Note: Descriptions are shown in the official language in which they were submitted.
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POLYVALENT CATION-SENSING RECEPTOR IN ATLANTIC SALMON
.10
BACKGROUND OF THE INVENTION
In nature, anadromous fish like salmon live most of their adulthood in
seawater, but swim upstream to freshwater for the purpose of breeding. As a
result,
anadromous fish hatch from their eggs and are born in freshwater. As these
fish
grow, they swim downstream and gradually adapt to the seawater.
Currently, wild Atlantic salmon are classified as endangered species in
multiple areas of their native habitats. Among the reasons for their decline
has been
man made alterations in freshwater conditions in their native streams that
have
produced multiple problems with their migration, spawning, smoltification and
survival. One problem complicating the effective restoration of wild Atlantic
salmon is the lack of a fundamental understanding of how these deleterious
environmental conditions effect the salmon's ability to home to freshwater
streams
from the ocean, interchangeably adapt to freshwater and seawater as well as
feed and
grow in both salinity environments.
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Despite the decline of wild populations, the global aquaculture industry has
utilized Atlantic salmon as one of chief fish species for large-scale marine
farming
operations. At the present time, large scale breeding programs of Atlantic
salmon
provide for high quality fish used in production by selection of specific
traits among
them rapid growth, seawater adaptability, flesh quality and taste.
However, fish hatcheries have experienced some difficulty in raising salmon
because the window of time in which the pre-adult salmon adapts to seawater
(e.g.,
undergoes smoltification) is short-lived, and can be difficult to pinpoint. As
a result,
these hatcheries can experience significant morbidity and mortality when
transferring salmon from freshwater to seawater. Additionally, many of the
salmon
that do survive the transfer from freshwater to seawater are stressed, and
consequently, experience decreased feeding, and increased susceptibility to
disease.
Therefore, salmon often do not grow-well after they-are transferred to
seawater.
The aquaculture industry loses millions of dollars each year due to problems
it encounters in transferring salmon from freshwater to seawater. Therefore, a
need
exists to gain a better understanding of the biological processes of salmon
that are
related to smoltification and adaptation to varying salinities, including
seawater. In
particular, a need exists to identify genes that play an important role in
these areas.
SUMMARY OF THE INVENTION
The present invention relates to genes that allow fish to sense and adapt to
ion concentrations in the surrounding environment. Modulating one or More of
these genes allow anadromous fish like salmon to better adapt to seawater
during
smoltification, which in turn allows salmon to grow faster and stronger after
transfer
to seawater. A gene, called a PolyValent Cation-sensing Receptor (PVCR), has
been
isolated in several species of fish, and in particular, in Atlantic Salmon. In
fact, four
foinis of the PVCR have been isolated in Atlantic Salmon, and have been
termed,
"SalmoKCall" genes and individually referred to as "SalmoKCaR#1",
"SalmoKCaR#2", "SalmoKCaR#3" and "SalmoKCaR#4". "PVCR" and
"SalmoKCaR" are used interchangeably when referring to Atlantic Salmon. These
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four genes work together to alter the salmon's Sensitivity to the surrounding
ion
concentrations, as further described herein.
The invention embodies nucleic acid molecules (e.g., RNA, genomic DNA
and cDNA) having nucleic acid sequences of SalmoKCaR#1 (SEQ ED NO: 7),
SalmoKCaR#2 (SEQ JD NO: 9), SalmoKCaR#3 (SEQ JD NO: 11), or
SalmoKCaR#4 (SEQ ID NO: 13). The invention also embodies polypeptide
molecules having amino acid sequences of SalmoKCaR#1 (SEQ II) NO: 8),
SahnoKCaR#2 (SEQ ID NO: 10), SalmoKCaR#3 (SEQ ID NO: 12), or
SalmoKCaR#4 (SEQ JD NO: 14). The present invention, in particular, encompasses
isolated nucleic acid molecules having nucleic acid sequences of SEQ ID NO: 7,
9,
11, or 13; the complementary strand thereof; the coding region of SEQ ID NO:
7, 9,
11, or 13; or the complementary strand thereof. The present invention also
embodies
nucleic acid molecules that encode polypeptides having an amino acid sequence
of
SEQ ID NO: 8, 10, 12, or 14. The present invention, in another embodiment,
includes isolated polypeptide molecules having amino acid sequences that
comprise
SEQ ID NO: 8, 10, 12, or 14; or amino acid sequences encoded by the nucleic
acid
sequence of SEQ JD NO: 7,9, 11, or 13.
In one embodiment, the present invention pertains to isolated nucleic acid
molecules that have a nucleic acid sequence with at least about 70% (e.g.,
75%,
80%, 85%, 90%, or 95%) identity with SEQ ID NO: 7, 9, 11, or 13, or the coding
- region of SEQ ID NO: 7, 9, 11, or 13. Such a nucleic acid sequence (
e.g., SEQ ID
NO: 7, 9, 11, or 13) encodes a polypeptide that allows for or assists in one
or more
of the following functions in Atlantic Salmon: sensing at least one SalmoKCaR
modulator in serum or in the surrounding environment; adapting to at least one
SalmoKCaR modulator present in the serum or surrounding environment;
imprinting
with an odorant; altering water intake; altering water absorption; or altering
urine
output.
The present invention further includes nucleic acid molecules that hybridize,
preferably under high stringency conditions, with SalmoKCaR#1, SalmoKCaR#2,
SalmoKCaR#3, or SalmoKCaR #4 but not to the Shark Kidney Calcium Receptor
related protein (SKCaR) nucleic acid sequence (SEQ ID NO: 1, shown in Figure
1)
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and/or the Fugu pheromone receptor (described in Naito, T. et al., PNAS,
95:5178-
5181 (1998), hereinafter "Naito" and deposited as GenBank Accession Nos:
AB008857 (Fugu CaR (01), SEQ ID NO: 31), A1B008858 (Fugu Ca02.1, SEQ ID
NO: 32), AB008859 (Fugu Ca09, SEQ JD NO: 33), AB008860 (Fugu Ca12, SEQ ID
NO: 34), AB008861 (Fugu Ca13, SEQ ID NO: 35), AB008862 (Fugu Ca15.1, SEQ
ID NO: 36), AB008863 (Fugu mGluR1, SEQ ID NO: 37), AB008864 (Fugu
mGluR2, SEQ ID NO: 38), AB008865 (Fugu mGluR7, SEQ ID NO: 39), and
AB008866 (Fugu mGluR8, SEQ ID NO: 40)). The sequences described in the Naito
including those deposited under these GenBank Accession Numbers are
incorporated by referenced in their entirety, and are referred to herein as
the "fugu
pheromone receptor" or "fugu receptor." SKCaR is a PVCR isolated from dogfish
shark. Specifically, the present invention relates to an isolated nucleic acid
molecule
that contains a nucleic acid sequence that hybridizes under high stringency
conditions to SEQ ID NO: 7, 9, 11, or 13; or the coding region of SEQ ID NO:
7, 9,
11, or 13; but excluding those that hybridize to SEQ ID NO: 1 or the nucleic
acid
sequence of the fugu pheromone receptor, as described herein, under the same
conditions.
The present invention also includes probes, vectors, viruses, plasmids, and
_
host cells that contain the nucleic acid sequences, as described herein. In
particular,
the present invention includes probes (e.g., nucleic acid probes or DNA
probes)
having a sequence from or hybridizes to SEQ ID NO: 7, 9, 11, or 13, but not
SEQ ID
NO: 1 or the nucleic acid sequence of the fagu pheromone receptor, as
described
herein. The present invention encompasses nucleic acid or peptide molecules
purified or obtained from clones deposited with American Type Culture
Collection
(ATCC), Accession No: PTA-4190, PTA-4191, PTA-4192, or (to be added).
In another embodiment, the present invention includes isolated polypeptide
molecules having at least about 70% (e.g., 75%, 80%, 85%, 90%, or 95%)
identity
with SEQ ID NO: 8, 10, 12, or 14; or an amino acid sequence encoded by the
nucleic
acid sequence of SEQ ID NO: 7, 9, 11, or 13. These polypeptide molecules
(e.g.,
SEQ ID NO: 8, 10, 12, or 14; or those encoded by the nucleic acid sequence of
SEQ
ID NO: 7, 9, 11, or 13) have one or more of the following functions in
Atlantic
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Salmon: sensing at least one SalmoKCaR modulator in serum or in the
surrounding
environment; adapting to at least one SalmoKCaR modulator present in the serum
or
surrounding environment; imprinting with an odorant; altering water intake;
altering
water absorption; or altering urine output.
Additionally, the present invention relates to antibodies that specifically
bind
to or are produced in reaction to polypeptide molecules described herein. The
invention further includes fusion proteins that contain one of the polypeptide
molecules described herein, and a portion of an immunoglobulin.
The present invention also pertains to assays for determining the presence or
absence of a SalmoKCaR in a sample by contacting the sample to be tested with
an
antibody specific to at least a portion of the SalmoKCaR polypeptide
sufficiently to
allow formation of a complex between SalmoKCaR and the antibody, and detecting
the presence or absence of the complex_ formation. Another assay for
determining
the presence or absence of a nucleic acid molecule that encodes SalmoKCaR in a
sample involves contacting the sample to be tested with a nucleic acid probe
that
hybridizes under high stringency conditions to a nucleic acid molecule having
a
sequence of SEQ JD NO: 7, 9, 11, or 13, sufficiently to allow hybridization
between
the sample and the probe; and detecting the SalmoKCaR nucleic acid molecule in
the sample. Such assay methods also include methods for detefinining whether a
compound is a modulator of SalmoKCaR. These methods include contacting a
compound to be tested with a cell that contains SalmoKCaR.nucleic acid
molecules
and/or expresses SalmoKCaR proteins, and determining whether compounds are
modulators by measuring the expression level or activity (e.g.,
phosphorylation,
dimerization, proteolysis or intracellular signal transduction) of SalmoKCaR
proteins. In one embodiment, one can measure changes that occur in one or more
intracellular signal transduction systems that are altered by activation of
the
expressed proteins coded for by a single or combination of nucleic acids. Such
methods can also encompass contacting a compound to be tested with a cell that
comprises one or more of SalmoKCaR nucleic acid molecules; and determining the
level of expression of said nucleic acid molecule. An increase or decrease in
the
expression level, as compared to a control, indicates that the compound is a
modulator.
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Lastly, the present invention relates to transgenic fish encoding a
SalmoKCaR polypeptide or having one or more nucleic acid molecules that
contain
the SalmoKCaR nucleic acid sequence, as described herein.
The present invention allows for a number of advantages, including the
ability to more efficiently grow Atlantic Salmon, and in particular, transfer
them to
seawater with increased growth and reduce mortality. The technology of the
present
invention also allows for assaying or testing these salmon to determine if
they are
ready for transfer to seawater, so that they can be transferred at the best
time. The
technology of the present invention provides for the imprinting of salmon with
an
odorant so that the salmon, once imprinted, can later more easily recognize
and/or
distinguish the odorant. For example, an attractant that has been used to
imprint
salmon can be added to feed so that the salmon will consume more feed and grow
at
a faster rate. A number of additional advantages for the present invention
exist and
are apparent from the description provided herein.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A-E show the annotated nucleotide sequence (SEQ ID NO: 1) and
the deduced amino acids sequence (SEQ ID NO: 2) of SKCaR with the Open
Reading Frame (ORF) starting at nucleotide (nt) 439 and ending at 3516.
Figure 2 is a graphical representation showing a normalized calcium
response (%) against the amount of Calcium (mM) of the SKCaR-I protein when
modulated by altei-nations in extracellular NaC1 concentrations.
Figure 3 is a graphical representation showing a normalized calcium
response (%) against the amount of magnesium(mM) of the SKCaR-I protein in
increasing amounts of extracellular NaC1 concentrations.
Figure 4 is a graphical representation showing the EC50 for calcium
activation of shark CaR (mM) against the amount of sodium (mM) of the SKCaR-I
protein in increasing amounts of extracellular NaC1 concentrations.
Figure 5 is a graphical representation showing the EC50 for magnesium
activation of shark CaR (mM) against the amount of sodium (mM) of the SKCaR-I
protein in increasing amounts of extracellular NaC1 concentrations.
Figure 6 is a graphical representation showing the EC50 for magnesium
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activation of shark CaR (mM) against the amount of sodium (mM) of the SKCaR-I
protein in increasing amounts of extracellular NaC1 concentrations and added
amounts of calcium (3mM).
Figures 7A and 7B show an annotated partial nucleotide sequence (SEQ JD
NO: 3) and the deduced amino acids sequence (SEQ ID NO: 4) of an Atlantic
salmon polyvalent cation-sensing receptor protein.
Figures 8A-8C show a second annotated partial nucleotide sequence (SEQ
ED NO: 5) and the deduced amino acids sequence (SEQ ID NO: 6) of an Atlantic
salmon polyvalent cation-sensing receptor protein.
Figures 9A-E show the nucleic acid (SEQ ID NO: 7) and amino acid (SEQ
ID NO: 8) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#1 with
the ORF starting at nt 180 and ending at 3005.
Figures 10A-E show the nucleic acid (SEQ ID NO: 9) and amino acid (SEQ
ID NO: 10) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#2 with
the ORF starting at nt 270 and ending at 3095.
Figures 11A-D show the nucleic acid (SEQ ID NO: 11) and amino acid (SEQ
ID NO: 12) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#3 with
the ORF starting at nt 181 and ending at 2733.
Figures 12A-E show the nucleic acid (SEQ ID NO: 13) and amino acid (SEQ
ID NO: 14) sequences of a full length Atlantic Salmon PVCR, SalmoKCaR#4 with
the ORF starting at nt 181 and ending at 3006.
Figures 13A-L are an alignment showing nucleic acid sequences of two
partial Atlantic Salmon Clones (SEQ ID NO: 3 and 5), SalmoKCaR#1 (SEQ ID NO:
7), SalmoKCaR#2 (SEQ ID NO: 9), and SalmoKCaR#3 (SEQ ID NO: 11).
Figures 14A-C are an alignment showing amino acid sequences of two
partial Atlantic Salmon Clones (SEQ ID NO: 4 and 6), SalmoKCaR#1 (SEQ ID NO:
8), SalmoKCaR#2 (SEQ ID NO: 10), and SalmoKCaR#3 (SEQ ID NO: 12).
Figure 15A is photograph showing a Southern blot in which SalmoKCaR#1,
2, and 3 hybridize to nucleic acid derived from SKCaR.
Figure 15B is a photograph showing a Western blot protein produced by
HEK cells transiently transfected with SalmoKCaR#1 and #3 constructs. In
particular, the left panel shows an immunoblotting analysis with SDD antiserum
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while the right panel shows immunoblotting analysis for Sall for the
following: a
standard, 5001 HEK (human) cells, HEK 293 mock transfected cells, SalmoKCaR#1
HEK cells, SalmoKCaR#3 HEK cells, and Salmon SW Kidney.
Figures 16A-H are an alignment of the full length nucleic acid sequences of
SalmoKCaR#1, 2, and 3 (SEQ ID NO: 7, 9, and 11, respectively). Alignment
obtained using Clustal method with weighted residue weight table.
Figures 17A-L are an alignment showing nucleic acid sequences of
SalmoKCaR#1 (SEQ ID NO: 7), SalmoKCaR#2 (SEQ ID NO: 9), SalmoKCaR#3
(SEQ ID NO: 11), and SalmoKCaR#4 (SEQ ID NO: 13). Alignment was obtained
using Clustal method with weighted residue weight table.
Figures 18A-C are an alignment showing amino acid sequences of
SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), SalmoKCaR#3
(SEQ ID NO: 12), and SalmoKCaR#4 (SEQ ID NO: 14).
Figures 19A-D are an alignment of the full length amino acid sequences of
Human Parathyroid Calcium Receptor (HuPCaR) (SEQ JD NO: 30), SKCaR (SEQ
ID NO: 2), SalmoKCaR#1 (SEQ ID NO: 8), SalmoKCaR#2 (SEQ ID NO: 10), and
SalmoKCaR#3 (SEQ ID NO: 12), and SalmoKCaR#4 (SEQ ID NO: 14). Alignment
obtained using Clustal method with PAM250 residue weight table.
Figures 20A-F are graphical representations comparing six photographs of
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis of
freshwater
(Figures 20B, D and F) and seawater (Figures 20A, C and E) adapted Atlantic
salmon tissues (gill, nasal lamellae, urinary bladder, kidney, stomach,
pyloric caeca,
proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb,
liver and
muscle) using either degenerate PVCR (Figures 20A-D) or salmon actin PCR
primers (Figures 20E,F). Wells 1-14 for Figures 20A-F, top row, are designated
as
follows: ladder, gill, nasal lamellae, urinary bladder, kidney, stomach,
pyloric caeca,
proximal intestine, distal intestine, brain, pituitary gland, olfactory bulb,
liver and
muscle, respectively. Wells 1, 2, 7, 9, and 12, bottom row, for Figures 20A,
C, and
E are designated as ladder, water, SalmoKCaR #1, SalmoKCaR#2 and
SalmoKCaR#3, respectively, and wells 1, 2, 3, 7, 9, and 12, bottom row, for
Figures
20B,D, and F are designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2
and SalmoKCaR#3, respectively.
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Figure 21A is photograph of a RT-PCR analysis using degenerate primers of
steady state SalmoKCaR mRNA transcripts from kidney tissue of Atlantic Salmon
adapted to freshwater, after 9 weeks of Process II treatment or 26 days after
transfer
to seawater. Process II treatment is defined in the Exemplification.
Figure 21B is a photograph of a RT-PCR analysis showing increased steady
state expression of SalmoKCaR transcripts in pyloric caeca of Process II
treated and
seawater fish as compared to freshwater Atlantic salmon smolt. Using
degenerate
(SEQ ID Nos 15 and 16) or actin (SEQ ID No 24 and 25) primers, samples of
either
freshwater (Panel A Lanes 3 and 6), Process II treated (Panel A Lanes 4 and 7)
or
seawater adapted (Panel A Lanes 5 and 8) Atlantic salmon smolt were analyzed
by
RT-PCR. To control for differences in sample loading, these identical samples
were
subjected to PCR analysis using actin specific primer (Panel A, Lanes 3-5).
Note
that both ethidium bromide stained gel (Panel A) and its corresponding
Southern
blot (Panel C) show increased amounts of SalmoKCaR transcripts in pyloric
caeca
from Process II and seawater adapted fish as compared to freshwater. As a
control,
Panel B demonstrates that these degenerate primers amplify SalmoKCaR #1 (Lane
1), SalmoKCaR #2 (Lane 2) and SalmoKCaR #3 (Lane 3) transcripts.
Figure 21C is a photograph of RT-PCR analysis showing expression of
SalmoKCaR transcripts in various stages of Atlantic salmon embryo development.
Using degenerate (SEQ ID Nos. 15 and 16) or actin (SEQ ID No 24 and 25)
primers,
RNA obtained from samples of whole Atlantic salmon embryos at various stages
of
develoiment were analyzed for expression of SalmoKCaRs using RT-PCR.
Ethidium bromide staining of samples from dechorionated embryos (Lane 1), 50%
hatched (Lane 2), 100% hatched (Lane 3), 2 weeks post hatched (Lane 4) and 4
weeks post hatched (Lane 5) shows that SalmoKCaR transcripts are present in
Lanes
1-4). Southern blotting of the same gel (Panel C) confirms expression of
SalmoKCaRs in embryos from very early stages up to 2 weeks after hatching. No
expression of SalmoKCaR was observed in embryos 4 weeks after hatching. Panel
B shows the series of controls where PCR amplification of actin content of
each of
the 5 samples shows they are approximately equal (lanes 1-5).
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Figure 22 is a photograph of a RNA blot containing 5 micrograms of poly A+
RNA from kidney tissue dissected from either freshwater adapted (FW) or
seawater
adapted (SW) Atlantic salmon probed with full length SalmoKCaR #1 clone.
Figures 23A-F are graphical representations comparing six photographs
showing RT-PCR analysis of freshwater (Figures 23B, D and F) and seawater
(Figures 23A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR
#3
specific PCR (Figures 23A-D) primers or salmon actin PCR primers (Figures
23E,F). Wells 1-14 for Figures 23A-F, top row, are designated as follows:
ladder,
gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca,
proximal
intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and
muscle,
respectively. Wells 1, 2, 8, 11, and 14, bottom row, for Figures 23A, C, and E
are
designated as ladder, water, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3,
respectively, and wells '1, 2, 3, 8, 11, and 14, bottom row, for Figures
23B,D, and F
are designated as ladder, water, ovary, SalmoKCaR #1, SalmoKCaR#2 and
Sahnol(CaR#3, respectively.
Figures 24A-F are graphical representations comparing six photographs
showing RT-PCR analysis of freshwater (Figures 248, D and F) and seawater
(Figures 24A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR
#1
specific PCR primers or salmon actin PCR primers. Wells 1-14 for Figures 24A-
F,
top row, are designated as follows: ladder, gill, nasal lamellae, urinary
bladder,
kidney, stomach, pyloric caeca, proximal intestine, distal intestine, brain,
pituitary
gland, olfactory bulb, liver and muscle, respectively. Wells 1, 2,3, 5, 6, and
7.
bottom row, for Figures 24A, C, and E are designated as ladder, water, Kidney-
RT,
SalmoKCaR #1, SalmoKCaR#2 and Sahnol(CaR#3, respectively, and wells 1, 2, 3,
5, 6, and 7, bottom row, for Figures 24B, D, and F are designated as ladder,
water,
ovary, SalmoKCaR #1, SalmoKCaR#2 and SalmoKCaR#3, respectively.
Figures 25A-F are graphical representations comparing six photographs
showing RT-PCR analysis of freshwater (Figures 25B, D and F) and seawater
(Figures 25A, C and E) adapted Atlantic salmon tissues using either SalmoKCaR
#2
specific PCR primers (Figures 25A-D) or salmon actin PCR primers (Figures
25E,F). Wells 1-14 for Figures 25A-F, top row, are designated as follows:
ladder,
gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca,
proximal
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intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver and
muscle,
respectively. Wells 1, 2,3, 5, 6, and 7. bottom row, for Figures 25A, C, and E
are
designated as ladder, water, Kidney-RT, SalmoKCaR #1, SalmoKCaR#2 and
SalmoKCaR#3, respectively, and wells 1, 2, 3, 5, 6, and 7, bottom row, for
Figures
25B, D, and F are designated as ladder, water, ovary, SalmoKCaR #1,
SalmoKCaR#2 and SalmoKCaR#3, respectively.
Figure 26 is a schematic diagram illustrating industry practice for salmon
aquaculture production, prior to the discovery of the present invention. The
diagram
depicts key steps in salmon production for SO (75 gram) and Si (100 gram)
smolts.
The wavy symbol indicates freshwater while the bubbles indicate seawater.
Figure 27A is a graphical representation comparing the weekly feed
consumption on a per fish basis betvveen Process I treated smolts weighing
approximately 76.6 gm vs industry standard smolt weighing approximately 95.8
gm.
These data are derived from individual netpens of fish containing about 10,000-
50,000 fish per pen. As shown, fish treated with Process I consumed
approximately
twice as much feed per fish during their first week after seawater transfer as
compared to the large industry standard smolts weekly food consumption after
30
days. Process I treatment is defined in the Exemplification.
Figure 27B is a graphical representation illustrating length (cm) and weight
(gm) of Process I Smolts 50 days after ocean netpen placement. Process I
smolts
had an average weight of 76.6 gram, when placed in seawater and were sampled
after
50 days.
