U.S. patent application number 11/086832 was filed with the patent office on 2005-08-18 for polyvalent cation-sensing receptor in atlantic salmon.
This patent application is currently assigned to MariCal, Inc.. Invention is credited to Betka, Marlies, Harris, H. William JR., Nearing, Jacqueline.
Application Number | 20050181427 11/086832 |
Document ID | / |
Family ID | 46280508 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181427 |
Kind Code |
A1 |
Harris, H. William JR. ; et
al. |
August 18, 2005 |
Polyvalent cation-sensing receptor in atlantic salmon
Abstract
The present invention encompasses three 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, and SalmoKCaR#3. 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 and/or 3.
Inventors: |
Harris, H. William JR.;
(Portland, ME) ; Nearing, Jacqueline; (N.
Yarmouth, ME) ; Betka, Marlies; (Portland,
ME) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
MariCal, Inc.
Portland
ME
|
Family ID: |
46280508 |
Appl. No.: |
11/086832 |
Filed: |
March 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11086832 |
Mar 22, 2005 |
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10125792 |
Apr 18, 2002 |
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10125792 |
Apr 18, 2002 |
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10121441 |
Apr 11, 2002 |
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10121441 |
Apr 11, 2002 |
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PCT/US01/31704 |
Oct 11, 2001 |
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60240392 |
Oct 12, 2000 |
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60240003 |
Oct 12, 2000 |
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Current U.S.
Class: |
435/6.1 ;
536/24.3 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 2319/00 20130101; A01K 2227/40 20130101; A61K 38/00
20130101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A probe that hybridizes under high stringency conditions to a
nucleic acid molecule that comprises a nucleic acid sequence having
SEQ ID NO: 11; or the coding region of SEQ ID NO: 11; but not to
SEQ ID NO: 1 or the coding region of SEQ ID NO: 1 under said
conditions.
2. A probe having a sequence from SEQ ID NO: 11, but not SEQ ID NO:
1.
3. A nucleic acid probe having a sequence from SEQ ID NO: 11, but
not SEQ ID NO: 1.
4. A DNA probe having a sequence from SEQ ID NO: 11, but not SEQ ID
NO: 1.
Description
RELATED APPLICATIONS
[0001] This is a Divisional application of Ser. No. 10/125,792,
filed Apr. 18, 2002, which is a continuation-in-part of U.S.
application Ser. No. 10/121,441, filed Apr. 11, 2002, now
abandoned, which is a continuation-in-part of International
Application No. PCT/US01/31704 (WO02/031149),which designated the
United States, filed Oct. 11, 2001, now abandoned, which claims the
benefit of U.S. Provisional Application No. 60/240,392, filed on
Oct. 12, 2000, and U.S. Provisional Application No. 60/240,003,
filed on Oct. 12, 2000. The entire teachings of the above
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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
[0007] 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, three forms of the PVCR
have been isolated in Atlantic Salmon, and have been termed,
"SalmoKCaR" genes and individually referred to as "SalmoKCaR#1",
"SalmoKCaR#2" and "SalmoKCaR#3." "PVCR" and "SalmoKCaR" are used
interchangeably when referring to Atlantic Salmon. These three
genes work together to alter the salmon's sensitivity to the
surrounding ion concentrations, as further described herein.
[0008] The invention embodies nucleic acid molecules (e.g., RNA,
genomic DNA and cDNA) having nucleic acid sequences of SalmoKCaR#1
(SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), or SalmoKCaR#3 (SEQ ID
NO: 11). The invention also embodies polypeptide molecules having
amino acid sequences of SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2
(SEQ ID NO: 10), or SalmoKCaR#3 (SEQ ID NO: 12). The present
invention, in particular, encompasses isolated nucleic acid
molecules having nucleic acid sequences of SEQ ID NO: 7, 9, or 11;
the complementary strand thereof; the coding region of SEQ ID NO:
7, 9, or 11; 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, or
12. The present invention, in another embodiment, includes isolated
polypeptide molecules having amino acid sequences that comprise SEQ
ID NO: 8, 10, or 12; or amino acid sequences encoded by the nucleic
acid sequence of SEQ ID NO: 7, 9, or 11.
[0009] 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, or 11, or the coding region of SEQ ID NO: 7,
9, or 11. Such a nucleic acid sequence encodes a polypeptide that
allows for or assists in one or more of the following fluctions:
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 Atlantic Salmon with an odorant; altering water intake;
altering water absorption; or altering urine output.
[0010] The present invention further includes nucleic acid
molecules that hybridize with SalmoKCaR#1, SalmoKCaR#2, or
SalmoKCaR#3, but not to the Shark Kidney Calcium Receptor Related
Protein (SKCaR) nucleic acid sequence. 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, or 11; or the coding region of SEQ ID NO: 7, 9, or 11;
but excluding those that hybridize to SEQ ID NO: 1 under the same
conditions.
[0011] 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 SEQ ID NO: 7, but not SEQ ID NO: 1. 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, or
PTA-4192.
[0012] 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, or 12;
or an amino acid sequence encoded by the nucleic acid sequence of
SEQ ID NO: 7, 9, or 11. These polypeptide molecules have one or
more of the following functions: 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 Atlantic Salmon with an
odorant; altering water intake; altering water absorption; or
altering urine output.
[0013] 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.
[0014] 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 ID NO: 7, 9, or 11, sufficiently to allow
hybridization between the sample and the probe; and detecting the
SalmoKCaR nucleic acid molecule in the sample. Such assay methods
also include methods for determining whether a compound is a
modulator of SalmoKCaR. These methods include contacting a compound
to be tested with a cell that contains SalmoKCaR nucleic acid
molecules and/or expresses SalmoKCaR proteins, and determining
whether compounds are modulators by measuring the expression level
or activity (e.g., phosphorylation, dimerization, proteolysis or
intracellular signal transduction) of SalmoKCaR proteins. In one
embodiment, one can measure changes that occur in one or more
intracellular signal transduction systems that are altered by
activation 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.
[0015] 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.
[0016] 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 optimal 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
[0017] FIGS. 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.
[0018] FIG. 2 is a graphical representation showing a normalized
calcium response (%) against the amount of Calcium (mM) of the
SKCaR-I protein when modulated by alternations in extracellular
NaCl concentrations.
[0019] FIG. 3 is a graphical representation showing a normalized
calcium response (%) against the amount of magnesium (mM) of the
SKCaR protein in increasing amounts of extracellular NaCl
concentrations.
[0020] FIG. 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
NaCl concentrations.
[0021] FIG. 5 is a graphical representation showing the EC50 for
magnesium activation of shark CaR (mM) against the amount of sodium
(mM) of the SKCaR protein in increasing amounts of extracellular
NaCl concentrations.
[0022] FIG. 6 is a graphical representation showing the EC50 for
magnesium activation of shark CaR (mM) against the amount of sodium
(mM) of the SKCaR protein in increasing amounts of extracellular
NaCl concentrations and added amounts of calcium (3 mM).
[0023] FIGS. 7A and 7B show an annotated partial nucleotide
sequence (SEQ ID NO: 3) and the deduced amino acids sequence (SEQ
ID NO: 4) of an Atlantic salmon polyvalent cation-sensing receptor
protein.
[0024] FIGS. 8A-8C show a second annotated partial nucleotide
sequence (SEQ ID NO: 5) and the deduced amino acids sequence (SEQ
ID NO: 6) of an Atlantic salmon polyvalent cation-sensing receptor
protein.
[0025] FIGS. 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.
[0026] FIGS. 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.
[0027] FIGS. 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.
[0028] FIGS. 12A-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).
[0029] FIGS. 13A-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).
[0030] FIG. 14 is photograph showing a Southern blot in which
SalmoKCaR#1, 2, and 3 hybridize to nucleic acid derived from
SKCaR.
[0031] FIGS. 15A-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.
[0032] FIGS. 16A-D are an alignment of the full length amino acid
sequences of Human Parathyroid Calcium Receptor (HuPCaR) (SEQ ID
NO: 28), SKCaR (SEQ ID NO: 2), SalmoKCaR#1 (SEQ ID NO: 8),
SalmoKCaR#2 (SEQ ID NO: 10) and SalmoKCaR#3 (SEQ ID NO: 12).
Alignment obtained using Clustal method with PAM250 residue weight
table.
[0033] FIGS. 17A-F are graphical representations comparing six
photographs of Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR) analysis of freshwater (FIGS. 17B, D and F) and seawater
(FIGS. 17A, 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 (FIGS. 17A-D)
or salmon actin PCR primers (FIGS. 17E,F). Wells 1-14 for FIGS.
17A-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 FIGS. 17A, 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 FIGS. 17B,D, and F are
designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and
SalmoKCaR#3, respectively.
[0034] FIG. 18A 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.
[0035] FIG. 18B 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
13 and 14) or actin (SEQ ID No 22 and 23) 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.
[0036] FIG. 18C 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. 13 and 14)
or actin (SEQ ID No 22 and 23) 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 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).
[0037] FIG. 19 is a photograph of a RNA blot containing 5
micrograms of poly A.sup.+ RNA from kidney tissue dissected from
either freshwater adapted (FW) or seawater adapted (SW) Atlantic
salmon probed with full length SalmoKCaR #1 clone.
[0038] FIGS. 20A-F are graphical representations comparing six
photographs showing RT-PCR analysis of freshwater (FIGS. 20B, D and
F) and seawater (FIGS. 20A, C and E) adapted Atlantic salmon
tissues using either SalmoKCaR #3 specific PCR (FIGS. 20A-D)
primers or salmon actin PCR primers (FIGS. 20E,F). Wells 1-14 for
FIGS. 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, 8, 11,
and 14, bottom row, for FIGS. 20A, 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
FIGS. 20B,D, and F are designated as ladder, water, ovary,
SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.
[0039] FIGS. 21A-F are graphical representations comparing six
photographs showing RT-PCR analysis of freshwater (FIGS. 21B, D and
F) and seawater (FIGS. 21A, C and E) adapted Atlantic salmon
tissues using either SalmoKCaR #1 specific PCR primers or salmon
actin PCR primers. Wells 1-14 for FIGS. 21A-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
FIGS. 21A, 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 FIGS. 21B, D, and F are
designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and
SalmoKCaR#3, respectively.
[0040] FIGS. 22A-F are graphical representations comparing six
photographs showing RT-PCR analysis of freshwater (FIGS. 22B, D and
F) and seawater (FIGS. 22A, C and E) adapted Atlantic salmon
tissues using either SalmoKCaR #2 specific PCR primers (FIGS.
22A-D) or salmon actin PCR primers (FIGS. 22E,F). Wells 1-14 for
FIGS. 22A-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 FIGS. 22A, 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 FIGS. 22B, D, and F are designated as ladder, water,
ovary, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.
[0041] FIG. 23 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 S0 (75 gram) and S1 (100 gram) smolts. The wavy
symbol indicates freshwater while the bubbles indicate
seawater.
[0042] FIG. 24A is a graphical representation comparing the weekly
feed consumption on a per fish basis between Process I treated
smolts weighing approximately 76.6 gm vs industry standard smolt
weighing approximately 95.8 gm. These data are derived from
individual netpens of fish containing about 10,000-50,000 fish per
pen. As shown, fish treated with Process I consumed approximately
twice as much feed per fish during their first week after seawater
transfer as compared to the large industry standard smolts weekly
food consumption after 30 days. Process I treatment is defined in
the Exemplification.
[0043] FIG. 24B 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.
[0044] FIG. 25 is a graphical representation illustrating length
(cm) and weight (gm) of representative Process I smolts prior to
transfer to seawater.
[0045] FIG. 26 is a graphical representation illustrating length
(cm) and weight (gm) of Process I smolts before transfer, and
mortalities after transfer to ocean netpens.
[0046] FIG. 27 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.
[0047] FIG. 28 is a graphical representation illustrating the
length (cm) and weight (gm) of St. John/St. John Process II smolts
prior to seawater transfer. Process II is defined in the
Exemplification Section.
[0048] FIGS. 29A and 29B 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).
[0049] FIGS. 30A-G are photographs of immunocytochemistry of
epithelia of the proximal intestine of Atlantic Salmon illustrating
SalmoKCaR localization and expression.
[0050] FIG. 31 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 preimmune)
illustrating SalmoKCaR expression.
[0051] FIGS. 32A-C are photographs of immunolocalization of the
SalmoKCaR in the epidermis of salmon illustrating SalmoKCaR
localization and expression.
[0052] FIG. 33 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.
[0053] FIG. 34 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.
[0054] FIG. 35 is a series of photographs of immunocytochemistry
showing the SalmoKCaR localization of Atlantic Salmon Olfactory
Bulb Nerve and Lamellae using an anti-SalmoKCaR antibody.
[0055] FIG. 36 is a schematic illustrating the effect of external
and internal ionic concentrations on the olfactory lamellae in
response to SalmoKCaR modulators.
[0056] FIG. 37A 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.
[0057] FIG. 37B 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.
[0058] FIG. 37C is a photograph showing localization of Sal ADD
antiserum by immunocytochemistry. Panel A shows the pattern of
immunostaining of immune anti-Sal ADD serum as compared to lack of
reactivity displayed by preimmune anti-Sal ADD serum when exposed
to identical kidney tissue sections (Panel B). 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).
[0059] FIG. 38 is a photograph of immunocytochemistry showing the
PVCR localization in nasal lamellae of dogfish shark using an
anti-PVCR antibody.
[0060] FIG. 39 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.
[0061] FIG. 40 is a histogram illustrating the amount of SalmoKCaR
protein, as determined by an ELISA (ng) for various tissue samples
(gill, liver, heart, muscle, stomach, olfactory epithelium, kidney,
urinary bladder, brain, pituitary gland, olfactory bulb, pyloric
ceacae, proximal intestine, and distal intestine).
[0062] FIG. 41 shows the raw and integrated recordings from high
resistance electrodes of freshwater adapted Atlantic Salmon when
exposed to 500 .mu.M L-alanine, 1 mmol calcium, 50 .mu.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.
[0063] FIG. 42 is a graph showing the response data for freshwater
adapted Atlantic salmon nasal lamellae for calcium, magnesium,
gadolinium, and sodium chloride normalized to the signal obtained
with 10 mM Calcium.
[0064] FIG. 43 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.
[0065] FIG. 44 shows the raw recordings from high resistance
electrodes of freshwater adapted Atlantic Salmon in response to a
series of repeated stimuli (L-alanine or NaCl) in 2 minute
intervals. The figure shows that the olfactory nerve impulse to the
attractant is reversibly altered in the presence of a SalmoKCaR
agonist
[0066] FIG. 45 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.sup.2+).
[0067] FIG. 46 is a graphical representation of the fractional
Ca.sup.2+ response, as compared to the extracelluar Ca.sup.2+ (mM)
for the PVCR in Ca.sup.2+ only, Phenylalanine, Isoleucine, or AA
Mixture (a variety of L-isomers in various concentrations).
DETAILED DESCRIPTION OF THE INVENTION
[0068] The present invention relates to three novel isolated
sequences from PVCR genes, SalmoKCaR#1, SalmoKCaR#2, and
SalmoKCaR#3, in Atlantic Salmon. These genes encode three
polypeptide sequences that are also the subject of the present
invention. These polypeptide sequences allow for or assist in
several functions 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 Atlantic Salmon with an
odorant; altering water intake; altering water absorption; or
altering urine output.
USES OF THE PRESENT INVENTION
[0069] 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 SalmoKCaR
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.
[0070] 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 (Al.sup.3+, Zn.sup.2+, Cu.sup.2+) that contaminate
their native rivers in both the US and other countries such as
Norway. These highly deleterious effects on salmon are manifested
principally in rivers with low natural Ca.sup.2+ concentrations.
Thus, treatment of wild strains of Atlantic salmon produced in
restocking hatcheries with either Process I or Process II 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.
[0071] 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, radioimmunoassays (RIAs),
southern blots and RT-PCR assays, which are described herein in
detail. The salmon are subjected to either Process I or Process II
for a period of time in freshwater before being 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 determine the optimal
amount of time for subjecting the salmon to either Process I or
Process II before transferring to seawater.
[0072] Additionally, comparison of the SalmoKCaR #3 sequence with
data generated from site directed mutagenesis studies of mammalian
CaRs indicates that the SalmoKCaR #3 protein likely generates a
dominant negative effect on the other SalmoKCaR #1 and #2 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 or #2 proteins and SalmoKCaR #3 would cause
a marked reduction in the sensitivity of the SalmoKCaR #1 or #2
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.
[0073] 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 normal and
altered receptors; and nucleotide sequences to be used as target
sites for antisense nucleic acids, ribozymes, or PCR amplification
primers; (3) as hybridization detection probes to detect the
presence of a native receptor and/or a related receptor in a
sample, as further described herein to determine the presence or
level of SalmoKCaR in a sample for, e.g., assessing whether salmon
are ready for transfer to seawater; (4) as PCR primers to generate
particular nucleic acid sequence sequences, for example, to
generate sequences to be used as hybridization detection probes;
and (5) for determining and isolating additional aquatic PVCR
homologs in other species.
[0074] Another use for nucleic acid sequences of SalmoKCaRs #1, #2
or #3 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 or #3 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.
[0075] 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.
[0076] Cell lines expressing SalmoKCaR 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
#1-3 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
#1-3 proteins either alone or in various combinations can also be
used in screening methods to screen for specific water conditions
including pH, ionic strength and composition of various compounds
dissolved in the water to alter the function of SalmoKCaR proteins
and thus lead to improved salinity responses in various life stages
of Atlantic salmon. Such assays would be designed to determine the
interactions and effects of these conditions on SalmoKCaR proteins
without having to test the effects of such compounds on either
whole living fish or some tissue explants.
