttgatgagat aattttcatt acatgcaatg 2520 agggctctat gatggcgctt ggcttcctaa ttgggtacac atgcctgctg gcagccatat 2580 gcttcttctt tgcatttaaa tcacgaaaac tgccagagaa ctttactgag gctaagttca 2640 , tcaccttcag catgctcatc ttcttcatcg tctggatctc tttcatccct gcctacttca 2700 gcacttacgg aaagtttgtg tcggctgtgg aggtcatcgc catactagcc tccagctttg 2760 gcctgctggc ctgtattttc ttcaataaag tctacatcat cctcttcaaa ccgtccagga 2820 acactataga ggaggttcgc tgtagcactg cggcccattc tttcaaagtg gcagccaagg 2880 ccactctgag acacagctca gcctccagga agaggtccag cagtgtgggg ggatcctgtg 2940 cctcaactcc ctcctcatcc atcagcctca agaccaatga caatgactcc ccatcaggtc 3000 agcagagaat ccataagcca agagtaagct ttggaagtgg aacagttact ctgtccttga 3060 gctttgagga gtccagaaag aattctatga agtagggaag tgtcttttgg tgggccgaga 3120 gccttgtcaa aacctgagtt ggtgttgcat tctttgttgg ctgggtagtt ggagcagaaa 3180 ttatgatatt aaaagctttg atgtattcag aatggtgaca cagcataggt ggccaagatt 3240 ccattatatt acaataatct gtgttgttca ttatgaggac atttcaaaat gctgaaaatc 3300 atcaaataca taatttactg agttttcttg ataatcttga gaatagaata gcctattcaa 3360 gtcatcgttg agcagacatt aattaacaat gatgtaatac tttccatacc tattttcttt 3420 aacaatagat tcacattgtt aaagttcaac tatgacctgt aaaatacatg aggtataaca 3480 ggagacaata aaactatgca tatcctagct tctgggcctg agtagcaggc agtttactct 3540 gggcacgctt ttcatccaaa cttccgaatg ctgcccccaa tcctagtgag gttaaaggcc 3600 cagtgcagtc atatcttttc tctaggcacg cttttcatcc aaacttccga atgcggctat 3660 atcagtctct ttcctactgt ctttttcatt aggccagtgt ttaacaaccc tggtccttaa 3720 gtacacacaa cagagcacat ttttgttgta gccctggaca atcactcctc actcagctca 3780 ttgagggcct gatgattagt tgacaagttg agtcgggtgt gcttgtccag ggttacgata 3840 cagatgtgta ctgttggggg tgctcgagga ccaggattgg gaaacattac attaggacta 3900 ctgtaggttc ttcaatatgg tgtcatacgg tcatatggtg tcatatggtg tctggttgtt 3960 ttctgcatat gtgtatttca ccaagttact gcacatgtta gacctataca ctggaataaa 4020 catttttttt c <210> 10 <211> 941 <212> PRT
<213> Salmo salar <400> 10 Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gin Lys Thr Gly Asp Ile Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp Gin Asp Leu Ala Ala Arg Pro Glu Ser Thr Glu Cys Val Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser Ser Thr Leu Leu Pro Asn Ile Thr Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Thr Asp His Ile Pro Ser Thr Ile Ala Val Val Gly Ala Ser Gly Ser Ala Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gin Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gin Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His Gin Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gin Trp Asn Trp Val Ile Ala Val Ala Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu Lys Phe Glu Lys Glu Met Glu Glu Arg Asp Ile Cys Ile His Leu Ser Glu Leu Ile Ser Gin Tyr Phe Glu Glu Trp Gin Ile Gin Gly Leu Val Gly Arg Ile Glu Asn Ser Ser Ala Lys Val Ile Val Val Phe Ala Ser Gly Pro Asp Ile Glu Pro Leu Ile Lys Glu Met Val Arg Arg Asn Ile Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp Ala Thr Thr Ser Leu Ile Ala Lys Pro Glu Tyr Leu Asp Val Val Val Gly Thr Ile Gly Phe Ala Leu Arg Ala Gly Glu Ile Pro Gly Phe Lys Asp Phe Leu Gin Glu Val Thr Pro Lys Lys Ser Ser His Asn Glu Phe Val Arg Glu Phe Trp Glu Glu Thr Phe Asn Cys Tyr Leu Glu Asp Ser Gin Arg Leu Arg Asp Ser Glu Asn Gly Ser Thr Ser Phe Arg Pro Leu Cys Thr Gly Glu Glu Asp Ile Met Gly Ala Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val Tyr Val Ala Val His Ser Ile Ala Gin Ala Leu Gin Asp Ile Leu Thr Cys Ile Pro Gly Arg Gly Leu Phe Ser Asn Asn Ser Cys Ala Asp Ile Lys Lys Ile Glu Ala Trp Gin Val Leu Lys Gin Leu Arg His Leu Asn Phe Ser Asn Ser Met Gly Glu Lys Val His Phe Asp Glu Asn Ala Asp Pro Ser Gly Asn Tyr Thr Ile Ile Asn Trp His Arg Ser Pro Glu Asp Gly Ser Val Val Phe Glu Glu Val Gly Phe Tyr Asn Met Arg Ala Lys Arg Gly Val Gin Leu Phe Ile Asp Asn Thr Lys Ile Leu Trp Asn Gly Tyr Asn Thr Glu Val Pro Phe Ser Asn Cys Ser Glu Asp Cys Glu Pro Gly Thr Arg Lys Gly Ile Ile Glu Ser Met Pro Thr Cys Cys Phe Glu Cys Thr Glu Cys Ser Glu Gly Glu Tyr Ser Asp His Lys Asp Ala Ser Val Cys Thr Lys Cys Pro Asn Asp Ser Trp Ser Asn Glu Asn His Thr Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe lie Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ser Phe Lys Val Ala Ala Lys Ala Thr Leu Arg His Ser Ser Ala Ser Arg Lys Arg Ser Ser Ser Val Gly Gly Ser Cys Ala Ser Thr Pro Ser Ser Ser Ile Ser Leu Lys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gin Gin Arg Ile His Lys Pro Arg Val Ser Phe Gly Ser Gly Thr Val Thr Leu Ser Leu Ser Phe Glu Glu Ser Arg Lys Asn Ser Met Lys <210> 11 <211> 3824 <212> DNA
<213> Salmo salar <400> 11 gttccaacag catatttttg ttgtatttgc tttggtttgt ctgaaatcaa gcattatcaa 60 gatcaagaac agcatgagtc agaaacaagg cgacagccag agtcactgga ggggacaaga 120 ctgaggttaa ctctgaagtc taatgtgctg agaggacaag gccctcctga gagctgaacg 180 atgagatttt acctgtatta cctggtgctt ttgggcttca gttctgtcat ctccacctat 240 gggcctcatc agagagcaca gaagactggg gatattctgc tgggcgggct gtttccaatg 300 cactttggtg ttacctccaa agaccaagac ctggcagcgc ggccagaatc cacagagtgt 360 gttaggtaca atttccgggg attccgttgg cttcaggcca tgatttttgc aatagaggag 420 atcaacaaca gcagtactct cctgcccaac atcacactgg gctacaggat ctttgacacc 480 tgcaacaccg tgtccaaggc cctggaggct accctcagtt tcgtagcaca gaataagatt 540 gactctctga acttggatga attctgtaac tgcactgatc acatcccatc gactatagca 600 gtggtggggg cttctgggtc agcggtctcc actgctgttg ccaatctgtt gggccttttc 660 tacatcccac agatcagcta tgcctcttcc agtcgcctac taagcaacaa gaaccagttc 720 aaatccttca tgaggaccat tcccacagat gagcaccagg ccactgccat ggcagatatc 780 atcgactact tccaatggaa ttgggtcatt gcagttgcgt ctgatgatga gtatggacgt 840 ccggggattg aaaaatttga gaaagagatg gaagaacgag acatttgtat ccatctgagt 900 gagctgatct ctcagtactt tgaggagtgg cagatccaag gattggttga ccgtattgag 960 aactcctcag ctaaagttat agtcgttttc gccagtgggc ctgacattga gcctcttatt 1020 aaagagatgg tcagacggaa catcaccgac cgcatctggt tggccagcga ggcttgggca 1080 accacctccc tcatcgccaa accagagtac cttgatgttg tagttgggac cattggcttt 1140 gctctcagag caggcgaaat acctggcttc aaggacttct tacaagaggt cacaccaaag 1200 aaatccagcc acaatgaatt tgtcagggag ttttgggagg agacttttaa ctgctatctg 1260 gaagacagcc agagactgag agacagtgag aatgggagca ccagtttcag accattgtgt 1320 actggcgagg aggacattat gggtgcagag accccatatc tggattacac tcatcttcgt 1380 atttcctata atgtgtatgt tgcagttcac tccattgcac aggccctaca ggacattctc 1440 acctgcattc ctggacgggg ttttttttcc aacaactcat gtgcagatat aaagaaaata 1500 gaagcatggc aggttctcaa gcagctcaga catttaaact tctcaaacag tatgggagaa 1560 aaggtacatt ttgatgagaa tgctgatccg tcaggaaact acaccattat caattggcac 1620 cggtctcctg aggatggttc tgttgtgttt gaagaggtcg gtttctacaa catgcgagct 1680 aagagaggag tacaactttt cattgataac acaaagattc tatggaatgg atataatact 1740 gaggttccat tctctaactg tagtgaagat tgtgaaccag gcaccagaaa ggggatcata 1800 gaaagcatgc caacgtgttg ctttgaatgt acagaatgct cagaaggaga gtatagtgat 1860 cacaaagatg ccagtgtttg taccaagtgt cccaatgact catggtctaa tgagaaccac 1920 acatcttgtt tcctgaagga gatagagttt ctgtcttgga cagagccctt tgggatcgcc 1980 ttggcattat gctctgtgct gggggtattc ttgacagcat tcgtgatggg agtgtttatc 2040 aaatttcgca acaccccaat tgttaaggcc acaaacagag agctatccta cctcctcctg 2100 ttctcactca tctgctgttt ctccagttcc ctcatcttca ttggtgaacc ccaggactgg 2160 acatgccgtc tacgccagcc tgcattcggg ataagttttg ttctctgcat ctcctgcatc 2220 ctggtaaaaa ctaaccgagt acttctagtg ttcgaagcca agatccccac cagtctccat 2280 cgtaagtggt gggggctaaa cttgcagttc ctgttagtgt tcctgttcac atttgtgcaa 2340 gtgatgatat gtgtggtctg gctttacaat gctcctccgg cgagctacag gaaccatgac 2400 attgatgaga taattttcat tacatgcaat gagggctcta tgatggcgct tggcttccta 2460 attgggtaca catgcctgct ggcagccata tgcttcttct ttgcatttaa atcacgaaaa 2520 ctgccagaga actttactga ggctaagttc atcaccttca gcatgctcat cttcttcatc 2580 gtctggatct ctttcatccc tgcctacttc agcacttacg gaaagtttgt gtcggctgtg 2640 gaggtcatcg ccatactagc ctccagcttt ggcctgctgg cctgtatttt cttcaataaa 2700 gtctacatca tccatcagcc tcaagaccaa tgacaatgac tccccatcag gtcagcagag 2760 aatccataag ccaagagtaa gctttggaag tggaacagtt actctgtcct tgagctttga 2820 ggagtccaga aagaattcta tgaagtaggg aagtgtcttt tggtgggccg agagccttgt 2880 caaaacctga gttggtgttg cattctttgt tggctgggta gttggagcag aaattatgat 2940 attaaaagct ttgatgtatt cagaatggtg acacagcata ggtggccaag attccattat 3000 attacaataa tctgtgttgt tcattatgag gacatttcaa aatgctgaaa atcatcaaat 3060 acataattta ctgagttttc ttgataatct tgagaataga atagcctatt caagtcatcg 3120 ttgagcagac attaattaac aatgatgtaa tactttccat acctattttc tttaacaata 3180 gattcacatt gttaaagttc aactatgacc tgtaaaatac atgaggtata acaggagaca 3240 ataaaactat gcatatccta gcttctgggc ctgagtagca ggcagtttac tctgggcacg 3300 cttttcatcc aaacttccga atgctgcccc caatcctagt gaggttaaag gcccagtgca 3360 gtcatatctt ttctctaggc acgcttttca tccaaacttc cgaatgcggc tatatcagtc 3420 tctttcctac tgtctttttc attaggccag tgtttaacaa ccctggtcct tgagtacaca 3480 caacagggca catttttgtt gtagccctgg acaatcactc ctcactcagc tcattgaggg 3540 cctgatgatt agttgacaag ttgggtcagg tgtgcttgtc cagggttaca atacagatgt 3600 gtgctgttgg gggtactcga ggaccaggat tgggaaacat tacattagga ctactgtagg 3660 ttcttcaata tggtgtcata cggtcatatg gtgtcatatg gtgtctggtt gttttctgca 3720 tatgtgtatt tcaccaagtt actgcacatg ttagacctat acactggaat aaacattttt 3780 tttcacaatg catccaatga caataaaatc accatatgcc aatg 3824 <210> 12 <211> 850 <212> PRT