Figure 28 is a graphical representation illustrating length (cm) and weight
(gm) of representative Process I smolts prior to transfer to seawater.
Figure 29 is a graphical representation illustrating length (cm) and weight
(gm) of Process I smolts before transfer, and mortalities after transfer to
ocean
netp ens.
Figure 30 is a three dimensional graph illustrating the survival over 5 days
of
Arctic Char in seawater after being maintained in freshwater, Process I for 14
days,
and Process I for 30 days.
Figure 31 is a graphical representation illustrating the length (cm) and
weight
(gin) of St. John/St. John Process II smolts prior to seawater transfer.
Process II is
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defined in the Exemplification Section.
Figures 32A and 32B are graphical representations illustrating weight (gm)
and length (cm) of Process II smolt survivors and mortalities 5 days after
transfer to
seawater tanks (A), and 96 hours after transfer to ocean netpens (B).
Figures 33A-G are photographs of immunocytochemistry of epithelia of the
proximal intestine of Atlantic Salmon illustrating SalmoKCaR localization and
expression.
Figure 34 is a photograph of a Western Blot of intestinal tissue from salmon
subjected to Process I for immune (lane marked CaR, e.g., a SalmoKCaR) and
preimmune (lane marked preinunune) illustrating SalmoKCaR expression.
Figures 35A-C are photographs of immunolocalization of the SalmoKCaR in
the epidermis of salmon illustrating SalmoKCaR localization and expression.
Figure 36 is a graphical representation quantifying the Enzyme-Linked
ImmunoSorbent Assay (ELISA) protein (ng) for various tissue samples (e.g.,
gill,
liver, heart, muscle, stomach, olfactory epithelium, kidney, urinary bladder,
brain,
pituitary gland, olfactory bulb, pyloric ceacae, proximal intestine, and
distal
intestine) from a single fish.
Figure 37 is a photograph of a RT-PCR amplification of a partial
SalmoKCaR mRNA transcript from various tissues (gill, nasal lamellae, urinary
bladder, kidney, intestine, stomach, liver, and brain (Wells 1-8,
respectively)) of
Atlantic Salmon. RT-PCR reactions were separated by gel electrophoresis and
either
stained in ethidium bromide (EtBr) or transferred to a membrane and Southern
Blotted (SB) using a 32P-labeled 653 basepair (bp) genomic DNA fragment from
the Atlantic salmon SalmoKCaR gene. Wells 9 and 10 are water (blank) and
positive
control, respectively.
Figure 38 is a series of photographs of immunocytochemistry showing the
SalmoKCaR localization of Atlantic Salmon Olfactory Bulb Nerve and Lamellae
using an anti-SalmoKCaR antibody.
Figure 39 is a schematic illustrating the effect of external and internal
ionic
concentrations on the olfactory lamellae in response to SalmoKCaR modulators.
Figure 40A is a photograph of immunocytochemistry showing the
SalmoKCaR protein expression in the developing nasal lamellae (Panel A) and
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olfactory bulb (Panel B) after hatching of Atlantic salmon using an anti-
SalmoKCaR
antibody.
Figure 40B is a photograph of immunocytochemistry of Atlantic salmon or
trout larval fish using Sal-I antiserum shows abundant PVCR protein expression
by
selected cells. Specific binding of Sal-I antiserum denoting the presence of
PVCR
protein is shown by the dark reaction product. Staining of myosepta between
various muscle bundles of larval fish is shown by asterisks (panel A). Panel B
shows the head of a trout larvae in cross section where abundant PVCR protein
is
present in the skin (asterisks) and developing nasal lamellae (open
arrowhead).
Panel C shows PVCR expression in the developing otolith as well as localized
PVCR protein in epithelial cells immediately adjacent to it. Panels D and E
show
high magnification views of myosepta shown in Panel A. Note the pattern of
localized expression of PVCR protein where some cells contain large amounts of
PVCR protein while those immediately adjacent to them have little or no
expression.
Panel F shows a corresponding H+E section where myosepta (open arrowheads) can
be clearly distinguished from intervening muscle bundles.
Figure 40C is a photograph showing localization of Sal ADD antiserum by
inununocytochemistry. Panel A shows the pattern of immunostaining of immune
anti-Sal ADD serum as compared to lack of reactivity displayed by preimmune
anti-
Sal ADD serum when exposed to identical kidney tissue sections (Panel B). Note
that anti-Sal ADD reactivity (denoted by arrows) is similar if not identical
to that
displayed by Sal-I antiserum. Corresponding kidney tubules exposed to
preimmune
antiserum show no reactivity (denoted by asterisks).
Figure 41 is a photograph of immunocytochemistry showing the PVCR
localization in nasal lamellae of dogfish shark using an anti-PVCR antibody.
Figure 42 is a photograph of a Southern blot of RT-PCR analyses of tissues
from Atlantic Salmon showing the presence of SalmoKCaR mRNA in nasal
lamellae of freshwater adapted fish. Wells 1-10 are designated as follows:
gill, nasal
lamellae, urinary bladder, kidney, intestine, stomach, liver, brain, water
(blank) and
positive control, respectively.
Figure 43 is a histogram illustrating the amount of SalmoKCaR protein, as
detemined by an ELISA (ng) for various tissue samples (gill, liver, heart,
muscle,
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stomach, olfactory epithelium, kidney, urinary bladder, brain, pituitary
gland,
olfactory bulb, pyloric ceacae, proximal intestine, and distal intestine).
Figure 44 shows the raw and integrated recordings from high resistance
electrodes of freshwater adapted Atiantic Salmon when exposed to 500 M L-
alanine, 1 mmol calcium, 50 p.M Gadolinium, and 250 mmol of NaCl. The figures
show the existence of an olfactory recording in response to L-alanine,
calcium,
gadolinium, and NaCl.
Figure 45 is a graph showing the response data for freshwater adapted
Atlantic salmon nasal lamellae for calcium, magnesium, gadolinium, and sodium
chloride normalized to the signal obtained with10 mM Calcium.
Figure 46 shows raw recording from high resistance electrodes of olfactory
nerve impulse in the presence of a repellant (finger rinse) and in the
presence of a
SalmoKCaR agonist (gadolinium) and a repellant (finger rinse). The figure
shows
that the olfactory nerve impulse to the repellant is reversibly altered in the
presence
of a SalmoKCaR agonist.
Figure 47 shows the raw recordings from high resistance electrodes of
freshwater adapted Atlantic Salmon in response to a series of repeated stimuli
(L-
alanine or NaC1) in 2 minute intervals. The figure shows that the olfactory
nerve
impulse to the attractant is reversibly altered in the presence of a SalmoKCaR
agonist
Figure 48 is a graphical representation of the ratio from FURA-2 cells
expressing a PVCR in the presence or absence of 10 mM L-Isoleucine in various
concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 20.0 mM) of extracellular calcium
(Ca').
Figure 49 is a graphical representation of the fractional Ca' response, as
compared to the extracelluar Ca' (mM) for the PVCR in Ca' only, Phenylalanine,
Isoleucine, or AA Mixture (a variety of L-isomers in various concentrations).
Figure 50 shows the nucleic acid sequence for a Fugu receptor, Fugu CaR
(01), (SEQ ID NO: 31) deposited under GenBank Accession Nos: AB008857.
Figures 51A-B show the nucleic acid sequence for a Fugu receptor, Fugu
Ca02.1, (SEQ ID NO: 32) deposited under GenBank Accession Nos: AB008858.
Figures 52A-B show the nucleic acid sequence for a Fugu receptor, Fugu
Ca09, (SEQ ID NO: 33) deposited under GenBank Accession Nos: AB008859.
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Figures 53A-B show the nucleic acid sequence for a Fugu receptor, Fugu
Ca12, (SEQ ID NO: 34) deposited under GenBank Accession Nos: AB008860.
Figures 54A-B show the nucleic acid sequence for a Fugu receptor, Fugu
Ca13, (SEQ ID NO: 35) deposited under GenBank Accession Nos: AB008861.
Figures 55A-B show the nucleic acid sequence for a Fugu receptor, Fugu
Ca15.1, (SEQ ID NO: 36) deposited under GenBank Accession Nos: A1B008862.
Figure 56 shows the nucleic acid sequence for a Fugu receptor, Fugu
mGluR1, (SEQ ID NO: 37) deposited under GenBank Accession Nos: AB008863.
Figure 57 shows the nucleic acid sequence for a Fugu receptor, Fugu
mGluR2, (SEQ ID NO: 38) deposited under GenBank Accession Nos: AB008864.
Figure 58 shows the nucleic acid sequence for a Fugu receptor, Fugu
mGluR7, (SEQ ID NO: 39) deposited under GenBank Accession Nos: AB008865.
Figure 59 shows the nucleic acid sequence for a Fugu receptor, Fugu
mGluR8, (SEQ ID NO: 40) deposited under GenBank Accession Nos: AB008866.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to at least four novel isolated sequences from
PVCR genes, SalmoKCaR#1, SalmoKCaR#2, SalmoKCaR#3, and SalmoKCaR#4
in Atlantic Salmon. These genes encode four polypeptide sequences that are
also the
subject of the present invention. These polypeptide sequences allow for or
assist in
several functions in Atlantic Salmon including sensing at least one SalmoKCaR
modulator in serum or in the surrounding environment; adapting to at least one
SalmoKCaR modulator present in the serum or surrounding environment;
imprinting
with an odorant; altering water intake; altering water absorption; or altering
urine
output.
Uses of the Present Invention
One use of the present invention relates to methods for improving the raising
of salmon and/or methods for preparing salmon for transfer from freshwater to
seawater. These methods involve adding one or more PVCR (e.g., SalmoKCaR)
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modulators to the freshwater (e.g., calcium and/or magnesium), and adding a
specially made or modified feed to the freshwater for consumption by the fish.
The
feed contains a sufficient amount of sodium chloride NaCl)( and/or a
SalmoKCaR
modulator (e.g., an amino acid like tryptophan) to significantly increase
levels of the
SalmoKCaR modulator in the serum. During this process, the serum level of the
SalmoKCaR modulator significantly increases in the salmon, and causes
modulated
(e.g., increased and/or decreased) SalmoKCaR expression and/or altered
SalmoKCaR sensitivity. This process prepares salmon for transfer to seawater,
so
that they can better adapt to seawater once they are transferred. The details
of how
to carry out this process is described in the Exemplification Section. In
particular,
the Exemplification describes two processes. Briefly, Process I involves
adding
calcium and magnesium to the water, and providing feed containing NaCl; and
Process II includes adding calcium and magnesium-to the water, and providing
feed
having both NaCl and tryptophan. Studies performed and described in Example 7
show that Atlantic Salmon maintained in freshwater and subjected to Process I
had a
survival rate of 91%, and those Atlantic Salmon subjected to Process II had a
survival rate of 99%; as compared to control fish having a survival rate of
only 67%
after transfer to seawater. Similarly, in the same experiment, five days after
transfer
to seawater, Atlantic Salmon subjected to Process I had a survival rate of
90%, while
Atlantic Salmon subjected to Process II had a survival rate of 99%. The
control fish
had a survival rate of only 50% after being transferred to seawater.
Furthermore,
experiments described in Example 6 demonstrate that modulated expression of
one
or more SahnoKCaR genes occurs in various tissues during Process I and Process
II.
Process I and II, as described herein, modulate the SalmoKCaR genes and allow
for
increased food consumption, growth and survival; and decreased morbidity and
susceptibility to disease.
Process I and II likely have further utility in restoration of wild Atlantic
salmon populations. Since a major cause of mortality of wild Atlantic salmon
smolt
is loss or capture by predators as they are adapting to seawater in river
estuaries,
treatment of wild Atlantic salmon produced in large numbers, as part of river
restocking programs would boost the productivity and survival of fish produced
in
such programs. Moreover, several studies have shown that salmon smolt are also
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poisoned by exposure to heavy metals (A13+, Zn21, Cu2+) that contaminate their
native
rivers in both the US and other countries such as Norway. These highly
deleterious
effects on salmon are manifested principally in rivers with low natural Ca'
concentrations. Thus, treatment of wild strains of Atlantic salmon produced in
restocking hatcheries with either Process I or Process 11 would render these
treated
smolt less susceptible to the effects of heavy metals since the smoltification
process
in these treated smolt was much further advanced that in untreated fish. Use
of
Process I or II to treat Atlantic salmon that would be released into rivers
also have
commercial utility in large-scale ocean ranching programs where large numbers
of
salmon smolt are released and captured for human consumption upon their return
from 1-3 years in the ocean.
Similarly, since expression of the SalmoKCaR genes changes during Process
- I and Process-II, assaying these genes allows one to determine if the salmon
are
ready for transfer to seawater. Examples of such assays are ELISAs,
radioinununoassays (RIAs), southern blots and RT-PCR assays, which are
described
herein in detail. The salmon are subjected to either Process I or Process II
for a
period of time in freshwater before being transferred to seawater. The
SalmoKCaR
genes, or polypeptides encoded by these genes, can be assayed for determining
the
optimal time period for maintaining the salmon in the freshwater, before
transfer to
seawater. Using methods described herein, salmon can be assayed to determine
if
modulated levels of.the SalmoKCaR genes and/or polypeptides have occurred, as
compared to controls. For example, when fish that are maintained in freshwater
and
subjected to either Process I or Process II and changes in one or more of
SalmoKCaR genes and/or polypeptide levels in at least one tissue are modulated
such that they mimic changes in the same genes and/or polypeptide levels that
would
be seen in fish adapted to seawater, then this group of fish are ready to be
transferred
to seawater. In one experiment, the increased expression of SalmoKCaR genes in
the kidney of Atlantic Salmon subjected to Process II was similar to the
increased
expression in the same tissue for Atlantic Salmon already adapted to seawater,
but
dissimilar to expression to Atlantic Salmon adapted to freshwater (i.e., no
increased
expression in the kidney water fish was seen). See Example 6. When levels of
SalmoKCaR genes and/or polypeptide encoded by these genes are similar to those
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levels seen in fish that have been transferred to seawater, then in the
experiments
described herein, the transfer of these salmon result in several benefits
including
increased survival and growth. Also, the optimal time periods for subjecting
salmon
to Process I or Process II are generally between 4-6 weeks, but vary depending
on
the strain of salmon or process used. Hence, the assays described herein can
be used
to deteiniine the optimal amount of time for subjecting the salmon to either
Process I
or Process II before transferring to seawater.
Additionally, comparison of the SalmoKCaR #3 sequence with data
generated from site directed mutagenesis studies of mammalian CaRs indicates
that
the Sahnol(CaR #3 protein likely generates a dominant negative effect on the
other
SalmoKCaR #1, #2 and #4 proteins when they are expressed together in the same
cell. This dominant negative effect of SalmoKCaR #3 occurs since it lacks that
necessary carboxyl terminal domain to propagate signals generated by the
binding of
PVCR agonists. Interactions between the fully functional SalmoKCaR #1, #2 or
#4
proteins and SalmoKCaR #3 would cause a marked reduction in the sensitivity of
the SalmoKCaR #1, #2 or #4 proteins. In one experiment, it was found that
increased expression of SalmoKCaR#3 was seen in tissues readily exposed to
high
concentrations of calcium and magnesium in the surrounding environment (e.g.,
gill
and nasal lamellae) or tissues that excrete high concentrations of calcium and
magnesium (e.g., urinary bladder and kidney). Therefore, such assays can be
used to
determine levels of the individual SalmoKCaR genes, and compare expression
levels
to one another, and to individual levels of these genes of seawater adapted
salmon to
determine whether the salmon being tested are ready for transfer to seawater.
Uses of nucleic acids of the present invention include one or more of the
following: (1) producing receptor proteins which can be used, for example, for
structure determination, to assay a molecule's activity, and to obtain
antibodies
binding to the receptor; (2) being sequenced to determine a receptor's
nucleotide
sequence which can be used, for example, as a basis for comparison with other
receptors to determine one or more of the following: conserved sequences;
unique
nucleotide sequences for noinial and altered receptors; and nucleotide
sequences to
be used as target sites for antisense nucleic acids, ribozymes, or PCR
amplification
primers; (3) as hybridization detection probes to detect the presence of a
native
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receptor and/or a related receptor in a sample, as further described herein to
determine the presence or level of SalmoKCaR in a sample for, e.g., assessing
whether salmon are ready for transfer to seawater; (4) as PCR primers to
generate
particular nucleic acid sequence sequences, for example, to generate sequences
to be
used as hybridization detection probes; and (5) for determining and isolating
additional aquatic PVCR homologs in other species.
Another use for nucleic acid sequences of SalmoKCaRs #1, #2, #3, or #4 is
as probes for the screening of Atlantic salmon broodstock, eggs, sperm,
embryos or
larval and juvenile fish as part of breeding programs. Use of SalmoKCaR probes
would enable identification of desirable traits such as enhanced salinity
responsiveness, homing, growth in seawater or freshwater or improve the feed
utilization that were due to or associated with naturally occurring or induced
mutations of SalmoKCaR genes. Nucleic -acid sequences- of SalmoKCaRs #1, #2,
#3, or #4 can also be used as probes for screening of wild Atlantic salmon in
various
regions as a tool to identify specific strains of fish from both sea run and
land locked
strains. Such strains could then be used to interbreed with existing
commercial
strains to produce further improvements in fish performance.
The structural-functional data generated via study of recombinant
SalmoKCaRs after their expression in cells as functional proteins can be used
to
identify desirable alternations in the function of SalmoKCaR proteins that
could then
be screened for as part of genetic selection-broodstock enhancement program.
Cell lines expressing SahnoKCaR proteins, either individually or in various
combinations, would have utility and value as a means to assay various
compounds,
chemicals and water conditions that occur both in the natural and commercial
environments. Utilization of transfected cells expressing SalmoKCaR #1-4
proteins
either alone or in various combinations can be used in screening methods to
identify
both naturally occurring and commercially synthesized compounds that would
enhance the performance of wild or commercially produced Atlantic salmon
including salinity adaption, feeding, growth and maturation, flesh quality,
homing to
areas of spawning, recognition of specific odorants as part of imprinting,
utilization
of nutrients with improved efficiency and altered behavior. Such screening
assay
would be a vast improvement over existing assays where large numbers of fish
are
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required and their end response (e.g., behavior, feeding, growth, survival or
appearance is altered) to a given compound produce complicated assays that
have
many problems with data interpretation. Transfected cells expressing SalmoKCaR
#1-4 proteins either alone or in various combinations can also be used in
screening
methods to screen for specific water conditions including pH, ionic strength
and
composition of various compounds dissolved in the water to alter the function
of
SalmoKCaR proteins and thus lead to improved salinity responses in various
life
stages of Atlantic salmon. Such assays would be designed to detellnine the
interactions and effects of these conditions on SalmoKCaR proteins without
having
to test the effects of such compounds on either whole living fish or some
tissue
explants.
Fragments of recombinant SalmoKCaR proteins also provide a utility as
modulators of PVCR function that could be added to water, applied to tissue
surfaces such as gills or skin or injected into fish via standard techniques.
The
present invention is also useful in immunization of any one of the various
life stages
of Atlantic salmon (eggs, embryo, larval or juvenile or adult fish) with
either whole
or fragments of recombinant SalmoKCaR proteins to create antibody responses
that
would, in turn, alter SalmoKCaR mediated functions of fish.
The present invention is not limited to the uses described in this section.
Based on the data and information described herein, additional uses of the
present
invention may be readily appreciated by one of skill in the art.
The SalmoKCaR Polypeptides and its Function
The present invention relates to isolated polypeptide molecules that have
been isolated in Atlantic Salmon including four full length sequences. The
present
invention includes polypeptide molecules that contain the sequence of any one
of the
full length SalmoKCaR amino acid sequence (SEQ ID NO: 8, 10, 12, or 14). See
Figures 9, 10, 11, and 12. The present invention also pertains polypeptide
molecules
that are encoded by nucleic acid molecules having the sequence of any one of
the
isolated full length SalmoKCaR nucleic acid sequences (SEQ ID NO: 7, 9, 11, or
13).
SalmoKCaR polypeptides referred to herein as "isolated" are polypeptides
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that separated away from other proteins and cellular material of their source
of
origin. Isolated SalmoKCaR proteins include essentially pure protein, proteins
produced by chemical synthesis, by combinations of biological and chemical
synthesis and by recombinant methods. The proteins of the present invention
have
been isolated and characterized as to its physical characteristics using
laboratory
techniques common to protein purification, for example, salting out,
immunoprecipation, column chromatography, high pressure liquid chromatography
or electrophoresis. SalmoKCaR proteins are found in many tissues in fish
including
gill, nasal lamellae, urinary bladder, kidney, stomach, pyloric caeca,
proximal
intestine, distal intestine, brain, pituitary gland, olfactory bulb, liver,
muscle, skin
and brain.
The present invention also encompasses SalmoKCaR proteins and
polypeptides having amino acid sequences analogous to the amino acid sequences
of
SalmoKCaR polypeptides. Such polypeptides are defined herein as SalmoKCaR
analogs (e.g., homologues), or mutants or derivatives. "Analogous" or
"homolgous"
amino acid sequences refer to amino acid sequences with sufficient identity of
any
one of the SalmoKCaR amino acid sequences so as to possess the biological
activity
of any one of the native SalmoKCaR polypeptides. For example, an analog
polypeptide can be produced with "silent" changes in the amino acid sequence
wherein one, or more, amino acid residues differ from the amino acid residues
of any
one of the SalmoKCaR protein, yet still possesses the function or biological
activity
of the SalmoKCaR. Examples of such differences include additions, deletions or
substitutions of residues of the amino acid sequence of SalmoKCaR. Also
encompassed by the present invention are analogous polypeptides that exhibit
greater, or lesser, biological activity of any one of the SalmoKCaR proteins
of the
present invention. Such polypeptides can be made by mutating (e.g.,
substituting,
deleting or adding) one or more amino acid or nucleic acid residues to any of
the
isolated SalmoKCaR molecules described herein. Such mutations can be performed
using methods described herein and those known in the art. In particular, the
present
invention relates to homologous polypeptide molecules having at least about
70%
(e.g., 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID NO: 8,
10,
12, or 14. Percent "identity" refers to the amount of identical nucleotides or
amino
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acids between two nucleotides or amino acid sequences, respectfully. As used
herein, "percent similarity" refers to the amount of similar or conservative
amino
acids between two amino acid sequences. Each of the SalmoKCaR polypeptides are
homologous to one another.
The percent identity when comparing one SalmoKCaR amino acid sequence to
another are as follows:
Percent Identity for Amino Acid Sequences*
Query SalmoKCaR#1 SalmoKCaR#2 SalrnoKCaR#3 SalmoKCaR#4
Sequence
SalmoKCaR#1 N/A 99.9% 89.6% 100%
SalmoKCaR#2 99.9% N/A 89.5% 99.9%
SalmoKCaR#3 99.2% 99.1% N/A 99.2%
SalmoKCaR#4 100% 99.9% 99.2% N/A
* Note that the percentages are based on the number of aa's in the target
sequence.
The polypeptides of the present invention, including the full length
sequences, the partial sequences, functional fragments and homologues, that
allow
for or assist in one or more of the following functions (e.g., in Atlantic
Salmon):
_
sensing at least one SalmoKCaR modulator in serum or in the surrounding
environment; adapting to at least one SalmoKCaR modulator present in the serum
or
surrounding environment; imprinting with an odorant; altering water intake;
altering
water absorption; altering urine output. These and additional functions of the
polypeptides are further described herein, and illustrated by the
Exemplification.
The tem "sense" or "sensing" refers to the SalmoKCaR's ability to alter its
expression and/or sensitivity in response to a SalmoKCaR modulator.