[0077] 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.
[0078] 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.
[0079] The SalmoKCaR Polypeptides and its Function
[0080] The present invention relates to isolated polypeptide
molecules that have been isolated in Atlantic Salmon including
three 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, or
12). See FIGS. 9, 10 and 11. 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, or 11).
[0081] 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.
[0082] 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, or 12. 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:
1 Percent Identity for Amino Acid Sequences* Query Sequence
SalmoKCaR#1 SalmoKCaR#2 SalmoKCaR#3 SalmoKCaR#1 N/A 99.9% 89.6%
SalmoKCaR#2 99.9% N/A 89.5% SalmoKCaR#3 99.2% 99.1% N/A *Note that
the percentages are based on the number of aa's in the target
sequence.
[0083] The polypeptides of the present invention, including the
full length sequences, the partial sequences, functional fragments
and homologues, allow for or assist in one or more of the following
functions: 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 Atlantic Salmon 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 term "sense" or
"sensing" refers to the SalmoKCaR's ability to alter its expression
and/or sensitivity in response to a SalmoKCaR modulator.
[0084] 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).
[0085] 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: 15 and 27),
or part of the full-length amino acid sequence (SEQ ID NO: 8, 10,
or 12), 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.
[0086] 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 NaCl 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 SalmoKCaR modulator
significantly increases in the salmon, and causes modulated (e.g.,
increased and/or decreased) SalmoKCaR expression and/or altered
SalmoKCaR sensitivity. One function or activity 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.
[0087] 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 II
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, U.S. Pat.
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 .mu.M
and about 100 .mu.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 .mu.M. Organic polycations include, but are not
limited to, aminoglycosides such as neomycin or gentamicin in
concentrations of between about 1 and about 8 gm/kg feed as well as
organic polycations including polyamines (e.g., polyarginine,
polylysine, polyhistidine, polyornithine, spermine, spermidine,
cadaverine, putrescine, copolymers of poly arginine/histidine, poly
lysine/arginine in concentrations of between about 10 .mu.M 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., SalmoKCaR modulator bound to negatively charged particles
including glass, proteins, or plastic surfaces). Any combination of
these modulators can be added to the water or to the feed (in
addition to the NaCl, as described herein), so long as the
combination modulates expression and/or sensitivity of one or more
of the SalmoKCaR genes.
[0088] 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 gene; and adding feed for
fish consumption to the freshwater. The feed contains at least one
an attractant (e.g., alanine); an amount of NaCl sufficient to
contribute to a significantly increased level 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
SalmoKCaR proteins and detection of SalmoKCaR expression using
RT-PCR in various organs involved in the imprinting process
including olfactory lamellae, olfactory bulb and brain is provided
for both larval (Example 13) and smolt stages (FIGS. 34 and 35).
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.
[0089] 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
monophosphate), 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 SalmoKCaR 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.
[0090] 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.
[0091] 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.
[0092] "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 10 mM Ca, about 40 mM 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 salinity.
[0093] 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.
[0094] 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
{fraction (1/10)}th seawater (100 mOsm/kg) by reduction in salinity
from 450 mM NaCl to 45 mM NaCl over an interval of 8 hrs. Summer
and Winter Flounder can be maintained in 1/10 or twice the salinity
for over a period of 6 months. After a 10 day interval where the
Summer and Winter Flounder were fed a normal diet, the distribution
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 as 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 formed 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 NaCl absorbed from the
urine are critical to salinity regulation in fish.
[0095] 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 euryhaline
adaptation of fish to occur, and improve the adaption. More
specifically, methods are now available to regulate salinity
tolerance in fish by modulating (e.g., alternating, activating and
or expressing) the activity 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 increased by activating the
PVCR, for example, by increasing the expression of PVCR in selected
epithelial cells, resulting in the secretion of ions and seawater
adaption. Specifically, this would involve regulatory events
controlling the conversion of epithelial cells of the gill,
intestine and kidney. In the kidney, PVCR activation facilitates
excretion of divalent metal ions including calcium and magnesium by
renal tubules. In the gill, PVCR activation reduces reabsorption of
ions by gill cells that occurs in fresh water and promote the net
excretion of ions by gill epithelia that occurs in salt water. In
the intestine, PVCR activation will permit reabsorption of water
and ions across the G.I. tract after their ingestion by fish.
[0096] Alternatively, the salinity tolerance of fish adapted to
seawater can be deceased by modulating one or more of the SalmoKCaR
polypeptides, for example by decreasing the expression of one or
more of the SalmoKCaR genes while others may be increased. The net
result of these changes would be alterations in the absorption of
ions that facilitate the adaption to freshwater conditions.
[0097] In another example, Winter and Summer Flounder were
maintained in at least twice the normal salinity or {fraction
(1/10)} the normal salinity. See Exemplification. These fish can be
maintained in these environments for long periods of time (e.g.,
over 3 months, over 6 months, or over 1 year). These limits were
defined by decreasing or increasing the ionic concentrations of
calcium, magnesium, and sodium, keeping a constant ratio between
the ions. These salinity limits can be further defined by
increasing and/or decreasing an individual ion concentration,
thereby changing the ionic concentration ratio among the ions.
Increasing and/or decreasing individual ion concentrations can
increase and/or decrease salinity tolerance. "Hypersalinity" or
"above normal salinity" levels refers to a level of at least one
ion concentration that is above the level found in normal salinity.
"Hyposalinity" or "below normal salinity" levels refers to a level
of at least one ion concentration that is below the level found in
normal salinity.
[0098] 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).
[0099] 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 normal seawater) for extended periods in
recycling water, exposure of summer flounder to hypersalinity
conditions might be used as a "biological" remediation process to
ensure that no Diphyllobothrium species are present in the GI tract
of summer flounder prior to their sale as product.
[0100] Data from Cole et al., (J. 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 coli are
killed by high concentrations of NaCl. In contrast, low
concentrations of NaCl (<300 mM NaCl) allow E. coli to grow and
under these conditions pleurocidin presumably helps to kill them.
These data provide evidence of NaCl killing of E. coli, as well as
highlight possible utility of bacterial elimination in fish.
[0101] 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).
[0102] 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.
[0103] 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 subfornical 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.
[0104] 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.
[0105] 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, et al., Proc. Nat. Acad. Sci USA, 92:131-135
(1995); and Sands, et al., J. Clinical Investigation 99:1399-1405
(1997). The biological activity 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.
[0106] 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. Pat. 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.
[0107] 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. Pat. 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.
[0108] SalmoKCaR Nucleic Acid Sequences, Plasmids, Vectors and Host
Cells
[0109] 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 ID NO: 9) or
SalmoKCaR#3 (SEQ ID NO: 11). See FIGS. 9, 10, and 11. 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.
[0110] 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, or 12. The SalmoKCaR full length
nucleic acid sequences encode polypeptides that allow or assist in
one or more of the following functions: 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 Atlantic Salmon with an
odorant; altering water intake; altering water absorption; or
altering urine output.
[0111] The present invention encompasses the SalmoKCaR full length
nucleic acid sequences, SalmoKCaR#1 (SEQ ID NO: 7), SalmoKCaR#2
(SEQ ID NO: 9), and SalmoKCaR#3 (SEQ ID NO: 11), or polypeptides
encoded by these sequences, which were deposited under the Budapest
Treaty with the ATCC, 10801 University Boulevard, Manassas, Va.
20110-2209, USA on Mar. 29, 2002, under Accession Numbers PTA-4190,
PTA-4191, and PTA-4192, 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.
[0112] 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.
[0113] 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: 8, 10, or 12. Each
of the SalmoKCaR genes are homologues to one another. The percent
identity for the SalmoKCaR nucleic acid sequences are as
follows:
2 Percent Identity for Nucleic Acid Sequences Query Sequence
SalmoKCaR#1 SalmoKCaR#2 SalmoKCaR#3 SalmoKCaR#1 N/A 99.8% 95.8%
SalmoKCaR#2 97.6% N/A 93.6% SalmoKCaR#3 98.7% 98.7% N/A
[0114] 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: 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 Atlantic Salmon 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.
[0115] 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
stringency 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.
[0116] 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).
[0117] SalmoKCaR genes were isolated by Polymerase Chain Reaction
(PCR) of genomic DNA with degenerate primers (SEQ ID NOS: 13 and
14) 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 permit
selective amplification of a sequence (nucleotides 2279-2934 of
SKCaR) that is highly conserved in both mammalian and shark kidney
calcium receptors. See Exemplification. The degenerate primers (SEQ
ID NOS: 13 and 14) 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,
SalmoKCaR genes can be isolated by Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR) after isolation of poly A+ RNA from aquatic
species with the same or similar degenerate primers. Methods of PCR
and RT-PCR are well characterized in the art (See generally, PCR
Technology: Principles and Applications for DNA Amplification (ed.
H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A
Guide to Methods and Applications (Eds. Innis, et al., Academic
Press, San Diego, Calif., 1990); Mattila, et al., Nucleic Acids
Res., 19:4967 (1991); Eckert, et al., PCR Methods and Applications,
1:17 (1991); PCR (eds. McPherson, et al., IRL Press, Oxford); and
U.S. Pat. No. 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 full-length cDNA from other species. Moreover, in
one embodiment, this DNA fragment serves as a basis for specific
assay kits for detection of SalmoKCaR expression in various tissues
of Atlantic Salmon.
[0118] 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.
[0119] The present invention embodies nucleic acid molecules (e.g.,
probes or primers) that hybridize to SEQ ID NO: 7, 9, or 11 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, or 11) 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.
[0120] Typically the nucleic acid probe comprises a nucleic acid
sequence (e.g. SEQ ID NO: 7, 9, or 11) 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 determined 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.
[0121] 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.
[0122] 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 such 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.degree. C. decrease in the melting temperature (T.sub.m) for
any chosen SSC concentration. Generally, a doubling of the
concentration of SSC results in an increase in the T.sub.m of about
17.degree. C. Using these guidelines, the washing temperature can
be determined empirically, depending on the level of mismatch
sought. Hybridization and wash conditions are explained in Current
Protocols in Molecular Biology (Ausubel, F. M. et al., eds., John
Wiley & Sons, Inc., 1995, with supplemental updates) on pages
2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
[0123] High stringency conditions can employ hybridization at
either (1) 1.times.SSC-(10.times.SSC=3 M NaCl, 0.3 M
Na.sub.3-citrate.multidot.2H.su- b.2O (88 g/liter), pH to 7.0 with
1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured
calf thymus DNA at 65.degree. C., (2) 1.times.SSC, 50% formamide,
1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42.degree. C., (3)
1% bovine serum albumin (fraction V), 1 mM Na.sub.2-EDTA, 0.5 M
NaHPO.sub.4 (pH 7.2) (1 M NaHPO.sub.4=134 g
Na.sub.2HPO.sub.4.7H.sub.2O, 4 ml 85% H.sub.3PO.sub.4 per liter),
7% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 65.degree. C., (4)
50% formamide, 5.times.SSC, 0.02 M Tris-HCl (pH 7.6), 1.times.
Denhardt's solution (100.times.=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.degree. C., (5) 5.times.SSC, 5.times. Denhardt's
solution, 1% SDS, 100 .mu.g/ml denatured calf thymus DNA at
65.degree. C., or (6) 5.times.SSC, 5.times. Denhardt's solution,
50% formamide, 1% SDS, 100 .mu.g/ml denatured calf thymus DNA at
42.degree. C., with high stringency washes of either (1)
0.3-0.1.times.SSC, 0.1% SDS at 65.degree. C., or (2) 1 mM
Na.sub.2EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1% SDS at 65.degree. 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.degree. C. below that of the calculated
T.sub.m of the hybrid, where T.sub.m in .degree. C.=(2.times. the
number of A and T bases)+(4.times. the number of G and C bases).
For hybrids believed to be about 18 to about 49 base pairs in
length, the T.sub.m in .degree. C.=(81.5.degree.
C.+16.6(log.sub.10M)+0.4- 1(% G+C)-0.61 (% formamide)-500/L), where
"M" is the molarity of monovalent cations (e.g., Na.sup.+), and "L"
is the length of the hybrid in base pairs.
[0124] Moderate stringency conditions can employ hybridization at
either (1) 4.times.SSC, (10.times.SSC=3 M NaCl, 0.3 M
Na.sub.3-citrate.2H.sub.2O (88 g/liter), pH to 7.0 with 1 M HCl),
1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus
DNA at 65.degree. C., (2) 4.times.SSC, 50% formamide, 1% SDS, 0.1-2
mg/ml denatured calf thymus DNA at 42.degree. C., (3) 1% bovine
serum albumin (fraction V), 1 mM Na.sub.2.EDTA, 0.5 M NaHPO.sub.4
(pH 7.2) (1 M NaHPO.sub.4=134 g Na.sub.2HPO.sub.4.7H.sub.2O, 4 ml
85% H.sub.3PO.sub.4 per 0.1-2 mg/ml denatured calf thymus DNA at
65.degree. C., (4) 50% formamide, 5.times.SSC, 0.02 M Tris-HCl (pH
7.6), 1.times. Denhardt's solution (100.times.=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.degree. C., (5) 5.times.SSC,
5.times. Denhardt's solution, 1% SDS, 100 .mu.g/ml denatured calf
thymus DNA at 65.degree. C., or (6) 5.times.SSC, 5.times.
Denhardt's solution, 50% formamide, 1% SDS, 100 .mu.g/ml denatured
calf thymus DNA at 42.degree. C., with moderate stringency washes
of 1.times.SSC, 0.1% SDS at 65.degree. 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.degree. C. below that of the calculated T.sub.m of the hybrid,
where T.sub.m in .degree. C.=(2.times.the number of A and T
bases)+(4.times.the number of G and C bases). For hybrids believed
to be about 18 to about 49 base pairs in length, the T.sub.m in
.degree. C.=(81.5.degree. C.+16.6(log.sub.10M)+0.41(% G+C)-0.61 (%
formamide)-500/L), where "M" is the molarity of monovalent cations
(e.g., Na.sup.+), and "L" is the length of the hybrid in base
pairs.
[0125] Low stringency conditions can employ hybridization at either
(1) 4.times.SSC, (10.times.SSC=3 M NaCl, 0.3 M
Na.sub.3.citrate.2H.sub.2O (88 g/liter), pH to 7.0 with 1 M HCR),
1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus
DNA at 50.degree. C., (2) 6.times.SSC, 50% formamide, 1% SDS, 0.1-2
mg/ml denatured calf thymus DNA at 40.degree. C., (3) 1% bovine
serum albumin (fraction V), 1 mM Na.sub.2.EDTA, 0.5 M NaHPO.sub.4
(pH 7.2) (1 M NaHPO.sub.4=134 g Na.sub.2HPO.sub.4.7H.sub.2O, 4 ml
85% H.sub.3PO.sub.4 per liter), 7% SDS, 0.1-2 mg/ml denatured calf
thymus DNA at 50.degree. C., (4) 50% formamide, 5.times.SSC, 0.02 M
Tris-HCl (pH 7.6), 1.times. Denhardt's solution (100.times.=10 g
Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin
(fraction V), water to 500 ml), 10% dextran sulfate,1% SDS, 0.1-2
mg/ml denatured calf thymus DNA at 40.degree. C., (5) 5.times.SSC,
5.times. Denhardt's solution, 1% SDS, 100 .mu.g/ml denatured calf
thymus DNA at 50.degree. C., or (6) 5.times.SSC, 5.times.
Denhardt's solution, 50% formamide, 1% SDS, 100 .mu.g/ml denatured
calf thymus DNA at 40.degree. C., with low stringency washes of
either 2.times.SSC, 0.1% SDS at 50.degree. C., or (2) 0.5% bovine
serum albumin (fraction V), 1 mM Na.sub.2EDTA, 40 mM NaHPO.sub.4
(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.degree. C. below that of the
calculated T.sub.m of the hybrid, where T.sub.m in .degree.
C.=(2.times.the number of A and T bases)+(4.times.the number of G
and C bases). For hybrids believed to be about 18 to about 49 base
pairs in length, the T.sub.m in .degree. C.=(81.5.degree.
C.+16.6(log.sub.10M)+0.41(% G+C)-0.61 (% formamide)-500/L), where
"M" is the molarity of monovalent cations (e.g., Na.sup.+), and "L"
is the length of the hybrid in base pairs.
[0126] 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).
[0127] 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 for identifying a
nucleic acid by hybridization are routine in the art (see Current
Protocols In Molecular Biology, Ausubel, F. M. et al., Eds., John
Wiley & Sons: New York, N.Y., (1997). The present method can
optionally include a labeled SalmoKCaR probe.
[0128] 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 Ausubel, F. M., et al., Current Protocols in Molecular
Biology, (Current Protocol, 1994) and Sambrook et al., "Molecular
Cloning: A Laboratory Manual," 2nd ED. (1989).
[0129] 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.
[0130] 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-7 cells). Host cells suitable in the present invention also
include a fish cell, a mammalian cell, a bacterial cell, a yeast
cell, an insect cell, and a plant cell.
[0131] 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 refers 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.
[0132] 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.
[0133] 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.degree.-40.degree. C.