<213> Salmo salar <400> 12 Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gin Lys Thr Gly Asp Ile Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp Gin Asp Leu Ala Ala Arg Pro Glu Ser Thr Glu Cys Val Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser Ser Thr Leu Leu Pro Asn Ile Thr Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Thr Asp His Ile Pro Ser Thr Ile Ala Val Val Gly Ala Ser Gly Ser Ala Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gin Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gin Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His Gin Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gin Trp Asn Trp Val Ile Ala Val Ala Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu Lys Phe Glu Lys Glu Met Glu Glu Arg Asp Ile Cys Ile His Leu Ser Glu Leu Ile Ser Gin Tyr Phe Glu Glu Trp Gin Ile Gin Gly Leu Val Asp Arg Ile Glu Asn Ser Ser Ala Lys Val Ile Val Val Phe Ala Ser Gly Pro Asp Ile Glu Pro Leu Ile Lys Glu Met Val Arg Arg Asn Ile Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp Ala Thr Thr Ser Leu Ile Ala Lys Pro Glu Tyr Leu Asp Val Val Val Gly Thr Ile Gly Phe Ala Leu Arg Ala Gly Glu Ile Pro Gly Phe Lys Asp Phe Leu Gln Glu Val Thr Pro Lys Lys Ser Ser His Asn Glu Phe Val Arg Glu Phe Trp Glu Glu Thr Phe Asn Cys Tyr Leu Glu Asp Ser Gln Arg Leu Arg Asp Ser Glu Asn Gly Ser Thr Ser Phe Arg Pro Leu Cys Thr Gly Glu Glu Asp Ile Met Gly Ala Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val Tyr Val Ala Val His Ser Ile Ala Gln Ala Leu Gln Asp Ile Leu Thr Cys Ile Pro Gly Arg Gly Phe Phe Ser Asn Asn Ser Cys Ala Asp Ile Lys Lys Ile Glu Ala Trp Gln Val Leu Lys Gln Leu Arg His Leu Asn Phe Ser Asn Ser Met Gly Glu Lys Val His Phe Asp Glu Asn Ala Asp Pro Ser Gly Asn Tyr Thr Ile Ile Asn Trp His Arg Ser Pro Glu Asp Gly Ser Val Val Phe Glu Glu Val Gly Phe Tyr Asn Met Arg Ala Lys Arg Gly Val Gln Leu Phe Ile Asp Asn Thr Lys Ile Leu Trp Asn Gly Tyr Asn Thr Glu Val Pro Phe Ser Asn Cys Ser Glu Asp Cys Glu Pro Gly Thr Arg Lys Gly Ile Ile Glu Ser Met Pro Thr Cys Cys Phe Glu Cys Thr Glu Cys Ser Glu Gly Glu Tyr Ser Asp His Lys Asp Ala Ser Val Cys Thr Lys Cys Pro Asn Asp Ser Trp Ser Asn Glu Asn His Thr Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gln Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr Ile Ile His Gin Pro Gln Asp Gin <210> 13 <211> 3988 <212> DNA
<213> Salmo salar <400> 13 gttccaacag catatttttg ttgtatttgc tttggtttgt ctgaaatcaa gcattatcaa 60 gatcaagaac agcatgagtc agaaacaagg cgacagccag agtcactgga ggggacaaga 120 ctgaggttaa ctctgaagtc taatgtgctg agaggacaag gccctcctga gagctgaacg 180 atgagatttt acctgtatta cctggtgctt ttgggcttca gttctgtcat ctccacctat 240 gggcctcatc agagagcaca gaagactggg gatattctgc tgggcgggct gtttccaatg 300 cactttggtg ttacctccaa agaccaagac ctggcagcgc ggccagaatc cacagagtgt 360 gttaggtaca atttccgggg attccgttgg cttcaggcca tgatttttgc aatagaggag 420 atcaacaaca gcagtactct cctgcccaac atcacactgg gctacaggat ctttgacacc 480 tgcaacaccg tgtccaaggc cctggaggct accctcagtt tcgtagcaca gaataagatt 540 gactctctga acttggatga attctgtaac tgcactgatc acatcccatc gactatagca 600 gtggtggggg cttctgggtc agcggtctcc actgctgttg ccaatctgtt gggccttttc 660 tacatcccac agatcagcta tgcctcttcc agtcgcctac taagcaacaa gaaccagttc 720 aaatccttca tgaggaccat tcccacagat gagcaccagg ccactgccat ggcagatatc 780 atcgactact tccaatggaa ttgggtcatt gcagttgcgt ctgatgatga gtatggacgt 840 ccggggattg aaaaatttga gaaagagatg gaagaacgag acatttgtat ccatctgagt 900 gagctgatct ctcagtactt tgaggagtgg cagatccaag gattggttga ccgtattgag 960 aactcctcag ctaaagttat agtcgttttc gccagtgggc ctgacattga gcctcttatt 1020 aaagagatgg tcagacggaa catcaccgac cgcatctggt tggccagcga ggcttgggca 1080 accacctccc tcatcgccaa accagagtac cttgatgttg tagttgggac cattggcttt 1140 gctctcagag caggcgaaat acctggcttc aaggacttct tacaagaggt cacaccaaag 1200 aaatccagcc acaatgaatt tgtcagggag ttttgggagg agacttttaa ctgctatctg 1260 gaagacagcc agagactgag agacagtgag aatgggagca ccagtttcag accattgtgt 1320 actggcgagg aggacattat gggtgcagag accccatatc tggattacac tcatcttcgt 1380 atttcctata atgtgtatgt tgcagttcac tccattgcac aggccctaca ggacattctc 1440 acctgcattc ctggacgggg tcttttttcc aacaactcat gtgcagatat aaagaaaata 1500 gaagcatggc aggttctcaa gcagctcaga catttaaact tctcaaacag tatgggagaa 1560 aaggtacatt ttgatgagaa tgctgatccg tcaggaaact acaccattat caattggcac 1620 cggtctcctg aggatggttc tgttgtgttt gaagaggtcg gtttctacaa catgcgagct 1680 aagagaggag tacaactttt cattgataac acaaagattc tatggaatgg atataatact 1740 gaggttccat tctctaactg tagtgaagat tgtgaaccag gcaccagaaa ggggatcata 1800 gaaagcatgc caacgtgttg ctttgaatgt acagaatgct cagaaggaga gtatagtgat 1860 cacaaagatg ccagtgtttg taccaagtgt cccaatgact catggtctaa tgagaaccac 1920 acatcttgtt tcctgaagga gatagagttt ctgtcttgga cagagccctt tgggatcgcc 1980 ttggcattat gctctgtgct gggggtattc ttgacagcat tcgtgatggg agtgtttatc 2040 aaatttcgca acaccccaat tgttaaggcc acaaacagag agctatccta cctcctcctg 2100 ttctcactca tctgctgttt ctccagttcc ctcatcttca ttggtgaacc ccaggactgg 2160 acatgccgtc tacgccagcc tgcattcggg ataagttttg ttctctgcat ctcctgcatc 2220 ctggtaaaaa ctaaccgagt acttctagtg ttcgaagcca agatccccac cagtctccat 2280 cgtaagtggt gggggctaaa cttgcagttc ctgttagtgt tcctgttcac atttgtgcaa 2340 gtgatgatat gtgtggtctg gctttacaat gctcctccgg cgagctacag gaaccatgac 2400 attgatgaga taattttcat tacatgcaat gagggctcta tgatggcgct tggcttccta 2460 attgggtaca catgcctgct ggcagccata tgcttcttct ttgcatttaa atcacgaaaa 2520 ctgccagaga actttactga ggctaagttc atcaccttca gcatgctcat cttcttcatc 2580 gtctggatct ctttcatccc tgcctacttc agcacttacg gaaagtttgt gtcggctgtg 2640 gaggtcatcg ccatactagc ctccagcttt ggcctgctgg cctgtatttt cttcaataaa 2700 gtctacatca tcctcttcaa accgtccagg aacactatag aggaggttcg ctgtagcact 2760 gcggcccatt ctttcaaagt ggcagccaag gccactctga gacacagctc agcctccagg 2820 aagaggtcca gcagtgtggg gggatcctgt gcctcaactc cctcctcatc catcagcctc 2880 aagaccaatg acaatgactc cccatcaggt cagcagagaa tccataagcc aagagtaagc 2940 tttggaagtg gaacagttac tctgtccttg agctttgagg agtccagaaa gaattctatg 3000 aagtagggaa gtgtcttttg gtgggccgag agccttgtca aaacctgagt tggtgttgca 3060 ttctttgttg gctgggtagt tggagcagaa attatgatat taaaagcttt gatgtattca 3120 gaatggtgac acagcatagg tggccaagat tccattatat tacaataatc tgtgttgttc 3180 attatgagga catttcaaaa tgctgaaaat catcaaatac ataatttact gagttttctt 3240 gataatcttg agaatagaat agcctattca agtcatcgtt gagcagacat taattaacaa 3300 tgatgtaata ctttccatac ctattttctt taacaataga ttcacattgt taaagttcaa 3360 ctatgacctg taaaatacat gaggtataac aggagacaat aaaactatgc atatcctagc 3420 ttctgggcct gagtagcagg cagtttactc tgggcacgct tttcatccaa acttccgaat 3480 gctgccccca atcctagtga ggttaaaggc ccagtgcagt catatctttt ctctaggcac 3540 gcttttcatc caaacttccg aatgcggcta tatcagtctc tttcctactg tctttttcat 3600 taggccagtg tttaacaacc ctggtcctta agtgcacaca acagggcaca tttttgttgt 3660 ggccctggac aatcactcct cactcagctc attgagggcc tgatgattag ttgacaagtt 3720 gagtcaggtg tgcttgtccg gggttgcaat acagatgtgt actgttgggg gtgctcgagg 3780 accaggattg ggaaacatta cattaggact actgtaggtt cttcaatatg gtgtcatacg 3840 gtcatatggt gtcatatggt gtctggttgt tttctgcata tgtgtatttc accaagttac 3900 tgcacatgtt agacctatac actggaataa acattttttt tcacaatgca tccaatgaca 3960 ataaaatcac catatgccaa tgaaaaaa 3988 <210> 14 <211> 941 <212> PRT