Homologous polypeptides can be determined using methods known to those
of skill in the art. Initial homology searches can be performed at NCBI
against the
GenBank, EMBL and SwissProt databases using, for example, the BLAST network
service. Altschuler, S.F., et al., J Mol. Biol., 215:403 (1990), Altschuler,
S.F.,
Nucleic Acids Res., 25:3389-3402 (1998). Computer analysis of nucleotide
sequences can be performed using the MOTIFS and the FindPatterns subroutines
of
the Genetics Computing Group (GCG, version 8.0) software. Protein and/or
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nucleotide comparisons were performed according to Higgins and Sharp (Higgins,
D.G. and Sharp, P.M., Gene, 73:237-244 (1988) e.g., using default parameters).
The SalmoKCaR proteins of the present invention also encompass
biologically active or functional polypeptide fragments of the full length
SalmoKCaR proteins. Such fragments can include the partial isolated amino acid
sequences (SEQ ID NO: 3 and 5), or part of the full-length amino acid sequence
(SEQ ID NO: 8, 10, 12, or 14), yet possess the function or biological activity
of the
full length sequence. For example, polypeptide fragments comprising deletion
mutants of the SalmoKCaR proteins can be designed and expressed by well-known
laboratory methods. Fragments, homologues, or analogous polypeptides can be
evaluated for biological activity, as described herein.
In one embodiment, the function or biological activity relates to preparing
salmon for transfer to seawater. The method for preparing Atlantic Salmon for
transfer to seawater includes adding at least one SalmoKCaR modulator (e.g.,
PVCR
modulator) to the freshwater, and adding a specially made or modified feed to
the
freshwater for consumption by the fish. The feed contains a sufficient amount
of
sodium chloride NaCl)( (e.g.,
between about 1% and about 10% by weight, or about
10,000 mg/kg to about 100,000 mg/kg) to significantly increase levels of the
SalmoKCaR modulator in the serum. This amount of NaC1 in the feed causes or
induces the Atlantic Salmon to drink more freshwater. Since the freshwater
contains
= a SalmoKCaR modulator and the salmon ingest increased amounts of it, the
serum
level of the almoKCaR modulator significantly increases in the salmon, and
causes
modulated (e.g., increased and/or decreased) SalmoKCaR expression and/or
altered
SalmoKCaR sensitivity. One function or activity of the SalmoKCaR genes is to
sense SalmoKCaR modulators in the serum. The SalmoKCaR expression is altered
by the SalmoKCaR modulators in the serum, which provides the ability for the
salmon to better adapt to seawater, undergo smoltification, survive, grow,
consume
food and/or to be less susceptible to disease.
A "PVCR modulator" or "SalmoKCaR modulator" refers to a compound
which modulates (e.g., increases and/or decreases) expression of SalmoKCaR, or
alters the sensitivity or responsiveness of SalmoKCaR genes. Such compounds
include, but are not limited to, SalmoKCaR agonists (e.g., inorganic
polycations,
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organic polycations and amino acids), Type II calcimimetics, and compounds
that
indirectly alter PVCR expression (e.g., 1,25 dihydroxyvitamin D in
concentrations of
about 3,000 ¨10,000 International Units /kg feed), cytokines such as
Interleukin
Beta, and Macrophage Chemotatic Peptide-1 (MCP-1)). Examples of Type IT
calcimimetics, which increase and/or decrease expression, and/or sensitivity
of the
SalmoKCaR genes, are, for example, NPS-R-467 and NPS-R-568 from NPS
Pharmaceutical Inc., (Salt Lake, Utah, Patent Nos. 5,962,314; 5,763,569;
5,858,684;
5,981,599; 6,001,884) which can be administered in concentrations of between
about 0.1 M and about 100 p.M feed or water. See Nemeth, E.F. et al., PNAS 95:
4040-4045 (1998). Examples of inorganic polycations are divalent cations
including
calcium at a concentration between about 2.0 and about 10.0 mM and magnesium
at
a concentration between about 0.5 and about 10.0 mM; and trivalent cations
including, but not limited to, gadolinium (Gd3+) at a concentration between
about 1
and about 500 [tM. Organic polycations include, but are not limited to,
aminoglycosides such as neomycin or gentamicin in concentrations of between
about
1 and about 8 gm/kg feed as well as organic polycations including polyamines
(e.g.,
polyarginine, polylysine, polyhistidine, polyomithine, spermine, spennidine,
cadaverine, putrescine, copolymers of poly arginine/histidine, poly
lysine/arginine in
_ _
concentrations of between about 10 [tM and 10 mM feed). See Brown, E.M. et
al.,
Endocrinology 128: 3047-3054 (1991); Quinn, S.J. et al., Am. J. Physiol. 273:
C1315-1323 (1997). Additionally, SalmoKCaR agonists include amino acids such
as L-Tryptophan L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-Arginine,
L-Histidine, L-Leucine, L-Isoleucine, L-Aspartic acid, L-Glutamic acid, L-
Glycine,
L-Lysine, L-Methionine, L-Asparagine, L-Proline, L-Glutamine, L-Threonine, L-
Valine, and L-Cysteine at concentrations of between about 1 and about 10 gm/kg
feed. See Conigrave, A.D., et al., PNAS 97: 4814-4819 (2000). Amino acids, in
one
embodiment, are also defined as those amino acids that can be sensed by at
least one
SalmoKCaR in the presence of low levels of extracellular calcium (e.g.,
between
about 1 mM and about 10 mM). In the presence of extracellular calcium, the
SalmoKCaR in organs or tissues such as the intestine, pyloric caeca, or kidney
can
better sense amino acids. The molar concentrations refer to free or ionized
concentrations of the SalmoKCaR modulator in the freshwater, and do not
include
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amounts of bound SalmoKCaR modulator (e.g., SahnoKCaR modulator bound to
negatively charged particles including glass, proteins, or plastic surfaces).
Any
combination of these modulators can be added to the water or to the feed (in
addition
to the NaC1, as described herein), so long as the combination modulates
expression
and/or sensitivity of one or more of the SalmoKCaR genes.
Another function of the SalmoKCaR polypeptides involves imprinting
Atlantic Salmon with an odorant (e.g., an attractant or repellant). Atlantic
Salmon
can be imprinted with an odorant so that, when the fish are later exposed to
the
odorant, they can more easily distinguish the odorant or are sensitized to the
odorant.
The SalmoKCaR polypeptides can work, for example, with one or more olfactory
receptors to modify the generation of the nerve impulse during sensing of an
odorant. Generation of this nerve impulse occurs upon binding of the odorant
to the
olfactory lamellae in the-fish. The SalmoKCaR modulator alters the olfactory
sensing of the salmon to the odorant. In some cases, the presence of a (e.g.,
at least
one) SalmoKCaR modulator in freshwater reversibly reduces or ablates the
fish's
ability to sense certain odorants. In other cases it can be heightened or
increased.
By exposing the salmon in freshwater having a SalmoKCaR modulator to an
odorant, the fish have an altered response which depending on the modulator
would
consist of either a decreased or heightened response to the odorant. Briefly,
these
imprinting methods involve adding at least one SalmoKCaR modulator (e.g.,
calcium and magnesium) to-the freshwater in .an amount sufficient to modulate
expression and/or sensitivity of at least one SalmoKCaR genre; and adding feed
for
fish consumption to the freshwater. The feed contains at least one an
attractant (e.g.,
alanine); an amount of NaC1 sufficient to contribute to a significantly
increased level
of the SalmoKCaR modulator in serum of the Atlantic Salmon; and optionally a
SalmoKCaR modulator (e.g., tryptophan). The odorant can also be added to the
water, instead of the feed. Salmon that has been imprinted with an attractant
consume more feed having this attractant and, as a result, grow faster. The
imprinting process occurs during various developmental stages of salmon
including
the larval stage and the smoltification stage. Localizations of SahnoKCaR
proteins
and detection of SalmoKCaR expression using RT-PCR in various organs involved
in the imprinting process including olfactory lamellae, olfactory bulb and
brain is
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provided for both larval (Example 13) and smolt stages (Figures 37 and 38).
The
process of imprinting the salmon with an odorant refers to creating a lasting
effect or
impression on the fish so that the fish are sensitized to the odorant or can
distinguish
the odorant. Being sensitized to the odorant refers to the fish's ability to
more easily
recognize or recall the odorant. Distinguishing an odorant refers to the
fish's ability
to differentiate among one or more odorants, or have a preference for one
odorant
over another.
An odorant is a compound that binds to olfactory receptors and causes fish to
sense odorants. Generation of an olfactory nerve impulse occurs upon binding
of the
odorant to the olfactory lamellae. A fish odorant is either a fish attractant
or fish
repellant. A fish attractant is a compound to which fish are attracted. The
sensitivity
of the attractant is modulated, at least in part, by the sensitivity and/or
expression of
the SalmoKCaR genes in the olfactory apparatus of the fish in response to a
SalmoKCaR modulator. Examples of attractants in some fish include amino acids
(e.g., L-Tryptophan L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-
Arginine,
L-Histidine, L-Leucine, L-Isoleucine, L-Aspartic acid, L-Glutamic acid, L-
Glycine,
L-Lysine, L-Methionine, L-Asparagine, L-Proline, L-Glutamine, L-Threonine, L-
Valine, and L-Cysteine), nucleotides (e.g., inosine tnonophosphate), organic
compounds (e.g., glycine-betaine and trimethylamine oxide), or a combination
thereof. Similarly, a fish repellant is a compound that fish are repelled by,
and the
sensitivity of the fish to the repellant is altered through expression and/or
sensitivity
of a SafmoKCaR gene in the olfactory apparatus of the fish in the presence of
a
SalmoKCaR modulator. An example of a repellant is a "finger rinse" which is a
mixture of mammalian oils and fatty acids produced by the epidermal cells of
the
skin, and is left behind after human fingers are rinsed with an aqueous
solution.
Methods for performing a finger rinse is known in the art and is described in
more
detailed in the Exemplification Section.
Additionally, the function of SalmoKCaR polypeptides includes its ability to
sense or adapt to ion concentrations in the surrounding environment. The
SalmoKCaR polypeptides sense various SalmoKCaR modulators including calcium,
magnesium and/or sodium. The SalmoKCaR polypeptides are modulated by varying
ion concentrations. For instance, any one of the SalmoKCaR polypeptides can be
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modulated (e.g., increased or decreased) in response to a change in ion
concentration
(e.g., calcium, magnesium, .or sodium). Responses to changes in ion
concentrations
of Atlantic Salmon containing the SalmoKCaR polypeptides include the ability
to
adapt to the changing ion concentration. Such responses include the amount the
fish
drinks, the amount of urine output, and the amount of water absorption.
Responses
also include changes in biological processes that affect its ability to
excrete
contaminants.
More specifically, methods are available to regulate salinity tolerance in
fish
by modulating (e.g., increasing, decreasing or maintaining the expression) the
activity of one or more of the SalmoKCaR proteins present in cells involved in
ion
transport. For example, salinity tolerance of fish adapted (or acclimated) to
freshwater can be increased by activating one or more of the SalmoKCaR
polypeptides, for example, by increasing the expression of one or more of
SalmoKCaR genes, resulting in the secretion of ions and seawater adaption.
Alternatively, the salinity tolerance of fish adapted to seawater can be
decreased by
inhibiting one or more of the SalmoKCaR proteins, resulting in alterations in
the
absorption of ions and freshwater adaption.
"Salinity" refers to the concentration of various ions in a surrounding
aquatic
_
environment. In particular, salinity refers to the ionic concentration of
calcium,
magnesium and/or sodium (e.g., sodium chloride). "Normal salinity" levels
refers to
the range of ionic concentrations of typical water environment in which an
aquatic
species naturally lives. Normal salinity or normal seawater concentrations are
about
10mM Ca, about 40mM Mg, and about 450 mM NaCl. "Salinity tolerance" refers to
the ability of a fish to live or survive in a salinity environment that is
different than
the salinity of its natural environment. Modulations of the PVCR allows fish
to live
in about four times and one-fiftieth, preferably, twice and one-tenth the
normal
The ability of anadromous fish (Atlantic salmon, trout and Arctic char) as
well as euryhaline fish (flounders, alewives, eels) to traverse from
freshwater to
seawater environments and back again is of key importance to their lifecycles
in the
natural environment. Both types of fish have to undergo similar physiological
changes including alterations in their urine output, altering water intake and
water
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absorption. Both types of fish utilize environments of either freshwater
(Atlantic
salmon) or partial salinity (flounders) to spawn and allow for the development
of
larval fish into juvenile forms that then undergo changes to migrate into full
strength
seawater. Both types of fish utilize PVCRs to sense when adult fish have
arrived in
a salinity environment suitable for spawning and to guide their return back to
full
strength seawater. Similarly, their resulting offspring utilize PVCRs to
control
various organs allowing for their normal development in fresh or brackish
(partial
strength seawater) water and subsequently to regulate the physiological
changes that
permit these fish to migrate into full strength seawater.
The following experiment was done in Summer and Winter Flounder, but is
applicable to Atlantic Salmon because both species of fish have PVCRs which
respond to ion concentrations in a similar manner. Summer and Winter Flounder
were adapted to live in 1/10th seawater (100 mOsm/kg) by reduction in salinity
from
450 mM NaCl to 45 mM NaCl over an interval of 8 hrs. Summer and Winter
Flounder can be maintained in 1/10 or twice the salinity for over a period of
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 ás in punctate sequences
throughout the cell. These data are consistent with previous Northern data
where
more PVCR protein is present in the urinary bladders of seawater fish vs fish
adapted to brackish water. These data show that PVCR protein is expressed in
epithelial cells that line the urinary bladder where the PVCR protein comes
into
direct contact with the urine that is being foimed by the kidney. Due to its
location
in the cell membrane of these epithelial cells, the PVCR proteins can "sense"
changes in the urine's composition on a continuous basis. Depending on the
specific
ionic concentrations of the urine, the PVCR protein alters the transport of
ions
across the epithelium of the urinary bladder and, in this way, determines the
final
composition of the urine. This composition and the amount of water and NaCI
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absorbed from the urine are critical to salinity regulation in fish.
As urinary magnesium and calcium concentrations increase when fish are
present in full strength sea water, activation of apical PVCR protein causes
reduction
in urinary bladder water transport. The invention provides methods to
facilitate
methods are now available to regulate salinity tolerance in fish by modulating
(e.g.,
alternating, activating and or expressing) the activity of the PVCR protein
present in
epithelial cells involved in ion transport, as well as in endocrine and
nervous tissue.
For example, salinity tolerance of fish adapted (or acclimated) to fresh water
can be
PVCR in selected epithelial cells, resulting in the secretion of ions and
seawater
adaption. Specifically, this would involve regulatory events controlling the
conversion of epithelial cells of the gill, intestine and kidney. In the
kidney, PVCR
activation facilitates excretion of divalent metal ions including calcium and
ions by gill cells that occurs in fresh water and promote the net excretion of
ions by
gill epithelia that occurs in salt water. In the intestine, PVCR activation
will permit
reabsorption of water and ions across the G.I. tract after their ingestion by
fish.
Alternatively, the salinity tolerance of fish adapted to seawater can be
In another example, Winter and Summer Flounder were maintained in at
These fish can be maintained in these environments for long periods of time
(e.g.,
over 3 months, over 6 months, or over 1 year). These limits were defined by
decreasing or increasing the ionic concentrations of calcium, magnesium, and
sodium, keeping a constant ratio between the ions. These salinity limits can
be
thereby changing the ionic concentration ratio among the ions. Increasing
and/or
decreasing individual ion concentrations can increase and/or decrease salinity
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tolerance. "Hypersalinity" or "above nomial salinity" levels refers to a level
of at
least one ion concentration that is above the level found in normal salinity.
"Hyposalinity" or "below normal salinity" levels refers to a level of at least
one ion
concentration that is below the level found in normal salinity.
Maintaining fish in a hypersalinity environment also results in fish with a
reduced number of parasites or bacteria. Preferably, the parasites and/or
bacteria are
reduced to a level that is safe for human consumption, raw or cooked. More
preferably, the parasites and/or bacteria are reduced to having essentially no
parasites
and few bacteria. These fish must be maintained in a hypersalinity environment
long
enough to rid the fish of these parasites or bacteria, (e.g., for at least a
few days or at
least a few weeks).
The host range of many parasites is limited by exposure to water salinity.
For example, Diphyllobothrium species commonly known as fish tapeworms, is
encountered in the flesh of fish, primarily fresh water or certain euryhaline
species.
Foodbome Pathogenic Microorganisms and Natural Toxins Handbook. 1991. US
Food and Drug Administration Center for Food Safety and Applied Nutrition, the
teachings of which are incorporated herein by reference in their entirety. In
contrast,
its presence in the flesh of completely marine species is much reduced or
absent.
Since summer flounder can survive and thrive at salinity extremes as high as
58 ppt
(1.8 times nonnal seawater) for extended periods in recycling water, exposure
of
summer flounder to hypersalinity conditions might be used as a-"biological"
remediation process to ensure that no Diphyllobothrium species are present in
the GI
tract of summer flounder prior to their sale as product.
Data from Cole et al., (.1 Biol. Chem. 272:12008-12013 (1997)), show that
winter flounder elaborate an antimicrobial peptide from their skin to prevent
bacterial infections. Their data reveals that in the absence of pleurocidin,
Escherichia coil are killed by high concentrations of NaCl. In contrast, low
concentrations of NaC1 (<300mM NaC1) allow E. colt to grow and under these
conditions pleurocidin presumably helps to kill them. These data provide
evidence
of NaC1 killing of E. coil, as well as highlight possible utility of bacterial
elimination
in fish.
Similarly, maintaining fish in a hyposalinity environment results in a fish
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with a reduced amount of contaminants (e.g., hydrocarbons, amines or
antibiotics).
Preferably, the contaminants are reduced to a level that is safe for human
consumption, raw or cooked fish. More preferable, the contaminants are reduced
to
having essentially very little contaminants left in the fish. These fish must
be
maintained in a hyposalinity environment long enough to rid the fish of these
contaminants, (e.g., for at least a few days or a few weeks).
Organic amines, such as trimethylamine oxide (TMAO) produce a "fishy"
taste in seafood. They are excreted via the kidney in flounder. (Krogh, A.,
Osmotic
Regulation in Aquatic Animals, Cambridge University Press, Cambridge, U.K. pgs
1-233 (1939), the teachings of which are incorporated herein by reference in
their
entirety). TMAO is synthesized by marine organisms consumed by fish that
accumulate the TMAO in their tissues. Depending on the species of fish, the
muscle
content of TMAO and organic amines is either large accounting for the "strong"
taste
of bluefish and herring or small such as in milder tasting flounder.
The presence of SalmoKCaR in brain reflects both its involvement in basic
neurotransmitter release via synaptic vesicles (Brown, E.M. et al., New
England J. of
Med., 333:234-240 (1995)), as well as its activity to trigger various hormonal
and
behavioral changes that are necessary for adaptation to either fresh water or
marine
environments. For example, increases in water ingestion by fish upon exposure
to
salt water is mediated by SalmoKCaR activation in a manner similar to that
described for humans where PVCR activation by hypercalcemia in the subfomical
organ of the brain cause an increase in water drinking behavior (Brown, E.M.
et al.,
New England J. of Med., 333:234-240 (1995)). In fish, processes involving both
alterations in serum hormonal levels and behavioral changes are mediated by
the
brain. These include the reproductive and spawning activities of euryhaline
fish in
fresh water after their migration from salt water as well as detection of
salinity of
their environment for purposes of feeding, nesting, migration and spawning.
The
key events for successful reproduction in Atlantic salmon are to migrate to a
specific
streambed for spawning after 1-3 years of free-swimming existence on the open
ocean. Successful achievement of this challenge depends on the combination of
adult salmon being able to remember and navigate their way back to this
original
location as well as successful imprinting of larval and juvenile Atlantic
salmon to
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=
odors present in freshwater in the freshwater streambed as well as the
characteristics
of the mouth of the river as the fish exit the river and enter the ocean.
Sensing of
salinity by PVCR and its modulation of the odorant detection system of salmon
for
detecting various odorants is critical to the achievement of these processes.
Data obtained recently from mammals now suggest that PVCR activation
plays a pivotal role in coordinating these events. For example, alterations in
plasma
cortisol have been demonstrated to be critical for changes in ion transport
necessary
for adaptation of salmon smolts from fresh water to salt water (Veillette,
P.A., et al.,
. Gen. and Comp. Physiol., 97:250-258 (1995)). As demonstrated recently in
humans,
plasma Adrenocorticotrophic Hormone (ACTH) levels that regulate plasma
cortisol
levels are altered by PVCR activation.
Additionally, the function or biological activity of the SalmoKCaR
polypeptide or protein is defined, in one aspect, to mean the osmoregulatory
activity
of SalmoKCaR protein. Assay techniques to evaluate the biological activity of
SalmoKCaR proteins and their analogs are described in Brown, et al., New Eng.
J
Med., 333:243 (1995); Riccardi, etal., Proc. Nat. Acad. Sci USA, 92:131-135
(1995); and Sands, et al., J. Clinical Investigation 99:1399-1405 (1997). The
biological activity also includes the ability of the SalmoKCaR to modulate
signal
transduction pathways in specific cells. Thus, depending on the distribution
and
nature of various signal transduction pathway proteins that are expressed in
cells,
biologically active SalmoKCaR proteins can modulate cellular functions in
either an
inhibitory or stimulatory manner.
Biologically active derivatives or analogs of the above described
SalmoKCaR polypeptides, referred to herein as peptide mimetics can be designed
and produced by techniques known to those of skill in the art. (see e.g., U.S.
Patent
Nos. 4,612,132; 5,643,873 and 5,654,276). These mimetics can be based, for
example, on a specific SalmoKCaR amino acid sequence and maintain the relative
position in space of the corresponding amino acid sequence. These peptide
mimetics possess biological activity similar to the biological activity of the
corresponding peptide compound, but possess a "biological advantage" over the
corresponding SalmoKCaR amino acid sequence with respect to one, or more, of
the
following properties: solubility, stability and susceptibility to hydrolysis
and
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proteolysis.
Methods for preparing peptide mimetics include modifying the N-terminal
amino group, the C-terminal carboxyl group, and/or changing one or more of the
amino linkages in the peptide to a non-amino linkage. Two or more such
modifications can be coupled in one peptide mimetic molecule. Modifications of
peptides to produce peptide mimetics are described in U.S. Patent Nos.
5,643,873
and 5,654,276. Other forms of the SalmoKCaR polypeptides, encompassed by the
present invention, include those which are "functionally equivalent." This
term, as
used herein, refers to any nucleic acid sequence and its encoded amino acid,
which
mimics the biological activity of the SalmoKCaR polypeptides and/or functional
domains thereof.
SalmoKCaR Nucleic Acid Sequences, Plasmids, Vecfors and Host Cells
The present invention, in one embodiment, includes an isolated full length
nucleic acid molecule having a sequence of SalmoKCaR#1 (SEQ ID NO: 7),
SalmoKCaR#2 (SEQ lD NO: 9), SalmoKCaR#3 (SEQ ID NO: 11), or
SalmoKCaR#4 (SEQ ID NO: 13). See Figures 9, 10, 11, and 12. The present
invention includes sequences to the full length SalmoKCaR nucleic acid
sequences,
as well as the coding regions thereof. As shown in these figures, the ORF
SalmoKCaR#1 begins at nt 180 and ends at nt 3005. For SalmoKCaR#2, it begins
at
nt 270 and ends at nt 3095, and for SalmoKCaR#3, the ORF=begins at nt 181 and
ends at nt 2733. SahnoKCaR#4 has an OFR that begins at nt181 and ends at nt
3006.