[0134] Antibodies, Fusion Proteins and Methods of Assessment of the
SalmoKCaR Nucleic Acid and Amino Acid Molecules
[0135] The present invention includes methods of detecting the
levels of the SalmoKCaR nucleic acid levels (mRNA levels) and/or
polypeptide levels to determine whether fish are ready for transfer
from freshwater to seawater. The present invention also includes
methods for assaying compounds that modulate 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 polypeptides,
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
SalmoKCaR levels include several suitable assays. Suitable assays
encompass immunological methods, such as FACS analysis,
radioimmunoassay, flow cytometry, immunocytochemistry,
enzyme-linked immunosorbent assays (ELISA) and chemiluminescence
assays. Additionally, antibodies, or antibody fragments, can also
be used to detect the presence of SalmoKCaR proteins and homologs
in other tissues using standard immunohistological methods. For
example, immunohistochemical studies were performed using the 1169
antibody which was raised against a portion 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.
[0136] 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.
[0137] 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.
[0138] Anti-SalmoKCaR antibodies can be raised against appropriate
immunogens, such as isolated and/or recombinant SalmoKCaR, analogs
or portion thereof (including synthetic molecules, such as
synthetic peptides). 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.
[0139] 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. Pat. No.
4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);
Current Protocols In Molecular Biology, Vol. 2 (Supplement 27,
Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons:
New York, N.Y.), 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.
[0140] 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 proteins and thereby alter their
function or specificity.
[0141] 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.
[0142] In particular, tissue samples, e.g., gill tissue samples,
can be taken from fish after they are anaesthetized with MS-222.
The tissue samples are fixed by immersion in 2% paraformaldehyde in
appropriate Ringers solution corresponding to the osmolality of the
fish, washed in Ringers, then frozen in an embedding compound,
e.g., O.C.T..TM. (Miles, Inc., Elkahart, Ind., 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:
ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO.: 24) A small peptide with the
sequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO.: 25) or
ARSRNSADGRSGDDLPC (SEQ ID NO.: 26) can also be used to make
antisera containing antibodies to SalmoKCaRs. Such antibodies can
be monoclonal, polyclonal or chimeric.
[0143] Suitable labels can be detected directly, such as
radioactive, fluorescent or chemiluminescent labels. They can also
be indirectly detected using labels such as enzyme labels and other
antigenic or specific binding partners like biotin. Examples of
such labels include fluorescent labels such as fluorescein,
rhodamine, chemiluminescent labels such as luciferase, radioisotope
labels such as .sup.32P, .sup.125I, .sup.131I, enzyme labels such
as horseradish peroxidase, and alkaline phosphatase,
.beta.-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, N.Y.).
[0144] 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 tissues, 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 NaCl diet.
For example, FIGS. 18 and 19 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.
[0145] 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.
[0146] The SalmoKCaR polypeptides can be in the form of a conjugate
or a fusion protein, which can be manufactured by known methods.
Fusion proteins can be manufactured according to known methods of
recombinant DNA technology. For example, fusion proteins can be
expressed from a nucleic acid molecule 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.
[0147] 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 three distinct mRNA species of
about 7 kb, 4.2 kb and 2.6 kb.
[0148] 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 or 11) 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 T.sub.m of approx. 80.degree. C.
(assuming 2.degree. C. for each A or T and 4 degrees for each G or
C).
[0149] 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.
[0150] The present invention encompasses detection of SalmoKCaRs
with PCR methods using primers disclosed or derived from sequences
described herein. For example, SalmoKCaRs can be detected by PCR
using SEQ ID Nos: 13 and 14, as described in Example 6. PCR is the
selective amplification of a target sequence by repeated rounds of
nucleic acid replication utilizing sequence-specific primers and a
thermostable polymerase. PCR allows recovery of entire sequences
between two ends of known sequence. Methods of PCR are described
herein and are known in the art.
[0151] 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, N.Y.,
1992); PCR Protocols: A Guide to Methods and Applications (Eds.
Innis, et al., Academic Press, San Diego, Calif., 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. Pat. No. 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 determined, for example, by agarose gel
electrophoresis. Examples of SalmoKCaR-specific primers are SEQ ID
NO: 16-21. 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
Taqman.RTM. (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.
[0152] 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.
[0153] Transgenic Fish
[0154] 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, T T et al., Transgenic Fish,
Trends in Biotechnology 8:209-215 (1990).
[0155] The present invention is further and more specifically
illustrated by the following Examples, which are not intended to be
limiting in any way.
[0156] Exemplification
[0157] 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." APS stands for "AquaBio Products
Sciences.RTM., L.L.C." 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 II 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.
[0158] Process I: Pre-adult anadromous fish (this includes both
commercially produced S0, S1 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.degree. C. Fish are exposed
to a constant photoperiod for the duration of
[0159] Process I. A Fluorescent Light is Used for the
Photoperiod.
[0160] Process II: Pre-adult anadromous fish (this includes both
commercially produced S0, S1 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 Magnesium ions. This
water is prepared by addition of calcium carbonate and/or chloride
and magnesium chloride to the freshwater. Fish are fed with feed
pellets containing 7% (weight/weight) NaCl and either 2 gm or 4 gm
of L-Tryptophan per kg of feed. Fish are exposed to or maintained
in this regimen of water mixture and feed for a total of 30-45 days
using standard hatchery care techniques. Water temperatures vary
between 10-16.degree. 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)
[0161] A shark .lambda.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 .sup.32P-labeled full length rat kidney CaR (RaKCaR) cDNA
probe under intermediate stringency conditions (0.5.times.SSC, 0.1%
SDS, 50.degree. 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, FIG. 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 FIG. 1.
Northern analyses were performed 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 PIR.
EXAMPLE 2
Expression/Activation Studies of SKCaR in Human Embryonic Kidney
(HEK) Cells
[0162] 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 NaCl, Ca2+ and Mg2+ in
extracellular fluid bathing the HEK cells. The ionic concentrations
encompassed the range which includes the transition from freshwater
to seawater. 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.
[0163] In particular, the following was shown:
[0164] 1. SKCaR encodes a functional ion receptor that is sensitive
to both Mg2+ and Ca2+ as well as alterations in NaCl.
[0165] 2. SKCaR's sensitivity to Ca2+, Mg2+ and NaCl occur in the
range that is found in marine environments and is consistent with
SKCaRs role as a salinity sensor.
[0166] 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.
[0167] 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 transfected cells was verified by
western blotting. Activation of SKCaR by extracellular Ca2+, Mg2+
or NaCl was quantified using a well characterized FURA 2 based
assay where increases in intracellular Ca2+ produced by SKCaR
activation are detected using methodology published previously by
Bai, M., S. Quinn, S. Trvedi, O. Kifor, S. H. S. Pearce, M. R.
Pollack, K. Krapcho, S. C. Hebert and E. M. Brown. Expression and
Characterization of Inactivating and Activating Mutations in the
Human Ca2+-sensing receptor. J Biol. Chem., 32:19537-19545 (1996);
and expressed as % normalized intracellular calcium response to
receptor activation.
[0168] SKCaR is a finctional extracellular Ca2+ sensor where its
sensitivity is modulated by alterations in extracellular NaCl
concentrations. As shown in FIG. 2, SKCaR is activated by
increasing concentrations of extracellular Ca2+ where half maximal
activation of SKCaR ranges between 1-15 mM depending on the
extracellular concentration of NaCl. These are the exact ranges of
Ca2+ (1-10 mM present in marine estuarian areas). Note that
increasing concentrations of NaCl reduce the sensitivity of SKCaR
to Ca2+. This alteration in SKCAR sensitivity to Ca2+ was not
observed after addition of an amount of sucrose sufficient to alter
the osmolality of the extracellular medium. This control experiment
shows it is not alterations in cell osmolality effecting the
changes observed.
[0169] The half maximal activation (EC.sub.50) by Ca2+ for SKCaR is
reduced in increased concentrations of extracellular NaCl. See FIG.
4. The EC.sub.50 for data shown on FIG. 4 is displayed as a
function of increasing extracellular NaCl concentrations. Note the
EC.sub.50 for Ca2+ increases from less than 5 mM to approximately
18 mM as extracellular NaCl concentrations increase from 50 mM to
550 mM.
[0170] SKCaR is a functional extracellular Mg2+ sensor where its
sensitivity is modulated by alterations in extracellular NaCl
concentrations. As shown in FIG. 3, SKCaR is activated in the range
of 5-40 mM extracellular Mg2+ and is modulated in a manner similar
to that shown in FIGS. 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.
[0171] The half maximal activation (EC.sub.50) by Mg2+ for SKCaR is
reduced in increased concentrations of extracellular NaCl. See FIG.
5. The EC.sub.50 for data shown on FIG. 5 is displayed as a
finction of increasing extracellular NaCl concentrations. Note the
EC.sub.50 for Mg2+ increases from less than 20 mM to approximately
80 mM as extracellular NaCl concentrations increase from 50 mM to
550 mM.
[0172] Addition of 3mM Ca2+ alters the sensitivity of SKCaR to Mg2+
and NaCl. See FIG. 6. The EC.sub.50 for Mg2+ of SKCaR is modulated
by increasing concentrations of NaCl as shown both in this FIG. 6
and in FIG. 5. Addition of 3 mM Ca2+ to the extracellular solution
alters the sensitivity characteristics of SKCaR as shown. Note the
3 mM Ca2+ increases the sensitivity of SKCaR to Mg2+ as a function
of extracellular NaCl concentrations.
[0173] 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. FIGS. 16A-D show the amino
acid sequences and alignment for the PVCRs from three full length
Atlantic salmon clones (SalmoKCar #1, #2, and #3) 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.
[0174] 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 perform 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.
[0175] 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.
[0176] Salinity survival limits for winter and summer flounder with
a constant ratio of divalent and monovalent ions were determined.
The survival limit of both winter and summer flounder in waters of
salinities greater than normal seawater (10 mM Ca2+, 50 mM Mg2+ and
450 mM NaCl) is water containing twice (20 mM Ca2+, 50 mM Mg2+ and
900 mM NaCl) the normal concentrations of ions present in normal
seawater. In contrast, the survival limit of both winter and summer
flounder in waters of salinity less than normal seawater is 10%
seawater (1 mM Ca2+, 5 mM Mg2+and 45 mM NaCl).
[0177] Use of a fully recycling water system permits growth of
flounder at vastly different salinities. Groups of flounder (n=10)
were adapted over a 15 day interval and maintained at either low
salinity (LS) (e.g., at 10% normal seawater), normal seawater (NS)
or hypersalinity (HS) (e.g., 2.times. 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
[0178] 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 to
design degenerate oligonucleotide primers, dSK-F3 (SEQ ID NO: 13)
and dSK-R4 (SEQ ID NO: 14), to highly conserved regions in the
transmembrane domain of polyvalent cation receptor proteins using
standard methodologies (See G M 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.
[0179] 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) (FIG. 7).
[0180] Primer Sequences for PCR of PVCR Clones:
3 (SEQ ID NO:13) dSK-F3 5'-TGT CKT GGA CGG AGC CCT TYG GRA TCG C-3'
(SEQ ID NO:14) dSK-R4 5'-GGC KGG RAT GAA RGA KAT CCA RAG RAT GAA
G-3'
[0181] 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
[0182] A second Atlantic salmon partial PVCR was isolated, as
described herein. An Atlantic salmon .lambda.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, Calif.) 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) (FIG. 8A)
containing an open reading frame encoding 388 amino acids (SEQ ID
NO: 6) (FIG. 8B). The open reading frame encoded by SEQ ID NO: 5
begins at nucleotide position 87.
EXAMPLE 6
Molecular Cloning of 3 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
Salinty
[0183] 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.
[0184] The data described in this Example show that the nt and
putative protein sequences of 3 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 3
full length PVCR clones from salmon kidney (SalmoKCar#1 (SEQ ID NO:
7), SalmoKCar#2 (SEQ ID NO: 9) and SalmoKCar#3 (SEQ ID NO: 11)) and
2) use of RT-PCR analysis with degenerate and clone specific
SalmoKCaR PCR primers to determine the tissue specific expression
of these 3 transcripts in seawater vs. freshwater as well as the
SuperSmolt.TM. 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 SuperSmolt.TM.
process.
[0185] Part 1. Isolation and Sequence of 3 Full Length PVCR Clones
from Salmon Kidney:
[0186] Materials and Methods: Total RNA was purified with Stat 60
reagent (Teltest B Friendswood, Tex.) and poly A.sup.+ purified
with the Micro FastTrack Kit (Invitrogen, Carlsbad, Calif.). cDNA
was then synthesized and fractionated whereby selected fractions
were ligated and packaged as .lambda.ZAP libraries (Stratagene, La
Jolla, Calif.). Library phage were then plated and duplicate filter
lifts performed that were screened under high stringency
(0.1.times.SSC, 0.1% SDS @55.degree. C.) with a .sup.32P-labeled
(RadPrime Kit, Invitrogen, Carlsbad, Calif.) genomic fragment of
Atlantic salmon PVCR (653 nt sequence) 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.).
[0187] Results: A total of seven cDNA clones containing PVCR
sequence were identified and purified from Atlantic Salmon kidney
and intestine libraries. A total of three of the seven contain full
length coding sequences for PVCR proteins together with 5' and 3'
regulatory elements. For convenience, these clones are designated
Salmo salar Kidney PVCRs (SalmoKCaRs) #1, #2 and #3 and their
aligned nt and putative protein sequences are shown in FIGS. 12 and
13, respectively. The remaining 4 positive clones were partial PVCR
clones very nearly identical to these 3 full-length SalmoKCaR
clones. Comparison of the different nt sequences of these 3 clones
reveals the following similarities and differences:
[0188] The SalmoKCaR #1 nucleic acid sequence (SEQ ID NO: 7)
consists of 3941 nts of 5' and 3' regulatory elements together with
full-length coding sequence for a 941 AA PVCR protein (SEQ ID NO:
8). See FIGS. 9A-E. The calculated molecular mass of this protein
is 106,125 Daltons.
[0189] The SalmoKCaR #2 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 FIGS. 10A-E. The calculated molecular mass of this protein
is 106,180 Daltons.
[0190] The SalmoKCaR #3 nucleic acid sequence (SEQ ID 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 FIGS. 11A-D. The calculated molecular mass of this protein
is 96,538 Daltons.
[0191] FIGS. 12A-L and 13A-C show an alignment of between the two
partial sequences of Atlantic Salmon PVCRs isolated and the 3 full
length clones for both the nucleic acid and amino acid sequences,
respectively. One partial nucleic acid sequence of an Atlantic
Salmon PVCR, SEQ ID NO: 3, can be found in all three SalmoKCaR
nucleic acid sequences between nt 1979 and 2572; nt 2069 and 2662;
and nt 1980 and 2573 of SEQ ID NO: 7, 9, and 11, respectively. The
second partial Atlantic Salmon clone, SEQ ID NO: 5, can also be
found in all three SalmoKCaR nucleic acid sequences: between nt
1753 and 3773; 1843 and 3863, and 1754 and 3616 of SEQ ID NO: 7, 9,
and 11, respectively. Similarly, the amino acid sequence of SEQ ID
NO: 4 is found between aa 601 and 797 of each of SEQ ID NO: 8, 10,
and 12. The amino acid sequence of the second Atlantic Salmon
Clone, SEQ ID NO: 6, is found in each of the polypeptides: between
aa 554 and 941 of SEQ ID NO: 8; between aa 554 and 941 of SEQ ID
NO: 10; and between aa 554 and 850 of SEQ ID NO: 12. Note that the
amino acid sequence of SEQ ID NO: 6 extends 91 aa past the end of
SEQ ID NO: 12.
[0192] 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:
[0193] nt 1893 change from G to A
[0194] nt 1970 change from G to A
[0195] nt 1973 change from G to A
[0196] nt 2001 change from G to A.
[0197] Table 1 compares the overall % identity of nucleotides (nt)
between cDNA clones that contain the SalmoKCaRs #1,2 and 3 vs.
shark kidney calcium receptor (SKCaR containing 4079 nts) or human
parathyroid CaR (HuPCaR containing 3783 nts). Note that all 3
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 3
SalmoKCaR clones and SKCaR, all 3 full length SalmoKCaR clones
hybridize to full length SKCaR clone under high stringency
conditions (0.5.times.SSC, 0.1% SDS @65.degree. C.) (See FIG.
14).
[0198] The percentage identities between the aligned nucleotide
sequences of the 3 full length SalmoKCaR clones (SEQ ID NO: 7, 9,
11) include:
[0199] 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.
[0200] 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.
[0201] 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.
4TABLE 1 Comparison of the % nucleotide (nt) identity of the
complete nt sequence of 3 SalmoKCaR clones #1, #2 and #3 (including
5' and 3' regulatory elements vs. either the SKCaR clone or the
clone HuPCaR clone. % NUCLEOTIDE IDENTITY SalmoKCaR .sup.#1
SalmoKCaR .sup.#2 SalmoKCaR .sup.#3 SKCaR vs. 56.2 56.5 57.2 HuPCaR
vs. 55.0 54.9 50.9
[0202] 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 3 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 3 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.
[0203] The percentage identities between the aligned amino acid
sequences of the 3 full length SalmoKCaR clones (SEQ ID NO: 8, 10,
or 12) include:
[0204] A total of 99.9% of the aa of SEQ ID NO: 8 are identical to
those corresponding aas in SEQ ID 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.
[0205] A total of 89.5% of the aa of SEQ ID 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.
[0206] A total of 89.6% of the aa of SEQ ID NO: 8 are identical to
those corresponding aas in SEQ ID NO: 12. A total of 99.2% ofthe aa
of SEQ ID NO: 12 are identical to those corresponding aa of SEQ ID
NO: 8.