<213> Salmo salar <400> 14 Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gin Lys Thr Gly Asp Ile Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp Gin Asp Leu Ala Ala Arg Pro Glu Ser Thr Glu Cys Val Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Asn Ser Ser Thr Leu Leu Pro Asn Ile Thr Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Thr Asp His Ile Pro Ser Thr Ile Ala Val Val Gly Ala Ser Gly Ser Ala Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gln Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gln Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His Gln Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gln Trp Asn Trp Val Ile Ala Val Ala Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu Lys Phe Glu Lys Glu Met Glu Glu Arg Asp Ile Cys Ile His Leu Ser Glu Leu Ile Ser Gln Tyr Phe Glu Glu Trp Gln Ile Gln Gly Leu Val Asp Arg Ile Glu Asn Ser Ser Ala Lys Val Ile Val Val Phe Ala Ser Gly Pro Asp Ile Glu Pro Leu Ile Lys Glu Met Val Arg Arg Asn Ile Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp Ala Thr Thr Ser Leu Ile Ala Lys Pro Glu Tyr Leu Asp Val Val Val Gly Thr Ile Gly Phe Ala Leu Arg Ala Gly Glu Ile Pro Gly Phe Lys Asp Phe Leu Gln Glu Val Thr Pro Lys Lys Ser Ser His Asn Glu Phe Val Arg Glu Phe Trp Glu Glu Thr Phe Asn Cys Tyr Leu Glu Asp Ser Gln Arg Leu Arg Asp Ser Glu Asn Gly Ser Thr Ser Phe Arg Pro Leu Cys Thr Gly Glu Glu Asp Ile Met Gly Ala Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val Tyr Val Ala Val His Ser Ile Ala Gln Ala Leu Gln Asp Ile Leu Thr Cys Ile Pro Gly Arg Gly Leu Phe Ser Asn Asn Ser Cys Ala Asp Ile Lys Lys Ile Glu Ala Trp Gln Val Leu Lys Gln Leu Arg His Leu Asn Phe Ser Asn Ser Met Gly Glu Lys Val His Phe Asp Glu Asn Ala Asp Pro Ser Gly Asn Tyr Thr Ile Ile Asn Trp His Arg Ser Pro Glu Asp Gly Ser Val Val Phe Glu Glu Val Gly Phe Tyr Asn Met Arg Ala Lys Arg Gly Val Gln Leu Phe Ile Asp Asn Thr Lys Ile Leu Trp Asn Gly Tyr Asn Thr Glu Val Pro Phe Ser Asn Cys Ser Glu Asp Cys Glu Pro Gly Thr Arg Lys Gly Ile Ile Glu Ser Met Pro Thr Cys Cys Phe Glu Cys Thr Glu Cys Ser Glu Gly Glu Tyr Ser Asp His Lys Asp Ala Ser Val Cys Thr Lys Cys Pro Asn Asp Ser Trp Ser Asn Glu Asn His Thr Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ser Phe Lys Val Ala Ala Lys Ala Thr Leu Arg His Ser Ser Ala Ser Arg Lys Arg Ser Ser Ser Val Gly Gly Ser Cys Ala Ser Thr Pro Ser Ser Ser Ile Ser Leu Lys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gin Gin Arg Ile His Lys Pro Arg Val Ser Phe Gly Ser Gly Thr Val Thr Leu Ser Leu Ser Phe Glu Glu Ser Arg Lys Asn Ser Met Lys <210> 15 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 15 tgtcktggac ggagccctty ggratcgc 28 <210> 16 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 16 ggckggratg aargakatcc aracratgaa g 31 <210> 17 <211> 16 <212> PRT
<213> Artificial Sequence <220>
<223> peptide for Sal-1 antibody production <400> 17 Cys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gin Gin Arg Ile His Lys <210> 18 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 18 caagcattat caagatcaag 20 <210> 19 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 19 ctcagagtgg ccttggc 17 <210> 20 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 20 cagttctctc tttaatggac 20 <210> 21 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 21 ctcagagtgg ccttggc 17 <210> 22 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 22 agtctacatc atccatcagc c 21 <210> 23 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 23 gattttattg tcattggatg c 21 <210> 24 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 24 tggaagatga aatcgccgc 19 <210> 25 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 25 gtggtggtga aactgtaacc gc 22 <210> 26 <211> 23 <212> PRT
<213> Artificial Sequence <220>
<223> peptide for 4641 antibody production <400> 26 Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys <210> 27 <211> 22 <212> PRT
<213> Artificial Sequence <220>
<223> peptide for 4641 antibody production <400> 27 Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile <210> 28 <211> 17 <212> PRT
<213> Artificial Sequence <220>
<223> peptide for SKCaR antibody production <400> 28 Ala Arg Ser Arg Asn Ser Ala Asp Gly Arg Ser Gly Asp Asp Leu Pro Cys <210> 29 <211> 18 <212> PRT
<213> Artificial Sequence <220>
<223> peptide for Sal-ADD antibody production <400> 29 Cys Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu Lys Phe Glu Lys Glu Met <210> 30 <211> 1078 <212> PRT
<213> Homo sapiens <400> 30 Met Ala Phe Tyr Ser Cys Cys Tip Val Leu Leu Ala Leu Thr Tip His Thr Ser Ala Tyr Ser Pro Ser Gin Pro Ala Gin Lys Lys Gly Asp Ile Ile Leu Gly Gly Leu Phe Pro Ile His Phe Gly Val Ala Ala Lys Asp Gin Asp Leu Lys Ser Arg Pro Glu Ser Val Glu Cys Ile Arg Tyr Asn Phe Arg Gly Phe Arg Trp Leu Gin Ala Met Ile Phe Ala Ile Glu Glu Ile Asn Ser Ser Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser Phe Val Ala Gin Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe Tyr Ile Pro Gin Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn Lys Asn Gin Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His Cys Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser Glu Leu Ile Ser Gin Tyr Ser Asp Glu Glu Glu Ile Gin Met Val Val Glu Val Ile Gin Asn Ser Thr Ala Lys Val Ile Val Val Phe Ser Ser Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val Pro Arg Asn Ile Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala Trp Ala Ser Ser Ser Leu Ile Ala Met Pro Gin Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe Ala Leu Lys Ala Gly Gin Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys Val His Pro Pro Lys Ser Val Asn Asn Gly Phe Ala Lys Glu Phe Trp Glu Glu Thr Phe Met Cys His Leu Gin Glu Gly Ala Lys Gly Pro Leu Pro Val Asp Thr Phe Leu Ala Gly His Glu Glu Ser Gly Asp Arg Phe Ser Asn Ser Ser Thr Ala Phe Pro Pro Leu Cys Thr Gly Asp Glu Asn Ile Ser Ser Val Glu Thr Pro Tyr Ile Asp Tyr Thr Asn Leu Arg Ile Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala Asn Ala Leu Gin Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe Thr Asn Gly Ser Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gin Val Leu Lys His Leu Arg Asn Leu Asn Phe Thr Asn Asn Met Gly Glu Gin Val Thr Phe Asp Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile Asn Trp His Leu Ser Pro Glu Asp Gly Ser Ile Val Phe Lys Glu Val Gly Tyr Tyr Asn Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe Ile Asn Glu Glu Lys Ile Leu Trp Ser Gly Phe Ser Arg Glu Val Pro Phe Ser Asn Cys Ser Arg Asp Cys Leu Ala Gly Thr Arg Lys Gly Ile Ile Glu Gly Glu Pro Thr Cys Cys Phe Glu Cys Val Glu Cys Pro Asp Gly Glu Tyr Ser Asp Glu Thr Asp Ala Ser Ala Cys Asn Lys Cys Pro Asp Asp Phe Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala Val Leu Gly Ile Ser Leu Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Phe Met Phe Lys Trp Trp Gly Leu Asn Leu Gin Phe Leu Leu Val Phe Leu Cys Thr Phe Asn Gin Ile Val Ile Cys Val Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg Asn Gin Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly Ser Leu Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala Tyr Ala Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala Asn Ala Phe Lys Val Ala Ala Arg Ala Thr Leu Arg Arg Ser Asn Val Ser Arg Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser Thr Pro Ser Ser Ser Ile Ser Ser Lys Ser Asn Ser Glu Asp Pro Phe Pro Arg Pro Glu Arg Gin Lys Gin Gin Gin Pro Leu Ala Leu Thr Gin Gin Glu Gin Gin Gin Gin Pro Leu Thr Leu Pro Gin Gin Gin Arg Ser Gin Gin Gin Pro Arg Cys Lys Gin Lys Val Ile Phe Gly Ser Gly Thr Val Thr Phe Ser Leu Ser Phe Asp Glu Pro Gin Lys Asn Ala Met Ala Asn Arg Asn Ser Thr Asn Gin Asn Ser Leu Glu Ala Gin Lys Ser Ser Asp Thr Leu Thr Ala Asn Gin Pro Leu Leu Pro Leu Gin Cys Gly Glu Thr Asp Leu Asp Leu Thr Val Gin Glu Thr Gly Leu Gin Gly Pro Val Gly Gly Asp Gin Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro Ala Leu Val Val Ser Ser Ser Gin Ser Phe Val Ile Ser Gly Gly Gly Ser Thr Val Thr Glu Asn Val Val Asn Ser <210> 31 <211> 3606 <212> DNA
<213> Fugu rubripes <400> 31 taatctcagg agagtttcgt ttccagagga aaccgaactt cttaaacgca ggtaggttgc 60 aagcgtcata tttcacagtg gaatcaaagg caagaaaacg ggtcatttca agagaaattt 120 gtcctccttt ctcccagtcg cagcaggaaa aacatgagtc tcctgaaaaa atgcagcagg 180 cagactgatg ggacaagata agagggaccg tatcacctta aaaaaattat ttttccaaaa 240 aaatccatta gaaagggcag gatggtgcga ctggtgctgc attatctcat acttctgggg 300 tccggttatg tcatttcaac atatgggcct aatcagagag cacagatgac tggcgatatt 360 ctgctgggag gacttttccc catacacttt ggcatttctt ccaaagacga aaacctcgct 420 gcccgaccgg aatccacaaa gtgtgtcagg taaaactcag cacttcatat gtaactgcat 480 gtaaatgctt aaatcacgtg tcagcactgt tgagcacttt caaaccttct ttccgcaggt 540 tcaatttccg tggtttccgc tggctgcagg ccatggtttt cgccatcgag gagatcaaca 600 acagcagcag cctcctgccc aacatcaccc tcggctacag gatcttcgac acgtgcaaca 660 cggtttcaaa agccctggag gctacgctga gcttcgtggc ccaaaacaag atcgactccc 720 tgaatttgga tgaattttgc aactgcacgg atcacatccc ggcaaccatc gcggtggtgg 780 gagcggcagg gtctgcggtc tctacggcag tcgcaaacct gctgagtctc ttttatattc 840 ctcaggtgag tgacggaatt atctttacac tccagttaaa accttaaagt caccgcaatc 900 ctctatcaca ccagatcagc tatgcgtcct ccagccgctt gctgagcaac aagaatcaat 960 acaagtcctt catgagaacc atccctacag atgagcacca ggccacagcc atggccgacg 1020 tcatcgagta cttccagtgg aactgggtca ttgccgtggc ctctgatgac gattatgggc 1080 ggcctgggat agaaaagttt gagaaggaga tggaagagcg agacatttgc atccatctga 1140 acgaactcat ctctcagtac ttcgaggact gtgaaatcaa agctctcgtg gacagaattg 1200 aaaactccac agccaaagtc atcgttgtgt ttgccagcgg ccctgatatc gagcctctga 