The present invention also encompasses isolated nucleic acid sequences that
encode SalmoKCaR polypeptides, and in particular, those which encode a
polypeptide molecule having an amino acid sequence of SEQ ID NO: 8, 10, 12, or
14. The SalmoKCaR full length nucleic acid sequences encode polypeptides that
allow or assist in one or more of the following functions in Atlantic Salmon:
sensing
at least one SalmoKCaR modulator in serum or in the surrounding environment;
adapting to at least one SalmoKCaR modulator present in the serum or
surrounding
environment; imprinting with an odorant; altering water intake; altering water
absorption; or altering urine output.
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The present invention encompasses the SalmoKCaR full length nucleic acid
sequences, SalmoKCaR#1 (SEQ IT) NO: 7), SalmoKCaR#2 (SEQ _________ NO: 9),
SalmoKCaR43 (SEQ ID NO: 11), and SalmoKCaR #4 (SEQ ID NO: 13), or
polypeptides encoded by these sequences, which were deposited under the
Budapest
Treaty with the ATCC, 10801 University Boulevard, Manassas, VA 20110-2209,
USA, under Accession Numbers PTA-4190 (March 29, 2002), PTA-4191 (March
29, 2002), and PTA-4192 (March 29, 2002), (deposited by MariCal, LLC, 400
Commercial Street, Portland, Maine USA) for SalmoKCaR#1 (SEQ ID NO: 7),
SalmoKCaR#2 (SEQ ID NO: 9), and SalmoKCaR#3 (SEQ ID NO: 11)
respectively. These clones are plasmid DNA which can be transformed into E.
Coli
and cultured. The viability of the clones can be tested with ampicillin
resistance.
The sequences of the present invention can be purified from these deposits
using
techniques known in the art.
As used herein, an "isolated" gene or nucleotide sequence which is not
flanked by nucleotide sequences which normally (e.g., in nature) flank the
gene or
nucleotide sequence (e.g., as in genomic sequences) and/or has been completely
or
partially purified from other transcribed sequences (e.g., as in a cDNA or RNA
library). Thus, an isolated gene or nucleotide sequence can include a gene or
nucleotide sequence which is synthesized chemically or by recombinant means.
Nucleic acid constructs contained in a vector are included in the definition
of =
"isolated!! as used herein. Also, isolated nucleotide sequences include
recombinant
nucleic acid molecules and heterologous host cells, as well as partially or
=
substantially or purified nucleic acid molecules in solution. in vivo and in
vitro
RNA transcripts of the present invention are also encompassed by "isolated"
nucleotide sequences. Such isolated nucleotide sequences are useful for the
manufacture of the encoded SalmoKCaR polypeptide, as probes for isolating
homologues sequences (e.g., from other mammalian species or other organisms),
for
gene mapping (e.g., by in situ hybridization), or for detecting the presence
(e.g., by
Southern blot analysis) or expression (e.g., by Northern blot analysis) of
related
genes in cells or tissue.
The SalmoKCaR nucleic acid sequences of the present invention include
homologues nucleic acid sequences. "Analogous" or "homologous" nucleic acid
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sequences refer to nucleic acid sequences with sufficient identity of any one
of the
SalmoKCaR nucleic acid sequences, such that once encoded into polypeptides,
they
possess the biological activity of any one of the native SalmoKCaR
polypeptides.
For example, an analogous nucleic acid molecule can be produced with "silent"
changes in the sequence wherein one, or more, nt differ from the nt of any one
of the
SalmoKCaR protein, yet, once encoded into a polypeptide, still possesses the
function or biological activity of any one of the native SalmoKCaR. Examples
of
such differences include additions, deletions or substitutions. Also
encompassed by
the present invention are nucleic acid sequences that encode analogous
polypeptides
that exhibit greater, or lesser, biological activity of the SalmoKCaR proteins
of the
present invention. In particular, the present invention is directed to nucleic
acid
molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity
with SEQ ID NO: 7, 9, 11, or 13. Each of the SalmoKCaR genes are homologues to
-
one another.
The percent identity for the SalmoKCaR nucleic acid sequences are as follows:
Percent Identity for Nucleic Acid Sequences
Query SalmoKCaR#1 SalmoKCaR#2 SalmoKCaR#3 SalmoKCaR#4
Sequence _ _
S almoKCaR#1 N/A 99.8% 95.8% 99%
SalmoKCaR#2 97.6% N/A 93.6% 98%
SalmoKCaR#3 98.7% 98.7% N/A 97%
SalmoKCaR#4 99% 98% 97% N/A
The nucleic acid molecules of the present invention, including the full length
sequences, the partial sequences, functional fragments and homologues, once
encoded into polypeptides, allow for or assist in one or more of the following
functions in Atlantic Salmon: sensing at least one SalmoKCaR modulator in
serum
or in the surrounding environment; adapting to at least one SalmoKCaR
modulator
present in the serum or surrounding environment; imprinting with an odorant;
altering water intake; altering water absorption; or altering urine output.
The
homologous nucleic acid sequences can be determined using methods known to
those of skill in the art, and by methods described herein including those
described
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for determining homologous polypeptide sequences.
Also encompassed by the present invention are nucleic acid sequences, DNA
or RNA, which are substantially complementary to the DNA sequences encoding
the
SalmoKCaR polypeptides and which specifically hybridize with their DNA
sequences under conditions of stringency known to those of skill in the art.
As
defined herein, substantially complementary means that the nucleic acid need
not
reflect the exact sequence of the SalmoKCaR sequences, but must be
sufficiently
similar in sequence to permit hybridization with SalmoKCaR nucleic acid
sequence
under high stringency conditions. For example, non-complementary bases can be
interspersed in a nucleotide sequence, or the sequences can be longer or
shorter than
the SalmoKCaR nucleic acid sequence, provided that the sequence has a
sufficient
number of bases complementary to the SalmoKCaR sequence to allow hybridization
therewith. Conditions for stlingency are described in e.g., Ausubel, F.M., et
al.;
Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown,
et al.,
Nature, 366:575 (1993); and further defined in conjunction with certain
assays.
The SalmoKCaR sequence, or a fragment thereof, can be used as a probe to
isolate additional homologues. Nucleic acids encoding SalmoKCaR polypeptides
were identified by screening a cDNA library with a SalmoKCaR-specific probe
under conditions known to those of skill in the art to identify homologous
receptor
proteins. For example, the full length sequences were isolated by screening
Atlantic
Salmon intestinal and kidney cDNA libraries with a probe consisting of a 653
nt
PCR amplified genomic sequence (SEQ ID NO: 3), Techniques for the preparation
and screening of a cDNA library are well-known to those of skill in the art.
For -
example, techniques such as those described in Riccardi, et al., Proc. Nat.
Acad. Sci.
USA, 92:131-135 (1995), can be used. Positive clones can be isolated,
subcloned
and their sequences determined. Using the sequences of either a full length or
several over-lapping partial cDNAs, the complete nucleotide sequence of the
SalmoKCaR cDNA were obtained and the encoded amino acid sequence deduced.
The sequences of the SalmoKCaRs can be compared to each other and other
aquatic
PVCRs to determine differences and similarities. Methods for screening and
identifying homologues genes as described in e.g., Ausubel, F.M., et al.,
Current
Protocols in Molecular Biology, (Current Protocol, 1994).
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SalmoKCaR genes were isolated by Polymerase Chain Reaction (PCR) of
genomic DNA with degenerate primers (SEQ ID NOS: 15 and 16) specific to a
highly conserved sequence of calcium receptors that does not contain introns.
For
example, partial Atlantic Salmon clones were obtained by using degenerate
primers
that peiniit selective amplification of a sequence (nucleotides 2279-2934 of
SKCaR)
that is highly conserved in both mammalian and shark kidney calcium receptors.
SalmoKCaR #4 was isolated using the full length SalmoKCaR#2, SEQ ID NO. :9 as
a probe. See Exemplification. The degenerate primers (SEQ ID NOS: 15 and 16)
amplify a sequence of 653 base pairs that is present in the extracellular
domain of
calcium receptors. This 653 nt sequence refers to SEQ ID NO: 3 with the
addition
of the sequence of the primers. The resulting amplified 653 bp fragment was
ligated
into a cloning vector and transformed into bacterial cells for growth,
purification and
sequencing. Additionally, SahnoKCaR genes can be isolated by Reverse
Transciiptase-Polymerase Chain Reaction (RT-PCR) after isolation of poly A+
RNA
from aquatic species with the same or similar degenerate primers. Methods of
PCR
and RT-PCR are well characterized in the art (See generally, PCR Technology:
Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman
Press,
NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis,
et al., Academic Press, San Diego, CA, 1990); Mattila, et al., Nucleic Acids
Res.,
19:4967 (1991); Eckert, et al., PCR Methods and Applications, 1:17 (1991); PCR
(eds. McPherson, et al., 1RL Press, Oxford); and U.S. Patent 4,683,202. Poly
A+
RNA can be isolated from any tissue which contains one or more of SalmoKCaR
polypeptides by standard methods as described. Preferred tissue for polyA+RNA
isolation can be determined using an antibody which is specific for the highly
conserved sequence of calcium receptors, by standard methods. The partial
genomic
or cDNA sequences derived from a SalmoKCaR gene are unique and, thus, can be
used as a unique probe to isolate the fall-length cDNA from other species.
Moreover, in one embodiment, this DNA fragment serves as a basis for specific
assay kits for detection of SalmoKCaR expression in various tissues of
Atlantic
Salmon.
Also encompassed by the present invention are nucleic acid sequences,
genomic DNA, cDNA, RNA or a combination thereof, which are substantially
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complementary to the DNA sequences encoding SalmoKCaR nucleic acid molecules
and which specifically hybridize with the SalmoKCaR nucleic acid sequences
under
conditions of sufficient stringency (e.g., high stringency) to identify DNA
sequences
with substantial nucleic acid identity. In another embodiment, the invention
includes nucleic acid sequences that hybridize to the SalmoKCaR sequences, SEQ
ID NO: 7,9, 11, or 13, but not to SEQ ID NO: 1 (SkCaR) or 31-40 (Fugu) under
the
same conditions.
The present invention embodies nucleic acid molecules (e.g., probes or
primers) that hybridize to SEQ ID NO: 7, 9, 11, or 13 under high stringency
conditions, as defined herein. In one aspect, the present invention includes
molecules that hybridize to at least about 200 contiguous nucleotides or
longer in
length (e.g., 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,
1500,
1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,
2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000).
Such
molecules hybridize to one of the SalmoKCaR nucleic acid sequences (SEQ ID NO:
7, 9, 11, or 13) under high stringency conditions. The present invention
includes
those molecules that hybridize with SalmoKCaR nucleic acid molecules and
encode
a polypeptide that has the functions or biological activity described herein.
Typically the nucleic acid probe comprises a nucleic acid sequence (e.g. SEQ
ID NO: 7,9, 11, or 13) and is of sufficient length and complementarity to
specifically hybridize to a nucleic acid sequence that encodes a SalmoKCaR
polypeptide. For example, a nucleic acid probe can be at least about 5%, 10%,
20%,
30%, 40%, 50%, 60%, 70%, 80% or 90% the length of the SalmoKCaR nucleic acid
sequence. The requirements of sufficient length and complementarity can be
easily
detefinined by one of skill in the art. Suitable hybridization conditions
(e.g., high
stringency conditions) are also described herein. Additionally, the present
invention
encompasses fragments that are biologically active SalmoKCaR polypeptides or
nucleic acid sequences that encodes biologically active SalmoKCaR
polypeptides, as
described further herein.
Such fragments are useful as probes for assays described herein, and as
experimental tools, or in the case of nucleic acid fragments, as primers. A
preferred
embodiment includes primers and probes which selectively hybridize to the
nucleic
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acid constructs encoding any one of the SalmoKCaR proteins. For example,
nucleic
acid fragments which encode any one of the domains described herein are also
implicated by the present invention.
Stringency conditions for hybridization refers to conditions of temperature
and buffer composition which permit hybridization of a first nucleic acid
sequence
to a second nucleic acid sequence, wherein the conditions determine the degree
of
identity between those sequences which hybridize to each other. Therefore,
"high
stringency conditions" are those conditions wherein only nucleic acid
sequences
which are very similar to each other will hybridize. The sequences can be less
similar to each other if they hybridize under moderate stringency conditions.
Still
less similarity is needed for two sequences to hybridize under low stringency
conditions. By varying the hybridization conditions from a stringency level at
which
no hybridization occurs:to a level at which hybridization is first observed,
conditions can be determined at which a given sequence will hybridize to those
sequences that are most similar to it. The precise conditions determining the
stringency of a particular hybridization include not only the ionic strength,
temperature, and the concentration of destabilizing agents such as formamide,
but
also factors_suc_h as the length of the nucleic acid sequences, their base
composition,
the percent of mismatched base pairs between the two sequences, and the
frequency
of occurrence of subsets of the sequences (e.g., small stretches of repeats)
within
other non-identical sequences. Washing is the step in which conditions are set
so as
to determine a minimum level of similarity between the sequences hybridizing
with
each other. Generally, from the lowest temperature at which only homologous
hybridization occurs, a 1% mismatch between two sequences results in a 1 C
decrease in the melting temperature (Tm) for any chosen SSC concentration.
Generally, a doubling of the concentration of SSC results in an increase in
the Tm of
about 17 C. Using these guidelines, the washing temperature can be detettnined
empirically, depending on the level of mismatch sought. Hybridization and wash
conditions are explained in Current Protocols in Molecular Biology (Ausubel,
F.M.
et al., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) on
pages
2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
High stringency conditions can employ hybridization at either (1) lx SSC
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(10x SSC = 3 M NaC1, 0.3 M Na3-citrate=2H20 (88 g/liter), pH to 7.0 with 1 M
HC1), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA
at 65 C, (2) lx SSC, 50% fon-namide, 1% SDS, 0.1 - 2 mg/ml denatured calf
thymus
DNA at 42 C, (3) 1% bovine serum albumin (fraction V), 1 m1VI Na2=EDTA, 0.5 M
NaHPO4 (pH 7.2) (1 M NaHPO4 = 134 g Na2HPO4.7H20, 4 ml 85% H3PO4 per
liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 65 C, (4) 50%
foimamide, 5x SSC, 0.02 M Tris-HC1 (pH 7.6), lx Denh.ardt's solution (100x =
10 g
Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V),
water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf
thymus DNA at 42 C, (5) 5x SSC, 5x Denhardt's solution, 1% SIDS, 100 g/ml
denatured calf thymus DNA at 65 C, or (6) 5x SSC, 5x Denhardt's solution, 50%
formamide, 1% SDS, 100 pg/ml denatured calf thymus DNA at 42 C, with high
stringency washes of either (1) 0.3 - 0.1x SSC, 0.1% SDS at 65 C, or (2) 1 mM
Na2EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS at 65 C. The above conditions are
intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the
hybrid is believed to be less than 18 base pairs in length, the hybridization
and wash
temperatures should be 5 - 10 C below that of the calculated Tm of the hybrid,
where
Tm in C = (2 x the number of A and T bases) + (4 x the number of G and C
bases).
For hybrids believed to be about 18 to about 49 base pairs in length, the Tm
in C
(81.5 C + 16.6(log10M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where
"M"
is the molarity of monovalent cations (e.g., Nat), and "L" is the length of
the hybrid
in base pairs.
Moderate stringency conditions can employ hybridization at either (1) 4x
SSC, (10x SSC = 3 M NaC1, 0.3 M Na3-citrate=2H20 (88 g/liter), pH to 7.0 with
1 M
HC1), 1% SDS (sodium dodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus DNA
at 65 C, (2) 4x SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf
thymus
DNA at 42 C, (3) 1% bovine serum albumin (fraction V), 1 mM Na2=EDTA, 0.5 M
NaHPO4 (pH 7.2) (1 M NaHPO4 -= 134 g Na2HPO4.7H20, 4 ml 85% H3PO4 per
liter), 7% SDS, 0.1 - 2 mg/ml denatured calf thymus DNA at 65 C, (4) 50%
foiniamide, 5x SSC, 0.02 M Tris-HC1 (pH 7.6), lx Denhardt's solution (100x =
10 g
Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V),
water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1 - 2 mg/ml denatured calf
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thymus DNA at 42 C, (5) 5x SSC, 5x Denhardt's solution, 1% SDS, 100 p.g/m1
denatured calf thymus DNA at 65 C, or (6) 5x SSC, 5x Denhardt's solution, 50%
formamide, 1% SDS, 100 p.g/m1 denatured calf thymus DNA at 42 C, with moderate
stringency washes of lx SSC, 0.1% SDS at 65 C. The above conditions are
intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the
hybrid is believed to be less than 18 base pairs in length, the hybridization
and wash
temperatures should be 5 - 10 C below that of the calculated Tin of the
hybrid, where
Tin in C = (2 x the number of A and T bases) + (4 x the number of G and C
bases).
For hybrids believed to be about 18 to about 49 base pairs in length, the Tin
in C --
(81.5 C + 16.6(log10M) + 0.41(% G + C) - 0.61 (% fonnamide) - 500/L), where
"M"
is the molarity of monovalent cations (e.g., Na), and "L" is the length of the
hybrid
in base pairs.
Low stringency conditions can employ hybridization-at either (1) 4x SSC,
(10x SSC = 3 M NaCl, 0.3 M Na3-citrate2.H20 (88 g/liter), pH to 7.0 with 1 M
HC1), 1% SOS (sodium clodecyl sulfate), 0.1 - 2 mg/ml denatured calf thymus
DNA
at 50 C, (2) 6x SSC, 50% formamide, 1% SDS, 0.1 - 2 mg/ml denatured calf
thymus
DNA at 40 C, (3) 1% bovine serum albumin (fraction V), 1 mM Na2=EDTA, 0.5 M
NaHPO4 (pH 7.2) (1 M NaHPO4= 134 g Na,HPO4.7H20, 4m1 85% H3PO4 per
liter), 7% SDS, 0.1 - 2 mg/nil denatured calf thymus DNA at 50 C, (4) 50%
foithamide, 5x SSC, 0.02 M Tris-HC1 (pH 7.6), lx Denhardt's solution (100x =
10 g
Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V),
water to 500 ml), 10% dextra.n sulfate, 1% SDS, 0.1 - 2 mg/m1 denatured calf
thymus DNA at 40 C, (5) 5x SSC, 5x Denhardt's solution, 1% SOS, 100 pz/m1
denatured calf thymus DNA at 50 C, or (6) 5x SSC, 5x Denhardt's solution, 50%
formamide, 1% SDS, 1001.1g/m1 denatured calf thymus DNA at 40 C, with low
stringency washes of either 2x SSC, 0.1% SDS at 50 C, or (2) 0.5% bovine serum
albumin (fraction V), 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. The
above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs
or
longer. Where the hybrid is believed to be less than 18 base pairs in length,
the
hybridization and wash temperatures should be 5 - 10 C below that of the
calculated
Tin of the hybrid, where Tin. in C = (2 x the number of A and T bases) + (4 x
the
number of G and C bases). For hybrids believed to be about 18 to about 49 base
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pairs in length, the Tm in C = (81.5 C + 16.6(log10M) + 0.41(% G + C) -0.61
(%
formamide) - 500/L), where "M" is the molarity of monovalent cations (e.g.,
Na),
and "L" is the length of the hybrid in base pairs.
The SalmoKCaR nucleic acid sequence, or a fragment thereof, can also be
used to isolate additional aquatic PVCR homologs. For example, a cDNA or
genomic DNA library from the appropriate organism can be screened with labeled
SalmoKCaR nucleic acid sequence to identify homologous genes as described in
e.g., Ausebel, et al., Eds., Current Protocols In Molecular Biology, John
Wiley &
Sons, New York (1997).
In another embodiment, the present invention pertains to a method of
isolating a SalmoKCaR nucleic acid comprising contacting an isolated nucleic
acid
with a SalmoKCaR -specific hybridization probe and identifying an aquatic
PVCR.
Methods foi- identifying a -nucleic acid by hybridization are routine in the
art (see
'Current Protocols In Molecular Biology, Ausubel, F.M. et al., Eds., John
Wiley &
Sons: New York, NY, (1997). The present method can optionally include a
labeled
SalmoKCaR probe.
The invention also provides vectors, plasmids or viruses containing one or
_ more of the SalmoKCaR nucleic acid molecules. Suitable vectors for use in
eukaryotic and prokaryotic cells are known in the art and are commercially
available
or readily prepared by a skilled artisan. Additional vectors can also be
found, for
example, in Au-subel, F.M., et al:, Current Protocols in Molecular Biology,
(Current
Protocol, 1994) and Sambrook et al., "Molecular Cloning: A Laboratory Manual,"
2nd ED. (1989).
Uses of plasmids, vectors or viruses containing the cloned SalmoKCaR
receptors or receptor fragments include one or more of the following; (1)
generation
of hybridization probes for detection and measuring level of SalmoKCaR in
tissue or
isolation of SalmoKCaR homologs; (2) generation of SalmoKCaR mRNA or protein
in vitro or in vivo; and (3) generation of transgenic non-human animals or
recombinant host cells.
In one embodiment, the present invention encompasses host cells
transformed with the plasmids, vectors or viruses described above. Nucleic
acid
molecules can be inserted into a construct which can, optionally, replicate
and/or
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integrate into a recombinant host cell, by known methods. The host cell can be
a
eukaryote or prokaryote and includes, for example, yeast (such as Pichia
pastorius
or Saccharomyces cerevisiae), bacteria (such as E. coli or Bacillus subtilis),
animal
cells or tissue, insect Sf9 cells (such as baculoviruses infected SF9 cells)
or
mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK)
cells, Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey COS-
7
cells). Host cells suitable in the present invention also include a fish cell,
a
mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant
cell.
The nucleic acid molecule can be incorporated or inserted into the host cell
by known methods. Examples of suitable methods of transfecting or transforming
cells include calcium phosphate precipitation, electroporation,
microinjection,
infection, lipofection and direct uptake. "Transformation" or "transfection"
as used
herein fefers to the acquisition of new or altered genetic features by
incorporation of
additional nucleic acids, e.g., DNA. "Expression" of the genetic information
of a
host cell is a term of art which refers to the directed transcription of DNA
to
generate RNA which is translated into a polypeptide. Methods for preparing
such
recombinant host cells and incorporating nucleic acids are described in more
detail
in Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition
(1989) and Ausubel, et al. "Current Protocols in Molecular Biology," (1992),
for
example.
The host cell is then maintained under suitable conditions for expression and
recovery of SalmoKCaR protein. Generally, the cells are maintained in a
suitable
buffer and/or growth medium or nutrient source for growth of the cells and
expression of the gene product(s). The growth media are not critical to the
invention, are generally known in the art and include sources of carbon,
nitrogen and
sulfur. Examples include Luria broth, Superbroth, Dulbecco's Modified Eagles
Media (DMEM), RPMI-1640, M199 and Grace's insect media. The growth media
can contain a buffer, the selection of which is not critical to the invention.
The pH
of the buffered Media can be selected and is generally one tolerated by or
optimal for
growth for the host cell.
The host cell is maintained under a suitable temperature and atmosphere.
Alternatively, the host cell is aerobic and the host cell is maintained under
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atmospheric conditions or other suitable conditions for growth. The
temperature
should also be selected so that the host cell tolerates the process and can be
for
example, between about 13 -40 C.