5TABLE 2 Comparison of % amino acid (AA) identities of 3 SalmoKCaR
proteins vs. AA sequence of shark kidney CaR (SKCaR-Upper Half) and
human parathyroid CaR (HuPCaR-Lower Half). SalmoKCaR SalmoKCaR
SalmoKCaR .sup.#1 .sup.#2 .sup.#3 % AA Identity to SKCaR Overall
Protein 68.4 68.3 63.3 Domains N-terminal 70.0 69.8 70.0
Extracellular Ion Binding Domain 7 Transmembrane 87.2 87.2 86.4
Region Carboxyl 31.8 31.8 0.0 Terminal Intra- Cellular Domain % AA
Identity to HuPCaR Overall Protein 66.3 66.3 61.4 Domains
N-terminal 71.9 71.9 72.1 Extracellular Ion Binding Domain 7
Transmembrane 89.2 89.2 88.4 Region Carboxyl 24.1 24.1 0 Terminal
Intra- Cellular Domain
[0207] FIG. 14 shows all 3 unique SalmoKCaR clones hybridize to
full length shark kidney CaR (SKCaR) under high stringency
conditions (0.5.times.SSC, 0.1% SDS @65.degree. C.). Representative
autoradiogram of Southern blot was exposed for 30 min.
[0208] 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. All 3 SalmoKCaRs possess Cys at
AAs corresponding to HuPCaR AA#101 and AA#236 and presumably
functions as dimers in a manner similar to mammalian CaRs.
[0209] Nucleotide Sequence Differences in the 5' and 3'
Untranslated Regions or UTRs of SalmoKCaRs #1,'#2 and #3:
[0210] FIG. 15 displays the aligned nucleotide sequences of
SalmoKCaR clones #1, #2, and 3. As compared to SalmoKCaR #1 and #3,
SalmoKCaR #2 possesses an 89 nt insert in its 5' UTR. Differences
between the 3' UTRs of the 3 SalmoKCaRs include a 36 nt insert just
prior to the poly A tail in SalmoKCaR #3 as well as other single nt
differences listed below where each difference is compared to the 2
other SalmoKCaR clones:
[0211] SalmoKCaR #1: nt 3660 A to G; nt 3739 A to G; nt 3745 A to
G
[0212] SalmoKCaR #2: nt 3837 A to G; nt 3862 A to G
[0213] SalmoKCaR #3: nt 3472 A to G; nt 3487 A to G; nt 3564 A to
G;
[0214] nt3568 Gto A; nt 3603Ato G; nt 3786Ato C.
[0215] 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 3 SalmoKCaR proteins.
[0216] Sequence Differences in the Coding Regions of SalmoKCaRs #1,
#2 and #3:
[0217] FIG. 16 displays the aligned AA sequences of SalmoKCaRs #1,
#2 and #3 as well as the Shark SKCaR protein and HuPCaR proteins.
As compared to SalmoKCaR #1, SalmoKCaR #2 possesses 2 different
AA's present at AA#257 and AA#941 of its AA sequence. In contrast
to SalmoKCaR #1 that possesses an Asp in AA#257, SalmoKCaR #2
possesses a Gly. The negative charge in this location may be
important since both SKCaR and Fugu PVCR possess Asp at #257 while
the mammalian CaRs, HuPCaR and RaKCaR possess a Glu. SalmoKCaR #3
also contains a Asp at AA#257.
[0218] At AA #443, SalmoKCaR #1 and #2 both possess a Leu whereas
SalmoKCaR #3 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
SalmoKCaRs #1 or 2, SalmoKCaR #3 possesses a truncated carboxyl
terminus as described below.
[0219] Sequence Differences in the Coding Regions of SalmoKCaRs #1,
#2 and #3 as Compared to Mammalian CaRs.
[0220] The putative AA sequences of SalmoKCaR #1, #2 and #3
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 FIG. 16). 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 await expression studies by MariCal.
[0221] Differences between SalmoKCaR proteins vs. mammalian and
other fish PVCRs include:
[0222] All 3 SalmoKCaRs 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.
[0223] 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 EC.sub.50 Ca.sup.2+ from
4.0 mM to 9.3 mM but not Gd.sup.3+ sensitivity. In contrast to
mammalian CaRs and SKCaR, all 3 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 3 SalmoKCaRs and Fugu.
[0224] Lastly, all 3 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.
[0225] SalmoKCaR #3 possesses a truncated carboxyl terminal domain
as compared to either SalmoKCaRs #1 or #2. The number of AA that
comprise the carboxyl terminal domains of the 3 SalmoKCaRs are
different and include: SalmoKCaR #1-96 AA; SalmoKCaR #2-97 AA and
SalmoKCaR #3-5 AA. Reduction in the 91-92AA's in SalmoKCaR #3 vs.
SalmoKCaRs #1 or #2 would reduce its estimated molecular mass by
9,600 Daltons.
[0226] 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.sup.2+ and Mg.sup.2+. 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.sup.2+ as well
as a decrease in the apparent cooperativity of Ca.sup.2+ 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
SalmoKCaR #3 protein is either inactive or exhibits a greatly
reduced functional affinity for Ca.sup.2+. Significant expression
of SalmoKCaR #3 together with other SalmoKCaRs #1 or #2 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.sup.2+ via combinations between
SalmoKCaR #3 and SalmoKCaR #1/#2 to reduce the sensitivity of the
latter PVCRs via cooperative interactions (dimers and higher
oligomers) with them.
[0227] 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. FIG. 16 shows that AA #888 is
a Thr in all wild type CaR and PVCR proteins including HuPCaR,
RaKCaR, SKCaR, BoPCaR and SalmoKCaR #1 and #2. 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 and #2. 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#918-921 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 and #2 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 and #2 await expression
studies by MariCal.
[0228] The Presence of Multiple Differences in the Nucleotide and
Putative Protein Sequences of SalmoKCaR Clones #1-#3 Strongly
Suggest the Presence of Multiple PVCR Genes Within Atlantic
Salmon:
[0229] 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.
[0230] The presence of single nucleotide substitutions together
with specific large scale alterations in the sequence of SalmoKCaR
clones #1-3 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.
[0231] Detailed studies in mammals including mice and humans show
the presence of a single functional PVCR 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 acknowledged 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
salmonids particularly Atlantic salmon.
[0232] 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 #1-3 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.
[0233] Part 2: Use of RT-PCR and Northern Analysis to Determine the
Expression of SalmoKCaR Clones #1, #2 and #3 in Various Tissues of
Atlantic Salmon:
BACKGROUND
[0234] 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 SalmoKCaR #1 or #2 or
#3 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 SalmoKCaR #1 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).
[0235] Materials and Methods:
[0236] Primers:
[0237] Degenerate Primers
[0238] DSK-F3 and DSK-R4 primers are shown in Example 4.
6 SalmoKCaR SalmoKCaR #1 Specific Primers #1 nts AS1-F17 5'-CAA GCA
TTA TCA AGA TCA nt 47-66 AG-3' (SEQ ID NO:16) AS2-R14 5'-CTC AGA
GTG GCC TTG GC-3' nt 2800-2784 (SEQ ID NO:17)
[0239] Product from amplification=2754 nt. The SalmoKCaR #1 primer
pair consists of a forward primer (AS1-F 17) spanning the 5' UTR
insertion in SalmoKCaR #2, and a reverse primer (AS2-R14) within
the 158 bp deleted from SalmoKCaR #3.
7 SalmoKCaR SalmoKCaR #2 Specific Primers #2 nts AS2-F13 5'-CAG TTC
TCT CTT TAA TGG nt 109-128 AC-3' (SEQ ID NO:18) AS2-R14 5'-CTC AGA
GTG GCC TTG GC-3' nt 2890-2874 (SEQ ID NO:19)
[0240] 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).
8 SalmoKCaR SalmoKCaR #3 Specific Primers #3 nts AS5-F11 5'-AGT CTA
CAT CAT CCA TCA nt 2700-2720 GCC-3' (SEQ ID NO:20) AS5-R12 5'-GAT
TTT ATT GTC ATT GGA nt 3810-3790 TGC-3' (SEQ ID NO:21)
[0241] 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.
9 Salmon Actin Primers SA-F1 5'-TGG AAG ATG AAA TCG CCG C-3' nt
2-20 (SEQ ID NO:22) SA-R2 5'GTG GTG GTG AAA CTG TAA CCG nt 608-587
C-3' (SEQ ID NO:23)
[0242] 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.
[0243] RNA Blotting Analysis and RT-PCR of Atlantic Salmon and
Elasmobranch Tissues:
[0244] Total RNA was purified with Stat 60 reagent (Teltest B
Friendswood, Tex.) DNAse (Introgen, Carlsbad, Calif.) treated and
used for RT-PCR after cDNA production with cDNA Cycle Kit
(Invitrogen,Carlsbad, Calif.). The cDNA was amplified (30 cycles of
1 min @94.degree. C., 1 min (57.degree. C., 3' @72.degree. 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, Mass.) and probed with a
.sup.32P-atlantic salmon genomic PCR product (653 bp sequence
identical to that shown in SEQ ID NO: 3 with added nt sequences,
washed (0.1.times.SSC, 0.1% SDS @55.degree. 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:
[0245] SalmoKCaR #1 amplification conditions and primer set: PCR: 1
min @ 94.degree. C., 1 min @50.degree. C., 3 min ( 72.degree. C.,
35 cycles. Amplification products attached to membrane were probed
with full length SalmoKCaR #1 clone and washed (0.1.times.SSC, 0.1%
SDS @55.degree. C.) and autoradiographed for 48 hr.
[0246] SalmoKCaR #2 amplification conditions and primer set: PCR: 1
min (94.degree. C., 1 min @50.degree. C., 3 min ( 72.degree. C., 35
cycles. Amplification products attached to membrane were probed
with full length SalmoKCaR #2 clone and washed (0.1.times.SSC, 0.1%
SDS @55.degree. C.) and autoradiographed for 168 hr.
[0247] SalmoKCaR #3 amplification conditions and primer set: PCR: 1
min ( 94.degree. C., 1 min @52.degree. C., 3 min @72.degree. C., 35
cycles. Amplification products attached to membrane were probed
with full length SalmoKCaR #3 clone and washed (0.1.times.SSC, 0.1%
SDS @55.degree. C.) and autoradiographed for 72 hr.
[0248] Results:
[0249] Analysis of Atlantic Salmon Tissues from Freshwater vs.
Seawater Adapted Fish Using Degenerate Primers:
[0250] FIG. 17 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 (866 gm and 41 cm in length) from a group
of 10 fish of average weight of 678 gm. 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 (112 gm and 21.5 cm)
selected from a group of 10 fish with an average weight of 142.8
gm. 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 or #3 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 #1-3. Thus, amplified
RT-PCR products are referred to as PVCR products since use of these
degenerate primers do not distinguish between various PVCR
species.
[0251] Analysis of panels A-D of FIG. 17 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 .sup.32P-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
[0252] FIG. 17 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 .sup.32P-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.
[0253] 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 performed 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.
[0254] 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 FIG. 17 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
#1-3.
[0255] RT-PCR Analysis Using Degenerate Primers Shows that Steady
State Content of Kidney PVCRs is Increased by the SuperSmolt.TM.
Process Similar to that Produced by Transfer of Atlantic Salmon to
Seawater.
[0256] FIG. 18A 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 SuperSmolt.TM. process in freshwater (lane 2) or
transferred to seawater and maintained for 26 days. FIG. 18B shows
RT-PCR analysis of a single representative experiment using pyloric
caeca from the same fish shown in FIG. 18A. Note the significant
increase in amplified PVCR product present in kidney (FIG. 18A) and
pyloric caeca (FIG. 18B) for both SuperSmolt.TM. (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 SuperSmolt.TM. 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
SuperSmolt.TM. process or transfer of Atlantic salmon to
seawater.
[0257] FIG. 18c 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 13 and 14) or actin (SEQ ID No 22 and 23) 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 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.
[0258] Northern Blotting of Kidney Poly A.sup.+ RNA with SalmoKCaR
#1 Reveals an Increase in PVCR Expression in Seawater-Adapted vs.
Freshwater-Adapted Atlantic Salmon.
[0259] To both confirm the size of SalmoKCaR transcripts and test
for changes in SalmoKCaR expression in fish exposed to different
salinities, poly A.sup.+ RNA from kidney of either freshwater
adapted (FW) or seawater adapted (SW) Atlantic salmon were probed
with SalmoKCaR #1. As shown in FIG. 19, kidney RNA contains a 4.2
kb band that corresponds to the 3.9-4.0 kb sizes of SalmoKCaR #1-3
as determined by nucleotide sequence analysis. Because of the high
degree of nucleotide identities between SalmoKCaR #1-3, the 4.2 kb
band is actually derived from the combination of all 3 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 4.2 kb SalmoKCaR band in SW
adapted fish as compared to their FW adapted counterparts.
[0260] FIG. 19 shows a RNA blot containing 5 micrograms of poly
A.sup.+ 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.
[0261] Use of RT-PCR with SalmoKCaR #3 Specific Primers
Demonstrates that Tissue Specific Alterations in the Steady State
Tissue Content of SalmoKCaR #3 mRNA in Freshwater vs. Seawater
Adapted Atlantic Salmon.
[0262] To determine whether specific SalmoKCaRs #3 are modulated by
exposure to different salinities, nucleotide primer sets that
allows for the specific amplification of SalmoKCaR transcripts were
designed. FIG. 20 shows RT-PCR analysis of freshwater (Panels B, D
and F) and seawater (Panels A, C and E) adapted Atlantic salmon
tissues using either SalmoKCaR #3 specific PCR primers or salmon
actin PCR primers. Total RNA from 13 (seawater adapted) and 14
(freshwater adapted) tissues of Atlantic salmon identical to those
shown in FIG. 17 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 .sup.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 FIG. 20. 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). 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.
[0263] 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 Ca.sup.2+ and Mg.sup.2+ are low and require a high degree of
sensitivity from SalmoKCaRs to sense changes in concentration, the
concentrations of Ca.sup.2+ and Mg.sup.2+ in seawater are 10 fold
and 50 fold higher and thus may require reduction of the high
sensitivity of SalmoKCaRs #1 and #2 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.sup.2+ and Mg.sup.2+ content of seawater (gill, nasal lamellae)
or experience high Ca.sup.2+ and Mg.sup.2+ concentrations as the
result of the excretion of these divalent cations (urinary bladder,
kidney).
[0264] Use of RT-PCR with SalmoKCaR #1 Specific Primers
Demonstrates Tissue Specific Alterations in the Steady State Tissue
Content of SalmoKCaR #1 mRNA in Freshwater vs. Seawater Adapted
Atlantic Salmon.
[0265] FIG. 21 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
FIGS. 17 and 20 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 .sup.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. As shown in lower halves of
Panels A and B of FIG. 21, 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). 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.
[0266] 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.
[0267] FIG. 22 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. DNA 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 .sup.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. FIG. 22 shows
data obtained using SalmoKCaR #2 specific primers and the identical
tissue RT and plasmid samples as shown in FIGS. 17, 20, and 21.
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
SalmoKCaR # 1 in both freshwater and seawater adapted salmon.
EXAMPLE 7
Survival and Growth of Pre-Adult Anadromous Fish by Modulating
PVCRS
[0268] 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 anadromous fish
including Atlantic Salmon, can either be utilized both to improve
the ocean netpen transfer of standard 70-110 grams smolt as well as
permit the successful ocean netpen transfer of smaller smolts
weighing, for example, only 15 grams. 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.degree. 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.
[0269] FIG. 23 shows in schematic form 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 S0 smolt (70 gram) during year 01. In contrast,
slower growing parr are smoltified in year 02 and placed in netpens
as S1 smolt (100 gram). In both S0 and S1 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 S1 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.
[0270] Standard smolts that are newly placed in ocean netpens are
not able to grow optimally during their first 40-60 day interval in
seawater because 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.
[0271] Methods
[0272] The smolt were subjected to the steps of Process I and II,
as described herein.
[0273] Results and Discussion:
[0274] SECTION I: Demonstration of the Benefits of the Process I
For Atlantic Salmon
[0275] Demonstration of the Benefits of the Process I For Atlantic
Salmon:
[0276] Process I increases the survival of small Atlantic Salmon S2
like smolt after their transfer to seawater when compared to
matched freshwater controls. Optimal survival is achieved by using
the complete process consisting of both the magnesium and calcium
water mixture as well as NaCl diet. In contrast, administration of
calcium and magnesium either via the food only or without NaCl
dietary supplementation does not produce results equivalent to
Process I.
[0277] 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.degree. 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 10 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. 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.
10TABLE 3 Comparison of the Survival of Atlantic Salmon S2 like
Smolts After Various Treatments Process I Parameter Control
Ca2+/Mg2+ Process I Water + NaCl Sampled Freshwater Diet Water Only
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 I mixture # of fish 22 2 60 128 % of fish 50% 6% 90% 99%
surviving 5 days after transfer to seawater .sup.1Survival
percentages expressed as rounded whole numbers
[0278] Application of the Process I to the Placement of 70-100 gm
Smolts in Seawater.
[0279] 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.
[0280] Experimental Protocol:
[0281] Smolts derived from the St. John strain of Atlantic salmon
produced by the Connors Brothers Deblois Hatchery located in
Cherryfield, Me., 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. S1 smolt were purchased during Maine's year
2000 smolt window and smolt deliveries were taken between the dates
of 29 Apr. 2000-15 May 2000. Smolts were either transferred
directly to Polar Circle netpens (24 m 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 (15 m.times.15 m.times.5 m)
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 I) or 120 days or greater (Control) fish. In both
cases, fish were removed from the netpens and multiple analyses
performed as described below.