1260 tcaaagaaat ggtgaggagg aacatcaccg atcggatctg gttagccagt gaagcgtggg 1320 caagctcctc cctcatcgct aaaccagaat atctcgatgt tgtggagggc acaatcggct 1380 ttgttttgaa agcagggaac atacctggtt tcagggagtt cttacagcag gtccagccaa 1440 agagaggcag ccacaacgaa tttgtcaggg agttttggga ggaaaccttc aactgttacc 1500 tggaggacag cccgagactg caagaaagtg agaatggtag cgacagtttc aggccgcttt 1560 gcaccagcga ggaagacatc acgagtgtgg agaccccgta cctcgaccac acacacctcc 1620 gtatctccta caatgtctat gttgcagttt actccatcgc tcacgccctc caggacatac 1680 tctcctgcac tcctggacat ggactctttg ctaacaactc ttgcgcggat ataaagaaaa 1740 tggaagcatg gcaggtaaaa actgctcttt tgcccacaat gttctacttt gtcagcctca 1800 aagggtcaaa tgtgtttgca ggtcctgaag cagctcagac atttgaacta caccaacagc 1860 atgggggaaa aggttcattt tgatgagaac gcagacatgg aagcaaacta caccatcata 1920 aactggcacc ggtctgctga ggacggctca gtggcgttca gggaggtggg ctactaccac 1980 atgcacgcca ggcggggagc caaactgctc attgataaca caaagatgat gtggaatgca 2040 tacagttcag aggtcagtca tcttcagggc aatttcctgt ttgtcctgat gtacagtgtc 2100 tctgttcctg acatcattta cgtggcaggg acaccaaatg tggtgtcatg tgagcatcta 2160 agggtttgca tctgttgtcc tttaaggtgc cgttttctaa ctgcagcgag gactgcgaac 2220 ctgggacgag gaagggcatc attgacagca tgcccacgtg ctgctttgaa tgtaccgagt 2280 gctcagacgg cgagtacagc gatcataaag gtagtagcgt ttgtttactg cttcctgtta 2340 ctgtccacga acactcaggt aatcctcaca tgcatgtttc agacgccagc atttgcacca 2400 agtgtccaaa caactcctgg tccagtggga accacacctt ctgctttctg aaggaaattg 2460 agttcctggc ctggtcagaa ccctttggga tcgctttggc catctgcgcg gtcctggggg 2520 ttctcctgac ggcatttgtg atgggagtct ttgtcagatt tcgcaacact ccgatcgtga 2580 aggcctcaaa ccgagaactg tcctacgtcc tcttgttgtc actcatctgc tgtttttcca 2640 gctcactcat cttcatcggg gagccccaag actggacctg ccgtttacgc cagcctgcct 2700 tcgggatcag ttttgtcctt tgcatctcct gcatcctcgt gaaaaccaac agggttcttt 2760 tggtgtttga agccaagatc cccaccagca tccatcgtaa atggtggggc ctgaacctgc 2820 agttcctcct ggtgtttctg tgcaccttcg ttcaggtcat gatatgtgtg gtctggcttt 2880 acaacgcccc cccctccagc tacaggaatc atgacatcga tgagatcatt ttcatcacct 2940 gtaatgaggg atccgtgatg gcgctcgggt ttctcattgg ctacacgtgc ttactagcgg 3000 ccatatgctt cttctttgcg tttaagtcaa ggaaacttcc cgaaaacttc acggaggcca 3060 agttcatcac tttctgcatg ctcatcttct ttattgtttg gatttctttt atccctgcgt 3120 acttcagtac ttacggcaag tttgtttcag cggtagaggc cattgccatc ctggcatcca 3180 gctacgggat gctagcttgt attttcttta ataaggtcta catcatcctg ttcaaaccct 3240 gtaggaacac catagaagag gtcaggtgca gcaccgcggc ccacgctttc agagtggccg 3300 ccaaagctac attaaaacac agaaccactg tgaggaaaaa gtcgaacagc atcggctcta 3360 ccgcctccac tccatcatca tcaattagcc tgaagaccaa cagcaacgac tgcgactccg 3420 cctcagggag gcataggccg agggtgagct tcggcagcgg aactgtcatg ctgtccttga 3480 gctttgagga gtccaggagg agctctctga tgtgacacac aaaccagttt catccacccg 3540 tcctttcagc ctctaatcca tctgtccacc cttctaatta ccttgattat taacctgctt 3600 aataaa 3606 <210> 32 <211> 4856 <212> DNA
<213> Fugu rubripes <400> 32 cttgtggctg gggtcagtag aagaacctag actagcggta catgaaccgt atcaaaagtg 60 taacggtgaa acattagcac aactgtgtgg gggaggaaat agagtcattg catagtttct 120 taaaccagtt gtgcagtacc tgagtgacaa actttgtttt gtccatttta tttttgcatt 180 aaacaaaagc gattttattg gaaattgcct cgatagatgt caataaatct cacatgatgt 240 tatcagcact tcattacagg tatcataaag atataatagg aacaactgta acttgtattt 300 gttctaaagg ttataaccct tggggaacag ccagataagg accacgtaat gttttgtctg 360 actgattaat aataataagc tgacatgagc agaaccaaca cctcagtcta taaaagcatt 420 tgtgcagaat gctccgagac atttttagga tggagatctc tgttatcttc ctttgcttca 480 tttctctgtt tgatttaaac tcagctggtg acctgaaggc tcctgagaac agtctgaaac 540 aacaggttgg accgagggag gacacaaccg gtgccgcagc tccgtctgac atatgtcgcc 600 tccagggttc tgctcgccta cctgctttct caaaggatgg agactttgtc atcggggggg 660 ttttctccat acaccgctac acagtgacgg tgaaccacaa ctacaccacc atgcctgagc 720 catttcgctg cagagtgtgc ggagaagaaa atgtttgatt gtttcatttc tggagtatat 780 atgatgttca cattatgatg cattttttat ttatacctac acttgtgaat gtttgttttc 840 tgtcatgatg tgcagcatcg accatcatga attgcaactg tctcatgcaa tggtcttcgc 900 catcgaggag atcaacaaca gcacggagct gcttcctgga atcaaactcg gttaccagat 960 ccacgactcg tgcgcagccg tgcccatcgc agtaaatgtg gccttccaac tgttaaacac 1020 tctggaccct gtgtttgtca caggtgacaa ctgttcacag tccggtatgg tgatggctgt 1080 cgtcggcgag tctggttcca ctccgtccat cagcatatcc cgcgtcatcg gctccttcga 1140 cattccacta gtgaatattt tcaactttta ccaaagcaca tgaacacgta atattaacca 1200 tcactgtaat gacacaatgt tcaacttcag gtgagccact ttgccacctg tgcatgcctg 1260 tctgataaac agaagtaccc aagtttcttc agaacgattc ccagtgacca gttccaggcc 1320 gacgctttgg tcaaactcat caaacacttt ggctggactt ggataggtgc tgtgtgctca 1380 gattcagact atggcaataa tggcatggca gcgttcctgc atgcagctca gaaggagggc 1440 atctgtgtgg agtactcgga atccttttat cgcacccacc cgcacagcag gatcaagaga 1500 gtggctgatg tcatccgcag gtgtatagat ctatttttgc ttcatgattc atgaaggtat 1560 ttgtgtttgt ttgaaatgac gcatatactt gtgagttttc tgcaggtcaa cagctgtggt 1620 ggttgtagcc tttacagcat ctacagaaat gatgatcctg ctcgaggaac tgtctcatga 1680 accctcacca cctcgccagt ggataggcag tgaatcctgg gtaactgacc cagacttgct 1740 gaggttcagc ttctgtgctg gaactattgg atttgccatt cagaggtctg tcatcccagg 1800 cctgagagac ttcctgctgg atctctcccc ctcgaaagtg gcttcatctc ctgtgctcac 1860 tgagttctgg gaggactcat tcaactgccg gttgggaaaa ggtgacagaa gttatacctc 1920 ttcttaattt actgtggata gattttaaat tagtgtccaa atctaataac aaaggctttc 1980 aacagatctc ctgtgttgtc agttgctgtt gcaggtgagc gcatgtgtga tggatctgaa 2040 gacataatga ctctccagag tccgtacact gacacttctg agctccgcat taccaacatg 2100 gtgtacaagg ctgtttatgc aatagctcat gccattcata atgcagtgtg tcaggacaca 2160 aatgctacca ctcggtgtag caaattcacc acgattaacc ccaaaaaggt caaacctaaa 2220 gaattgcctc tgtaactact aacatggttt attacaagtg tttcagctga agtctttcct 2280 ctgactctct gtttcattca attcaggttc tcactcagct gaagacagta aatttttcac 2340 aaaatggtta tgctgtatcc tttgatgcca acggggatcc tgtggcatca tatgagctgg 2400 taaactggaa aaagagtggg agcgggagca tcgaagttgt gccagtagga tactatgatg 2460 cttcactgcc agaaggccaa gagttccgta tcttcaggga catcacatgg gtggatggca 2520 gaaagcaagt acgacagcca gaataattaa taagacagta attcaggcat gaaaacacaa 2580 catctctgaa ttaaaggctt ttttgttcaa ttgtgactct atgtaaaggc accaacggca 2640 gttctgttaa aatctttttc aactggtggg tgtgtatgtc tcaggtgcca gtgtcagtgt 2700 gcagcgacag ctgtcctcaa ggaactcgta aggtactgca gaaaggaaaa cccatctgct 2760 gttatgactg tgtacaatgt ccagagggag agattagcaa tgttacaggt agttatctag 2820 atttcaaaga cagcagggct aaataaataa tttgtttaaa ccctacatga tttttgtttc 2880 ctagattctc ctgaatgcat cccttgtctt gatgacttct ggcctaaccc agagagaaat 2940 gcctgtttcc ccaaacctgt ggagtttctt tcctttaacg aggttctagg aatcatccta 3000 gctgtgttct cagtcggtgg tgcctgtctg gctgttatca cagcagctgt atttttccat 3060 cacaggacct ctcctattgt cagagccaac aactctgagt tgagcttcct gttgctcttc 3120 tctttgactc tgtgcttctt atgttcactc actttcattg gtgctccctc tcacctgtcc 3180 tgcatgctgc gccacacagc gtttgggatc acctttgtcc tttgcatctc atgtgtttta 3240 gggaaaactg tggtggtgtt gatggccttc agagcgactc tccccgggag taatgtcatg 3300 aagtggttcg gtccacctca gcaaaggatg actgttgtga ctttcacgtc tatccaggtt 3360 ttaatatgca ttgtttggtt ggttgtcaat cccccgtttc cagtgagaaa tctgaccacg 3420 tacaaggaga gaatcatcct ggagtgtgcg ttaggctcat ctgttggatt ctgggctgta 3480 ctcgggtaca tcggccttct ggcagctgtt tgtttagttc tagccgtcct cgcccgaaaa 3540 ctccccgata atttcaacga agccaaaatg atcaccttca gcatgctgat attctgtgcc 3600 gtctggatca ccttcatccc cgcttacgtc agctctcctg ggaaattcac cgtggccgtg 3660 gagatctttg ccattttggc ctccagtttt ggactcatcc tgtgcatttt tgctccaaaa 3720 tgttttatta ttttatttaa gccagagaaa aatagcaaaa aacacttgat gaacaagaaa 3780 taatcagaat gtctgcaata tgttgggtga gtttaacgac tggaagtatg ggtccgtaca 3840 gcagcaatag cccagtctgt agatgagtga gctgtgggtt tgagggtatc tggttcaagt 3900 tccatttggg aacaaagatt aggaaatgga cctgtagaag gagatgtgca gggacatatt 3960 aagaacccca ctatggtatc attaagcaaa ataccaaacc cccaaatgat aagattgcct 4020 ttatctctct tcatgattgc atgtgcatgt gtttattgta gcttcatgtt taaccgcttg 4080 taacttgtgt gtaacaaagt gtaaacacag acatttcccc tttgtgcttt taatgtatgc 4140 tttttttctt ctcttgtttt acactgtact gccattcctc ccaacgttat gtgacaggct 4200 agattaggct gcatgatgta tgagttttag aaaatctcac cctttacaac acaagtccgc 4260 tgatacagtt ttccatctct cctcccttat gtccaccata acctcttttc tattttctat 4320 gtgattgtta aacaattaca cttttcattg ggaatgttat tgataaatgc aagtaaaact 4380 actgtgatat ctgtgtagat tctcaatcat ccaggtcatg gtcatccaat gcagtttgct 4440 ttgtcaaatg gactgttttt cttctctttg aagttctgga gtgtgatgta ttcaaaacat 4500 cattaaagtt ataaagtggg aacgactgta acagatttgt tcaaaaggtt ataacccttg 4560 gggaacagcc agataaagac cacataattg cagaatgctc caagacattt ttaggatgga 4620 gatctgttct cttcctttgc ttcatttctc tgtgtgattt gaactcagct ggtgacctga 4680 aggttcctgg gaacagtctg aaacaacagc ttggactgag ggaggacaca accgctgccg 4740 cggctccgtc tgtcatatgt cggctccagg gttttgctcg cctacctgcc ttctcaaaga 4800 atggagactt tgtcatcggg gggggttttc tccatatacc actacaaaat gacagt 4856 <210> 33 <211> 4981 <212> DNA