Antibodies, Fusion Proteins and Methods of Assessment of the SalmoKCaR Nucleic
Acid and Amino Acid Molecules
The present invention includes methods of detecting the levels of the
SalmoKCaR nucleic acid levels (mRNA levels) and/or polypeptide levels to
deteindne whether fish are ready for transfer from freshwater to seawater. The
present invention also includes methods for assaying compounds that modulate
SalmoKCaR nucleic acid levels, expression levels or activity of SalmoKCaR
polypeptides. Activity of SalmoKCaR polypeptides includes, but is not limited
to,
phosphorylation of one or more of the SalmoKCaR polyp-eptides, dimerization of
one of the SalmoKCaR polypeptides with a second SalmoKCaR polypeptide,
proteolysis of one or more of the SalmoKCaR polypeptides, and/or increase or
decrease in the intracellular signal transduction system or pathway of one or
more of
the SalmoKCaR polypeptides. The present invention also includes assaying
activities, as known in the art._ Methods that measure SahnoKCaR levels
include
several suitable assays. Suitable assays encompass immunological methods, such
as
FACS analysis, radiohninunoassay, flow cytometry, immunocytochemistry,
enzyme-linked immunosorbent assays (ELISA) and chemiluminescence assays.
Additionally, antibodies, or antibody fragments, can also be used to detect
the r
presence of SalmoKCaR proteins and homologs in other tissues using standard
immunohistological methods. For example, immunohistochemical studies were
performed using the 1169 antibody which was raised against a portion of the
shark
kidney calcium receptor demonstrating localized expression in the olfactory
organ.
Antibodies are absorbed to determine the SalmoKCaR protein levels. Antibodies
could be used in a kit to monitor the SalmoKCaR protein level of fish in
aquaculture. Any method known now or developed later can be used for measuring
SalmoKCaR expression.
Antibodies reactive with any one of the SalmoKCaR or portions thereof can
be used. In a preferred embodiment, the antibodies specifically bind with
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SalmoKCaR polypeptides or a portion thereof. The antibodies can be polyclonal
or
monoclonal, and the term antibody is intended to encompass polyclonal and
monoclonal antibodies, and functional fragments thereof. The terms polyclonal
and
monoclonal refer to the degree of homogeneity of an antibody preparation, and
are
not intended to be limited to particular methods of production.
In several of the preferred embodiments, immunological techniques detect
SalmoKCaR levels by means of an anti-SalmoKCaR antibody (i.e., one or more
antibodies). The term "anti-SalmoKCaR" antibody includes monoclonal and/or
polyclonal antibodies, and mixtures thereof.
Anti-SalmoKCaR antibodies can be raised against appropriate immunogens,
such as isolated and/or recombinant SahnoKCaR, analogs or portion thereof
(including synthetic molecules, such as synthetic peptides). In one
embodiment,
antibodies are raised against an isolated and/or recombinant SalmoKCaR or
portion
thereof (e.g., a peptide) or against a host cell which expresses recombinant
SalmoKCaR. In addition, cells expressing recombinant SalmoKCaR, such as
transfected cells, can be used as immunogens or in a screen for antibody which
binds
receptor.
Any suitable technique can prepare the immunizing antigen and produce
polyclonal or monoclonal antibodies. The art contains a variety of these
methods
(see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:
511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al.,
U.S.
Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current
Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel,
F.M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)).
Generally, fusing a suitable immortal or myeloma cell line, such as SP2/0,
with
antibody producing cells can produce a hybridoma. Animals immunized with the
antigen of interest provide the antibody producing cell, preferably cells from
the
spleen or lymph nodes. Selective culture conditions isolate antibody producing
hybridoma cells while limiting dilution techniques produce them. Researchers
can
use suitable assays such as ELISA to select antibody producing cells with the
desired
specificity.
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Other suitable methods can produce or isolate antibodies of the requisite
specificity. Examples of other methods include selecting recombinant antibody
from
a library or relying upon immunization of transgenic animals such as mice.
Such
methods include immunization of various lifestages of Atlantic salmon to
produce
antibodies to native PVCR protein' s and thereby alter their function or
specificity.
According to the method, an assay can determine the level of SalmoKCaR in
a biological sample. In determining the amounts of SalmoKCaR, an assay
includes
combining the sample to be tested with an antibody having specificity for the
SalmoKCaR, under conditions suitable for formation of a complex between
antibody
and the SalmoKCaR, and detecting or measuring (directly or indirectly) the
formation of a complex. The sample can be obtained directly or indirectly, and
can
be prepared by a method suitable for the particular sample and assay format
selected.
In particular, tissue samples, e.g., gill tissue samples, can be taken from
fish
after they are anaesthetized with MS-222. The tissue samples are fixed by
immersion in 2% parafonnaldehyde in appropriate Ringers solution corresponding
to
the osmolality of the fish, washed in Ringers, then frozen in an embedding
compound, e.g., 0.C.T.Tm (Miles, Inc., Elkahart, Indiana, USA) using
methylbutane
cooled with liquid nitrogen. After cutting 8-10 micron tissue sections with a
cryostat, individual sections are subjected to various staining protocols. For
example, sections are: 1) blocked with goat serum or serum obtained from the
same
species of fish, 2) incubated with rabbit anti-CaR or anti-SalmoKCaR
antiserum, and
3) washed and incubated with peroxidase-conjugated affinity-purified goat
antirabbit
antiserum. The locations of the bound peroxidase-conjugated goat antirabbit
antiserum are then visualized by development of a rose-colored
aminoethylcarbazole
reaction product. Individual sections are mounted, viewed and photographed by
standard light microscopy techniques. The anti-CaR antiserum used to detect
fish
SalmoKCaR protein is raised in rabbits using a 23-mer peptide corresponding to
amino acids numbers 214-236 localized in the extracellular domain of the
RaKCaR
protein. The sequence of the 23-mer peptide is:
ADDDYGRPGIRKFREEABERDIC (SEQ ID NO.: 26) A small peptide with the
sequence DDYGRPGIEKFREEABERDICI (SEQ ID NO.: 27) or
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ARSRNSADGRSGDDLPC (SEQ ID NO.: 28) can also be used to make antisera
containing antibodies to SalmoKCaRs. Such antibodies can be monoclonal,
polyclonal or chimeric.
Suitable labels can be detected directly, such as radioactive, fluorescent or
chemiluminescent labels. They can also be indirectly detected using labels
such as
enzyme labels and other antigenic or specific binding paitners like biotin.
Examples
of such labels include fluorescent labels such as fluorescein, rhodamine,
chemiluminescent labels such as luciferase, radioisotope labels such as 32P,
125I, 131I,
enzyme labels such as horseradish peroxidase, and alkaline phosphatase,
f3-galactosidase, biotin, avidin, spin labels, magnetic beads and the like.
The
detection of antibodies in a complex can also be done immunologically with a
second antibody which is then detected (e.g., by means of a label).
Conventional
methods-or other suitable methods can directly or indirectly label an
antibody.
Labeled primary and secondary antibodies can be obtained commercially or
prepared
using methods know to one of skill in the art (see Harlow, E. and D. Lane,
1988,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring
Harbor, NY).
Using the immunocytochemistry method, the levels of SalmoKCaR in
various tissues can be detected and examined as to whether they change in
comparison to control. Modulated levels or the presence of SalmoKCaR
expression
in various tis-sues, as compared to a control, indicate that the fish or the
population of
fish from which a statistically significant amount of fish were tested, are
ready for
transfer to freshwater. A control refers to a level of SalmoKCaR, if any, from
a fish
that is not subjected to the steps of the present invention, e.g., not
subjected to
freshwater having a SalmoKCaR modulator and/or not fed a NaC1 diet. For
example, Figures 21 and 22 show that fish not subjected to the present
invention had
no detectable SalmoKCaR level, whereas fish that were subjected to the steps
of the
invention had SalmoKCaR levels that were easily detected.
In determining whether compounds are modulators, one can measure changes
that occur in the expression levels of one or more the SalmoKCaR genes, or
those
that occur in one or more intracellular signal transduction systems or
pathways. A
signal transduction pathway is a pathway involved in the sensing and/or
processing
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of stimuli. In particular, such pathways are altered by activation of the
expressed
proteins coded for by a single or combination of nucleic acids of the present
invention.
The SalmoKCaR polypeptides can be in the foul.' of a conjugate or a fusion
protein, which can be manufactured by known methods. Fusion proteins can be
manufactured according to known methods of recombinant DNA technology. For
example, fusion proteins can be expressed from a nucleic acid molecule
comprising
sequences which code for a biologically active portion of the SalmoKCaR
polypeptide and its fusion partner, for example a portion of an immunoglobulin
molecule. For example, some embodiments can be produced by the intersection of
a
nucleic acid encoding immunoglobulin sequences into a suitable expression
vector,
phage vector, or other commercially available vectors. The resulting construct
can
be introduced into a suitable host cell for expression. Upon expression, the
fusion
proteins can be isolated or purified from a cell by means of an affinity
matrix. By
measurement of the alternations in the functions of transfected cells
occurring as a
result of expression of recombinant SalmoKCaR proteins, either the cells
themselves
or SalmoKCaR proteins produced from the cells can be utilized in a variety of
screening assays that all have a high degree of utility over screening methods
involving tests on the same PVCR proteins in whole fish.
The SalmoKCaRs can also be assayed by Northern blot analysis of mRNA
from tissue samples. Northern blot analysis from various shark tissues has
revealed
that the highest degree of PVCR expression is in gill tissue, followed by the
kidney
and the rectal gland. There appear to be at least four distinct mRNA species
of about
7 kb, 4.2 kb and 2.6 kb.
The SalmoKCaRs can also be assayed by hybridization, e.g., by hybridizing
one of the SalmoKCaR sequences provided herein (e.g., SEQ ID NO: 7,9, 11, or
13)
or an oligonucleotide derived from one of the sequences, to a DNA or RNA-
containing tissue sample from a fish. Such a hybridization sequence can have a
detectable label, e.g., radioactive, fluorescent, etc., attached to allow the
detection of
hybridization product. Methods for hybridization are well known, and such
methods
are provided in U.S. Pat. No. 5,837,490, by Jacobs et al., the entire
teachings of
which are herein incorporated by reference in their entirety. The design of
the
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oligonucleotide probe should preferably follow these parameters: (a) it should
be
designed to an area of the sequence which has the fewest ambiguous bases
("N's"),
if any, and (b) it should be designed to have a Tni of approx. 80 C (assuming
2 C for
each A or T and 4 degrees for each G or C).
Additionally, the above probes could be used in a kit to identify SalmoKCaR
homologs and their expression in various fish tissue. The present invention
also
encompasses the isolation of SalmoKCaR homologs and their expression in
various
fish tissues with a kit containing primers specific for conserved sequences of
SalmoKCaR nucleic acids and proteins.
The present invention encompasses detection of Sah-noKCaRs with PCR
Methods using primers disclosed or derived from sequences described herein.
For
example, SalmoKCaRs can be detected by PCR using SEQ ID Nos: 15 and 16, as
described in Example 6. PCR is the selective amplification of a talget
sequence by
repeated rounds of nucleic acid replication utilizing sequence-specific
primers and a
thermostable polymerase. PCR allows recovery of entire sequences between two
ends of known sequence. Methods of PCR are described herein and are known in
the art.
In particular, the levels of SalmoKCaR nucleic acid can be determined in
various tissues by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
after isolation of poly A+ RNA from aquatic species. Methods of PCR and RT-PCR
are well characterized in the art (See generally, PCR Technology: Principles
and
Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY,
1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al.,
Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res.,
19:4967
(1991); Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds.
McPherson et al., IRL Press, Oxford); Ausebel, F. M. et al., Current Protocols
in
Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience 1987, &
Supp.
49, 2000; and U.S. Patent 4,683,202). Briefly, mRNA is extracted from the
tissue of
interest and reverse transcribed. Subsequently, a PCR reaction is performed
with
SalmoKCaR-specific primers and the presence of the predicted SalmoKCaR product
is deteiinined, for example, by agarose gel electrophoresis. Examples of
SalmoKCaR-specific primers are SEQ ID NO: 18-23. The product of the RT-PCR
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reaction that is performed with SalmoKCaR-specific primers is referred to
herein as
a RT-PCR product. The RT-PCR product can include nucleic acid molecules having
part or all of the SalmoKCaR sequence. The RT-PCR product can optionally be
radioactively labeled and the presence or amount of SalmoKCaR product can be
determined using autoradiography. Two examples of commercially available
fluorescent probes that can be used in such an assay are Molecular Beacons
(Stratagene) and Taqman0 (Applied Biosystems). Alternative methods of labeling
and quantifying the RT-PCR product are well known to one of skill in the art
(see
Ausebel, F. M. et al., Current Protocols in Molecular Biology, Greene
Publishing
Assoc. and Wiley-Interscience 1987, & Supp. 49, 2000. Poly A+ RNA can be
isolated from any tissue which contains at least one SalmoKCaR by standard
methods. Such tissues include, for example, gill, nasal lamellae, urinary
bladder,
kidney, intestine, stomach, liver and brain. -
Hence, the present invention includes kits for the detection of SalmoKCaR or
the quantification of SalmoKCaR having either antibodies specific for
SalmoKCaR
or a portion thereof, or a nucleic acid sequence that can hybridize to the
nucleic acid
of SalmoKCaR.
Transgenic Fish
Alterations in the expression or sensitivity of SalmoKCaRs could also be
accomplished by introduction of a suitable transgene. Suitable transgenes
would
include either the SalmoKCaR genes itself or modifier genes that would
directly or
indirectly influence SalmoKCaR gene expression. Methods for successful
introduction, selection and expression of the transgene in fish oocytes,
embryos and
adults are described in Chen, TT et al., Transgenic Fish, Trends in
Biotechnology
8:209-215 (1990).
The present invention is further and more specifically illustrated by the
following Examples, which are not intended to be limiting in any way.
EXEMPLIFICATION
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The following examples refer to Process I and Process II throughout.
Process I is also referred to herein as "SUPERSMOLT TM I Process" or "APS
Process I." A "Process I" fish or smolt refers to a fish or smolt that has
undergone
the steps of Process I. A Process I smolt is also referred to as a "SUPERSMOLT
TM
I" or an "APS Process I" smolt. Likewise, Process ll is also referred to
herein as
"SUPERSMOLT TM II Process" or "Process II." A "Process II" fish or smolt
refers
to a fish or smolt that has undergone the steps of Process II. A Process II
smolt is
also referred to as a "SUPERSMOLT TM II" or an "APS Process II" smolt.
Process I: Pre-adult anadromous fish (this includes both commercially
produced SO, Si or S2 smolts as well as smaller parr/smolt fish) are exposed
to or
maintained in freshwater containing either 2.0-10.0 mM Calcium and 0.5-10.0 mM
Magnesium ions. This water is prepared by addition of calcium carbonate and/or
chloride and magnesium chloride to the freshwater. Fish are fed with feed
pellets
containing 7% (weight/weight) NaCl. Fish are exposed to or maintained in this
regimen of water mixture and feed for a total of 30-45 days, using standard
hatchery
care techniques. Water temperatures vary between 10-16 C. Fish are exposed to
a
constant photoperiod for the duration of Process I. A fluorescent light is
used for the
photoperiod.
Process II: Pre-adult anadromous fish (this includes both commercially
produced SO, Si or S2 smolts as well as smaller parr/smolt fish) are exposed
to or
maintained in freshwater containing 2.0-10.0 mM Calcium and 0.5-10.0 mM
MagnesicUm ions. This water is prepared by addition of calcium carbonate
and/or
chloride and magnesium chloride to the freshwater. Fish are fed with feed
pellets
containing 7% (weight/weight) NaCI and either 2 gm or 4 gm of L-Tryptophan per
kg of feed. Fish are exposed to or maintained in this regimen of water mixture
and
feed for a total of 30-45 days using standard hatchery care techniques. Water
temperatures vary between 10-16 C. Fish are exposed to a constant photoperiod
for
the duration of Process II. A fluorescent light is used for the photoperiod.
EXAMPLE 1: MOLECULAR CLONING OF SHARK KIDNEY CALCIUM
RECEPTOR RELATED PROTEIN (SKCaR)
A shark )ZAP cDNA library was manufactured using standard commercially
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available reagents with cDNA synthesized from poly A+ RNA isolated from shark
kidney tissue as described and published in Siner et al. Am. J. Physiol.
270:C372-C381, 1996. The shark cDNA library was plated and resulting phage
plaques screened using a 32P-labeled full length rat kidney CaR (RaKCaR) cDNA
probe under intermediate stringency conditions (0.5X SSC, 0.1% SDS, 50 C.).
Individual positive plaques were identified by autoradiography, isolated and
rescued
using phagemid infections to transfer cDNA to KS Bluescript vector. The
complete
nucleotide sequence, Figure 1, (SEQ ID NO: 1) of the 4.1 kb shark kidney PVCR
related protein (SKCaR) clone was obtained using commercially available
automated sequencing service that performs nucleotide sequencing using the
dideoxy chain termination technique. The deduced amino acid sequence (SEQ ID
NO: 2) is shown in Figure 1. Northern analyses were perfotnied as described in
Siner et. al. Am. J. Physiol. 270:C372-C381, 1996. The SKCAR-nucleotide -
sequence was compared to others CaRs using commercially available nucleotide
and
protein database services including GENBANK and SWISS MR.
EXAMPLE 2. EXPRESSION/ACTIVATION STUDIES OF SKCaR Th HL1MAN
EMBRYONIC KIDNEY (HEK) CELLS
PVCRs serve as salinity sensors in fish. These receptors are localized to the
apical membranes of various cells within the fish's body (e.g., in the gills,
intestine,
kidney) that are known to be responsible for osmoregulation. A full-length
cation
receptor (CaR, also referred to as "PVCR") from the dogfish shark has been
expressed in human HEK cells. This receptor was shown to respond to
alterations in
ionic compositions of NaC1, Ca2+ and Mg2+ in extracellular fluid bathing the
HEK
cells. The ionic concentrations encompassed the range which includes the
transition
from freshwater to seawater. Expression of PVCR mRNA is also increased in fish
after their transfer from freshwater to seawater, and is modulated by PVCR
agonists.
Partial genomic clones of PVCRs have also been isolated from other fish
species,
including winter and summer flounder and lumpfish, by using nucleic acid
amplification with degenerate primers.
In particular, the following was shown:
1. SKCaR encodes a functional ion receptor that is sensitive to both
Mg2+ and
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Ca2+ as well as alterations in NaCl.
2. SKCaR's sensitivity to Ca2+, Mg2+ and NaC1 occur in the range that
is
found in marine environments and is consistent with SKCaRs role as a
salinity sensor.
3. SKCaR's sensitivity to Mg2+ is further modulated by Ca2+ such that SKCaR
is capable to sensing various combinations of divalent and monovalent
cations in seawater and freshwater. These data can be used to design novel
electrolyte solutions to maintain fish in salinities different from those
present
in their natural environment.
SKCaR cDNA was ligated into the mammalian expression vector PCDNA II
and transfected into HEK cells using standard techniques. The presence of
SKCaR
protein in transfeeted cells was verified by western blotting. Activation of
SKCaR
by extracellular Ca2+, Mg2+ or NaC1 was quantified using a well characterized
FURA 2 based assay where increases in intracellular Ca2+ produced by SKCaR
activation are detected using methodology published previously by Bai, M., S.
Quinn, S. Trvedi, 0. Kifor, S.H.S. Pearce, M.R. Pollack, K. Krapcho, S.C.
Hebert
and E.M. Brown. Expression and characterization of inactivating and activating
_ _
mutations in the human Ca2+-sensing receptor. I Biol. Chem., 32:19537-19545
(1996); and expressed as % normalized intracellular calcium response to
receptor
activation.
SKCaR is a functional extracellular Ca2+ sensror where its sensitivity is
modulated by alterations in extracellular NaCl concentrations. As shown in
Figure
2, SKCaR is activated by increasing concentrations of extracellular Ca2+ where
half
maximal activation of SKCaR ranges between 1-15 mM depending on the
extracellular concentration of NaCl. These are the exact ranges of Ca2+ (1-10
mM
present in marine estuarian areas). Note that increasing concentrations of
NaC1
reduce the sensitivity of SKCaR to Ca2+. This alteration in SKCaR sensitivity
to
Ca2+ was not observed after addition of an amount of sucrose sufficient to
alter the
osmolality of the extracellular medium. This control experiment shows it is
not
alterations in cell osmolality effecting the changes observed.
The half maximal activation (EC50) by Ca2+ for SKCaR is reduced in
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increased concentrations of extracellular NaCl. See Figure 4. The EC50 for
data
shown on Figure 4 is displayed as a function of increasing extracellular NaC1
concentrations. Note the ECso for Ca2+ increases from less than 5 mM to
approximately 18 mM as extracellular NaC1 concentrations increase from 50 mM
to
550 mM.
SKCaR is a functional extracellular Mg2+ sensor where its sensitivity is
modulated by alterations in extracellular NaC1 concentrations. As shown in
Figure
3, SKCaR is activated in the range of 5-40 mM extracellular Mg2+ and is
modulated
in a manner similar to that shown in Figures 2 and 4 by increasing
concentrations of
extracellular NaCl. Similarly, this alteration in SKCaR sensitivity to Ca2+
was not
observed after addition of an amount of sucrose sufficient to alter the
osmolality of
the extracellular medium.
The half maximal activation (EC50) by Mg2+ for SKCaR is reduced in
increased concentrations of extracellular NaCl. See Figure 5. The EC50 for
data
shown on Figure 5 is displayed as a function of increasing extracellular NaC1
concentrations. Note the ECso for Mg2+ increases from less than 20 mM to
approximately 80 mM as extracellular NaC1 concentrations increase from 50mM to
550mM.
Addition of 3mM Ca2+ alters the sensitivity of SKCaR to Mg2+ and NaCl.
See Figure 6. The EC50 for Mg2+ of SKCaR is modulated by increasing
concentrations of NaC1 as shown both in this Figure 6 and in Figure 5.
Addition of
mM Ca2+ to the extracellular solution alters the sensitivity characteristics
of
SKCaR as shown. Note the 3mM Ca2+ increases the sensitivity of SKCaR to Mg2+
as a function of extracellular NaC1 concentrations.
This method was also used to isolate partial genomic clones of PVCRs for
Atlantic salmon and other species such as Arctic char and rainbow trout, as
described herein. Figures 19A-D show the amino acid sequences and alignment
for
the PVCRs from four full length Atlantic salmon clones (SalmKCar #1, #2, #3,
and
#4) relative to the PVCR from the kidney of the dogfish shark (Squalus
acanthias)
(SKCaR) and human parathyroid calcium receptor (HuPCaR).
EXAMPLE 3: DEFINING SALINITY LIMITS AS AN ASSAY TO IDENTIFY
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FISH WITH ENHANCED SALINITY RESPONSIVE AND ALTERED PVCR
FUNCTION. Both anadromous fish (Atlantic salmon, trout and Arctic
char)
and euryhaline fish (flounders, alewives, eels) traverse from freshwater to
seawater
environments and back again as part of their lifecycles in the natural
environment.
To successful accomplish this result; both types of fish have to undergo
similar
physiological changes including alterations in their urine output, altering
water
intake and water absorption. In some cases, naturally occurring mutations to
PVCR
would provide for altered salinity adaptation capabilities that would have
significant
value for both commercial and environmental restoration uses. For example,
identification of selective traits associated with PVCR mediated salinity
responses
might allow identification of new strains of fish for commercial aquaculture.
Similarly, identification of selected environmental parameters from a host of
natural
and man made variables that are the most important to improve the survival and
successful restocking and/or ocean ranching of either wild Atlantic salmon or
winter
flounder would also be of great utility. To permit the identification of
individual
fish possessing enhanced salinity responsive characteristics, assays must be
designed
that enable these fish to survive while others not possessing these
characteristics will
either die or perfoim poorly. As described below, such assays would take
advantage
_ _
of the ability of these anadromous and euryhaline fish to withstand a wide
range of
salinities. Fish that were identified using such assays would then be
propagated in
breeding-selection programs.