[0282] All calculations to obtain feed conversion ratio (FCR) or
specific growth rate (SGR) and growth factor (GF3) were performed
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.
11TABLE 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
[0283] Table 5 displays data obtained after seawater transfer of
Control S1 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 day salmon
aquaculture in Maine. Significant mortalities (average 3.3%) were
experienced after transfer into cool (10.degree. C.) seawater and
full feeding was achieved only after a significant interval
(.about.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.
[0284] In contrast to the immediate transfer of Control S1 smolt as
described above to ocean netpens (Table 5), a total of 10,600 S1
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.degree. 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 10.degree. C. and
were equivalent to treated smolts prior to transfer to
seawater.
12TABLE 5 Characteristics of St. John S1 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 May 1, 2000 May 5, 2000 May 14, 2000 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
.+-. 74 305.80 .+-. 64 298.90 .+-. 37.40 Length (cm) 33.4 .+-. 1.9
28.30 .+-. 9.0 30.40 .+-. 1.17 Condition Factor (k) 1.02 1.34 1.06
SGR 0.96 1.10 1.17 during initial 120 days
[0285] During the 45 day interval when S1 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 Jun. 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.degree. 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.
[0286] 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 (1.5 m.times.1.5 m.times.1.5 m) 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.
[0287] 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 5. Process I fish were sampled 51 days after
their seawater placement and compared to the Industry Standard
smolts from Table 5. As shown in Table 6, comparison of their
characteristics reveals dramatic differences between Industry
Standard smolts vs Process I.
13TABLE 6 Comparison of the characteristics of St. John S1 Process
I Smolts subjected to Process I treatment and then placed in ocean
netpens vs corresponding industry standard smolts. Averaged
Industry Standard Data from Table 5 in this Process I Smolts
Example Total Fish 10,600 150,577 Mean Date of Seawater Jun. 26,
2000 May 7, 2000 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
[0288] In summary, notable differences between Process I, Control
smolt and Industry Standard smolt include:
[0289] 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.degree. 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.degree. 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.
[0290] 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 full feeding, the Process I smolts
fed vigorously within 2 days. Moreover, the initial growth rate
(SGR 1.8) demonstrated by Process I smolts are significantly
greater than published data for standard smolt during their initial
50 days after seawater placement (published values (Stradmeyer, L.
Is feeding nonstarters a waste of time. Fish Farmer 3:12-13, 1991;
Usher, M L, C Talbot and F B 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 osmoregulatory stress which would prevent them from
feeding immediately.
[0291] 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 782 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.
[0292] 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.
FIG. 24A 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.
[0293] FIG. 24B provides data on the characteristics of Process I
smolts after seawater transfer. These experiments were carried out
for over 185 days.
[0294] Application of the Process I to Atlantic Salmon Pre-Adult
Fish that are Smaller than the Industry Standard "Critical Size"
Smolt.
[0295] 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, Me., USA
on 1 Aug. 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.degree. 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 gram smolts above, the
mortality, feed consumption, growth and overall health of these 30
gram Process I smolts were monitored closely.
[0296] FIG. 25 displays the characteristics of a representative
sample of a larger group of 1,209 Process I smolts immediately
prior to their transfer to seawater. These parameters included an
average weight of 26.6+8.6 gram, length of 13.1+1.54 cm and
condition factor of 1.12+0.06. After seawater transfer, Process I
smolts exhibited a low initial mortality despite the fact that
their average body weight is 26-38% of industry standard 70-100
gram S0-S1 smolts. As shown in Table 7, Process I smolts mortality
within the initial 72 hr after seawater placement was 1/140 or 0.7%
for the laboratory tank. Ocean netpen mortalities after placement
of Process I smolts were 143/1069 or 13.4%. FIG. 25 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 experiments are described in co-pending application Ser. No:
09/975,553, Attorney Docket No: 2213.1004-001.
14TABLE 7 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 Transfer Sep. 5, 2000 (40); Sep.
12, 2000 Sep. 12, 2000 (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 hrs
[0297] FIG. 26 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
occurring after seawater placement of 1,069 Process I smolts were
plotted on data obtained from a 100 fish sampling as shown
previously in FIG. 25. Note that the mortalities are exclusively
distributed among the smaller fish within the larger Process I
netpen population.
[0298] 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 FIG. 26. 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).
[0299] Application of Process I to Trout Pre-Adult Fish that are
Smaller than the Industry Standard "Critical Size" Smolt.
[0300] Table 8 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 90-120
gram.
15TABLE 8 Comparison of the Survival of Rainbow Trout (3-5 gram) in
Seawater After Various Treatments. Percent Survival of Fish.sup.1
Constant 14 Constant 23 Hours Post Constant 14 day day Seawater
Control day Photoperiod Photoperiod + Transfer Freshwater
Photoperiod Process I Process I 0 100 100 100 100 24 0 25 80 99 48
0 70 81 72 40 68 96 30 58 120 30 46 Number of 10 20 30 80 Fish Per
Experiment .sup.1Survival percentages expressed as rounded whole
numbers
[0301] 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.degree. 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.
[0302] Application of the Process I to Arctic Char Pre-Adult Fish
that are Smaller than the Industry Standard "Critical Size"
Smolt.
[0303] Although arctic char are salmonids and anadromous fish,
their tolerance to seawater transfer is far less as compared to
either salmon or trout. FIG. 27 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 FIG. 27 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.
[0304] FIG. 27 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.
[0305] SECTION II: The Use of the Process II to Permit Successful
Transfer of 10-30 gram Smolt into Seawater Netpens and Tanks.
[0306] 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 S0 smolt.
[0307] Application of Process II to Atlantic Salmon
Fingerlings:
[0308] 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 Apr. 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.degree. C. and a standard
freshwater parr diet (Moore-Clark Feeds). On 17 Jul. 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 Aug. 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
II smolts were transported to ocean netpens by conventional truck
transport and placed into seawater (15.2.degree. C.) in either a
single ocean netpen identical to that described for placement of
Process I smolts or into laboratory tanks (15.6.degree. C.) within
the research facility.
[0309] FIG. 28 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. FIG. 28 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+/-1.5 cm and condition factor of
1.19+/-0.09. The data displayed in Tables 9 and 10 show 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).
[0310] 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 9, a
separate seawater transfer of St John/St John juvenile Atlantic
salmon possessing an average weight of 21 gms 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.
16TABLE 9 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 Aug. 9, 2001 Water Temperature (.degree. C.) 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
[0311]
17TABLE 10 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 Aug. 31, 2000 Sep. 5, 2000
Water Temperature (.degree. 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 13 0; 0% 1; 0.08% grams or greater
Time to achieve feeding 48 hrs 48 hrs after transfer
[0312] 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.
[0313] FIG. 29 compares characteristics of survivors and
mortalities of Process II smolts after seawater transfer to either
laboratory tanks (FIG. 29A) or ocean netpens (FIG. 29B). FIG. 29A
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, FIG. 29B displays only mortality data from ocean
netpen. In both cases, only smaller Process II smolts experienced
mortality. Note differences in Y axis scales of FIGS. 29A-B.
[0314] 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+/-1.5 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 II 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.
[0315] Quantitation of Feeding and Growth of Process I and II
smolts after Seawater Transfer:
[0316] 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.
[0317] The St John/St John Process II smolts fed vigorously when
first offered food 48 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.
[0318] The growth rates of identical fish treated with either
Process I or II within laboratory seawater tanks has been
quantified. As shown in Table 11, 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 70-100 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.
18TABLE 11 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
weight/day) 0.60 0.68 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.
[0319] In smolts that were exposed to 10 mM Ca.sup.2+ and 5.2 mM
Mg.sup.2+, 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.
[0320] Tissues were taken from either Atlantic salmon or rainbow
trout, after anesthesitizing the animal with MS-222. Samples of
tissues were then obtained by dissection, fixed by immersion in 3%
paraformaldehyde, washing in Ringers then frozen in an embedding
compound, e.g., O.C.T..TM. (Miles, Inc., Elkahart, Ind., 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.
[0321] The results are shown in FIGS. 30A-30G, 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. 31, 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.
[0322] FIGS. 30A and 30B 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 (1440.times.) shown in
FIG. 30B 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. 30C 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 FIGS. 30A and
30B including apical membrane staining (small arrowheads) as well
as larger globular round cells (arrows). FIG. 30D shows a
1900.times. magnification of PVCR-stained intestinal epithelia from
another representative fish that was exposed to the Process I and
maintained in freshwater containing 10 mM Ca2+ and 5.2 mM Mg2+ for
50 days and fed 1% NaCl in the diet. 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 FIGS. 30A-D, FIGS. 30E (1440.times.) and 13F (1900.times.)
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. 30G (1440.times.) shows the
lack of any apparent PVCR staining upon the substitution of
preimmune serum on a section corresponding to that shown in FIG.
30A where anti-PVCR antiserum identified the PVCR protein. The lack
of any PVCR staining with preimmune antiserum is a control to
demonstrate the specificity of the anti-PVCR antiserum under these
immunocytochemistry conditions.
[0323] The relative amount of PVCR protein present in intestinal
epithelial cells of freshwater smolts (FIGS. 30E and 30F) 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. 30A
and 30B). 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. 30C and 30D). The AEC staining was specific for the presence
of the anti-PVCR antiserum, since substitution of the immune
antiserum by the preimmune eliminated all reaction product from
intestinal epithelial cell sections (FIG. 30G).
[0324] 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.
[0325] 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.
[0326] 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 hormones (e.g.,
5-hydroxytryptamine (5-HT), serotonin, or cholecystokinin (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.
[0327] 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,
NaCl 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.
[0328] To further demonstrate the specificity of the anti-CaR
antiserum to recognize salmon smolt PVCRs, FIG. 31 shows a Western
blot of intestinal protein from salmon smolt maintained in 10 mM
Ca2+, 5 mM Mg2+and fed 1% NaCl in the diet. Portions 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, Ill.) 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. 30G, and shows a complete lack of
bands. Taken together with immunocytochemistry data shown in FIG.
30, this immunoblot demonstrates that the antiserum 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.
[0329] 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.
[0330] Methods: Samples of the skin from juvenile Atlantic Salmon
resident in seawater for over 12 days were fixed in 3%
paraformaldehyde dissolved in buffer (0.1 M NaP04, 0.15M NaCl, 0.3M
sucrose pH 7.4), manually descaled, rinsed in buffer and frozen at
-80.degree. 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.
[0331] Results and Discussion: FIG. 32A shows that salmon epidermis
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. FIG. 32B 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 FIG. 32A. 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.
FIG. 32C 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.
[0332] 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
Demonstrationk 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.
[0333] 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 perform
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 PVCR content of 14 tissues from a
single juvenile Atlantic salmon, as shown in FIG. 33.
[0334] Description of Experimental Protocol:
[0335] 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 MgCl2, 10 mM KCl, 1 mM
Phenylmethylsulfonyl fluoride (PMSF), 0.5 dithiothreitol (DTT) and
1 mM benzamidine pH 8.8) and using a standard glass
Potter-Elvenhiem homogenizer with a rotary pestle. After
centrifugation at 2,550.times.g for 20 min. at 4.degree. C. to
remove larger debris, the supernatant was either used directly or
frozen at -80.degree. 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 KCl pH 8.0), primary
antiserum (either rabbit anti-PVCR immune or corresponding rabbit
pre-immune antiserum) at a 1:1500 dilution was added. After a 1 hr
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 405 nm. Relative amounts of
tissue PVCR content were determined after corrections for minimal
background and nonspecific antibody binding as measured by binding
of preimmune antiserum.
[0336] Results and Data Interpretation:
[0337] FIG. 33 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 of juvenile 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
Antibodies Made from the Carboxyl Terminal Portion of an Atlantic
Salmon PVCR Protein are Effective in Immunocytochemistry and
Immunoblotting Assays to Determine the Presence, Absence or Amount
of the PVCR Protein
[0338] Degenerate primers, dSK-F3 (SEQ ID NO: 13) and dSK-R4 (SEQ
ID NO: 14), 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.
[0339] 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 using .sup.32P-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 frame 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.
[0340] 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:
[0341] The peptide sequence for antibody production is as
follows:
19 Peptide #1: Ac-CTNDNDSPSGQQRIHK-amide (SEQ ID NO.:15)
[0342] producing rabbit antiserum SAL-1
[0343] The peptide was derivatized to carrier proteins and utilized
to raise peptide specific antiserum in two rabbits using methods
for making a polyclonal antibody.
[0344] 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
immunoblot was taken showing protein bands that were recognized by
antisera raised against peptides containing either SAL-1 (SEQ ID
NO.: 15) 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-SalI, 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 antiserum 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.
[0345] The Sal I antiserum is also useful in localization of
SalmoKCaR proteins in larval Atlantic salmon (See FIG. 37B). 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.
[0346] 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.
[0347] A second peptide sequence was used for antibody
production:
20 Peptide #2: CSDDEYGRPGIEKFEKEM. (SEQ ID NO: 27)
[0348] 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 I. (See
FIG. 37C). Since both SalmoKCaR #1 and #2 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 and #2 but not #3 within the
kidney of Atlantic salmon.
[0349] In contrast, the Salmo ADD antibody binds to a peptide
sequence present in the extracellular domain of all 3 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
[0350] In Example 4, 2 degenerate primers, dSK-F3 (SEQ ID NO: 13)
and dSK-R4 (SEQ ID NO: 14), 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:
[0351] 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.
[0352] 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.
[0353] 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.
[0354] RT-PCR Method:
[0355] Total RNA was purified from selected tissues using Teltest B
reagent (Friendswood, Tex.) and accompanying standard protocol. A
total of 5 micrograms of total RNA was reverse transcribed with
oligo dT primers using Invitrogen's cDNA Cycle Kit (Invitrogen Inc,
Madison, Wis.). The resulting cDNA product was denatured and a
second round of purification was performed. Two microliters of the
resulting reaction mixture was amplified in a PCR reaction (30
cycles of 1 min. @94.degree. C., 2 min. @57.degree. C., 3 min.
@72.degree. C.) using degenerate primers dSK-F3 (SEQ ID NO: 13) and
dSK-R4 (SEQ ID NO: 14). 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.
[0356] DNA Sequencing of RT-PCR Products were Performed as
Follows:
[0357] 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 HR spin columns (Amersham Inc,
Piscataway, N.J.). Purified DNA was sequenced using degenerate PVCR
primers (SEQ ID NO.: 13 and 14) as sequencing primers. Automated
sequencing was performed using an Applied Biosystems Inc. Model
373A Automated DNA Sequencer (University of Maine, Orono, Me.). The
resulting DNA sequences were aligned using MacVector (GCG) and
LaserGene (DNA STAR) sequence analysis software.
[0358] Detection of Amplified RT-PCR cDNA products by Southern
Blotting:
[0359] Alternatively, the presence of amplified PVCR products was
detected by Southern blotting analyses of gel fractionated RT-PCR
products using a .sup.32P-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, Mass.). After
crosslinking of the DNA, blots were prehybridized and then probed
overnight (68.degree. C. in 6.times.SSC, 5.times. Denhardt's
Reagent, 0.5% SDS, 100 ug/ml calf thymus DNA) with the 653 bp
Atlantic salmon PCR product (labeled with RadPrime DNA Labeling
System, Gibco Life Sciences). Blots were then washed with
0.1.times.SSC, 0.1% SDS @55.degree. C. and subjected to
autoradiography under standard conditions.
[0360] FIG. 34 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
.sup.32P-labeled 653 bp genomic DNA fragment from the Atlantic
salmon PVCR gene. FIG. 34 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] 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 environment) 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.
[0365] One feature of this biological system is alteration in the
sensitivity of the PVCR protein for divalent cations such as
Ca.sup.2+ and Mg.sup.2+ by changes in the NaCl concentration of the
water. Thus, PVCRs in fish olfactory organs have different apparent
sensitivity to Ca.sup.2+ 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.
[0366] 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:
[0367] 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
[0368] 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 olfactory apparatus of salmon
possess the capacity of modulating responses to various
odorants.
[0369] 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 spermidine) are attractants to
marine organisms including fish and crustaceans, these data provide
for another means by which PVCR proteins would serve not only as
modulators of olfaction in fish but also as sensors of amino acids
and polyamines themselves. PVCR proteins in other organs 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.
[0370] Description of Experimental Results and Data
Interpretation:
[0371] 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:
[0372] FIG. 35 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.
[0373] 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 FIG. 36, 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. FIG. 37 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 (FIG. 37B). 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. SalmoKCaR 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.
[0374] 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 (FIG. 38).
Use of other methodology including RT-PCR using specific degenerate
primers (FIG. 39) and ELISA methods (FIG. 40) 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.
[0375] Measurement of Extracellular Electrical Potentials (EEG's)
from Olfactory Nerve from Freshwater Adapted Atlantic Salmon
Reveals the Presence of Functional PVCR Proteins:
[0376] FIG. 41 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, P C, et al., Olfactory sensitivity to changes
in environmental Ca2+ in the marine teleost Sparus Aurata, J. 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.