<213> Fugu rubripes <400> 33 gtgtatttgt ttatgcattc ataacgaagc aacaaactca tattctgcaa gttttgatgg 60 atttgctggc attattaata cttacaatta gattagatta gattagatta gattagatta 120 gataaaagaa attcaggaag ggaaatgcag acattatatt taccaatcct atgaactatc 180 caaatcttgc atagccttaa attaaacatg gtctagatga taaaagtaac atgtatgaag 240 tgaacgaatc tgacgtcatc ggtcacacct tgatgggtgg tgtcatcaga catggtgaag 300 acacaactct gcatataaaa tatgcagtag agatgaagag aacctgaggt ggaggaagga 360 ggaagagtga tggctgacat cactggcaca ttgggcctct tcttcaccct catcacattg 420 tttgtctcct cttccacctc cttcaatgct ccgacttgca agctatggag gaaatttcag 480 cttaatgaga tgcacgaacc gggcgatgtg cttctaggcg ggctgtttca agttcattac 540 agctctgttt ttcccgagtg gacgtttaca tctgaaccac atcagcctgt ctgcactagg 600 taagtttcat gcacaccaca aacaatgatt caagctcaga ttggtcctgg aagaaaaatg 660 tcaattaaat gtttgtgatg ggtgaatcca tccataaagg gtgtatctgg agccagtttc 720 accaggttct tgttaatggt tttggtattt tgcatgtcag gtttgacatt ctaggcttcc 780 gtcacgctat gaccatggcc ttcgctgttc aggagataaa caagaaccct gacctgcttc 840 ctaatctgac tctgggatac cgtctttatg acaactgcgg agcccttgtg gttggattca 900 gtggagcgtt agcactggca agtggtcagg aggaggcgtt tgctcttcag ggaggctgtg 960 ctgggagccc accggtcctg gggatcgtgg gtgattcact ctctacgttc actattgctt 1020 ctgccagtgt tttaggcttg tacaaaatac ccatggtcag tgattagtgg cactaaagtt 1080 tgtgttttgt atgtgaatta taaactttca tcttttgaat aatattgtaa aacactggaa 1140 gaaatgtgat tggttatgta gagaaatgga tgtcatctac tgaagacata taaataatta 1200 ttacattaat ttgatcaagt tcataaatta accctttttc aaatgaaaaa gtatttttta 1260 ccaaacaaaa actgcaaaat gaacttaaat cttagttaaa tttagtgtaa aagtgatact 1320 cacgcttcgg atcagagtac agacgcagac agctaatctt tggagaaaat ctaagtgcac 1380 tgagatttaa aatttttgtt taaacattca aatgtctttt tcttttcttt tctgttttta 1440 aaaacgttga tctctcttac acagttaaat ctgttaaaat aaaagaataa tttcatgttc 1500 atgctgagtg ctccggaaat ctttaatgct cgaaaacata tttaaaacat ttgggcttgt 1560 gctgtttaat ttcctatttt taggtaagct attttgctac atgttcctgt ctgaccaatc 1620 gtcagcgctt tccgtccttt tttagaacca tcccgagcga tgatttccag gtaaaatatt 1680 cctttcaagc tgagaatcta cagagattaa aaaaaactcc ccaccctcaa attcaaacaa 1740 atattactta gtatcagatt acaaactgtg ggaactaaaa cactcaattg tctggactga 1800 tcccccattc aggtacgtgc aatgatccag atcctgaaac actttggatg gacttgggtg 1860 ggactgctgg tcagtgatga cgattatggg ctccatgtgg cccgttcgtt ccagtctgac 1920 ctggtccagt cagggcaagg ttgtctggcc tacttggagg ttttgccctg ggacaattat 1980 ctgagtgaaa acaggaggat tgtgcacgtg ataaaggaat caacggctcg cgtgctgatg 2040 gtgtttgcac accagtccca catgattcat ctcatggagg aggtttgatg tgcacagaaa 2100 aaactgcagt gaacacgcta aagaatgcta tttctggtat taataatgat atgtacaggt 2160 ggtgagacag aaggtgacag gccttcagtg gttagcaagt gaagcttgga ctggaacgac 2220 cttcctccag acgccggact tcatgccgta cctgaatggc acgctgggca tcgccatccg 2280 tcgaggtgaa ataacaggtc tcagagattt cctcctacga atacggcctg gccagagtag 2340 caacaacact agttatgata tggtaattat ttcttactat tttaactccc agaggaactg 2400 ttcatttaaa aaataaatga tatattttaa tcttttttca ggttcaacag ttttgggaat 2460 actcattcca gtgtaaattt ggtgcgtcag gttcagcaga agcctgcaca ggagatgaaa 2520 atatccagca ggtggacgct gagtttttgg acgtgtctaa cctcagacca gagtataaca 2580 tttacaaagc tgtgtacgct ttggcgtacg cgcttgatga catgctgcag tgtgagccag 2640 ggagggggcc gttcagtgga ggcagctgcg cggatattca caaactggag ccctggcagg 2700 tgagacctga cactggaaaa taacaagcgc ttcccctttt agtgtgagcc tttgaaattt 2760 agattcaccc agtggtccct cagacctgat tactgtcaac ctccatcagc tgttcaaatt 2820 agagcaacag ggtccagaat taggattcga cctaataagc ctgatgtggt attgtgcatt 2880 tgcagttcgt gcattatcta caacatgtca atttcaccac aacgtttggc gatcaagtat 2940 catttgatga aaatggggac gtcctaccca tctatgacat cctcaactgg cagtggctcc 3000 ctgatggaag aactcaggtt cagaatgtgg gtgaggtcaa gaggtcaccg tccagaggtg 3060 aagaactcca gatccatgaa gacaagatct tctggaactt tgaatctaat aaggttattc 3120 ttgtgtgctc ctgttatctg ttaaaagaaa tgcatgaaaa tcagatgttg atttttcctc 3180 ctgcagcccc cacactcagt gtgcagtgaa agctgtcctc ctggaacccg catgtccagg 3240 aagaaaggac aacctgtctg ctgctttgac tgtcttcttt gttctgaggg gaaaatcagc 3300 aacacaacag gttggtattt gatttcaaac actattttgg gcacattaat ctcaaagctg 3360 cgtccatgtt ttgcttcaga ctccatggag tgcaccagtt gtcctgagga cttctggtcc 3420 agcccccagc gggaccactg cgttcccaag aaaactgagt tcctctccta ccatgaacct 3480 ctgggtatct gtctgacagc cgcctccttg ttgggaacag tgatcagcgt ggttgtgttg 3540 ggcatcttca tccatcatcg cagtacacct gtagtacggg ccaacaattc cgagctcagc 3600 ttcctgctcc tggtgtccct taagctgtgt ttcctctgtt ctctgctgtt cattggtcgt 3660 cccaggctgt ggacgtgcca gctgagacac gcagcattcg ggatcagctt tgtgctctgt 3720 gtctcgtgca tcctggtgaa aaccatggtg gtcctggctg tcttcagggc ctccaagcca 3780 ggagggggcg ccactctgaa gtggtttggt gccgtgcagc agagaggaac agttctgggc 3840 cttacttcaa tccaggcagc aatctgcttt gcctggctat tgtcgtcttc accaaagcca 3900 cataaaaaca ttcagtacca caaagacaag atcgtttttg agtgtgtcgt tgggtccaca 3960 gtgggtttcg ctgtattgct cagttacatc ggtttactgg ccatcctcag tttcctgtta 4020 gcatttcttg cacggaatct tcccgacaac ttcaatgagg ccaaactcat cactttcagc 4080 atgctgatct tttgtgctgt gtgggtggcc tttgtcccag cttacatcaa ctcaccgggc 4140 aaatatgcag atgccgtcga ggtctttgct atcctcacct ccagttttgg cctcttggtg 4200 gcgctgtttg gaccaaaatg ttatataatc ctgtttcgtc ctgagaggaa cacaaaaaga 4260 gcaataatgg cccgttgaaa tcagaaccag cacttccaaa tcacaataac aaacactcat 4320 tgaagcatca acagcaaaag tacaaatgca aaaaaaatac aattaaaatt aaagcaattc 4380 accccctagt ggttaaagat tgcactgcat tacacgtttt ccccatttcc ccacctttcc 4440 atgtcacatt aacaatgtgc actgctgcat gtattccaac atccacgggg taagataaga 4500 ccatataaga cagaatctca gtcttcattt ttgaatgtca tcaataactg attcttctat 4560 ttctccacac tttctgtttt gcttctcttc ccttctcact gtatttcttg tgctgttatg 4620 ttaaacttta ttatgatgat caatgagggc agttgagctg tataatatgc tcactcttgt 4680 tttattgtgg taaataaatt tatatttaaa tctgccacat gcttttggtc agtaaaggtt 4740 tgtgaacgtt ttttttgtcc tgcaatatag aaaaaataat atgtaataaa gatttcaagg 4800 gtcaaacaga aaggtatgac tccatctatc tgtgtggaat agaaaaataa gtaacattta 4860 agcagcaatt aaatacattt ttatcacact tcacatacgc atacaattta aaaaaacaaa 4920 acaaaattcc agcccacgtg gctgaagccc tcacgaggga tgaggaagcc tgagtgactg 4980 a 4981 <210> 34 <211> 4781 <212> DNA
<213> Fugu rubripes <400> 34 cagccttcat aaacacaaat aggatatcct atagattggc atagtgaaat ttgaccactt 60 tcaacttgcc gtctggcatc tggagactag atccagacct gatcagtgcc aaggtcaaac 120 gctgtaacac agttaaccct tgaatagctg tctttcatgg tgggggaaac tcaaacttca 180 gtcaaacaga aaaagagaaa gttctaatca gactgatact cccttcatcc ctccactgca 240 acctggctta gcatccacag gtcaaacctt gtctttatgt tggggagggc acacacattc 300 catgcacaca gaagtacaat gtgctgattc tacatggaaa aaaattcagg aatctgacag 360 agattcagat tcagattcag attcagattc agattattga catgatgaaa aggtgataaa 420 tgttgttgtg ctaaactacc tatctatcat acctgtggcc acaaaacaat cactgcgctc 480 ttccaaatat catgacttaa agaaaactcc tcaagtatca cattttcatt tgaggtctga 540 catgacatga cagttctgag acattcttct cacacattca actactttca tctttaactt 600 ccaactaaac tcaatttaga ctatttacaa aacatgaact aataactcag tttgtttgtg 660 atagcggggt cattcattcc agtgcctgaa agctggctac cagttcatta attggtgacc 720 ctgaggagac aaattcagtt tatctaacca cttaatccta attaaggttt aaggagactg 780 aggcaggaaa aaaccctgga caggtcacct gtccaccagt aactgttcac tatcaactag 840 ggactattaa tcatcactga acctaatatg catgttttta tgctgtggaa gcaagggggg 900 acagagagaa cgcaaacttc gcgcagaaat gccagagtct ggagccctga atgctcctgc 960 tctgatattg gtgatcactg caccctcatg cagtctctac aatgacctcc agatgttttc 1020 aggggtttcc aaaaatattt ttttaatcca tctttatttt ctgcgatatt tacaaacaat 1080 gaatcatctc aacaaattct gcagtttttc ctcctgtagg atgaaggaaa atcttgctac 1140 ggcccaaagt tgtcccctgg gaaatgcagg atgcagagtt cttgatgcaa tagatgcaaa 1200 tctattagta agttgtcgtc atggtgatta tgtaatgaag tagaaaaagc aagttaaatg 1260 ttgaagagct gcaccctctg acatcagcaa tgagggcttt cttcttataa aaggatgtta 1320 aacattagga tggcctgcag gagtatataa ttcttctgtt ttcttctgtg agacattttt 1380 gcttacgatt gtgtgtgtca ggttccacca gatgcctgtc tgtgtctgtg taatgcttct 1440 gtttgcgctt ttccacggag cctttggagc cgaggacaat ctaaagtgta agatgttggg 1500 gagaccagag tttccactgt tatctcagga aggagacatc actattgggg gagccttcac 1560 tctccacagc caaatgtcaa aaccttctct ctcctttgaa gagacaccag aagatctcac 1620 atgctccagg taacatttat tttctactgt agtgctattg ttttaccttc agagaaccta 1680 aacctctgtt attttccttt caggattaat ttaagagaat ttcgctttgc ccaaacaatg 1740 atttttgcaa tagaggagat caacaacagc agctcgcttt tgccaaatat ttccattggt 1800 tataaagtat ttgacacgtg cggtttgacg ctgccttcta cacgagcagt aatggcctta 1860 atgaatggaa agacaagaac cccagaagga ggctgctcca gtcggacatc tgttcatgca 1920 attattggtg cttcggagtc gtcttcaacc attgtgatgc ttcaaatttc agggatattt 1980 caaataccgg tggtaataat gtgtcatatt tgtgattggt ttgcttttga ttctgtcttt 2040 ggtaaaaaaa atgaccagat tctgattcat gtattgcttt ccctcttgtt ttttcctctc 2100 