Winter and Summer Flounder can be grown and maintained in recycling
water systems. Groups of both winter (Pleuronectes americanus) and summer
(Paralichthus dentalus) flounder were maintained in multiple modular recycling
water system units that are composed of a single 1 meter fish tank maintained
by a 1
meter biofilter tank located directly above it. The upper tank of each unit
contains
168 sq. ft. of biofilter surface area that will support a maximum of 31 lbs of
flounder, while maintaining optimal water purity and oxygenation conditions.
Each
unit is equipped with its own pump and temperature regulator apparatus. Both
the
temperature and photo-period of each unit can be independently regulated using
black plastic curtains that partition each tank off from its neighbor. The
inventors
have a total of 12 independent modular units that permit 3 experiments each
with 4
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variables to be performed simultaneously. Using this experimental system, the
following data have been obtained.
Salinity survival limits for winter and summer flounder with a constant ratio
of divalent and monovalent ions were determined. The survival limit of both
winter
=
and summer flounder in waters of salinities greater than normal seawater (10
mM
Ca2+, 50 m_M Mg2+ and 450 mM NaC1) is water containing twice (20 mM Ca2+, 50
mM Mg2+ and 900 mM NaC1) the normal concentrations of ions present in noinial
seawater. In contrast, the survival limit of both winter and summer flounder
in
waters of salinity less than nonnal seawater is 10% seawater (1 mM Ca2+, 5 mM
Mg2+ and 45 mM NaC1).
Use of a fully recycling water system patinits growth of flounder at vastly
different salinities. Groups of flounder (n=10) were adapted over a 15 day
interval
and maintained at either low salinity (LS) (e.g., at 10% normal seawater),
normal
seawater (NS) or hypersalinity (HS) (e.g., 2X seawater) for intervals of 3
months,
under otherwise identical conditions. Survival among the 3 groups were
comparable
(all greater than 80%) and there were no differences in the electrolyte
content of
their respective sera.
EXAMPLE 4: ISOLATION OF PARTIAL ATLANTIC SALMON PVCRs
A partial PVCR gene of Atlantic Salmon was isolated as follows: sequences
of shark kidney calcium receptor together with the nucleotide sequence of
mammalian calcium receptors were used t6 design degenerate oligonucleotide
primers, dSK-F3 (SEQ ID NO: 15) and dSK-R4 (SEQ ID NO: 16), to highly
conserved regions in the transmembrane domain of polyvalent cation receptor
proteins using standard methodologies (See GM Preston, Polymerase chain
reaction
with degenerate oligonucleotide primers to clone gene family members, Methods
in
Mol. Biol. Vol. 58 Edited by A. Harwood, Humana Press, pages 303-312, 1993).
Using these primers, genomic DNA from the above species was amplified using
standard PCR methodology. The PCR product (653 nt) was then purified by
agarose
gel electrophoresis and ligated into appropriate plasmid vector that was then
transformed into a bacterial strain. After growth in liquid media, vectors and
inserts
are purified using standard techniques, analyzed by restriction enzyme
analysis and
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sequenced. Using this methodology, a total of 8 nucleotide sequences from 8
fish
species including Atlantic Salmon were amplified. Each clone is 594 nt (with-
out
primer sequences) and encodes a 197 amino acid sequence which corresponds to
the
conserved transmembrane domain of the calcium receptors.
Atlantic salmon partial PVCR nucleic acid sequence (SEQ ID NO: 3) is
composed of 594 nucleotides (nt) containing an open reading frame encoding 197
amino acids (SEQ ID NO: 4) (Figure 7).
Primer sequences for PCR of PVCR clones:
dSK-F3 (SEQ ID NO:15) CKT GGA CGG AGC CCT TYG GRA TCG C-3'
dSK-R4 (SEQ ID NO:16) 5f-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G-3'
I=deoxyinosine, N=A+C+T+G, R=A+G, Y=C+T, M=A+C, K=T+G, S=C+G, W=A+T, H=A+T+C,
B=T+C+G, D=A+T+G, V=A+C+G; Product from amplification = 653 nt
EXAMPLE 5: MOLECULAR CLONING OF A SECOND PARTIAL ATLANTIC
SALMON PVCR
A second Atlantic salmon partial PVCR was isolated, as described herein.
An Atlantic salmon ),ZAP cDNA library was manufactured using standard
commercially available reagents with cDNA synthesized from poly A+ RNA
isolated from Atlantic salmon intestine tissue according to manufacturers
instructions (Stratagene, La Jolla, CA) and screened using the Atlantic salmon
PCR
product as a probe. A partial Atlantic salmon PVCR cDNA (SEQ ID NO: 5) is
composed of 2021 nucleotides (nt) (Figure 8A) containing an open reading frame
encoding 388 amino acids (SEQ ID NO: 6) (Figure 8B). The open reading frame
encoded by SEQ ID NO: 5 begins at nucleotide position 87.
EXAMPLE 6: MOLECULAR CLONING OF 4 FULL LENGTH CDNA CLONES
FROM KIDNEY OF ATLANTIC SALMON (SALMO SALAR) AND
DETERMINATION OF THEIR TISSUE SPECIFIC EXPRESSION IN VARIOUS
SALMON TISSUES MODULATED BY WATER SALINITY.
In Example 5, a homology based approach was used to screen cDNA
libraries under moderate stringency conditions to obtain a full length shark
kidney
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PVCR clone (SKCaR). Using sequence information derived from Examples 4 and
5, both nucleotide (nt) and antibody probes were designed to detect PVCRs in
other
fish species. Using degenerate primers whose sequence was derived from
knowledge of the nt sequence of SKCaR, PCR was utilized to amplify a series of
genomic and cDNA (RT-PCR) sequences that contain partial nt and putative
protein
sequences of PVCRs from multiple fish including Atlantic salmon. =See Examples
1, 4, and 5.
The data described in this Example show that the nt and putative protein
sequences of 4 PVCR transcripts from Atlantic salmon kidney were isolated and
characterized. Additionally, their tissue specific expression and modulation
of tissue
expression levels by alterations in water salinity were determined. This
Example is
divided into 2 parts: 1) isolation and sequence of 4 full length PVCR clones
from
salmon kidney (Salm oKCar#1 (SEQ 1D NO: 7), SalmoKCar#2 (SEQ ft) NO: 9),
SalmoKCaR#3 (SEQ ID NO: 11), and SalmoKCaR#4 (SEQ ID NO: 13)) and 2) use
of RT-PCR analysis with degenerate and clone specific SalmoKCaR PCR primers to
determine the tissue specific expression of these 4 transcripts in seawater
vs.
freshwater as well as the SuperSmoltTM process. Taken together, these data
provide
the framework for achieving a fundamental understanding of both PVCRs in
salmonids as well as the their roles in the SuperSmoltTM process.
Part 1. Isolation and sequence of 4 full length PVCR clones from salmon
kidney:
Materials and Methods: Total RNA was purified with Stat 60 reagent (Teltest B
Friendswood, TX) and poly A+ purified with the Micro FastTrack Kit
(Invitrogen,Carlsbad, CA). cDNA was then synthesized and fractionated whereby
selected fractions were ligated and packaged as AZAP libraries (Stratagene, La
Jolla,
CA). For SalmoKCaR #1,2, and 3, library phage were then plated and duplicate
filter lifts performed that were screened under high stringency (0.1X SSC,
0.1% SDS
55 C) with a 32P-labeled (RadPrime Kit, Invitrogen, Carlsbad, CA) genomic
fragment of Atlantic salmon PVCR (653 nt sequence) amplified using protocols
and
reagents described in Examples 1, 4 and 5. For SalmoKCaR #4, library phage
were
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then plated and duplicate filter lifts performed that were screened under high
stringency (0.1X SSC, 0.1% SDS @ 55 C) with a 3213-labeled (RadPrime Kit,
Invitrogen, Carlsbad, CA) full length nucleic acid sequence of SalmoKCaR#2,
SEQ
ID NO: 9, amplified using protocols and reagents described in Examples 1, 4
and 5.
Primary positive plaques were purified, excised and sequenced using commercial
sequencing services (U. of Maine, Orono, ME) and their sequences compared with
those of other PVCRs using BLAST. (National Library of Medicine, Bethesda,
MD).
Results:
For isolation of SalmoKCaR#1,2, and 3:
A total of seven cDNA clones containing PVCR sequence were identified
and purified from Atlantic Salmon kidney and intestine libraries. A total of
three of
the seven contain full length coding sequences for PVCR proteins together with
5'
and 3' regulatory elements. For convenience, these clones are designated Salmo
salar Kidney PVCRs (SalmoKCaRs) #1,112 and '3 and their aligned nt and
putative
protein sequences are shown in Figures 9-11, respectively. The remaining 4
positive
clones were partial PVCR clones very nearly identical to these 3 full-length
SalmoKCaR clones.
For SalmoKCaR#4:
120,000 additional plaques were screened from the same cDNA library
which was used to isolate SalMoKCaRs 1-3. Five positive plaques were picked,
plated out and screened on secondary phage plates. Isolated plaques were
picked,
excised into bacterial plasmids, and transferred into bacterial cells as
previously
described. Plasmid DNA preparations of all 5 cDNAs showed strong hybridization
to SalmoKCaR#3 by Southern blot analysis. Sequence analysis revealed two full
length clones and three partials. One of the full length clones was a
duplicate of a
previously isolated SalmoKCaR, and the other was a new clone, SalmoKCaR4.
This clone was designated as SalmoKCaR#4, commensurate with the naming
of the SalmoKCaR#1-3 clones, above. The aligned nt and putative protein
sequence
for SalmoKCaR #4 is shown in Figure 12.
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Comparison of the different nt sequences of these 4 clones reveals the
following
similarities and differences:
- The SalmoKCaR 41 nucleic acid sequence (SEQ JD NO: 7) consists of 3941
nts of 5' and 3' regulatory elements together with full-length coding
sequence for a 941 AA PVCR protein (SEQ ID NO: 8). See Figures 9A-E.
The calculated molecular mass of this protein is 106,125 Daltons.
- The SalmoKCaR 42 nucleic acid sequence (SEQ ID NO: 9) consists of 4031
nts of 5' and 3' regulatory elements together with full-length coding
sequence for a 941 AA PVCR protein (SEQ ID NO: 10). See Figures 10A-
E. The calculated molecular mass of this protein is 106,180 Daltons.
- The SalmoKCaR '3 nucleic acid sequence (SEQ TD NO: 11) consists of
3824 nts of 5' and 3' regulatory elements together with full-length coding
sequence for a 850 AA PVCR protein (SEQ ID NO: 12). See Figures 11A-
D. The calculated molecular mass of this protein is 96,538 Daltons.
- The SalmoKCaR#4 nucleic acid sequence (SEQ ID NO: 13) consists of
3988 nt of 5' and 3' regulatory elements together with full-length coding
sequence for a 941 AA PVCR protein (SEQ ID NO: 13). See Figures 12A-
E. The calculated molecular mass of this protein is 106,131 Daltons.
Figures 13A-L and 14A-C show an alignment of between the two partial
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SEQ ID NO: 12. Note that the amino acid sequence of SEQ ID NO: 6 extends 91 aa
past the end of SEQ ID NO: 12.
Figures 17 and 18 show an alignment of between the two partial sequences of
Atlantic Salmon PVCRs isolated and all 4 full length clones, SalmoKCaR #1-4,
for
both the nucleic acid and amino acid sequences, respectively. One partial
nucleic
acid sequence of an Atlantic Salmon PVCR, SEQ ID NO: 3, can be found from 1980
to 2573 of SalmoKCaR#4, SEQ ID NO: 13. The second partial Atlantic Salmon
clone, SEQ ID NO: 5, can also be found in the SalmoKCaR#4 nucleic acid
sequence: between nt 1754 and 3774. Similarly, the amino acid sequence of SEQ
ID
NO: 4 is found between aa 601 and 797 of SEQ ID NO: 14. The amino acid
sequence of the second Atlantic Salmon Clone, SEQ II) NO: 6, is found between
aa
554 and 941 of SEQ ID NO: 14.
Additional differences between the partial Atlantic salmon PVCR (SEQ ID
NO: 5) and full length PVCR (SEQ ID NO: 7, 9, or 11) include: nt 1-112 do not
align with any corresponding sequence in SEQ ID NO: 7, 9, or 11. There are
also 4
single nt base pair substitutions that are present in SEQ ID NO: 5 that are
different
than corresponding nt in full length SEQ ID NO: 7, 9, or 11. These include:
_
nt 1893 change from G to A
nt 1970 change from G to A
nt 1973 change from G to A
nt 2001 change from G to A.
Table 1 compares the overall % identity of nucleotides (nt) between cDNA
clones that contain the SalmoKCaRs #1,2, 3, and 4 vs. shark kidney calcium
receptor (SKCaR containing 4079 nts) or human parathyroid CaR (HuPCaR
containing 3783 nts). Note that all 4 SalmoKCaR clones possess approximately a
56-57% nt identity to SKCaR and an approximately 50-55% nt identity to HuPCaR.
However, in spite of the rather low overall % nt identity between the 4
SalmoKCaR
clones and SKCaR, all 4 full length SalmoKCaR clones hybridize to full length
SKCaR clone under high stringency conditions (0.5XSSC, 0.1% SDS @ 65 C.)
(See Figure 15A).
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The percentage identities between the aligned nucleotide sequences of the 4
full length SahnoKCaR clones (SEQ ID NO: 7, 9, 11, 13) include:
A total of 99.8% of the nt of SEQ ID NO: 7 are identical to those of
corresponding SEQ ID NO: 9. A total of 97.6% of the nt of SEQ ID NO: 9
are identical to those corresponding nt of SEQ ID NO: 7.
A total of 93.6% of the nt of SEQ ID NO: 9 are identical to those
corresponding nt of SEQ ID NO: 11. A total of 98.7% of the nt of SEQ ID
NO: 11 are identical to the corresponding nt present in SEQ ID NO: 9.
A total of 95.8% of the nt of SEQ ID NO: 7 are identical to the
corresponding nt of SEQ ID NO: 11. A total of 98.7% of the nt of SEQ ID
NO: 11 are identical to those corresponding in SEQ ID NO: 7.
A total of 99% of the nt of SEQ ID NO: 7 are identical to corresponding nt of
SEQ ID NO: 13. A total of 98% of the lit of SEQ ID NO: 9 are identical to
corresponding nt of SEQ ID NO:13. A total of 97% of nt of SEQ ID NO: 13
are identical to corresponding nt of SEQ ID NO:11.
Table 1: Comparison of the % nucleotide (nt) identity of the complete nt
sequence of
SalmoKeaR clones #1, #2, #3, and #4 (including 5' and 3' regulatory elements)
vs.
either the SKCaR done or the clone HuPCaR clone.
% NUCLEOTIDE IDENTITY
SalmoKCaR #1 SalmoKCaR 42 SalmoKCaR #3SalmoKCaR #4
SKCaR vs. 56.2 56.5 57.2 56
HuPCaR vs. 55.0 54.9 50.9 55
Table 2 compares both the overall and domain-specific percent amino acid
(% AA) identity for each of the SalmoKCaR clones vs. shark kidney PVCR
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(SKCaR-upper half) and human parathyroid CaR (HuPCaR-lower half). When
compared to SKCaR, all 4 SalmoKCaR proteins possess approximately a 63-68%
overall AA identity to SKCaR.. However, their domain-specific identities show
significant degrees of variation with the carboxyl terminal domain of the
SalmoKCaR 3 being the most widely divergent. Not surprisingly, comparisons
between the 4 SalmoKCaR proteins vs. HuPCaR reveal that the 7 transmembrane
region possesses the highest degree of homology followed by the extracellular
domain and finally the intracellular carboxy terminal domain.
The percentage identities between the aligned amino acid sequences of the 4
full length SalmoKCaR clones (SEQ ID NO: 8, 10, 12, or 14) include:
A total of 99.9% of the aa of SEQ ID NO: 8 are identical to those
corresponding aas in SEQ D NO: 10. A total of 99.9% of the aa of SEQ ID
NO: 10 are identical to corresponding aa in SEQ ID NO: 8.
A total of 89.5% of the aa of SEQ JD NO: 10 are identical to those
corresponding aas in SEQ ID NO: 12. A total of 99.1% of the aa of SEQ ID
NO: 12 are identical to those corresponding aa in SEQ ID NO: 10.
A total of 89.6% of the aa of SEQ JD NO: 8 are identical to those
corresponding aas in SEQ ID NO: 12. A total of 99.2% of the aa of SEQ ID
NO: 12 are identical to those corresponding aa of SEQ ID NO: 8.
A total of 100% of the aa of SEQ ID NO: 8 are identical to those
corresponding aa's in SEQ ID NO: 14. A total of 99.9% of the aa of SEQ ID NO:
10 are identical to those corresponding aa's in SEQ ID NO: 14. A total of
99.2% of
the aa of SEQ ID NO: 12 are identical to those corresponding aa's in SEQ ID
NO:
14.
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Table 2: Comparison of % amino acid (AA) identities of 4 SalmoKCaR proteins
vs.
AA sequence of shark kidney CaR (SKCaR-Upper Half) and human parathyroid
CaR (HuPCaR -Lower Half).
% AA Identity to SKCaR
SalmoKCaR SalmoKCaR SalmoKCaR SalmoKCaR
112
Overall Protein 68.4 68.3 63.3 68.4
N-terininal 70.0 69.8 70.0 70.0
Extracellular
Ion Binding Domain
7 Transmembrane 87.2 87.2 86.4 87.2
Region
Carboxyl Terminal 31.8 31.8 0.0 31.8
luta-Cellular Domain
% AA Identity to HuPCaR
Overall Protein 66.3 66.3 61.4 66.3
N-terminal 71.9 - 71.9 72.1 71.9
Extracellular
Ion Binding Domain
7 Transmembrane 89.2 89.2 88.4 89.2
Region
Carboxyl Teaninal 24.1 24.1 0 24.1
Intra-Cellular Domain
Figure 15A shows unique SalmoKCaR#1,2, and 3 clones hybridize to full
length shark kidney CaR (SKCaR) under high stringency conditions (0.5XSSC,
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0.1% SDS @ 65 C). Representative autoradiogram of Southern blot was exposed
for 30 min. Since the SalmoKCaR nt sequence, SEQ ID NO:1, is 97-99% identical
to SalmoKCaR's #1, 2, and 3, and so it also would be expected to hybridize
under
these conditions.
Site directed mutagenesis studies of mammalian CaRs, notably HuPCaR,
have identified AAs that are particularly important in the various functions
of CaRs.
Cysteine AAs at AA#101 and AA#236 mediate dimerization of HuPCaR. HuPCaR
and native CaRs in rat kidney exist primarily as dimers within the cell
membrane
where disulfide bond-mediated dimerization is required for normal agonist-
mediated
CaR activation. SalmoKCaR#1-4 possess Cys at AAs corresponding to HuPCaR
AA#101 and AA#236 and presumably functions as dimers in a manner similar to
mammalian CaRs.
Nucleotide Sequence Differences in the 5' and 3' untranslated regions or UTRs
of
SalmoKCaRs 41, 42 43 and #4:
Figure 16 displays the aligned nucleotide sequences of SalmoKCaR clones
#1, #2, and #3, and Figure 17 displays the aligned nucleotide sequences of
SalmoKCaR #1-4. As compared to SalmoKCaR #1 and '3, SalmoKCaR 2 possesses
an 89 nt insert in its 5' UTR. SalmoKCaR#4 does not contain the 89 nt insert
in
. the 5' UTR that SalmoKCaR# 2 contains. It does not share the 158 nt deletion
in the
ORE of SalnioKCaR# 3. However, it does share a 39 nt insertion in the 3' UTR
just
prior to the poly A tail with SalmoKCaR# 3. This insertion is likely a
regulatory
element. SalmoKCaR #4 looks as though it would have the same structure and
function as SalmoKCaR #1, but would be co-regulated with SalmoKCaR 3.
Differences between the 3' UTRs of the 4 SahnoKCaRs include a 36 nt insert
just
prior to the poly A tail in SalmoKCaR #3 as well as other single nt
differences listed
below where each difference is compared to the 3 other SalmoKCaR clones:
SalmoKCaR #1: nt 3660 A to G; nt 3739 A to G; nt 3745 A to G
SalmoKCaR nt 1039 A to G; nt 3837 A to G; nt 3862 A to G
SalmoKCaR 43: nt 1462 C to T; nt 3472 A to G; nt 3487 A to G; nt
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3564 A to G; nt 3568 G to A; nt 3603 A to G; nt 3786
A to C.
SalmoKCaR 44: nt 3634 A to G; nt 3645 A to G; nt 3661 A to G,
nt
3726 G to A, nt 3740 A to G, nt 3746 A to G, nt
3773 A to G
Although the functional significance of each of these nt differences in the
5' or 3' UTRs is unknown at the present time, each nt difference either
individually or in combinations could represent a means for controlling
either the stability or processing of the RNA transcript or its translation
into each of the 4 SalmoKCaR proteins.
Sequence Differences in the Coding Regions of SalmoKCaRs 41, 42, 43
and #4:
Figure 19 displays the aligned AA sequences of SalmoKCaRs #1,
#2, #3, and #4 as well as the Shark SKCaR protein and HuPCaR
proteins. As compared to SalmoKCaR 41 and #4, SalmoKCaR
possesses 2 different AA's present at AA#257 and AA#941 of its AA
sequence. In contrast to SalmoKCaR 41 and #4 that possesses an Asp in
AA#257, SalmoKCaR 42 possesses a Gly. The negative charge in this
location may be important since both SKCaR and Fugu PVCR possess
Asp at #257 while the mammalian CaRs, HuPCaR and RaKCaR possess
a Glu. SalmoKCaR #3 also contains a Asp at AA#257.
At AA #443, SalmoKCaR 41, 42 and #4 both possess a Leu
whereas SalmoKCaR 43 contains a Phe. The conserved hydrophobic
nature of the AA at this position appears to be important since Fugu
PVCR also contains a Leu whereas SKCaR contains an Ile. As compared
to SahnoKCaRs 41, 2, or #4 SalmoKCaR 43 possesses a truncated
carboxyl teiminus as described below.
Sequence Differences in the Coding Regions of SahnoKCaRs 41, 42 43
and #4 as Compared to Mammalian CaRs.
The putative AA sequences of SalmoKCaR 41, 42, 43 and #4
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proteins possess multiple differences in AAs at various positions
throughout their extracellular, 7 transmembrane and carboxyl terminal
domains when compared to mammalian CaRs such as HuPCaR (see
aligned differences with HuPCaR in Figure 19). While many of the
differences between SalmoKCaR species and HuPCaR are conserved
substitutions that preserve the overall net charge or hydrophobicity
characteristics at that specific position in the PVCR protein, other
substitutions may have functional consequences as based on previous
structure-functional studies of mammalian CaRs. The actual functional
consequences of these AA differences in SalmoKCaR proteins are
described herein in the following expression study.
Expression _of SalmoKCaR #1 and #3 in Human Embryonic Kidney
(HEK) Cells
This express study demonstrates that SahnoKCaR #1 and #3 clones can
be expressed in HEK cells to yield corresponding PVCR proteins of
predicted molecular mass. It also is a demonstration of creation of HEK
cells expressing SalmoKCaRs #1 and #3 as tools for assay systems to
measure PVCR interactions and sensitivity to PVCR modulators for
another aquatic PVCR, SKCaR. Furthermore, it is a demonstration of the
use of SDD and_Sal-1 antisera, respectively, as reagents to detect either
both SalmoKCaRs #1 and #3, or selectively detest SalmoKCaR #1.