[0377] As shown in FIG. 41, irrigation of salmon olfactory
epithelium with distilled water produces minimal generation of
large signals in olfactory nerve. The data in FIG. 41 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 1 mM Ca.sup.2+ or 250 mM NaCl also
produce responses in EEG activity. To test for the presence of
functional PVCR protein, the olfactory epithelium was exposed to 50
micromolar gadolinium (Gd.sup.3+-a PVCR agonist) and also obtained
a response. FIG. 42 shows dose response data from multiple fish to
various PVCR agonists or modulators where the relative magnitudes
of individual olfactory nerve response were normalized relative to
the response produced by the exposure of the olfactory epithelium
to 10 mM Ca.sup.2+. As shown in FIG. 42, the olfactory epithelium
of freshwater adapted juvenile salmon is very sensitive to
Ca.sup.2+ where the half maximal excitatory response (EC.sub.50) is
approximately 1-10 micromolar. Similarly, exposure of olfactory
epithelium to the PVCR agonist Gd.sup.3+ produces responses of a
similar magnitude to those evoked by Ca.sup.2+ in a concentration
range of 1-10 micromolar. In contrast, olfactory epithelium
responses to Mg.sup.2+ do not occur until 10-100 micromolar
solutions are applied. These dose response curves (EC.sub.50
Gd.sup.+3.ltoreq.Ca.sup.2+<Mg.su- p.2+) are similar to those
obtained for PVCR modulated responses in other fish epithelium
(flounder urinary bladder NaCl-mediated water transport-see SKCaR
application).
[0378] In contrast, analysis of the olfactory epithelium responses
to NaCl exposure shows that it is unresponsive until a
concentration of 250 millimolar NaCl is applied. Since NaCl does
not directly activate PVCRs in a manner such as Gd.sup.+3 Ca.sup.2+
or Mg.sup.2+ 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 NaCl likely occurs via
other PVCR independent mechanisms.
[0379] 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.
[0380] 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:
[0381] FIG. 43 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 5 mM Ca.sup.2+ or 50 micromolar Gd.sup.3+
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 mammalian finger rinse. The ablation of responses to both
the PVCR agonists as shown in FIG. 42 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.
[0382] Addition of PVCR agonists such as Ca2+ or Gd3+ but not NaCl
to distilled water containing the well known salmon attractant
L-alanine reversibly ablates the response of the olfactory
epithelium to these stimuli:
[0383] FIG. 44 shows a time series of stimuli (2 min between each
stimulus in a single fish) similar to that displayed on FIG. 43
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 mM Ca.sup.2+ (recording #2) or 50 micromolar Gd.sup.3+
(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 Gd.sup.3+ 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 NaCl
resulted initially in a similar response (recordings #4 and #9)
followed by an enhanced response to L-Alanine alone (recordings #5
and #10).
[0384] In summary, the data displayed in FIGS. 43 and 44 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.sup.2+ enhances the response of SKCaR to ambient Ca.sup.2+
concentrations. Regardless of their nature, these negative
modulatory effects of PVCR agonists including Ca.sup.2+ 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.
[0385] Recombinant PVCR Protein SKCaR Possesses the Capability to
Sense Concentrations of Amino Acids After its Expression in Human
Embryonic Kidney (HEK) Cells:
[0386] 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.
[0387] FIG. 45 shows a comparison of fluorescence tracings of
FURA2-loaded cells stably expressing SKCaR that were bathed in
physiological saline (125 mM NaCl, 4 mM KCl, 0.5 mM CaCl.sub.2, 0.5
MgCl.sub.2, 20 mM HEPES (NaOH), 0.1% D-glucose pH 7.4) in the
presence or absence of 10 mM L-Isoleucine (L-Ile) before being
placed into the fluorimeter. Baseline extracellular Ca.sup.2+
concentration was 0.5 mM. Aliquots of Ca.sup.2+ were added to
produce final extracellular concentrations of 2.5 mM, 5 mM, 7.5 mM,
10 mM and 20 mM Ca.sup.2+ with changes in the fluorescence
recorded. Note that increases in cell fluorescence were greater in
the presence of 10 mM Phe for extracellular Ca.sup.2+
concentrations less than 10 mM.
[0388] FIG. 46 shows data plotted from multiple experiments as
described in FIG. 45 where the effects of 10 mM Phe, 10 mM 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 Gln, 125 Ser, 30
Glu, 250 Gly, 180 Pro, 250 Val, 30 Met, 10 Asp, 200 Lys, 100 Arg,
75 Ile, 150 Leu. Note that both 10 mM Phe and 10 mM Ile as well as
the mixture of amino acids increase SKCaR's response to a given
Ca.sup.2+ concentration. Thus, these data show that presence of
amino acids either alone or in combination increase the apparent
sensitivity to Ca.sup.2+ permitting SKCaR to "sense"amino acids in
the presence of physiological concentrations of Ca.sup.2+. These
data obtained for SKCaR are comparable to those obtained for the
human CaR.
[0389] The significance of these data for aquatic organisms stand
in marked contrast to the roles of human CaRs amino acid sensing
capabilities. FIG. 45 shows that SKCaR's maximal capability to
sense amino acids is confined to a range of Ca.sup.2+ 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 inform 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.sup.2+, Mg.sup.2+ and NaCl) 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.
[0390] 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.
[0391] Companion Patent Application Nos. (not yet assigned;
Attorney Docket No: 2213.1006-005 and 2213.1006-006), both entitled
"Polyvalent Cation-sensing Receptor in Atlantic Salmon," filed on
Apr. 18, 2002; patent application Ser. No. 09/687,373, entitled
"Growing Marine Fish in Fresh Water," filed on Oct. 12, 2000; PCT
Application No.: PCT/US01/31625, entitled "Growing Marine Fish in
Fresh Water," filed Oct. 11, 2001; patent application Ser. No.
09/687,476, entitled "Methods for Raising Pre-adult Anadromous
Fish," filed on Oct. 12, 2000; patent application Ser. No.
09/687,372, entitled "Methods for Raising Pre-adult Anadromous
Fish," filed on Oct. 12, 2000; patent application Ser. No.
09/687,477, entitled "Methods for Raising Pre-adult Anadromous
Fish," filed on Oct. 12, 2000; patent application Ser. No.
09/975,553, entitled "Methods for Raising Pre-adult Anadromous
Fish," filed on Oct. 11, 2001; International PCT Application No.
PCT/US01/31562, entitled, "Methods for Raising Pre-adult Anadromous
Fish," filed on Oct. 11, 2001; Provisional Patent Application No.
60/382,464, "Methods for Growing and Imprinting Fish Using an
Odorant," filed Oct. 11, 2001; are all hereby incorporated by
reference in their entirety.
[0392] Additionally, U.S. Pat. No 6,334,391, issued on Jan. 8,
2002, International PCT application No. PCT/US97/05031, filed on
Mar. 27, 1997, and application Ser. No. 08/622,738 filed Mar. 27,
1996, all entitled, "Polycation Sensing Receptor in Aquatic Species
and Methods of Use Thereof" are all hereby incorporated by
reference in their entirety.
[0393] All relevant portions of literature articles, references,
patent applications, patent publications, and patents cited herein
are hereby incorporated by referenced in their entirety.
[0394] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
28 1 4134 DNA Squalus acanthias 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 tgggaggtct cttcccaata cactttggag tagccgccaa ggatcaggac
600 ttaaaatcga gaccggaggc gacaaaatgt attcggtaca attttcgagg
cttccgatgg 660 ctccaggcga tgatattcgc aattgaagag attaacaaca
gtatgacttt cctgcccaat 720 atcaccctgg gatatcgcat atttgacacg
tgtaacaccg tgtccaaggc gctagaggca 780 acactcagct ttgtggccca
gaacaaaatc gactcgctga acttagatga gttctgtaac 840 tgctctgacc
atatcccatc cacaatagca gtggtcgggg caaccgggtc aggaatctcc 900
acggctgtgg ccaatctatt gggattattt tacattccac aggtcagcta tgcctcctcg
960 agcaggctgc tcagcaacaa gaatgagtac aaggccttcc tgaggaccat
ccccaatgat 1020 gagcaacagg ccacggccat ggccgagatc atcgagcact
tccagtggaa ctgggtggga 1080 accctggcag ccgacgatga ctatggccgc
ccaggcattg acaagttccg ggaggaggcc 1140 gttaagaggg acatctgtat
tgacttcagt gagatgatct ctcagtacta cacccagaag 1200 cagttggagt
tcatcgccga cgtcatccag aactcctcgg ccaaggtcat cgtggtcttc 1260
tccaatggcc ccgacctgga gccgctcatc caggagatag ttcggagaaa catcaccgat
1320 cggatctggc tggccagcga ggcttgggcc agctcttcgc tcattgccaa
gccagagtac 1380 ttccacgtgg tcggcggcac catcggcttc gctctcaggg
cggggcgtat cccagggttc 1440 aacaagttcc tgaaggaggt ccaccccagc
aggtcctcgg acaatgggtt tgtcaaggag 1500 ttctgggagg agaccttcaa
ctgctacttc accgagaaga ccctgacgca gctgaagaat 1560 tccaaggtgc
cctcgcacgg accggcggct caaggggacg gctccaaggc ggggaactcc 1620
agacggacag ccctacgcca cccctgcact ggggaggaga acatcaccag cgtggagacc
1680 ccctacctgg attatacaca cctgaggatc tcctacaatg tatacgtggc
cgtctactcc 1740 attgctcacg ccctgcaaga catccactct tgcaaacccg
gcacgggcat ctttgcaaac 1800 ggatcttgtg cagatattaa aaaagttgag
gcctggcagg tcctcaacca tctgctgcat 1860 ctgaagttta ccaacagcat
gggtgagcag gttgactttg acgatcaagg tgacctcaag 1920 gggaactaca
ccattatcaa ctggcagctc tccgcagagg atgaatcggt gttgttccat 1980
gaggtgggca actacaacgc ctacgctaag cccagtgacc gactcaacat caacgaaaag
2040 aaaatcctct ggagtggctt ctccaaagtg gttcctttct ccaactgcag
tcgagactgt 2100 gtgccgggca ccaggaaggg gatcatcgag ggggagccca
cctgctgctt tgaatgcatg 2160 gcatgtgcag agggagagtt cagtgatgaa
aacgatgcaa gtgcgtgtac aaagtgcccg 2220 aatgatttct ggtcgaatga
gaaccacacg tcgtgcatcg ccaaggagat cgagtacctg 2280 tcgtggacgg
agcccttcgg gatcgctctg accatcttcg ccgtactggg catcctgatc 2340
acctccttcg tgctgggggt cttcatcaag ttcaggaaca ctcccatcgt gaaggccacc
2400 aaccgggagt tgtcctacct gctgctcttc tccctcatct gctgcttctc
cagctcgctc 2460 atcttcatcg gcgagcccag ggactggacc tgtcggctcc
gccaaccggc ctttggcatc 2520 agcttcgtcc tgtgcatctc ctgcatcctg
gtgaagacca accgggtgct gctggtcttc 2580 gaggccaaga tccccaccag
cctccaccgc aagtgggtgg gcctcaacct gcagttcctc 2640 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 2 1027 PRT
Squalus acanthias 2 Met Ala Gln Leu His Cys Gln Leu Leu Phe Leu Gly
Phe Thr Leu Leu 1 5 10 15 Gln Ser Tyr Asn Val Ser Gly Tyr Gly Pro
Asn Gln Arg Ala Gln Lys 20 25 30 Lys Gly Asp Ile Ile Leu Gly Gly
Leu Phe Pro Ile His Phe Gly Val 35 40 45 Ala Ala Lys Asp Gln Asp
Leu Lys Ser Arg Pro Glu Ala Thr Lys Cys 50 55 60 Ile Arg Tyr Asn
Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe 65 70 75 80 Ala Ile
Glu Glu Ile Asn Asn Ser Met Thr Phe Leu Pro Asn Ile Thr 85 90 95
Leu Gly Tyr Arg Ile Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu 100
105 110 Glu Ala Thr Leu Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu
Asn 115 120 125 Leu Asp Glu Phe Cys Asn Cys Ser Asp His Ile Pro Ser
Thr Ile Ala 130 135 140 Val Val Gly Ala Thr Gly Ser Gly Ile Ser Thr
Ala Val Ala Asn Leu 145 150 155 160 Leu Gly Leu Phe Tyr Ile Pro Gln
Val Ser Tyr Ala Ser Ser Ser Arg 165 170 175 Leu Leu Ser Asn Lys Asn
Glu Tyr Lys Ala Phe Leu Arg Thr Ile Pro 180 185 190 Asn Asp Glu Gln
Gln Ala Thr Ala Met Ala Glu Ile Ile Glu His Phe 195 200 205 Gln Trp
Asn Trp Val Gly Thr Leu Ala Ala Asp Asp Asp Tyr Gly Arg 210 215 220
Pro Gly Ile Asp Lys Phe Arg Glu Glu Ala Val Lys Arg Asp Ile Cys 225
230 235 240 Ile Asp Phe Ser Glu Met Ile Ser Gln Tyr Tyr Thr Gln Lys
Gln Leu 245 250 255 Glu Phe Ile Ala Asp Val Ile Gln Asn Ser Ser Ala
Lys Val Ile Val 260 265 270 Val Phe Ser Asn Gly Pro Asp Leu Glu Pro
Leu Ile Gln Glu Ile Val 275 280 285 Arg Arg Asn Ile Thr Asp Arg Ile
Trp Leu Ala Ser Glu Ala Trp Ala 290 295 300 Ser Ser Ser Leu Ile Ala
Lys Pro Glu Tyr Phe His Val Val Gly Gly 305 310 315 320 Thr Ile Gly
Phe Ala Leu Arg Ala Gly Arg Ile Pro Gly Phe Asn Lys 325 330 335 Phe
Leu Lys Glu Val His Pro Ser Arg Ser Ser Asp Asn Gly Phe Val 340 345
350 Lys Glu Phe Trp Glu Glu Thr Phe Asn Cys Tyr Phe Thr Glu Lys Thr
355 360 365 Leu Thr Gln Leu Lys Asn Ser Lys Val Pro Ser His Gly Pro
Ala Ala 370 375 380 Gln Gly Asp Gly Ser Lys Ala Gly Asn Ser Arg Arg
Thr Ala Leu Arg 385 390 395 400 His Pro Cys Thr Gly Glu Glu Asn Ile
Thr Ser Val Glu Thr Pro Tyr 405 410 415 Leu Asp Tyr Thr His Leu Arg
Ile Ser Tyr Asn Val Tyr Val Ala Val 420 425 430 Tyr Ser Ile Ala His
Ala Leu Gln Asp Ile His Ser Cys Lys Pro Gly 435 440 445 Thr Gly Ile
Phe Ala Asn Gly Ser Cys Ala Asp Ile Lys Lys Val Glu 450 455 460 Ala
Trp Gln Val Leu Asn His Leu Leu His Leu Lys Phe Thr Asn Ser 465 470
475 480 Met Gly Glu Gln Val Asp Phe Asp Asp Gln Gly Asp Leu Lys Gly
Asn 485 490 495 Tyr Thr Ile Ile Asn Trp Gln Leu Ser Ala Glu Asp Glu
Ser Val Leu 500 505 510 Phe His Glu Val Gly Asn Tyr Asn Ala Tyr Ala
Lys Pro Ser Asp Arg 515 520 525 Leu Asn Ile Asn Glu Lys Lys Ile Leu
Trp Ser Gly Phe Ser Lys Val 530 535 540 Val Pro Phe Ser Asn Cys Ser
Arg Asp Cys Val Pro Gly Thr Arg Lys 545 550 555 560 Gly Ile Ile Glu
Gly Glu Pro Thr Cys Cys Phe Glu Cys Met Ala Cys 565 570 575 Ala Glu
Gly Glu Phe Ser Asp Glu Asn Asp Ala Ser Ala Cys Thr Lys 580 585 590