tttttgttat cttttttgtt cagataagcc actttgccac ctgtgcatgc ctcagtaaca 2160 ggaaggagta cccctccttc ttcagaacca tccccagtga cttctatcag agcagagctc 2220 tggctaaact agtgaaacat ttcgggtgga cgtgggtcgg ggctgtgaaa agtgacaatg 2280 attatgggaa caatggcctg gcgacattta ttatggctgc tgagcaggaa ggggtctgtg 2340 ttgagtactc ggagggcttt tcgtggacag accccagcga gcagattgcg agggtggtca 2400 cagtcattaa aagtggcagc gcaagggttt tagtcgcctt ccttgcacag agtgagatgt 2460 ccgctctgct ggaggaggct gtaaagcaga acctgacggg gctgcagtgg gttgggagcg 2520 agtcctggat cacagcaggt cacctggctc ttaagaaata ctcagcaatc ctgacagggt 2580 ccctgggctt caccatcaga aagacaaaga taactggcct tcaggagttt cttttacagg 2640 ttaacccaag tcagaaccct cagaataatc ttctcaaaga attctgggag accacatttg 2700 gttgtagttt ccagtctgat gtgcacgggg ccacccagtg ctctggtgtt gagaagctaa 2760 aggacattca gaatcctttt acagatgtgt cagaacttag gatctccaac aatgtgtata 2820 aagctgtgta cgctgtggct catgctatgc atagcatgct aaaatgtggc caaagtggcg 2880 aggcagtgaa tcagtcatgt accactaaaa aggattttga gctaaagcag gtaagatgtt 2940 gtctctaaga attatgtcca gctgaatttc tttacacccc ccattgacct gttctttctg 3000 acaggttgta gagcacctgc aatcagtgaa cttcactctt cagtcaggtg aaagagtgta 3060 ttttgatgac tatggagacc cggcggctac ttatgagctg gtgaactggc agagaagccc 3120 agaaggaaat acagtatttg tggtcgttgg gaactatgac gcttcacaac caaacgggag 3180 gcagtttacc atgaacaaca tcaatataac atgggctgcc aggctgcaga aggtacgacg 3240 catgttaaaa agcattgatt tacacgctga tgtttataca ggcatgggtt aaatggcttc 3300 atctatgtat tttgtatttt cgtcttaaaa cagagaccgc tgtctgtgtg cagtcagagt 3360 tgcattccag gcttccgaca ggctgtgatt aaggggaaac ccatctgctg tttcacctgc 3420 gtcgcctgtg ctgcaggaga gatcagcaac tccagcagta agttcaagtc ttcagagcag 3480 gcaatattta tcctgaaaca atctttttta ctctcttctg cttcaagctt tattcatggc 3540 acctttgatt tttagactct gcagagtgtt tgcagtgccc actggagttc tggtcaaacg 3600 aagatcacag ccagtgcgtt ccaaaggtga tcgagttctt atcatttgaa gaaaccatgg 3660 gggcccttct tgccgccgtt tcactgtttg gggctgcgtt aacgtcgctg gtgttttgtg 3720 tcttttttcg gttccgtcac acacctcttg tcaaagccag taactccgag ctgagctttc 3780 ttcttctttt ctctctgact ctgtgcttcc tttgttccct cacattcatc gggaggccct 3840 caaggtggtc ctgcgtgctg cgacacacgg cgttcggcat cacctttgct ctctgcatgt 3900 cctgcgtcct ggcaaaaact gtggctgttt tgttcgcatt tacagcaaaa aggccaggaa 3960 acacagtttt ttactgttct gttccacttc agagaacaag tgtttttgcc tgcatcactt 4020 tgcaggttat aatttgtgtg ctctggttga cgcttgcccc accacaccct cacaaaaata 4080 ccgctcatgc caaagagcgc attattttag agtgtaattt aggttcccca gtgtggtttt 4140 gggtggtatt ggggtatatc gggctcttgg ctgtgatttg ctttatcctt gcttttcttg 4200 ctagaaagct gcctgataat tttaacgaag ctaaattcat caccttcagc atgctgatat 4260 tttgtgcagt ttgggtcact tttatcccag cgtacgtcag ctctcctggg aagttcacag 4320 ttgccgtgga gatctttgct atcctggcct caagttttgg gttacttttc tgtatatttg 4380 ccccaaaatg ttatatatta atattgaagc cagaaaaaaa cactaaaaaa cacatgatgg 4440 ggagaaacca tcaaaattag gctgaactga gaattgtatc acttaaataa cagtaatttt 4500 gttctttaat atttaagtcc agaaagtgtt ttattaatac agagcattat gtaagtgcac 4560 atatataaaa tgcaccttat gtcagtttga ttttgggatg gaattgactc acatgttaat 4620 gtcatttatc gattaaagct acaataaagt taaactacgt ggacagttgt ttaatagttt 4680 ccctgtcaag tattttaaaa gcaacaattg catgtaaaag caacacagct gtaataggcc 4740 cactcgtctg tcactcacag cagtgaggtg atgccattgg a 4781 <210> 35 <211> 4846 <212> DNA
<213> Fugu rubripes <400> 35 gatccaaact tgcgacaggg atttgtgcca gtatatcaac gccagaacgg cgtctggtat 60 ttgacagtta aggctgtgca gttgtgggag aaagtagatg ctttttaact gtttgctcta 120 gaatttgtgg acacttttag tcttaatgga tgtataatca atttcctaat ttctcttgaa 180 tctggcatat tgcaaacatt tgttgatcac cacaaacaca cacacacaca cacacgcaca 240 cacacacaca cacacacaca cacacataca cacacactac aaatctacga acttgagctg 300 cagcagtggg tgcacacgtt tgtattgggc tgtgagtctg ttcacagaat gagttggatg 360 ggatggattc cctcacaaag aggtgtacaa cttctctgcc ttctgtgctg catgataatt 420 cctgttgtca ttgccctcct ggatcagagc caacattgca gggtcatccc gggctccatg 480 tccctgccag tgctggagaa gaggggggac atcattttag gcggtctctt ctctcttcac 540 gacatggtgg tagagccaaa tctgcccttc acctctacac caccacccac tcagtgcact 600 aggtaaggta caataatccc ttctacactt ttattcatgt gtaaatttta agttttttgc 660 tgtaaatata gcagtatgca caattccttg tctcttactg acaagtcagc atcctgtttt 720 tcatttcttc ttcccagatt cagttttcgg acattccgtt ggatgcaaac catgatcttt 780 gccgtcgagg agatcaacag aaatgccgaa atccttccca atatcacact gggctacaag 840 atctacgact cgtgcagcac gccccatcag agtttgaagg ctgccatcga tttaatgggg 900 agtgagaaag attcccagtt tgagggaaag ttgcagaggg aaggctgcga tgggaacgtg 960 ccggccgtca tcggagacgg aggatctact cagtctctcg tcgtggcccg tttccttggg 1020 gtctttcatg taccgcaggt atctcgcatt gctgatgctt caaagggaac tcttctctca 1080 tgctagaaat catttgtttg ctagggttca cttaaaagca attatcactt atttttggtc 1140 gtgtcaggtc agttattttt ccagctgtgc ctgtctcagc gacaaaacgc agttcccagc 1200 ctttttaagg acgatgccca gcgatctctt tcaggtttga ggcagcagcc ttaaaacttt 1260 tttttttaaa tttaaagcta tgagagaacc ttcatctcct tcatttctag gtgggtgccc 1320 ttgtacagct cgtcaagtat ttcggctgga cttgggtcgg agtgattgca ggagacgacg 1380 cttacggccg tggcggagcg gccatattcg ctaatgaggt agtaatgaga acaaatggac 1440 atttcaatgc tttattgagc atcgtgcggg cactgaaaat taggattaat ggcatgtctt 1500 ctatctgggg taggtgagaa ggctcggggc ttgcattgct ctctatgaga tgatccccaa 1560 gacacaatca caggctgcca tttcatctat catttccaac atccgctctt ccggggctcg 1620 tgtagttctg gtgtttgctg ttgagcaaga cgtggccagg ttgttcgatg aggcagtcag 1680 gtatggcacc tgtgtatccg caaacgtaca aaaacaactc taagctattt agcttctcct 1740 aaatatagac agaagctgac tgggattcag tggttagcca gtgaggcgtg gagcacagct 1800 gccatcctct ccacccctaa aaggtaccac cacatcctgc aggggtctat gggatttgct 1860 attcggagag cggacatccc tggactgcaa gacttcctcc ttcgcttgca tccctcgagc 1920 gccgaggctg atgacgatcc tttcctgatc ccattctggg aggaggtgtt tcagtgtagc 1980 ctggatccgc acggccactc tgaggctaaa cgtccctgct ctgggacaga ggagcttagg 2040 agcgtgaaga acatctattc tgatgtatca cagcttagga tttcctacaa tgtctacaag 2100 gcggtttatg cccttgccta cgccatcaaa gccatgagaa gttgtgaaaa aggaagtggg 2160 ccattttctc aacaagcctg ccctgattta gacaatatcc acccgtggca ggtatgcaat 2220 taacaaaatt ccatgtgcag aatgacttgt gatgttggaa tcgaatcttc cgctcttttt 2280 tcgcagcttc accactacat aaaacaagtg aactacacga acagattcgg cgatgagatt 2340 aaatttgatg aaaacgggga tcccgccgcc atgtatgact tgattaactg gcagctgacg 2400 ccgggcgggg acatggactt cgtcaccgtt gggaaatttg atgacatagc cgggaccgga 2460 aggaagaacc tccacattga ggaagagaag atcgtgtgga atggcaacaa cacccaagta 2520 ggagctgtca ataaaatgct tcaacactca cgggtgactc agagatgtat ccttatgtga 2580 ccgtgtgctg tactctagtt tcctcttttg ctcaggttcc cttatcagta tgcagtagca 2640 tctgtccccc ggggacccgc aaggcaatcc gacctaatta ccctatctgc tgccatgact 2700 gtgtggtttg tacagcaggg gagattagca atcaaacagg tgagaagagg aggagaatgg 2760 tcacagtttt actcttccag acctctgtcc ataagccagt gaagtggatt tttgataaaa 2820 tgttccttta cgttttcctc tttttctgca gatgccatag aatgtgcccg ctgcctgccg 2880 gagttctggt ccaatgctga caggacagcc tgtgtcccca aacaagtgga gttcctctcc 2940 ttcggtgaca caataggcat tgctctgttg gttgtttccc tgatcggctc cttccttacc 3000 tgcgccgtgg ccctcgtatt cttctatcac aggacctccc ccatcgtcag agccaacaac 3060 tctgacctga gcttcctgct cctcttctct ctgactctgt gcttcctgtg ctctctgacc 3120 ttcatcagcc caccctccca gtggtcctgc atgctgcgac acacagcctt cgggatcacg 3180 tttgtcctct gcatctcgtg cattctgggg aaaacaattg tggtcttaat ggcgttcaga 3240 gccacacttc ctggtagtga tgtgatgaaa tggttcggac cagggaagca aaaggcaatt 3300 atcactttca gcacactggt ccaggtaagg gcttctttta agttgtctca aattgttacg 3360 gttgcatgta ttgaatgtaa catttctgat catttcctct tttgttgcag gttgttattt 3420 gcacggtgtg gctggttgtt gctcctccca ccccacgaca gtacatgcca cgtgaaagtg 3480 ctatcatcat tctcttatgt gacgaaggct caaccatagc cttctccctt gttttgggat 3540 acattggcgt tctggcctgt atgtgtttcc tcctagcctt cctggcgaga aaactgccag 3600 acaatttcaa cgaggccaga ctaatcgcct tcagcatgct cattttttgt gcagtctggg 3660 tggcctttgt cccagcttat atcagctctc cagggaaata ctccacgctc acggaaatct 3720 ttgccatctt ggcctccagt tacggactgc tgggctgcat ctttgcaccc aagtgctata 3780 taattctcat gaagtcagaa aagaacacaa ggaaacactt gatgtcaaaa agtgaaagat 3840 tttagagata tattattgct tgcgttgctg tgggttttga aatgaaatgg ggggacaact 3900 ttaaacgtcc gcgtcatgga tgaagtgtct cttttggcca ccagagggaa gtgttgtgct 3960 gctcatgcaa cactggttca ctgcttatac tggaaaaagc acaaaaagaa catgtattgt 4020 ttccacatac tacagtgtgt tcccgataaa acagtaaaat atatggttag cttataaatt 4080 catatagcaa tcatcccatt aatatattga agtgctcatt gtcattaaag tgcaatattc 4140 tcagtgacag tgtgaaggac agcttttttt ttatgacagt gggatttggc agcattttca 4200 gtgctgatat ggcttgaggt ggaatgaggc cctgaggtgt attctgtgga aatcccagct 4260 gttaaattaa atccatacac cccgacggcc ctttcacata aacataaggc cctggctgat 4320 tccctataat aacaaactaa aacgatatat ttcccgccct ccacagctgc tctcaggact 4380 gtatcgaccg ccttccgccg attgatctgt tgtccctccc gaagctgagg agggattgga 4440 aattgaaaga tggggagtca tctctgctgt ccatcaggaa atggcatatg agcacaatct 4500 atttacattc acttcttacc gtcattgttc ttaaatgtcc cactacagcc atctgtgccc 4560 tccccctctc cccatctcaa aagggattat gacaaagtga atttcccttc atagcatgtc 4620 catttcagga gtccttcagt ggctttgctg ttggtttgat tttaattaag gtgaacagga 4680 gagttcacct cagagggttt aatcatcgag tattagaaac acagtcaaag aaaagagtac 4740 aaaagagtca aatggctttc aaataacaat tcacaggaaa catatcagca gaaagtagag 4800 agttaaacaa aggccacgac ttttttagca cattaaaccc aataga 4846 <210> 36 <211> 4743 <212> DNA