Methods for the Transfection and HEK Expression of SalmoKCaRs:
Full length SalmoKCaR 1 and SalmoKCaR 3 cDNAs were
directionally cloned into the Kpnl and XbaI sites of the mammalian
expression vector pcDNA3. 1/Hyg- (Invitrogen) as described for the
transfection and transient expression of SKCaR PVCR. DNA was
prepared using the Wizard Midiprep Kit (Promega) and 15 jig of
sterilized Salm.oKCaR 1 or SalmoKCaR 3 DNA was diluted in 1 ml
Opti-Mem I reduced serum media (Gibco) and combined with 45111
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Lipofectamine 2000 (Gibco). The DNA/Lipofectamine mixture was
diluted with 8 ml Optio-Mem I and added to the flask. Flasks were
incubated for 5 hours at 37 C, 5% CO2 to allow transfer of DNA into the
cells. 10 ml of Opti-Mem I media supplemented with 20% FBS was
added to each flask, for a final concentration of 10% serum. Flasks were
incubated overnight at 37 C, 5% CO2. At 24 hours post transfection, the
media was replaced with 20 ml full growth media. Flasks were kept at
37 C, 5% CO2 for 24-48 hrs to optimize expression of protein.
Methods for Immunoblotting Analysis of HEK Expression of
SahnoKCaR #1 or #2 Using SDD and Sal-1.2 Antisera:
The transfected cells were then rinsed with PBS and treated with
trypsin-EDTA (Gibco) for 2 minutes at 37 C, 5% CO2 to detach cells
from the surface of the plate. The detached cells were added to 10 ml of
full growth media (serum deactivates trypsin) and pelleted at 250 x g for
10 minutes. The cells were resuspended in 3 ml homogenization buffer
(20 mM Tris, 250 mM sucrose, at pH 7.4 plus protease inhibitors,
including 1 g/m1pepstatin, .5 g/m1 leupeptin, and 1mM PMSF). The
cells were homogenized in 14 ml (Falcon) tubes using a polytron
motorized homogenizer. The homogenate was centrifuged at 3, 000 x g
for 15 minutes at 4 C twice, re-suspending pellet in 3 ml of
homogenization buffer between spins and pooling supernatant from both
spins into high speed centrifuge tubes. Mitochondrial material was then
sedimented from pooled supernatant at 22, 000 x g for 20 minutes at 4 C.
The supernatant was subsequently sedimented at 37, 000 x g for 30
minutes at 4 C to pellet the plasma membranes, and the resulting pellet
was solubilized with 100 I homogenization buffer. A fresh sample of
salmon kidney from a fish 7 weeks post transfer into salt water was
prepared using the same homogenization protocol, along with the mock
transfected HEK 293 cells and a flask of HEK cells stably transfected
with human calcium receptor (5001 HEK) to be used as positive and
negative controls on the western blots. Protein was quantified from the
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plasma membrane preparations using the Bio-Rad Protein Assay (Bio-
Rad Laboratories). For the gel shown in Figure 15B, 20 jig of protein
was loaded into each lane except the 5001 HEK of which 15 g was
loaded. Novex 10% Tris-Glycine pre-cast gels (Invitrogen) were used to
separate protein bands in each sample by gel electrophoresis. The protein
bands were subsequently electro-transferred to a Hybond-P PVDF
nitrocellulose membrane (Amersham-Pharmacia). The membranes were
blocked overnight at 4 C in a 5% block solution (Amersham-Pharmacia).
The membranes were washed in TBS-T and incubated for 1 hour at room
temperature in primary antibodies of SalmoSDD or Sal 1 or the
preimmunie serum to those antibodies, at 1:4,000 dilutions. The
secondary antibody used was an antirabbit horseradish peroxidase
(Amersham-Pharmacia) at 1:50,000 dilution, incubated for 1 hour at
room temperature. The protein bands shown in Figure 15B were detected
with an Enhanced Chemiluminescence system (Amersham-Phannacia)
and exposed to high performance chemiluminescence film (Amersham-
Phanuacia).
Figure 15B shows a Western blot analysis of PVCR proteins
produced by HEK cells transiently transfected with SalmoKCaR #1 and
#3 constructs. Both Right and Left Panels each containing 10 lanes were
loaded and electrophoresed with 20 micrograms of protein except for
HEK-5001 (HuPCaR control) that contained 15 micrograms. In Figure
15B, left panel, shows an immunoblotting analysis with SDD antiserum.
SDD antiserum recognizes an aa sequence in the extracellular domain of
PVCRs that is present in both HUPCaR, SalmoKCaR #1, SalmoKCaR
#3 and salmon SW kidney. Thus, HEK cells expressing HuPCaR
(Positive Control) display a prominent -115kDa band, as well as higher
molecular mass bands which represent oligomers of HuPCaR. In
contrast, mock transfected HEK cells (Negative Control) show minimal
inunuoreactivity similar to that present when lanes are exposed to
preimmune SDD antiserum. However, SDD antiserum displays intense
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reactivity to HEK cells transfect with SalmoKCaR #1 (SalmoKCaR #1)
or SahnoKCaR #3 (SalmoKCaR #3). Note the immunoreactive band
produced by SalmoKCaR #1 is slightly larger (higher molecular mass)
when compared to SalmoKCaR #3 as predicted by their nucleotide and
amino acid sequence analysis of corresponding clones. Note also the
presence of larger immunoreactive bands present in SalmoKCaR #3 lane
that corresponds to oligomeric complexes of SalmoKCaR protein. The
ability of SalmoKCaR #3 to farm oligomeric complexes could result in
formation of it exerting a dominant negative effect on the activity of
other PVCRs expressed in the same cell. The lane designated Salmon
SW kidney is a positive control showing that several bands in both
SalmoKCaR #1 and #3 lanes are an identical molecular mass to PVCR
proteins present in kidney tissue of Atlantic salmon.
Figure 15B, right panel, shows immunoblotting analysis with SAL1
antiserum. SAL1 antiserum recognizes an aa sequence in the carboxyl
terminal of SalmoKCaR #1 that is not present in either HuPCaR or
SalmoKCaR #3. As a result, minimal immunoreactivity is displayed in
either immune or preimmune lanes except for SalmoKCaR #1 and
Salmon SW Kidney lanes. Note the presence of higher molecular wight
immunoreactive SalmoKCaR proteins in both SalmoKCaR #1 and
Salmon SW Kidney. The absence of immunoreactivity of SalmoKCaR
#3 lane is expected since nucleotide and sequence analyses show that
SalmoKCaR #3 is missing the aa sequence that is recognized by Sal-1
antiserum.
Differences between SalmoKCaR proteins vs. mammalian and
other fish PVCRs include:
- SalmoKCaR #1, #2, #3 and #4 possess a deletion of 15 AA's
beginning at AA #369 as compared to either HuPCaR or
RaKCaR. Fugu PVCR also exhibits a 19 AA deletion at the same
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location. In contrast, SKCaR does not exhibit any deletion in this
area and thus is more similar to mammalian CaRs as compared to
either SalmoKCaR or Fugu in this regard.
- Another notable difference between SalmoKCaRs vs. mammalian CaRs
and SKCaR is differences in AA #227 where mutagenesis studies have
identified the presence of the positively charged Arg as important in CaR
sensitivity since its alteration in HuPCaR to a Leu results in over a 2 fold
reduction in EC50 Ca from 4.0mM to 9.3mM but not Gd' sensitivity. In
contrast to mammalian CaRs and SKCaR, all 4 SalmoKCaRs possess a
negatively charged Glu at AA#227. Fugu PVCR also exhibits the same Glu
at AA#227. interestingly, the AA sequence immediately following AA#227
is Glu-Glu-Ala in the mammalian HuPCaR and elasmobranch SKCaR
whereas it is Lys-Glu-Met in all 4 SalmoKCaRs and Fugu.
- Lastly, all 4 SalmoKCaR clones as well as Fugu possess an in frame
deletion of a single AA at position #757 (between TM4 and 5) as compared
to either mammalian CaRs or SKCaR.
- SalmoKCaR #3 possesses a truncated carboxyl terminal domain as
compared to either SalmoKCaRs #1, #2 or #4. The number of AA that
comprise the carboxyl terminal domains of the 4 SalmoKCaRs are different
and include: SalmoKCaR #1 ¨96 AA; SalmoKCaR #2 ¨ 97 AA,
SalmoKCaR #3 ¨5 AA, and SalmKCaR #4 -96 AA. Reduction in the 91-
92AA's in SalmoKCaR #3 vs. SalmoKCaRs #1, #2 or #4 would reduce its
estimated molecular mass by 9,600 Daltons.
Studies from multiple site directed mutagenesis studies of HuPCaR reveal
that alterations to the structure of the carboxyl terminal domain of PVCRs
have
profound effects on their function and sensitivity to ligands such as Ca" and
Mg".
Various truncations of the carboxyl terminal domain of HuPCaR have highlighted
the importance of HuPCaR AAs #860-910. Truncation of the carboxyl terminal
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domain of HuPCaR to AAs less than AA#870 produced either an inactive receptor
or a modified HuPCaR with a marked decrease in its affinity for extracellular
Ca' as
well as a decrease in the apparent cooperativity of Ca" dependent activation.
While
the exact functional characteristics of SalmoKCaR #3 remain to be determined
using
similar HEK transfection studies, these data derived from HuPCaR mutagenesis
studies suggest that SahnoKCaR #3 protein is either inactive or exhibits a
greatly
reduced functional affinity for Ca'. Significant expression of SalmoKCaR #3
together with other SalmoKCaRs #1, #2 or #4 could result in an overall
reduction in
the response to extracellular Ca2+ due to so called dominant negative effects.
These
dominant negative effects could occur where SalmoKCaR#3 reduces the overall
sensitivity of cells to Ca' via combinations between SalmoKCaR #3 and
SalmoKCaR #1/#2/#4 to reduce the sensitivity of the latter PVCRs via
cooperative
interactions (dimers and higher oligomers) with them. -
Certain mutagenesis studies also highlight the importance of the Threonine
AA at AA#888 in mediation of HuPCaR's sensitivity to Ca2+ and normal signal
transduction. Figure 19 shows that AA #888 is a Thr in all wild type CaR and
PVCR proteins including HuPCaR, RaKCaR, SKCaR, BoPCaR and SalmoKCaR
#1, #2, and #4. SalmoKCaR #3 is missing Thr #888 because of its truncated
tail.
Of interest is also the presence of consensus sites for receptor kinase
phosphorylation (Ser-Ser-Ser) that are present at AA#907-909 in HuPCaR,
RaKCaR, SKCaR BoPCaR and SalmoKCaR #1,- #2 and #4. In contrast, Fugu PVCR
possesses an Asn at AA#908 that would render its site nonrecongizable to
protein
kinases. A similar protein kinase site also appears in the region of AA#918-
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, #2 and #4 also have intact Ser-Ser-Ser motifs at position AA
#918-920 or #919-921. The exact functional significance of these Ser-Ser-Ser
sites
possessed by SalmoKCaR #1, #2 and #4 await expression studies.
The presence of multiple differences in the nucleotide and putative protein
sequences of SahnoKCaR clones #1-#4 strongly suggest the presence of multiag
PVCR genes within Atlantic salmon:
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Recent studies in rainbow trout provide direct evidence of the existence of
multiple genes encoding two different forms of a specific type of protein,
each of
which are differentially expressed in specific tissues of trout. These
proteins are aryl
hydrocarbon receptor Type 2 (AhRs). Detailed studies on AhRs have shown the
presence of 2 functional genes that produce different closely related AhR
proteins,
"Two forms of aryl hydrocarbon receptor type 2 in rainbow trout (Oncorhynchus
mykiss)," by Abnet, C. C., et al., J. of Biological Chemistry 274: 15159-
15166,
(1999). These two proteins are differentially expressed in various tissues
where they
perform closely related but distinct functions.
The presence of single nucleotide substitutions together with specific large
scale alterations in the sequence of SalmoKCaR clones #1-4 including the
gapping
of large numbers of nucleotides and alterations in reading frame of the
resulting
_ SalmoKCaR transcript are not readily explainable on the basis of
differential
splicing of RNA transcripts derived from a single gene, or perhaps some
complex
process where different alleles of a single gene are present in salmon.
Alternatively,
these data suggest that there are multiple PVCR genes present in Atlantic
salmon
that work in concert to enable Atlantic salmon and likely other salmonids to
carry
out their lifecycle stages that include hatching as well as development of
larval and
juvenile phases in freshwater followed by smoltification and migration into
seawater
with a subsequent return to freshwater for spawning.
Detailed studies in mammals including mice and humans show the presence
of a single functionarPVCR gene. However, multiple published reports provide
support for the possibility that multiple PVCR genes exist in fish, while only
a single
functional PVCR gene exists in mammals including humans. Support for multiple
PVCR genes is provided by detailed studies of well characterized genes that
have
demonstrated that teleost fish including salmonids possess multiple sets of
duplicated genes as compared to mammals. These duplicated genes have arisen as
a
result of either genomic duplication events occurring early in the
evolutionary
history of fishes with subsequent gene drop out or via more recent selective
duplication of genes or some combination of both. Moreover, it is widely
aclmowledged that salmonids are polyploid with respect to other teleost fish
and
have undergone an additional genome duplication. This additional genomic
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duplication further heightens the possibility that multiple functional PVCR
genes
exist in sahnonids particularly Atlantic salmon.
If the products of a duplicated gene are not important in the development,
growth or maintenance of an organism, the nonfunctional gene accumulates
natural
mutations and is either inactivated becoming a pseudogene or lost from the
genome
altogether. However, multiple authors have provided evidence that preservation
of
duplicated genes likely involves changes in the developmental or tissue
specific
expression pattern of the duplicated vs. original gene or formation of a new
functional gene protein product that would interact with the original gene
product in
novel ways. (See AhR data above). These data provide support for the possible
roles
of SalmoKCaR transcripts #1-4 as either differentially expressed in various
tissues
of Atlantic salmon as well as SalmoKCaR #3 exerting a dominant negative effect
on
the remaining functional SalmoKCaR proteins. As discussed below, such -
interactions amongst SalmoKCaR transcripts would provide Atlantic salmon and
perhaps all salmonids with the ability to exploit a wide variety of freshwater
and
seawater environments.
Part 2: Use of RT-PCR and northern analysis to deteimine the expression of
SalmoKCaR clones #1, #2 and 43 in various tissues of Atlantic salmon:
Background:
SalmoKCaR clones #1, #2 and #3 were originally isolated from a Atlantic _
salmon kidney cDNA library. To determine the pattern of tissue specific
expression
of these various SalmoKCaR clones, both degenerate (to amplify all Salmo PVCRs
species) and SalmoKCaR primers that will specifically amplify either
SalrnoKCaR
41 or 42 or 43 were utilized. As shown in "Materials and Methods" Section
below,
these primers amplify DNA products of different sizes that can be
distinguished by
agarose gel electrophoresis. PCR on specific cDNA clones confirms that these
primer pairs function exclusively on the clones for which they have been
designed.
Note that both the degenerate and SalmoKCaR #3 specific primers do not span an
intron and therefore RNA was treated with DNAse to ensure that there was not
amplification of contaminating genomic DNA in the results shown. Primers
specific
for SahnoKCaR 41 and #2 span introns and therefore DNAase treatment is not
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required to interpret these results. As a control, the amounts of mRNA added
to
each RT-PCR reaction was determined by separate amplification of actin using
primers designed from the published sequence of Atlantic salmon actin (Genbank
Accession #AF012125 Salmo salar beta actin mRNA). SalmoKCaR #4 could be
distinguished from SalmoKCaRs #1 or #2 by use of appropriate restriction
enzymes
that would identify single nt differences between SalmoKCaR's 41, #2 versus
#4.
Materials and Methods:
Primers:
Degenerate Primers
DSK-F3 and DSK-R4 primers are shown in Example 3.
SalmoKCaR #1 Specific Primers
SalmoKCaR #1
nts
AS1-F17 5'¨ CAA GCA TTA TCA AGA TCA AG 3' nt 47-
66
(SEQ ID NO:18)
AS2-R14 5' ¨ CTC AGA GTG GCC
TTG GC ¨3' nt 2800-
2784
(SEQ ID NO:19)
Product from amplification 2754 nt. The.SalmoKCaR #1 primer pair consists of a
forward primer (AS1-F17) spanning the 5' UTR insertion in SalmoKCaR #2, and a
reverse primer (AS2-R14) within the 158 bp deleted from SalmoKCaR #3.
SalmoKCaR #2 Specific Primers
SalmoKCaR #2
nts
AS2-F13 5'¨ CAG TTC TCT CTT TAA TGG AC 3' nt 109-128
(SEQ ID NO:20)
AS2-R14 5' ¨ CTC AGA GTG GCC TTG GC ¨3' nt 2890-2874
(SEQ ID NO:21)
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Product from amplification = 2782 nt. The SalmoKCaR #2 primer pair is a
forward
primer (AS2-F13) in the 5' UTR insertion in SalmoKCaR #2 clone, and the same
reverse primer as SalmoKCaR #1 primer (AS2-R14).
SalmoKCaR #3 Specific Primers
SalmoKCaR #3
nts
AS5-F'11 5' ¨ AGT CTA CAT CAT CCA TCA GCC ¨3' nt 2700-2720
(SEQ ID NO:22)
AS5-R12 5' ¨ GAT TTT ATT GTC ATT GGA TGC ¨3' nt 3810-3790
(SEQ ID NO:23)
Product from amplification = 1111 nt. The SalmoKCaR #3 primer pair consists of
a
forward primer (AS5-F11) which spans the 158 bp deletion, and a reverse primer
(AS5-R12) located in the 36 bp insertion at the 3' end of the SalmoKCaR #3
clone.
Salmon Actin Primers
SA-Fl 5' ¨ TGG AAG ATG AAA TCG CCG C ¨3' nt 2-20
(SEQ ID NO:24)
SA-R2 5' ¨ GTG GTG GTG AAA CTG TAA CCG C ¨3' nt 608-587
(SEQ ID NO:25)
Product from amplification = 607 nt. This primer set is used to amplify salmon
actin
mRNA that serves as a control to quantify differences in mRNA content.
RNA blotting analysis and RT-PCR of Atlantic salmon and elasmobranch tissues:
Total RNA was purified with Stat 60 reagent (Teltest B Friendswood, TX)
DNAse (Introgen, Carlsbad, CA) treated and used for RT-PCR after cDNA
production with cDNA Cycle Kit (Invitrogen,Carlsbad, CA). The cDNA was
amplified (30 cycles of lmin @ 94 C, lmin @ 57 C, 3' @72 C) using degenerate
primers [forward primer dSK-F3 (SKCaR nts 2279-2306) and reverse primer dSK-
R4 (SKCaR nts 2904-2934). Aliquots of PCR reactions were subjected to gel
electrophoresis and ethidium bromide (EtBr) staining or blotted onto
Magnagraph
membranes (Osmonics, Westboro, MA) and probed with a 32P-atlantic salmon
genomic PCR product (653 bp sequence identical to that shown in SEQ ED NO: 3
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with added nt sequences, washed (0.1x SSC, 0.1% SDS @ 55 C) and
autoradiographed. Selected amplified PCR products from Atlantic salmon tissues
were sequenced as described above. The following conditions were utilized for
each
of the SalmoKCaR specific primers and corresponding blots:
SalmoKCaR #1 amplification conditions and primer set: PCR: lmin @ 94 C, lmin
@ 50 C, 3min @ 72 C, 35 cycles. Amplification products attached to membrane
were probed with full length SalmoKCaR #1 clone and washed (0.1x SSC, 0.1%
SDS @ 55 C) and autoradiographed for 48 hr.
SalmoKCaR #2 amplification conditions and primer set: PCR: lmin @ 94 C, lmin
@ 50 C, 3min @ 72 C, 35 cycles. Amplification products attached to membrane
were probed with full length SalmoKCaR #2 clone and washed (0..lx SSC, 0.1% -
SDS @ 55 C) and autoradiographed for 168 hr.
SalmoKCaR #3 amplification conditions and primer set: PCR: lmin @ 94 C, linin
@ 52 C, 3min @ 72 C, 35 cycles. Amplification products attached to membrane
were probed with full length SalmoKCaR #3 clone and washed (0.1x SSC, 0.1%
SDS @ 55 C) and autoradiographed for 72 hr.
Results:
Analysis of Atlantic salmon tissues from freshwater vs. seawater adapted fish
using
degenerate primers:
Figure 20 shows data obtained from 14 tissues of freshwater or seawater
adapted Atlantic salmon using the degenerate primers described above. Samples
were obtained from a single representative seawater adapted salmon (866gm and
41cm in length) from a group of 10 fish of average weight of 678gm. Samples
from
nasal lamellae, urinary bladder, olfactory bulb and pituitary gland were all
pooled
samples from all 10 fish. The samples were from a representative single
freshwater
adapted fish (112gm and 21.5cm) selected from a group of 10 fish with an
average
weight of 142.8gm. In contrast, samples from nasal lamellae, urinary bladder,
olfactory bulb and pituitary gland were all pooled samples from all 10 fish.
Note
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that the amplification products from these degenerate primers do not
distinguish
between SalmoKCaR #1, #2, #3 or #4 since their nt sequences in the region
amplified by the primers are all identical (lanes 7, 9 and 12 Lower gel ¨
Panels A, B,
C and D). Moreover, these degenerate primers also possess the capacity to
amplify
additional PVCRs (if any are present) in salmon tissues that could be distinct
from
either SalmoKCaR #1-4. Thus, amplified RT-PCR products are referred to as
PVCR products since use of these degenerate primers do not distinguish between
various PVCR species.
Analysis of panels A-D of Figure 20 shows that the PVCR degenerate
primers yield PCR products in various tissues of both seawater and freshwater
adapted fish. These various bands are more visible in Southern blots (Panels
C, D)
of corresponding ethidium bromide gels (Panels A and B) because detection of
PVCR amplified products via hybridization of a 3213-PVCR probe is more
sensitive
as compared to ethidium bromide staining. Prominent ethidium bromide stained
bands are visible in urinary bladder (lane 4), kidney (lane 5) and muscle
(lane 14) in
seawater adapted fish (Panel A) while either faint or no bands are seen in
other
tissues. In contrast, ethidium bromide bands are also visible in nasal
lamellae (lane
3), urinary bladder (lane 4) and kidney (lane 5) as well as olfactory bulb
(lane 12) in
freshwater fish (Panel B). In summary, these data show differential tissue
expression of PVCRs
Figure 20 shows a RT-PCR analysis of freshwater (Panels B, D and F) and
seawater (Panels A, C and E) adapted Atlantic salmon tissues using either
degenerate PVCR or salmon actin PCR primers. Total RNA from 13 (seawater
adapted) and 14 (freshwater adapted) tissues of Atlantic salmon was first
treated
with DNAase to remove any genomic DNA contamination then used to synthesize
cDNA that was amplified using degenerate primers. (Panels A and B): Ethidium
bromide stained agarose gel. DNA markers in lane 1 of both Panels A and B were
used to indicate size of amplification products. (Panels C and D) Southern
blot of
gel in Top Panel using 3213-labeled Atlantic salmon genomic fragment. (Panels
E
and F) Ethidium bromide stained gels of RT-PCR amplification products using
Atlantic salmon beta actin primers as described above. These reactions serve
as
controls to ensure that samples contain equal amounts of RNA.