Cys Pro Asn Asp Phe Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala 595
600 605 Lys Glu Ile Glu Tyr Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala
Leu 610 615 620 Thr Ile Phe Ala Val Leu Gly Ile Leu Ile Thr Ser Phe
Val Leu Gly 625 630 635 640 Val Phe Ile Lys Phe Arg Asn Thr Pro Ile
Val Lys Ala Thr Asn Arg 645 650 655 Glu Leu Ser Tyr Leu Leu Leu Phe
Ser Leu Ile Cys Cys Phe Ser Ser 660 665 670 Ser Leu Ile Phe Ile Gly
Glu Pro Arg Asp Trp Thr Cys Arg Leu Arg 675 680 685 Gln Pro Ala Phe
Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu 690 695 700 Val Lys
Thr Asn Arg Val Leu Leu Val Phe Glu Ala Lys Ile Pro Thr 705 710 715
720 Ser Leu His Arg Lys Trp Val Gly Leu Asn Leu Gln Phe Leu Leu Val
725 730 735 Phe Leu Cys Ile Leu Val Gln Ile Val Thr Cys Ile Ile Trp
Leu Tyr 740 745 750 Thr Ala Pro Pro Ser Ser Tyr Arg Asn His Glu Leu
Glu Asp Glu Val 755 760 765 Ile Phe Ile Thr Cys Asp Glu Gly Ser Leu
Met Ala Leu Gly Phe Leu 770 775 780 Ile Gly Tyr Thr Cys Leu Leu Ala
Ala Ile Cys Phe Phe Phe Ala Phe 785 790 795 800 Lys Ser Arg Lys Leu
Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Thr 805 810 815 Phe Ser Met
Leu Ile Phe Phe Ile Val Trp Ile Ser Phe Ile Pro Ala 820 825 830 Tyr
Val Ser Thr Tyr Gly Lys Phe Val Ser Ala Val Glu Val Ile Ala 835 840
845 Ile Leu Ala Ser Ser Phe Gly Leu Leu Gly Cys Ile Tyr Phe Asn Lys
850 855 860 Cys Tyr Ile Ile Leu Phe Lys Pro Cys Arg Asn Thr Ile Glu
Glu Val 865 870 875 880 Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val
Ala Ala Arg Ala Thr 885 890 895 Leu Arg Arg Ser Ala Ala Ser Arg Lys
Arg Ser Ser Ser Leu Cys Gly 900 905 910 Ser Thr Ile Ser Ser Pro Ala
Ser Ser Thr Cys Gly Pro Gly Leu Thr 915 920 925 Met Glu Met Gln Arg
Cys Ser Thr Gln Lys Val Ser Phe Gly Ser Gly 930 935 940 Thr Val Thr
Leu Ser Leu Ser Phe Glu Glu Thr Gly Arg Tyr Ala Thr 945 950 955 960
Leu Ser Arg Thr Ala Arg Ser Arg Asn Ser Ala Asp Gly Arg Ser Gly 965
970 975 Asp Asp Leu Pro Ser Arg His His Asp Gln Gly Pro Pro Gln Lys
Cys 980 985 990 Glu Pro Gln Pro Ala Asn Asp Ala Arg Tyr Lys Ala Ala
Pro Thr Lys 995 1000 1005 Gly Thr Leu Glu Ser Pro Gly Gly Ser Lys
Glu Arg Pro Thr Thr 1010 1015 1020 Met Glu Glu Thr 1025 3 594 DNA
Salmo salar 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 4 197 PRT
Salmo salar misc_feature (171)..(171) Xaa = Any Amino Acid 4 Leu
Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met 1 5 10
15 Gly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr Asn
20 25 30 Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys
Phe Ser 35 40 45 Ser Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp
Thr Cys Arg Leu 50 55 60 Arg Gln Pro Ala Phe Gly Ile Ser Phe Val
Leu Cys Ile Ser Cys Ile 65 70 75 80 Leu Val Lys Thr Asn Arg Val Leu
Leu Val Phe Glu Ala Lys Ile Pro 85 90 95 Thr Ser Leu His Arg Lys
Trp Trp Gly Leu Asn Leu Gln Phe Leu Leu 100 105 110 Val Phe Leu Phe
Thr Phe Val Gln Val Met Ile Cys Val Val Trp Leu 115 120 125 Tyr Asn
Ala Pro Pro Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile 130 135 140
Ile Phe Ile Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu 145
150 155 160 Ile Gly Tyr Thr Cys Leu Leu Ala Ala Ile Xaa Phe Phe Phe
Ala Phe 165 170 175 Lys Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala
Lys Phe Ile Thr 180 185 190 Phe Ser Met Leu Ile 195 5 2021 DNA
Salmo salar 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 6 388 PRT Salmo salar 6 Tyr
Lys His Tyr Phe Ser Ile Pro Ala Asp Ala Ser Val Cys Thr Lys 1 5 10
15 Cys Pro Asn Asp Ser Trp Ser Asn Glu Asn His Thr Ser Cys Phe Leu
20 25 30 Lys Glu Ile Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile
Ala Leu 35 40 45 Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala
Phe Val Met Gly 50 55 60 Val Phe Ile Lys Phe Arg Asn Thr Pro Ile
Val Lys Ala Thr Asn Arg 65 70 75 80 Glu Leu Ser Tyr Leu Leu Leu Phe
Ser Leu Ile Cys Cys Phe Ser Ser 85
90 95 Ser Leu Ile Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg Leu
Arg 100 105 110 Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser
Cys Ile Leu 115 120 125 Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu
Ala Lys Ile Pro Thr 130 135 140 Ser Leu His Arg Lys Trp Trp Gly Leu
Asn Leu Gln Phe Leu Leu Val 145 150 155 160 Phe Leu Phe Thr Phe Val
Gln Val Met Ile Cys Val Val Trp Leu Tyr 165 170 175 Asn Ala Pro Pro
Ala Ser Tyr Arg Asn His Asp Ile Asp Glu Ile Ile 180 185 190 Phe Ile
Thr Cys Asn Glu Gly Ser Met Met Ala Leu Gly Phe Leu Ile 195 200 205
Gly Tyr Thr Cys Leu Leu Ala Ala Ile Cys Phe Phe Phe Ala Phe Lys 210
215 220 Ser Arg Lys Leu Pro Glu Asn Phe Thr Glu Ala Lys Phe Ile Thr
Phe 225 230 235 240 Ser Met Leu Ile Phe Phe Ile Val Trp Ile Ser Phe
Ile Pro Ala Tyr 245 250 255 Phe Ser Thr Tyr Gly Lys Phe Val Ser Ala
Val Glu Val Ile Ala Ile 260 265 270 Leu Ala Ser Ser Phe Gly Leu Leu
Ala Cys Ile Phe Phe Asn Lys Val 275 280 285 Tyr Ile Ile Leu Phe Lys
Pro Ser Arg Asn Thr Ile Glu Glu Val Arg 290 295 300 Cys Ser Thr Ala
Ala His Ser Phe Lys Val Ala Ala Lys Ala Thr Leu 305 310 315 320 Arg
His Ser Ser Ala Ser Arg Lys Arg Ser Ser Ser Val Gly Gly Ser 325 330
335 Cys Ala Ser Thr Pro Ser Ser Ser Ile Ser Leu Lys Thr Asn Asp Asn
340 345 350 Asp Ser Pro Ser Gly Gln Gln Arg Ile His Lys Pro Arg Val
Ser Phe 355 360 365 Gly Ser Gly Thr Val Thr Leu Ser Leu Ser Phe Glu
Glu Ser Arg Lys 370 375 380 Asn Ser Met Lys 385 7 3941 DNA Salmo
salar 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 960 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 8 941 PRT Salmo
salar 8 Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser
Val 1 5 10 15 Ile Ser Thr Tyr Gly Pro His Gln Arg Ala Gln Lys Thr
Gly Asp Ile 20 25 30 Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly
Val Thr Ser Lys Asp 35 40 45 Gln Asp Leu Ala Ala Arg Pro Glu Ser
Thr Glu Cys Val Arg Tyr Asn 50 55 60 Phe Arg Gly Phe Arg Trp Leu
Gln Ala Met Ile Phe Ala Ile Glu Glu 65 70 75 80 Ile Asn Asn Ser Ser
Thr Leu Leu Pro Asn Ile Thr Leu Gly Tyr Arg 85 90 95 Ile Phe Asp
Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu 100 105 110 Ser
Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe 115 120
125 Cys Asn Cys Thr Asp His Ile Pro Ser Thr Ile Ala Val Val Gly Ala
130 135 140 Ser Gly Ser Ala Val Ser Thr Ala Val Ala Asn Leu Leu Gly
Leu Phe 145 150 155 160 Tyr Ile Pro Gln Ile Ser Tyr Ala Ser Ser Ser
Arg Leu Leu Ser Asn 165 170 175 Lys Asn Gln Phe Lys Ser Phe Met Arg
Thr Ile Pro Thr Asp Glu His 180 185 190 Gln Ala Thr Ala Met Ala Asp
Ile Ile Asp Tyr Phe Gln Trp Asn Trp 195 200 205 Val Ile Ala Val Ala
Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu 210 215 220 Lys Phe Glu
Lys Glu Met Glu Glu Arg Asp Ile Cys Ile His Leu Ser 225 230 235 240
Glu Leu Ile Ser Gln Tyr Phe Glu Glu Trp Gln Ile Gln Gly Leu Val 245
250 255 Asp Arg Ile Glu Asn Ser Ser Ala Lys Val Ile Val Val Phe Ala
Ser 260 265 270 Gly Pro Asp Ile Glu Pro Leu Ile Lys Glu Met Val Arg
Arg Asn Ile 275 280 285 Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp
Ala Thr Thr Ser Leu 290 295 300 Ile Ala Lys Pro Glu Tyr Leu Asp Val
Val Val Gly Thr Ile Gly Phe 305 310 315 320 Ala Leu Arg Ala Gly Glu
Ile Pro Gly Phe Lys Asp Phe Leu Gln Glu 325 330 335 Val Thr Pro Lys
Lys Ser Ser His Asn Glu Phe Val Arg Glu Phe Trp 340 345 350 Glu Glu
Thr Phe Asn Cys Tyr Leu Glu Asp Ser Gln Arg Leu Arg Asp 355 360 365
Ser Glu Asn Gly Ser Thr Ser Phe Arg Pro Leu Cys Thr Gly Glu Glu 370
375 380 Asp Ile Met Gly Ala Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu
Arg 385 390 395 400 Ile Ser Tyr Asn Val Tyr Val Ala Val His Ser Ile
Ala Gln Ala Leu 405 410 415 Gln Asp Ile Leu Thr Cys Ile Pro Gly Arg
Gly Leu Phe Ser Asn Asn 420 425 430 Ser Cys Ala Asp Ile Lys Lys Ile
Glu Ala Trp Gln Val Leu Lys Gln 435 440 445 Leu Arg His Leu Asn Phe
Ser Asn Ser Met Gly Glu Lys Val His Phe 450 455 460 Asp Glu Asn Ala
Asp Pro Ser Gly Asn Tyr Thr Ile Ile Asn Trp His 465 470 475 480 Arg
Ser Pro Glu Asp Gly Ser Val Val Phe Glu Glu Val Gly Phe Tyr 485 490
495 Asn Met Arg Ala Lys Arg Gly Val Gln Leu Phe Ile Asp Asn Thr Lys
500 505 510 Ile Leu Trp Asn Gly Tyr Asn Thr Glu Val Pro Phe Ser Asn
Cys Ser 515 520 525 Glu Asp Cys Glu Pro Gly Thr Arg Lys Gly Ile Ile
Glu Ser Met Pro 530 535 540 Thr Cys Cys Phe Glu Cys Thr Glu Cys Ser
Glu Gly Glu Tyr Ser Asp 545 550 555 560 His Lys Asp Ala Ser Val Cys
Thr Lys Cys Pro Asn Asp Ser Trp Ser 565 570 575 Asn Glu Asn His Thr
Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser 580 585 590 Trp Thr Glu
Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly 595 600 605 Val
Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn 610 615
620 Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu
625 630 635 640 Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe
Ile Gly Glu 645 650 655 Pro Gln Asp Trp Thr Cys Arg Leu Arg Gln Pro
Ala Phe Gly Ile Ser 660 665 670 Phe Val Leu Cys Ile Ser Cys Ile Leu
Val Lys Thr Asn Arg Val Leu 675 680 685 Leu Val Phe Glu Ala Lys Ile
Pro Thr Ser Leu His Arg Lys Trp Trp 690 695 700 Gly Leu Asn Leu Gln
Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gln 705 710 715 720 Val Met
Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr 725 730 735
Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly 740
745 750 Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu
Ala 755 760 765 Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu
Pro Glu Asn 770 775 780 Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met
Leu Ile Phe Phe Ile 785 790 795 800 Val Trp Ile Ser Phe Ile Pro Ala
Tyr Phe Ser Thr Tyr Gly Lys Phe 805 810 815 Val Ser Ala Val Glu Val
Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu 820 825 830 Leu Ala Cys Ile
Phe Phe Asn Lys Val Tyr Ile Ile Leu Phe Lys Pro 835 840 845 Ser Arg
Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala His Ser 850 855 860
Phe Lys Val Ala Ala Lys Ala Thr Leu Arg His Ser Ser Ala Ser Arg 865
870 875 880 Lys Arg Ser Ser Ser Val Gly Gly Ser Cys Ala Ser Thr Pro
Ser Ser 885 890 895 Ser Ile Ser Leu Lys Thr Asn Asp Asn Asp Ser Pro
Ser Gly Gln Gln 900 905 910 Arg Ile His Lys Pro Arg Val Ser Phe Gly
Ser Gly Thr Val Thr Leu 915 920 925 Ser Leu Ser Phe Glu Glu Ser Arg
Lys Asn Ser Met Lys 930 935 940 9 4031 DNA Salmo salar 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 4031 10 941 PRT Salmo salar 10 Met Arg Phe Tyr
Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val 1 5 10 15 Ile Ser
Thr Tyr Gly Pro His Gln Arg Ala Gln Lys Thr Gly Asp Ile 20 25 30
Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp 35
40 45 Gln Asp Leu Ala Ala Arg Pro Glu Ser Thr Glu Cys Val Arg Tyr
Asn 50 55 60 Phe Arg Gly Phe Arg Trp Leu Gln Ala Met Ile Phe Ala
Ile Glu Glu 65 70 75 80 Ile Asn Asn Ser Ser Thr Leu Leu Pro Asn Ile
Thr Leu Gly Tyr Arg 85 90 95 Ile Phe Asp Thr Cys Asn Thr Val Ser
Lys Ala Leu Glu Ala Thr Leu 100 105 110 Ser Phe Val Ala Gln Asn Lys
Ile Asp Ser Leu Asn Leu Asp Glu Phe 115 120 125 Cys Asn Cys Thr Asp
His Ile Pro Ser Thr Ile Ala Val Val Gly Ala 130 135 140 Ser Gly Ser
Ala Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe 145 150 155 160
Tyr Ile Pro Gln Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165
170 175 Lys Asn Gln Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu
His 180 185 190 Gln Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gln
Trp Asn Trp 195 200 205 Val Ile Ala Val Ala Ser Asp Asp Glu Tyr Gly
Arg Pro Gly Ile Glu 210 215 220 Lys Phe Glu Lys Glu Met Glu Glu Arg
Asp Ile Cys Ile His Leu Ser 225 230 235 240 Glu Leu Ile Ser Gln Tyr
Phe Glu Glu Trp Gln Ile Gln Gly Leu Val 245 250 255 Gly Arg Ile Glu
Asn Ser Ser Ala Lys Val Ile Val Val Phe Ala Ser 260 265 270 Gly Pro
Asp Ile Glu Pro Leu Ile Lys Glu Met Val Arg Arg Asn Ile 275 280 285
Thr Asp Arg Ile Trp Leu Ala Ser Glu Ala Trp Ala Thr Thr Ser Leu 290
295 300 Ile Ala Lys Pro Glu Tyr Leu Asp Val Val Val Gly Thr Ile Gly
Phe 305 310 315 320 Ala Leu Arg Ala Gly Glu Ile Pro Gly Phe Lys Asp
Phe Leu Gln Glu 325 330 335 Val Thr Pro Lys Lys Ser Ser His Asn Glu
Phe Val Arg Glu Phe Trp 340 345 350 Glu Glu Thr Phe Asn Cys Tyr Leu
Glu Asp Ser Gln Arg Leu Arg Asp 355 360 365 Ser Glu Asn Gly Ser Thr
Ser Phe Arg Pro Leu Cys Thr Gly Glu Glu 370 375 380 Asp Ile Met Gly
Ala Glu Thr Pro Tyr Leu Asp Tyr Thr His Leu Arg 385 390 395 400 Ile
Ser Tyr Asn Val Tyr Val Ala Val His Ser Ile Ala Gln Ala Leu 405 410
415 Gln Asp Ile Leu Thr Cys Ile Pro Gly Arg Gly Leu Phe Ser Asn Asn
420 425 430 Ser Cys Ala Asp Ile Lys Lys Ile Glu Ala Trp Gln Val Leu
Lys Gln 435 440 445 Leu Arg His Leu Asn Phe Ser Asn Ser Met Gly Glu
Lys Val His Phe 450 455 460 Asp Glu Asn Ala Asp Pro Ser Gly Asn Tyr
Thr Ile Ile Asn Trp His 465 470 475 480 Arg Ser Pro Glu Asp Gly Ser
Val Val Phe Glu Glu Val Gly Phe Tyr 485 490 495 Asn Met Arg Ala Lys
Arg Gly Val Gln Leu Phe Ile Asp Asn Thr Lys 500 505 510 Ile Leu Trp
Asn Gly Tyr Asn Thr Glu Val Pro Phe Ser Asn Cys Ser 515 520 525 Glu
Asp Cys Glu Pro Gly Thr Arg Lys Gly Ile Ile Glu Ser Met Pro 530 535
540 Thr Cys Cys Phe Glu Cys Thr Glu Cys Ser Glu Gly Glu Tyr Ser Asp
545 550 555 560 His Lys Asp Ala Ser Val Cys Thr Lys Cys Pro Asn Asp
Ser Trp Ser 565 570 575 Asn Glu Asn His Thr Ser Cys Phe Leu Lys Glu