<213> Fugu rubripes <400> 36 ggacaggggt caacatgggc accctgactg gtctgtaact ggtccccatt acactgaaat 60 ctggcatcat gttgattatt acgatccagc tgtaaagcag ctgacggctc agaagatgaa 120 gtctgattgt tgatgtctga tgttccttct tagcccagtt tagctggtgt agtggtgaac 180 agatccctgt actccagggg aggggcccca tgtttctgga actggaccct gatcactgtg 240 actggtgggc tctatttttg cactaaataa taaatttaaa ggctgtatta cttttattta 300 ttattaaagt ttgatcttcc accatgatca taaaaaactg tatcacatga gatcagtgtg 360 aatttacaat gtggtttgaa aaacaattta atgaggcatc ttcataacga tcccatgatg 420 aaacaacaat gtgctgaggg gggcctggag gggccggagc ctgacaggcc ttctgggata 480 cgcctggtca ctgccctcca ggtgaacgct gcagtccctc tttcctctct ctctctctca 540 cacatgcaca cacatgaaca catgcacaca aatcactcag tcatttcatt attgtttttc 600 ttttgccttt gagagcggta atttgatgct gtgcagttct gtttactgcc acccctccct 660 gggctttaca gcagttataa atatcagcgc aggcctgctg ggcagtcata tgttcgctcc 720 gtgtcatgtg taaggagtga tgcgttgcag cgccgtcagg acatgcctgc gctccatact 780 ttgcctctgt cgtgatctgc tgtattggtt tgtttgagca aatgtaactt tccatttcca 840 ggctgttgtc tggtcattcc gtcggcgcga ggtgggctga acagcatcat ccatgtctca 900 gttatcacgg atcttcacct tgattgtggg cttcggtggc agggagctgg gactcggggg 960 ggtcttacag gtagttcagg ctctgacttg ttctcagtgg tccacaccaa ctgaacaggg 1020 cctcttccag gatgggcacg tggttgtcgg tgggctcttt aaccttcatt acacgcctcc 1080 agacacagcc aacaacttca ctcagcaatc gcattacaaa gcttgcaccg ggtacagctg 1140 caatctttca cgtcatttgt gtgaactgtt gtgacctttt cccctccaaa tccaatgttt 1200 acacttggat tctcctcatt cttggcttca ggctggaaaa cctccctttg cagtacattt 1260 acgccatggt gtttgcagtg gaggagatca atcacagcgc agcgctgctg ccaggcgtga 1320 agctcgggta ccacattcgc gatagctgcg ccctccaccc ctggaccact caggcggcgc 1380 tggcactggt cgcaggagac agtgccagct gtgaattggc aaccccagcg gactactctg 1440 cagagacgag tgaagaaaaa ggtactgatt aagccttgca atacctgatg aactcaaact 1500 tgatttaaaa ttgtaatttt taaaacaatt taggtgctgc ctccgttcct ttgattattg 1560 gtggcgcttc ttccaatgca gcaaaaatac tcctgggaac cctgagtcca ctatctgtac 1620 ctttagtgag ttttccgtcc tttcagttta tataaatcat atgacatgtt tttaaagcac 1680 acatgagcct gcggatgatg taattggatt ctatttataa tataataata ataataaagc 1740 tttaaggatc tttgtggttg ttcttccaga taagctacac agctagctgc ccttgcctga 1800 gtgacaggca ccgatatccc accttcttca ggaccatggc cagtgatatt taccaggctc 1860 aggctctcgc ccagcttgtg ttacgcttca actggacgtg gatcggggca gtggtggcaa 1920 acaatgatta cggtcatgtg gcagttaagg tgaaatattc tggctaagca tgcaagacat 1980 tctcctgtgg ataatcctac atgcacagct gcggtgtgtt tatctgtgta cgtcaggtgt 2040 ttcaagagca gactcagggg aaaggtgtgt gtctggcgtt tgtcgagact ctccagaggg 2100 agacgatcgt ggcagacgcc gtgcgtgcgg cgcgcacaat tcaggcctcg actgcgaggg 2160 tgattctggt ttttagctgg tacactgacg tagggcatct cttccgtcag ctgcagaaaa 2220 taaacgtgag aagttctgtg cagggatctg agtgtctaat cagtcagagg agttctgatc 2280 tttgaacgct gcctccgctg caggtgaccg acagacagtt tctggccagc gaggcctgga 2340 gcaccagtga ggttcttctc aaagatcctg acacttctac agtggcgagt ggagtcgtcg 2400 gcgtggccat cgcaagccaa cacatccctg ggtttgaccg tttcctcaga ggcctgaacc 2460 cgtctcttcg gcccagcgac aagtttttac aagaattctg ggaggaggaa tttggctgca 2520 gcccctcgcc tccttcttca gagacctctg gtgatttgaa cgcttcgctg cctccctgca 2580 gtggtgcaga gtctctggag ggagtgcagc atcccttcac cgatacctca cacctgaggg 2640 tgacgtataa cgtctacctg gctgtatatg ctgcggccaa cgcccttcac agccttctct 2700 cttgccccat tcataacagc ccttctggaa cttctcactg cacctccccg aagggcatta 2760 aaacaacaga ggtaaagaaa ataatacaca gatcaaataa acgcagtaaa atcacaaaat 2820 aataataagt ggcaacaccg tcgtgtttac agctgctgca acacttgagc aaagtgaatt 2880 tcaccacgcc gcagggcaaa cacttgtact tccgaggtgc agatattccg gcaatgtacg 2940 acctcattaa ctggcagagt ggcacagacg ggaccctcca gctcgtcctc atcggtgccg 3000 tggctggatt tgacctgcag ctcaatgagt cagaaattga gtggagcgcc aaatataatc 3060 aggtgattca gaggaagcat ctcaatcaca aaaatctgga tacatggaaa cgtatttaat 3120 atgctggggg tgtgcatgtc taggtgcctg tgtcagtgtg cagtgagagc tgcccccccg 3180 gcaccagaaa ggccaacagg aagggagaac ctctctgctg cttcgactgt atcccttgtg 3240 ctgacggcga gattagcaac acaagcggtg agggaaatat cacagcagct tctttctgtt 3300 ctgaactttg ggggtttgaa cgtctgactg tcactgttta ggttctcttc agtgtgaccg 3360 ttgccctcct gagttctggt ccaacgatgg acggactgct tgtgttcctc gacagctgga 3420 ttttctgtcc tttaatgaaa ccttgggcgt tgctctgacc gccgtggccg tgtccggcgc 3480 tgtggtgaca acagccgtgt ttgtggtgtt ccttcactat cgtcacacgc ccatggtgag 3540 aactcagagt cacgtcctgg aaaagaaacc actgaatctg tttaatcagt tcatcctaat 3600 ttccattgga aggttcgagc caacaactct gaattgagct tcctgctgct gctgtcactc 3660 aagctgtgtt tcctgtgctc gctggtgttc atcggtcgtc cgtccgtctg gtcctgtcgg 3720 ttccagcagg cggcttttgg gatcagcttc gtgctttgtg tttcctgcct ccaagtcaag 3780 actatagtgg ttctggcagc cttccgctcg gcccggccgg gcgcgggggc cctaatgaag 3840 tggttcggcc cgtcccaaca gagaggaagc gtttgcatct ttacttgtgt acaggcaaga 3900 gttacagatg gattaagaga aaacattgtt tttttaatgt cagtatgatt tatgtgatgt 3960 tatatgagtg tttgttttca cagtgttgag ctgatgttgc acaatgcttt ctttcatcaa 4020 ggttatcatc tgtattgttt ggctgtcact gagcccccca gtgccccaag ctgacttgga 4080 tgtgccgggc ttacaggtca ccctggagtg cgccatggcc tccgtggtgg gcttctctct 4140 ggtcctgggc tacatcggtc tgctggcctg cacctgcctc ctgttggcgt tcctggctcg 4200 gaaactcccc gacaacttca acgaggccaa gctgatcacc ttcagcatgc tgatcttctg 4260 cgccgtctgg gtggccttcg tccccgctta catcagctct cccggaaaat actcagttgc 4320 cgtggaaata tttgccatcc tggcttccag ttatggcctg ctcttctgta tctttgctcc 4380 aaagtgtttc atcatcctgt tgagacccga gaaaaacaca aagaaacacc tgatgatgcg 4440 atagcaaaca aaatatgaga tttatattcc ttttttagaa atatgcagtc ttaatatctg 4500 ctgctgtgtg ttatttatgc atctgcatca ataaaacgct gaaagaaatt gtaaaacaag 4560 taaaaacatt ctgattctta tttgctttca ttggctattt agaaaaaggt aatgcattat 4620 gggacattta aagcacaggg ttaataaaaa atatactata tcatgtgtct gttgtgtgta 4680 tattctaatg cagtttgatt atttattcat tatggattcc ttatcatgcc acctcaacag 4740 atc 4743 <210> 37 <211> 469 <212> DNA
<213> Fugu rubripes <400> 37 catcatcctc gctggaatct tcctgggcta tatttgtccc ttcaccctca tcgcccgccc 60 cactgtagct tcctgctacc tccagaggct cctcgtgggg ctctccgctg ccatatgcta 120 ctctgccctt gtcaccaaaa ccaatcgcat cgctcgtatt ctggcaggca gcaaaaagaa 180 gatttgcacc aggaaaccga ggttcatgag cgcctgggct caggtggtca tcgcctttat 240 cctgatcagt ctacagctca gtctggaggt caccctcatc gtcctggagc ctcctgaacc 300 catcaaatcg caccccagca taagagaggt tttcctcatt tgcaacacca gcaacatggg 360 cgtcgtggcg ccgctcggat acaacggcct gcttataatg agctgcacct actacgcctt 420 caagacgcgc aatgtacctg ccaactttaa cgaagccaaa tacatcgcc 469 <210> 38 , 4 <211> 318 <212> DNA
<213> Fugu rubripes <400> 38 gcccgcatct tcagcggcgt gaaagatgga gcccagcgac cgcgcttcat cagccccacc tcccagttag ccatctgcgg cgctcttatc tcctgccagt tgctggtggc gctgatctgg ttaatggtgg aggtgcccgg ggtccgcaag gaagtgagct cggagcggag gaacgtggtc atcctcaagt gcaacagcaa ggacagcagc atgctgatgt cgctgaccta caactgcgtc ctcatcatcc tctgcaccgt ctacgcgttc aagacgcgaa agtgccccga gaacttcaac gaggccaagt tcatcggc <210> 39 <211> 559 <212> DNA
<213> Fugu rubripes <220>
<221> misc_feature <222> (528)..(535) <223> n is a, c, g, or t <400> 39 cgtgttgctt acgggcattt tcctcatcta cctcatcacc ttcctcatga tcgccgagcc aagtgtggct gtgtgtgcct tccgcaggct gtttctgggg ctcggcatgt gcatcagcta ctcggccatg ctcaccaaga ccaaccggat ctaccggatc tttgagcagg gcaaaaagtc agtcacgccc ccgaaattta tcagccccac ctcccagctg attatcacct tcatactcat ctcagtgcag gtatacatgc gcattcacgt gcacgatctg ctgcgaagcc tctgctaacc ctagcccccg tgctccttct cgcagcttct cggggtcttc atctggtttg gcgtgatgcc tccgcacacc atcatcgact acgaagagca gaagcccccg aatccggagt ttgcccgcgg agtcctgaag tgcgacatgt ctgacctctc cctcatctta tgtctgagct acagtctggt gctgatgatc acctgcacgg tgtacgccat caagaacaga agggtccnnn nnnnnttcaa cgaagccaag cccatcggc <210> 40 <211> 499 <212> DNA
<213> Fugu rubripes <220>
<221> misc_feature <222> (85)..(85) <223> n is a, c, g, or t <400> 40 taccgcatct tcgagcaggg caagaggtcg gtgaccccgc ccaagttcat cagccccact 60 tcccagctca tcatcacctt cgtgntgatc tctgtgcagg tgagcctggt ggcctgctcc cacttcctgc ccgcctgaag gggctttttt tgaccttcgg ctggtgtttg tgtgcttcag gtcttcggcg tgttcgtgtg gtttgctgtg gtccctcctc acaccatcat cgattacgag gaactgcgtc ctccccagcc caacctggcc cggggcatcc tgaagtgcga catgtccgac ctgtccatca tctgctgcct cagctacagc atcgtgctca tggtaacaca gcgaccttca gccacgtcaa ctcccgctct tgtgaacgat aacaggaaat aagcgtggcg tgtcggtgtg ttgctaggtg acgtgtacgg tgtacgccgt gaagagccgc ggcgttccag agacgtttaa cgaagccaag cccatcggg

Claims (20)

-130-

1. An isolated nucleic acid molecule having a nucleic acid sequence that comprises:
a) SEQ ID NO: 7, 9, 11, or 13; or b) the complementary strand of a).

2. An isolated nucleic acid molecule that comprises a nucleic acid sequence having the coding region of SEQ ID NO: 7, 9, 11, or 13; or the complementary strand of the coding region of SEQ ID NO: 7, 9, 11, or 13.

3. An isolated nucleic acid molecule that comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence of SEQ ID NO:
8, 10, 12, or 14.

4. An isolated nucleic acid molecule that comprises a nucleic acid sequence that hybridizes under high stringency conditions of 1x SSC, wherein 10x SSC comprises about 3 M NaCI, about 0.3 M Na3-citrate.cndot.2H2O, about pH
7.0 and about 1 M HCl, about 1% sodium dodecyl sulfate (SDS), and 0.1 - 2 mg/ml denatured calf thymus DNA at about 65°C, to the complement of SEQ ID NO: 7, 9, 11, or 13; but not to the complement of SEQ ID NO: 1, or 31-40 under said conditions; said nucleic acid molecule encoding a polypeptide with the same biological activity as a polypeptide having the amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.

5. An isolated nucleic acid molecule that comprises a nucleic acid sequence that hybridizes under high stringency conditions of 1x SSC, wherein 10x SSC comprises about 3 M NaCl, about 0.3 M Na3-citrate.cndot.2H2O, about pH
7.0 and about 1 M HCl, about 1% sodium dodecyl sulfate (SDS), and 0.1 - 2 mg/ml denatured calf thymus DNA at about 65°C, to the complement of the coding region of SEQ ID NO: 7, 9, 11, or 13; but not to the complement of the coding region of SEQ ID NO: 1, or 31-40 under said conditions; said nucleic acid molecule encoding a polypeptide with the same biological activity as a polypeptide having the amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.

6. An isolated nucleic acid molecule that comprises a nucleic acid sequence that encodes SEQ ID NO: 8, 10, 12, or 14, wherein the nucleic acid molecule is an RNA molecule.

7. A probe that hybridizes under high stringency conditions of 1x SSC, wherein 10x SSC comprises about 3 M NaCl, about 0.3 M Na3-citrate.2H20, about pH 7.0 and about 1 M HCI, about 1% sodium dodecyl sulfate (SDS), and 0.1 - 2 mg/ml denatured calf thymus DNA at about 65°C, to a nucleic acid molecule that comprises a nucleic acid sequence having SEQ ID NO: 7, 9, 11, or 13; or the coding region of SEQ ID NO: 7, 9, 11, or 13; but not to SEQ ID NO: 1, or 31-40 or coding region thereof under said conditions.

8. A vector or plasmid that comprises a nucleic acid sequence having SEQ ID

NO: 7, 9, 11, or 13; or the coding region of SEQ ID NO: 7, 9, 11, or 13.

9. A vector or plasmid that comprises an isolated nucleic acid sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.

10. A vector or plasmid that comprises a nucleic acid sequence that hybridizes under high stringency conditions of lx SSC, wherein 10x SSC comprises about 3 M NaCl, about 0.3 M Na3-citrate.cndot.2H20, about pH 7.0 and about 1 M HCl, about 1% sodium dodecyl sulfate (SDS), and 0.1 - 2 mg/ml denatured calf thymus DNA at about 65°C, to the complement of SEQ ID
NO: 7, 9, 11, or 13 or the coding region thereof; but not to the complement of SEQ ID NO: 1, or 31-40 or coding region thereof under said conditions;
said nucleic acid sequence encoding a polypeptide with the same biological activity as a polypeptide having the amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.

11. A host cell transformed with a nucleic acid molecule that comprises a nucleic acid sequence having SEQ ID NO: 7, 9, 11, or 13; or the coding region of SEQ ID NO: 7, 9, 11, or 13.

12. A host cell transformed with a nucleic acid molecule that comprises an isolated nucleic acid sequence that encodes a polypeptide having an amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.

13. A host cell transformed with a nucleic acid molecule that comprises a nucleic acid sequence that hybridizes under high stringency conditions of 1x SSC, wherein 10x SSC comprises about 3 M NaCl, about 0.3 M Na3-citrate.cndot.2H2O, about pH 7.0 and about 1 M HCl, about 1% sodium dodecyl sulfate (SDS), and 0.1 - 2 mg/ml denatured calf thymus DNA at about 65°C, to the complement of SEQ ID NO: 7, 9, 11, or 13; or to the complement of the coding region of SEQ ID NO: 7, 9, 11, or 13; but not to the complement of SEQ ID NO: 1, or 31-40 or coding region thereof under said conditions;
said nucleic acid molecule encoding a polypeptide with the same biological activity as a polypeptide having the amino acid sequence of SEQ ID NO: 8, 10, 12, or 14.

14. An isolated polypeptide molecule having an amino acid sequence that comprises SEQ ID NO: 8, 10, 12, or 14.

15. An isolated polypeptide molecule having an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:
7, 9, 11, or 13.

16. An antibody that specifically binds to:
a) SEQ ID NO: 8, 10, 12, or 14; or b) an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 7, 9, 11, or 13.

17. An assay for determining the presence or absence of SalmoKCaR in a sample, that comprises:
a) contacting a sample to be tested with an antibody specific to at least a portion of the SalmoKCaR having an amino acid sequence of SEQ ID NO: 8, 10, 12, or 14, sufficiently to allow formation of a complex between SalmoKCaR and the antibody, and b) detecting the presence or absence of the complex formation.

18. An assay for determining the presence or absence of a nucleic acid molecule that encodes SalmoKCaR in a sample, that comprises:
a) contacting a sample to be tested with a nucleic acid probe that hybridizes under high stringency conditions of 1x SSC, wherein 10x SSC comprises about 3 M NaCl, about 0.3 M Na3-citrate.cndot.2H2O, about pH 7.0 and about 1 M HCI, about 1% sodium dodecyl sulfate (SDS), and 0.1 - 2 mg/ml denatured calf thymus DNA at about 65°C, to a nucleic acid molecule having a sequence of SEQ ID NO: 7, 9, 11, or 13, but not to the complement of SEQ ID NO: 1, or 31-40 or coding region thereof under said conditions, sufficiently to allow hybridization between the nucleic acid molecule and the probe; and b) detecting the SalmoKCaR nucleic acid molecule in the sample.

19. A method for determining whether a compound is a modulator of a nucleic acid molecule that encodes an amino acid sequence of SEQ ID NO: 8, 10, 12, or 14; wherein said method comprises:
a) contacting a compound to be tested with a cell that comprises said nucleic acid molecule; and b) determining the level of expression of said nucleic acid molecule;
wherein an increase or decrease in the expression level, as compared to a control, indicates that the compound is a modulator.

20. A method for determining whether a compound is a modulator of a polypeptide having an amino acid sequence that comprises SEQ ID NO: 8, 10, 12, or 14; wherein said method comprises:
a) contacting a compound to be tested with a cell that expresses SEQ ID NO: 8, 10, 12, or 14; and b) determining the level of expression or activity of SEQ ID
NO: 8, 10, 12, or 14;
wherein an increase or decrease in the expression level or activity, as compared to a control, indicates that the compound is a modulator.