Ile Glu Phe Leu Ser 580 585 590 Trp Thr Glu Pro Phe Gly Ile Ala Leu
Ala Leu Cys Ser Val Leu Gly 595 600 605 Val Phe Leu Thr Ala Phe Val
Met Gly Val Phe Ile Lys Phe Arg Asn 610 615 620 Thr Pro Ile Val Lys
Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu 625 630 635 640 Phe Ser
Leu Ile Cys Cys Phe Ser Ser Ser Leu Ile Phe Ile Gly Glu 645 650 655
Pro Gln Asp Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile Ser 660
665 670 Phe Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val
Leu 675 680 685 Leu Val Phe Glu Ala Lys Ile Pro Thr Ser Leu His Arg
Lys Trp Trp 690 695 700 Gly Leu Asn Leu Gln Phe Leu Leu Val Phe Leu
Phe Thr Phe Val Gln 705 710 715 720 Val Met Ile Cys Val Val Trp Leu
Tyr Asn Ala Pro Pro Ala Ser Tyr 725 730 735 Arg Asn His Asp Ile Asp
Glu Ile Ile Phe Ile Thr Cys Asn Glu Gly 740 745 750 Ser Met Met Ala
Leu Gly Phe Leu Ile Gly Tyr Thr Cys Leu Leu Ala 755 760 765 Ala Ile
Cys Phe Phe Phe Ala Phe Lys Ser Arg Lys Leu Pro Glu Asn 770 775 780
Phe Thr Glu Ala Lys Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile 785
790 795 800 Val Trp Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly
Lys Phe 805 810 815 Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser
Ser Phe Gly Leu 820 825 830 Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr
Ile Ile Leu Phe Lys Pro 835 840 845 Ser Arg Asn Thr Ile Glu Glu Val
Arg Cys Ser Thr Ala Ala His Ser 850 855 860 Phe Lys Val Ala Ala Lys
Ala Thr Leu Arg His Ser Ser Ala Ser Arg 865 870 875 880 Lys Arg Ser
Ser Ser Val Gly Gly Ser Cys Ala Ser Thr Pro Ser Ser 885 890 895 Ser
Ile Ser Leu Lys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gln Gln 900 905
910 Arg Ile His Lys Pro Arg Val Ser Phe Gly Ser Gly Thr Val Thr Leu
915 920 925 Ser Leu Ser Phe Glu Glu Ser Arg Lys Asn Ser Met Lys 930
935 940 11 3824 DNA Salmo salar 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 12 850 PRT
Salmo salar 12 Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe
Ser Ser Val 1 5 10 15 Ile Ser Thr Tyr Gly Pro His Gln Arg Ala Gln
Lys Thr Gly Asp Ile 20 25 30 Leu Leu Gly Gly Leu Phe Pro Met His
Phe Gly Val Thr Ser Lys Asp 35 40 45 Gln Asp Leu Ala Ala Arg Pro
Glu Ser Thr Glu Cys Val Arg Tyr Asn 50 55 60 Phe Arg Gly Phe Arg
Trp Leu Gln Ala Met Ile Phe Ala Ile Glu Glu 65 70 75 80 Ile Asn Asn
Ser Ser Thr Leu Leu Pro Asn Ile Thr Leu Gly Tyr Arg 85 90 95 Ile
Phe Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu 100 105
110 Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe
115 120 125 Cys Asn Cys Thr Asp His Ile Pro Ser Thr Ile Ala Val Val
Gly Ala 130 135 140 Ser Gly Ser Ala Val Ser Thr Ala Val Ala Asn Leu
Leu Gly Leu Phe 145 150 155 160 Tyr Ile Pro Gln Ile Ser Tyr Ala Ser
Ser Ser Arg Leu Leu Ser Asn 165 170 175 Lys Asn Gln Phe Lys Ser Phe
Met Arg Thr Ile Pro Thr Asp Glu His 180 185 190 Gln Ala Thr Ala Met
Ala Asp Ile Ile Asp Tyr Phe Gln Trp Asn Trp 195 200 205 Val Ile Ala
Val Ala Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu 210 215 220 Lys
Phe Glu Lys Glu Met Glu Glu Arg Asp Ile Cys Ile His Leu Ser 225 230
235 240 Glu Leu Ile Ser Gln Tyr Phe Glu Glu Trp Gln Ile Gln Gly Leu
Val 245 250 255 Asp Arg Ile Glu Asn Ser Ser Ala Lys Val Ile Val Val
Phe Ala Ser 260 265 270 Gly Pro Asp Ile Glu Pro Leu Ile Lys Glu Met
Val Arg Arg Asn Ile 275 280 285 Thr Asp Arg Ile Trp Leu Ala Ser Glu
Ala Trp Ala Thr Thr Ser Leu 290 295 300 Ile Ala Lys Pro Glu Tyr Leu
Asp Val Val Val Gly Thr Ile Gly Phe 305 310 315 320 Ala Leu Arg Ala
Gly Glu Ile Pro Gly Phe Lys Asp Phe Leu Gln Glu 325 330 335 Val Thr
Pro Lys Lys Ser Ser His Asn Glu Phe Val Arg Glu Phe Trp 340 345 350
Glu Glu Thr Phe Asn Cys Tyr Leu Glu Asp Ser Gln Arg Leu Arg Asp 355
360 365 Ser Glu Asn Gly Ser Thr Ser Phe Arg Pro Leu Cys Thr Gly Glu
Glu 370 375 380 Asp Ile Met Gly Ala Glu Thr Pro Tyr Leu Asp Tyr Thr
His Leu Arg 385 390 395 400 Ile Ser Tyr Asn Val Tyr Val Ala Val His
Ser Ile Ala Gln Ala Leu 405 410 415 Gln Asp Ile Leu Thr Cys Ile Pro
Gly Arg Gly Phe Phe Ser Asn Asn 420 425 430 Ser Cys Ala Asp Ile Lys
Lys Ile Glu Ala Trp Gln Val Leu Lys Gln 435 440 445 Leu Arg His Leu
Asn Phe Ser Asn Ser Met Gly Glu Lys Val His Phe 450 455 460 Asp Glu
Asn Ala Asp Pro Ser Gly Asn Tyr Thr Ile Ile Asn Trp His 465 470 475
480 Arg Ser Pro Glu Asp Gly Ser Val Val Phe Glu Glu Val Gly Phe Tyr
485 490 495 Asn Met Arg Ala Lys Arg Gly Val Gln Leu Phe Ile Asp Asn
Thr Lys 500 505 510 Ile Leu Trp Asn Gly Tyr Asn Thr Glu Val Pro Phe
Ser Asn Cys Ser 515 520 525 Glu Asp Cys Glu Pro Gly Thr Arg Lys Gly
Ile Ile Glu Ser Met Pro 530 535 540 Thr Cys Cys Phe Glu Cys Thr Glu
Cys Ser Glu Gly Glu Tyr Ser Asp 545 550 555 560 His Lys Asp Ala Ser
Val Cys Thr Lys Cys Pro Asn Asp Ser Trp Ser 565 570 575 Asn Glu Asn
His Thr Ser Cys Phe Leu Lys Glu Ile Glu Phe Leu Ser 580 585 590 Trp
Thr Glu Pro Phe Gly Ile Ala Leu Ala Leu Cys Ser Val Leu Gly 595 600
605 Val Phe Leu Thr Ala Phe Val Met Gly Val Phe Ile Lys Phe Arg Asn
610 615 620 Thr Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu
Leu Leu 625 630 635 640 Phe Ser Leu Ile Cys Cys Phe Ser Ser Ser Leu
Ile Phe Ile Gly Glu 645 650 655 Pro Gln Asp Trp Thr Cys Arg Leu Arg
Gln Pro Ala Phe Gly Ile Ser 660 665 670 Phe Val Leu Cys Ile Ser Cys
Ile Leu Val Lys Thr Asn Arg Val Leu 675 680 685 Leu Val Phe Glu Ala
Lys Ile Pro Thr Ser Leu His Arg Lys Trp Trp 690 695 700 Gly Leu Asn
Leu Gln Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gln 705 710 715 720
Val Met Ile Cys Val Val Trp Leu Tyr Asn Ala Pro Pro Ala Ser Tyr 725
730 735 Arg Asn His Asp Ile Asp Glu Ile Ile Phe Ile Thr Cys Asn Glu
Gly 740 745 750 Ser Met Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr Cys
Leu Leu Ala 755 760 765 Ala Ile Cys Phe Phe Phe Ala
Phe Lys Ser Arg Lys Leu Pro Glu Asn 770 775 780 Phe Thr Glu Ala Lys
Phe Ile Thr Phe Ser Met Leu Ile Phe Phe Ile 785 790 795 800 Val Trp
Ile Ser Phe Ile Pro Ala Tyr Phe Ser Thr Tyr Gly Lys Phe 805 810 815
Val Ser Ala Val Glu Val Ile Ala Ile Leu Ala Ser Ser Phe Gly Leu 820
825 830 Leu Ala Cys Ile Phe Phe Asn Lys Val Tyr Ile Ile His Gln Pro
Gln 835 840 845 Asp Gln 850 13 28 DNA Artificial Sequence primer 13
tgtcktggac ggagccctty ggratcgc 28 14 31 DNA Artificial Sequence
primer 14 ggckggratg aargakatcc aracratgaa g 31 15 16 PRT
Artificial Sequence peptide for Sal-1 antibody production 15 Cys
Thr Asn Asp Asn Asp Ser Pro Ser Gly Gln Gln Arg Ile His Lys 1 5 10
15 16 20 DNA Artificial Sequence primer 16 caagcattat caagatcaag 20
17 17 DNA Artificial Sequence primer 17 ctcagagtgg ccttggc 17 18 20
DNA Artificial Sequence primer 18 cagttctctc tttaatggac 20 19 17
DNA Artificial Sequence primer 19 ctcagagtgg ccttggc 17 20 21 DNA
Artificial Sequence primer 20 agtctacatc atccatcagc c 21 21 21 DNA
Artificial Sequence primer 21 gattttattg tcattggatg c 21 22 19 DNA
Artificial Sequence primer 22 tggaagatga aatcgccgc 19 23 22 DNA
Artificial Sequence primer 23 gtggtggtga aactgtaacc gc 22 24 23 PRT
Artificial Sequence peptide for 4641 antibody production 24 Ala Asp
Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu Glu 1 5 10 15
Ala Glu Glu Arg Asp Ile Cys 20 25 22 PRT Artificial Sequence
peptide for 4641 antibody production 25 Asp Asp Tyr Gly Arg Pro Gly
Ile Glu Lys Phe Arg Glu Glu Ala Glu 1 5 10 15 Glu Arg Asp Ile Cys
Ile 20 26 17 PRT Artificial Sequence peptide for SKCaR antibody
production 26 Ala Arg Ser Arg Asn Ser Ala Asp Gly Arg Ser Gly Asp
Asp Leu Pro 1 5 10 15 Cys 27 18 PRT Artificial Sequence peptide for
Sal-ADD antibody production 27 Cys Ser Asp Asp Glu Tyr Gly Arg Pro
Gly Ile Glu Lys Phe Glu Lys 1 5 10 15 Glu Met 28 1078 PRT Homo
sapiens 28 Met Ala Phe Tyr Ser Cys Cys Trp Val Leu Leu Ala Leu Thr
Trp His 1 5 10 15 Thr Ser Ala Tyr Ser Pro Ser Gln Pro Ala Gln Lys
Lys Gly Asp Ile 20 25 30 Ile Leu Gly Gly Leu Phe Pro Ile His Phe
Gly Val Ala Ala Lys Asp 35 40 45 Gln Asp Leu Lys Ser Arg Pro Glu
Ser Val Glu Cys Ile Arg Tyr Asn 50 55 60 Phe Arg Gly Phe Arg Trp
Leu Gln Ala Met Ile Phe Ala Ile Glu Glu 65 70 75 80 Ile Asn Ser Ser
Pro Ala Leu Leu Pro Asn Leu Thr Leu Gly Tyr Arg 85 90 95 Ile Phe
Asp Thr Cys Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu 100 105 110
Ser Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe 115
120 125 Cys Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Gly
Ala 130 135 140 Thr Gly Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu
Gly Leu Phe 145 150 155 160 Tyr Ile Pro Gln Val Ser Tyr Ala Ser Ser
Ser Arg Leu Leu Ser Asn 165 170 175 Lys Asn Gln Phe Lys Ser Phe Leu
Arg Thr Ile Pro Asn Asp Glu His 180 185 190 Cys Ala Thr Ala Met Ala
Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp 195 200 205 Val Gly Thr Ile
Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu 210 215 220 Lys Phe
Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp Phe Ser 225 230 235
240 Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile Gln Met Val Val
245 250 255 Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile Val Val Phe
Ser Ser 260 265 270 Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu Ile Val
Pro Arg Asn Ile 275 280 285 Thr Gly Lys Ile Trp Leu Ala Ser Glu Ala
Trp Ala Ser Ser Ser Leu 290 295 300 Ile Ala Met Pro Gln Tyr Phe His
Val Val Gly Gly Thr Ile Gly Phe 305 310 315 320 Ala Leu Lys Ala Gly
Gln Ile Pro Gly Phe Arg Glu Phe Leu Lys Lys 325 330 335 Val His Pro
Pro Lys Ser Val Asn Asn Gly Phe Ala Lys Glu Phe Trp 340 345 350 Glu
Glu Thr Phe Met Cys His Leu Gln Glu Gly Ala Lys Gly Pro Leu 355 360
365 Pro Val Asp Thr Phe Leu Ala Gly His Glu Glu Ser Gly Asp Arg Phe
370 375 380 Ser Asn Ser Ser Thr Ala Phe Pro Pro Leu Cys Thr Gly Asp
Glu Asn 385 390 395 400 Ile Ser Ser Val Glu Thr Pro Tyr Ile Asp Tyr
Thr Asn Leu Arg Ile 405 410 415 Ser Tyr Asn Val Tyr Leu Ala Val Tyr
Ser Ile Ala Asn Ala Leu Gln 420 425 430 Asp Ile Tyr Thr Cys Leu Pro
Gly Arg Gly Leu Phe Thr Asn Gly Ser 435 440 445 Cys Ala Asp Ile Lys
Lys Val Glu Ala Trp Gln Val Leu Lys His Leu 450 455 460 Arg Asn Leu
Asn Phe Thr Asn Asn Met Gly Glu Gln Val Thr Phe Asp 465 470 475 480
Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile Asn Trp His Leu 485
490 495 Ser Pro Glu Asp Gly Ser Ile Val Phe Lys Glu Val Gly Tyr Tyr
Asn 500 505 510 Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe Ile Asn Glu
Glu Lys Ile 515 520 525 Leu Trp Ser Gly Phe Ser Arg Glu Val Pro Phe
Ser Asn Cys Ser Arg 530 535 540 Asp Cys Leu Ala Gly Thr Arg Lys Gly
Ile Ile Glu Gly Glu Pro Thr 545 550 555 560 Cys Cys Phe Glu Cys Val
Glu Cys Pro Asp Gly Glu Tyr Ser Asp Glu 565 570 575 Thr Asp Ala Ser
Ala Cys Asn Lys Cys Pro Asp Asp Phe Trp Ser Asn 580 585 590 Glu Asn
His Thr Ser Cys Ile Ala Lys Glu Ile Glu Phe Leu Ser Trp 595 600 605
Thr Glu Pro Phe Gly Ile Ala Leu Thr Leu Phe Ala Val Leu Gly Ile 610
615 620 Ser Leu Thr Ala Phe Val Leu Gly Val Phe Ile Lys Phe Arg Asn
Thr 625 630 635 640 Pro Ile Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr
Leu Leu Leu Phe 645 650 655 Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu
Phe Phe Ile Gly Glu Pro 660 665 670 Gln Asp Trp Thr Cys Arg Leu Arg
Gln Pro Ala Phe Gly Ile Ser Phe 675 680 685 Val Leu Cys Ile Ser Cys
Ile Leu Val Lys Thr Asn Arg Val Leu Leu 690 695 700 Val Phe Glu Ala
Lys Ile Pro Thr Ser Phe Met Phe Lys Trp Trp Gly 705 710 715 720 Leu
Asn Leu Gln Phe Leu Leu Val Phe Leu Cys Thr Phe Asn Gln Ile 725 730
735 Val Ile Cys Val Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg
740 745 750 Asn Gln Glu Leu Glu Asp Glu Ile Ile Phe Ile Thr Cys Asn
Glu Gly 755 760 765 Ser Leu Met Ala Leu Gly Phe Leu Ile Gly Tyr Thr
Cys Leu Leu Ala 770 775 780 Ala Ile Cys Phe Phe Phe Ala Phe Lys Ser
Arg Lys Leu Pro Glu Asn 785 790 795 800 Phe Asn Glu Ala Lys Phe Ile
Thr Phe Ser Met Leu Ile Phe Phe Ile 805 810 815 Val Trp Ile Ser Phe
Ile Pro Ala Tyr Ala Ser Thr Tyr Gly Lys Phe 820 825 830 Val Ser Ala
Val Glu Val Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu 835 840 845 Leu
Ala Cys Ile Phe Phe Asn Lys Ile Tyr Ile Ile Leu Phe Lys Pro 850 855
860 Ser Arg Asn Thr Ile Glu Glu Val Arg Cys Ser Thr Ala Ala Asn Ala
865 870 875 880 Phe Lys Val Ala Ala Arg Ala Thr Leu Arg Arg Ser Asn
Val Ser Arg 885 890 895 Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly
Ser Thr Pro Ser Ser 900 905 910 Ser Ile Ser Ser Lys Ser Asn Ser Glu
Asp Pro Phe Pro Arg Pro Glu 915 920 925 Arg Gln Lys Gln Gln Gln Pro
Leu Ala Leu Thr Gln Gln Glu Gln Gln 930 935 940 Gln Gln Pro Leu Thr
Leu Pro Gln Gln Gln Arg Ser Gln Gln Gln Pro 945 950 955 960 Arg Cys
Lys Gln Lys Val Ile Phe Gly Ser Gly Thr Val Thr Phe Ser 965 970 975
Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala Met Ala Asn Arg Asn Ser 980
985 990 Thr Asn Gln Asn Ser Leu Glu Ala Gln Lys Ser Ser Asp Thr Leu
Thr 995 1000 1005 Ala Asn Gln Pro Leu Leu Pro Leu Gln Cys Gly Glu
Thr Asp Leu 1010 1015 1020 Asp Leu Thr Val Gln Glu Thr Gly Leu Gln
Gly Pro Val Gly Gly 1025 1030 1035 Asp Gln Arg Pro Glu Val Glu Asp
Pro Glu Glu Leu Ser Pro Ala 1040 1045 1050 Leu Val Val Ser Ser Ser
Gln Ser Phe Val Ile Ser Gly Gly Gly 1055 1060 1065 Ser Thr Val Thr
Glu Asn Val Val Asn Ser 1070 1075
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