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Southern blots (Panels C and D) of the corresponding gels shown in Panels A
and B reveal that amplified PVCR products are present in additional tissues
not
shown by simple ethidium bromide staining as described above. As shown in
Panel
C, PVCRs are present in tissues of seawater-adapted salmon including gill
(lane 2),
nasal lamellae (lane 3), urinary bladder (lane 4), kidney (lane 5), stomach
(lane 6),
pyloric caeca (lane 7), proximal (lane 8) and distal (lane 9) intestine,
pituitary gland
(11) and muscle (lane 14). Ovary tissue was not tested in seawater-adapted
fish. In
contrast, freshwater-adapted salmon possess amplified PVCR products in gill
(lane
2), nasal lamellae (lane 3), urinary bladder (lane 4), kidney (lane 5),
proximal
intestine (lane 8), brain (lane 10), pituitary (lane 11), olfactory bulb (lane
12), liver
(lane 13), muscle (lane 14) and ovary (lower lane 3). The intensity of
individual
actin bands shown in Panels E and F perfolured on identical aliquots of the RT-
PCR
reactions serve to quantify any differences in pools of cDNA from the
individual RT
reactions in each sample. Isolation and subcloning of the ethidium bromide
stained
bands from olfactory lamellae and urinary bladder show that nucleotide
sequences of
multiple subclones from these bands all are identical to the nucleotide
sequence
present in SalmoKCaR clones #1-3.
Close examination of the differences in Panel C (seawater adapted) vs. Panel
D (freshwater adapted) reveal differences in the apparent abundance of PVCR
mRNA in specific tissues. Apparent increases in tissue PVCR mRNA abundance in
seawater-adapted salmon vs. freshwater-adapted salmon are present in gill,
kidney,
stomach, pyloric caeca, distal intestine, and muscle. The increased expression
of
PVCRs in Atlantic salmon exposed to seawater is consistent with other data
that an
increase in PVCR expression in at least one tissue occurs upon transfer of
Atlantic
salmon from freshwater to seawater. In contrast, the abundance of PVCR mRNA
species in olfactory bulb tissue of seawater adapted salmon appears to be
reduced as
compared to olfactory bulbs of freshwater adapted counterparts (Lane 12 in
Panels C
vs. D). In other tissues such as nasal lamellae (Lane 3 in Panel C vs. D)
there is little
or no apparent change in the steady state PVCR mRNA content. In summary, these
data demonstrate tissue specific changes in the steady state expression of
PVCR
mRNA species in seawater adapted vs. freshwater adapted Atlantic salmon.
Depending on the tissue, steady state PVCR mRNA content is either increased,
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decreased or remains unchanged when freshwater adapted fish are compared to
seawater adapted counterparts. Since these analyses shown in Figure 20 use
PVCR
degenerate primers, it is not possible to determine from these experiments
whether
the alterations in steady state PVCR mRNA content are the result of changes in
individual SalmoKCaRs #1-4.
RT-PCR analysis using degenerate primers shows that steady state content of
kidney
PVCRs is increased by the SuperSmoltTM process similar to that produced by
transfer of Atlantic salmon to seawater.
Figure 21A shows RT-PCR analysis of a single representative experiment
where kidney tissue was harvested from Atlantic salmon that had either been
freshwater adapted (lane 1), exposed to 9 weeks of the SuperSmoltml process in
freshwater (lane 2) or transferred to seawater and maintained for 26 days.
Figure
21B shows RT-PCR analysis of a single representative experiment using pyloric
caeca from the same fish shown in Figure 21A. Note the significant increase in
amplified PVCR product present in kidney (Figure 21A) and pyloric caeca
(Figure
21B) for both SuperSmoltTM (lanes 2 and 7, respectively) and seawater adapted
(lanes 3 and 8, respectively) fish as compared to freshwater (lanes 1 and 6,
respectively). The increased expression of PVCRs in these 2 tissues of
Atlantic
salmon exposed to the SuperSmoltTM process where this increased PVCR
expression
mimics that produced after seawater transfer is consistent with earlier data
that an
increase in PVCR expression in at least one tissue occurs upon either
treatment with
the SuperSmoltTM process or transfer of Atlantic salmon to seawater.
Figure 21C shows RT-PCR analysis using the same degenerate primers to
detect expression of SalmoKCaR transcripts in various stages of Atlantic
salmon
embryo development. Using degenerate (SEQ ID Nos 15 and 16) or actin (SEQ ID
No 24 and 25) primers, RNA obtained from samples of whole Atlantic salmon
embryos at various stages of development were analyzed for expression of
SalmoKCaRs using RT-PCR. Ethidium bromide staining of samples from
dechorionated embryos (Lane 1), 50% hatched (Lane 2), 100% hatched (Lane 3), 2
weeks post hatched (Lane 4) and 4 weeks post hatched (Lane 5) shows that
SalmoKCaR transcripts are present in Lanes 1-4. Southern blotting of the same
gel
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(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.
Northern blotting of kidney poly .A+ RNA with SalmoKCaR 41 reveals an increase
in
PVCR expression in seawater-adapted vs. freshwater-adapted Atlantic salmon.
To both confirm the size of SalmoKCaR transcripts and test for changes in
SalmoKCaR expression in fish exposed to different salinities, poly A+ RNA from
kidney of either freshwater adapted (FW) or seawater adapted (SW) Atlantic
salmon
were probed with SalmoKCaR 41. As shown in Figure 22, kidney RNA contains a
4.2kb band that corresponds to the 3.9-4.0 kb sizes of SalmoKCaR 41-4 as
determined by nucleotide sequence analysis. Because of the high degree of
nucleotide identities between SalmoKCaR #1-4, the 4.2kb band is actually
derived
from the combination of all 4 SalmoKCaR species and any additional PVCR
species
in salmon kidney due to crosshybridization of SalmoKCaR #1. However, these
data
show an increase in the intensity of the 4.2kb SalmoKCaR band in SW adapted
fish
as compared to their FW adapted counterparts.
Figure 22 shows a RNA blot containing 5 micrograms of poly A+ RNA from
kidney tissue dissected from either freshwater adapted (FW) or seawater
adapted
(SW) Atlantic salmon probed with full length SalmoKCaR #1 clone.
Autoradiogram exposure after 7 days.
Use of RT-PCR with SalmoKCaR43 specific primers demonstrates that tissue
specific alterations in the steady state tissue content of SalmoKCaR 43 mRNA
in
freshwater vs. seawater adapted Atlantic salmon.
To determine whether specific SalmoKCaRs 43 are modulated by exposure to
different salinities, nucleotide primer sets that allows for the specific
amplification
of SalmoKCaR transcripts were designed. Figure 23 shows RT-PCR analysis of
freshwater (Panels B, D and F) and seawater (Panels A, C and E) adapted
Atlantic
salmon tissues using either SalmoKCaR 43 specific PCR primers or salmon actin
PCR primers. Total RNA from 13 (seawater adapted) and 14 (freshwater adapted)
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tissues of Atlantic salmon identical to those shown in Figure 20 were first
treated
with DNAase to remove any genomic DNA contamination, then used to synthesize
cDNA that was amplified using SalmoKCaR #3 primers. All RNA samples were
prepared from a single fish with the exception of olfactory bulb, pituitary,
urinary
bladder and nasal lamellae that are composed of RNA from pooled samples of
fish.
Selected reactions were subjected to primer amplification using SalmoKCaR#3
specific primers. DNA markers in lane 1 of both Panels A and B were used to
indicate size of amplification products. (Panels C and D) Southern blot of gel
in
Top Panel using 32P-labeled Atlantic salmon genomic fragment. (Panels E and F)
Ethidium bromide stained gel of RT-PCR amplification products using Atlantic
salmon beta actin primers as described above. These reactions serve as
controls to
ensure that samples contain equal amounts of RNA. The specificity of these
- SalmoKCaR#3 primers is demonstrated in the bottom half of Panels A and B
of
Figure 23. The specific SalmoKCaR #3 primers only amplify product from
SalmoKCaR #3 clone (lane 14) and not SalmoKCaR #1 (lane 8) or SalmoKCaR #2
(lane 11) or SalmoKCaR #4. Note that in the tissue sample lanes, ethidium
bromide
stained bands are present in the kidney of seawater adapted salmon (lane 5
upper
gel- Panel A) and only very faintly in urinary bladder of freshwater adapted
salmon
(lane 4 upper gel- Panel B). The corresponding Southern blots of freshwater
adapted
tissue samples (Panel D) reveal detectable SalmoKCaR #3 product only in
urinary
bladder (lane 4) and a small amount in kidney (lane 5). In contrast, in
seawater-
adapted salmon (Panel C) there are detectable increases in SalmoKCaR #3
product
in both urinary bladder (lane 4) and kidney (lane 5) as well as the presence
of
SalmoKCaR #3 amplified product in gill (lane 2), nasal lamellae (lane 3),
pyloric
caeca (lane 7) and muscle (lane 14) of seawater adapted fish.
As described above, the increase in tissue expression of SalmoKCaR #3
serves to provide for a possible means to reduce the overall tissue
sensitivity to
PVCR-mediated sensing via an action where SalmoKCaR #3 would act as a
dominant negative effector. In contrast to freshwater where the ambient water
concentrations of both Ca2+ and Me are low and require a high degree of
sensitivity
from SalmoKCaRs to sense changes in concentration, the concentrations of Ca2+
and
Mg2+ in seawater are 10 fold and 50 fold higher and thus may require reduction
of
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the high sensitivity of SalmoKCaRs #1, #2 and #4 by SalmoKCaR #3. It is of
interest that many of these specific tissues exhibiting significant SalmoKCaR
#3
expression are either exposed directly to the high Ca' and Mg" content of
seawater
(gill, nasal lamellae) or experience high Ca' and Mg' concentrations as the
result of
the excretion of these divalent cations (urinary bladder, kidney).
Use of RT-PCR with SalmoKCaR 41 specific primers demonstrates tissue specific
alterations in the steady state tissue content of SalmoKCaR 41 mRNA in
freshwater
vs. seawater adapted Atlantic salmon.
Figure 24 shows RT-PCR analysis of freshwater (Panels B, D and F) and
seawater (Panels A, C and E) adapted Atlantic salmon tissues using either
SalmoKCaR #1 specific PCR primers or salmon actin PCR primers. Total RNA
from 13 (seawater adapted) and 14 (freshwater adapted) tissues of Atlantic
salmon
identical to those shown in Figures 20 and 23 were used to synthesize cDNA
that
was amplified using SalmoKCaR #1 primers. All RNA samples were prepared from
a single fish with the exception of olfactory bulb, pituitary, urinary bladder
and nasal
lamellae that are composed of RNA from pooled samples of fish. As controls to
demonstrate primer specificity, selected reactions were subjected to primer
amplification of portions of individual SalmoKCaR clones or water alone
(Panels A
and B): Ethidium bromide stained agarose gel. DNA markers in lane 1 of both
Panels A and B were used to indicate size of amplification products. (Panels C
and
D) Southern blot of gel in Top Panel using 3213-labeled Atlantic salmon
genomic
fragment. (Panes E and F) Ethidium bromide stained gel of RT-PCR amplification
products using Atlantic salmon beta actin primers as described above. These
reactions serve as controls to ensure that samples contain equal amounts of
RNA.
As shown in lower halves of Panels A and B of Figure 24, PCR amplification
with
these primers yields an ethidium bromide staining band (lane5) when SalmoKCaR
#1 clone is used as a template but not either SalmoKCaR #2 (lane 6) or
SalmoKCaR
#3 (lane 7). Similar analysis could be performed on SalmoKCaR #4. Southern
blotting analysis of the gels shown in Panels A and B reveals that the
amplification
product of the SalmoKCaR #3 is highly positive (lanes 5) - - Panels C and D.
In the
various tissue samples, SalmoKCaR #1 product is amplified in selected tissues
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including urinary bladder (lane 4) and pyloric caeca (lane 7) in seawater-
adapted
salmon (Panel C) as compared to urinary bladder (lane 4) and kidney (lane 5)
in
freshwater-adapted salmon (Panel D). The exact nature of the smaller and
larger
than expected PCR amplification products present in gill (lane 2 ¨Panels C, D)
and
nasal lamellae (lane 3 ¨ Panel D) are not known at present. These data show
tissue
specific expression of SalmoKCaR #1 in both freshwater and seawater adapted
salmon.
Use of RT-PCR with SalmoKCaR #2 specific primers demonstrates tissue specific
alterations in the steady state tissue content of SalmoKCaR #2 mRNA in
freshwater
vs. seawater adapted Atlantic salmon.
Figure 25 shows RT-PCR analysis of freshwater (Panels B, D and F) and
seawater (Panels A, C and E) adapted Atlantic salmon tissues using either
SalmoKCaR #2 specific PCR primers or salmon actin PCR primers. Total RNA
from 13 (seawater adapted) and 14 (freshwater adapted) tissues of Atlantic
salmon
was used to synthesize cDNA that was amplified using SalmoKCaR #2 primers.
All RNA samples were prepared from a single fish with the exception of
olfactory
bulb, pituitary, urinary bladder and nasal lamellae that are composed of RNA
from
pooled samples of fish. As controls to demonstrate primer specificity,
selected
reactions were subjected to primer amplification with samples of portions of
individual SalmoKCaR clones or water alone (Panels A and B): Ethidium bromide
stained agarose gel. 6NA markers in lane 1 Panels A, B, E and F were used to
indicate size of amplification products. (Panels C and D) Southern blot of gel
in
Top Panel using 32P-labeled Atlantic salmon genomic fragment. (Panes E and F)
Ethidium bromide stained gel of RT-PCR amplification products using Atlantic
salmon beta actin primers as described above. These reactions serve as
controls to
ensure that samples contain equal amounts of RNA. Figure 25 shows data
obtained
using SalmoKCaR #2 specific primers and the identical tissue RT and plasmid
samples as shown in Figures 20, 23, and 24. Corresponding Southern blots shown
in
Panels C and D reveal the presence of SalmoKCaR #2 PCR amplification product
in
urinary bladder of seawater-adapted salmon (lane 4) as well as urinary bladder
(lane
4) and kidney (lane 5) of freshwater-adapted salmon. These data provide
evidence
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of the tissue specific expression of Sahnol(CaR #1 in both freshwater and
seawater
adapted salmon.
EXAMPLE 7: SURVIVAL AND GROWTH OF PRE-ADULT ANADROMOUS
FISH BY MODULATING PVCRS
An important feature of current salmon farming is the placement of smolt
from freshwater hatcheries to ocean netpens. Present day methods use smolt
that
have attained a critical size of approximately 70-110 grams body weight. The
methods described herein to modulate one or more PVCRs of the anadrornous fish
including Atlantic Salmon, can either be utilized both to improve the ocean
netpen
transfer of standard 70-110 grams smolt as well as permit the successful ocean
netpen transfer of smaller smolts weighing, for example, only 15 grams. As
shown
herein, one utility for the present invention is its use in conjunction with
transferring _
Atlantic Salmon from freshwater to seawater. For standard 70-110 gram smolt,
application of the invention eliminates the phenomenon known as "smolt window"
and permits fish to be maintained and transferred into ocean water at 15 C or
higher.
Use of these methods in 15 gram or larger smolt permits greater utilization of
freshwater hatchery capacities followed by successful seawater transfer to
ocean
_
netpens. In both cases, fish that undergo the steps described herein feed
vigorously
within a short interval of time after transfer to ocean netpens and thus
exhibit rapid
growth rates upon transfer to seawater.
Figure 26 shows in schematic foun the key features of current aquaculture of
Atlantic salmon in ocean temperatures present in Europe and Chile. Eggs are
hatched in inland freshwater hatcheries and the resulting fry grow into
fingerlings
and parr. Faster growing parr are able to undergo smoltification and placement
in
ocean netpens as SO smolt (70 gram) during year 01. In contrast, slower
growing
parr are smoltified in year 02 and placed in netpens as Si smolt (100 gram).
In both
SO and Si transfers to seawater, the presence of cooler ocean and freshwater
temperatures are desired to minimize the stress of osmotic shock to newly
transferred smolt. This is particularly true for 51 smolt since freshwater
hatcheries
are often located at significant distances from ocean netpen growout sites and
their
water temperatures rise rapidly during early summer. Thus, the combination of
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rising water temperatures and the tendency of smolt to revert or die when held
for
prolonged intervals in freshwater produces a need to transfer smolt into
seawater
during the smolt window.
Standard molts that are newly placed in ocean netpens are not able to grow
optimally during their first 40-60 day interval in seawater because of the
presence of
osmotic stress that delays their feeding. This interval of osmotic adaptation
prevents
the smolts from taking advantage of the large number of degree days present
immediately after either spring or fall placement. The combination of the
presence
of the smolt window together with delays in achieving optimal smolt growth
prolong
the growout interval to obtain market size fish. This is particularly
problematic for
SO's since the timing of their harvest is sometimes complicated by the
occurrence of
grilsing in maturing fish that are exposed to reductions in ambient
photoperiod.
Methods
The smolt were subjected to the steps of Process I and II, as described
herein.
RESULTS AND DISCUSSION:
SECTION I: Demonstration of the Benefits of the Process I For Atlantic Salmon
Demonstration of the Benefits of the Process I For Atlantic Salmon:
Process I increases the survival of small Atlantic Salmon Sllike smolt after
their transfer to seawater when compared to matched freshwater controls.
Optimal
survival is achieved by using the complete process consisting of both the
magnesium
and calcium water mixture as well as NaC1 diet. In contrast, administration of
calcium and magnesium either via the food only Or without NaC1 dietary
supplementation does not produce results equivalent to Process I.
Table 3 shows data obtained from Atlantic salmon S2 like smolts less than 1
year old weighing approximately 25 gm. This single group of fish was
apportioned
into 4 specific groups as indicated below and each were maintained under
identical
laboratory conditions except for the variables tested. All fish were
maintained at a
water temperature of 9-13 C and a continuous photoperiod for the duration of
the
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experiment. The control freshwater group that remained in freshwater for the
initial
45 day interval experienced a 33% mortality rate under these conditions such
that
only 67% were able to be transferred to seawater. After transfer to seawater,
this
group also experienced high mortality where only one half of these smolts
survived.
Inclusion of calcium (10 mM) and magnesium (5 mM) within the feed offered to
smolt (Ca2+/Mg2+diet) reduced survival as compared to controls both in
freshwater
(51% vs 67%) as well after seawater transfer (1% vs 50%). In contrast,
inclusion of
mM Ca2+ and 5 mM Mg2+ in the freshwater (Process I Water Only) improved
smolt survival in Process I water as well as after transfer of smolt to
seawater.
10 However, optimal results were obtained (99% survival in both the Process
I water
mixture as well as after seawater transfer) when smolt were maintained in
Process I
water mixture and fed a diet supplemented with 7% sodium chloride.
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Table 3: Comparison of the Survival of Atlantic Salmon S2 like Smolts After
Various Treatments
Parameter Control Ca2+/Mg2+ Process I Process I
Sampled Freshwater Diet Water Only Water + NaC1
Diet
Starting # of 66 70 74 130
fish
# of fish 44 36 67 129
% of fish 67% 51% 91% 99%
surviving
after 45 days
in freshwater
or Process
mixture
# of fish 22 2 60 128
% of fish 50% 6% 90% 99%
surviving 5
days after
transfer to
seawater
'Survival percentages expressed as rounded whole numbers
Application of the Process Ito the Placement of 70-100 gm smolts in seawater.
These data show that use of the Process I eliminates the "smolt window" and
provides for immediate smolt feeding and significant improvement in smolt
growth
rates.
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Experimental Protocol:
Smolts derived from the St. John strain of Atlantic salmon produced by the
Connors Brothers Deblois Hatchery located in Cherryfield, Maine, USA were
utilized for this large scale test. Smolts were produced using standard
practices at
this hatchery and were derived from a January 1999 egg hatching. All smolts
were
transferred with standard commercially available smolt trucks and transfer
personnel. Si smolt were purchased during Maine's year 2000 smolt window and
smolt deliveries were taken between the dates of 29 April 2000 - 15 May 2000.
Smolts were either transferred directly to Polar Circle netpens (24m diameter)
located in Blue Hill Bay Maine (Controls) or delivered to the treatment
facility
where they were treated with Process I for a total of 45 days. After receiving
the
Process I treatment, the smolt were then transported to the identical Blue
Hill Bay
netpen site and placed in an adjacent rectangular steel cage (15mX15mX5m) for
growout. Both groups of fish received an identical mixture of moist (38%
moisture)
and dry (10% moisture) salmonid feed (Connors Bros). Each of the netpens were
fed by hand or feed blower to satiation twice per day using camera
visualization of
feeding. Mort dives were performed on a regular basis and each netpen received
identical standard care practices established on this salmon farm. Sampling of
fish
_
for growth analyses was performed at either 42 days (Process l) or 120 days or
greater (Control) fish. In both cases, fish were removed from the netpens and
multiple analyses performed as described below.
All calculations to obtain feed conversion ratio (FCR) Or specific growth rate
(SGR) and growth factor (GF3) were perfatined using standard accepted formulae
(Willoughby, S. Manual of Salmonid Farming Blackwell Scientific, Oxford UK
1999) and established measurements of degree days for the Blue Hill Bay site
as
provided in Table 4 below. A degree day is calculated by multiplying the
number of
days in a month by the mean daily temperature in degrees Celsius.
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Table 4: Degree days for Blue Hill Bay Salmon Aquaculture Site
Month Degree Days
Jan 60
Feb 30
Mar 15
April 120
May 210
June 300
July 390
Aug 450
Sept 420
Oct 360
Nov 240
Dec 180
Table 5 displays data obtained after seawater transfer of Control Si smolt.
Smolt ranging from 75-125 gm were placed into 3 independent netpens and
subjected to normal farm practices and demonstrated characteristics typical
for
present rday salmon aquaculture in Maine. Significant mortalities (average
3.3%)
were experienced after transfer into cool (10 C) seawater and full feeding was
achieved only after a significant interval (-56 days) in ocean netpens. As a
result,
the average SGR. and GF3 values for these 3 netpens were 1.09 and 1.76
respectively
for the 105-121 day interval measured.
In contrast to the immediate transfer of Control Si smolt as described above
to ocean netpens (Table 5), a total of 10,600 Si smolt possessing an average
size of
63.6 grams were transported on 11 May 2000 from the Deblois freshwater
hatchery
to the research facility. While being maintained in standard circular tanks,
these fish
were held for a total of 45 days at an average water temperature of 11 C and
were
subjected to Process I. During this interval, smolt mortality was only 64 fish
(0.6%).
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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 C
and were equivalent to treated smolts prior to transfer to seawater.
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Table 6: Characteristics of St. John Si smolt subjected to immediate placement
in
ocean netpens after transport form the freshwater hatchery without Process I
or
Process II technology (the Control fish)
Netpen Number
#17 #18 #10
Total Fish 51,363 43,644 55,570
Mean Date of 5/1/00 5/5/00 5/14/00
Seawater Transfer
Average Size at (117.6)
Transfer (grams) 100-125 75-100 75-100
Mortalities after 30 1,785;_3.5% 728; 1.7% 2503; 4.5%
days (# and % total)
Time to achieve full 68 days 48 days 50 days
feeding after
transfer
Interval between 121 120 105
_
netpen placement
and analysis
Average size at
Analysis
Weight (gram) 376.8 174 305.80164 298.90137.40
Length (cm) 33.411.9 28.3019.0 30.4011.17
Condition Factor 1.02 1.34 1.06
(k)
SGR 0.96 1.10 1.17
during initial 120 days
During the 45 day interval when Si smolts were receiving Process I, fish grew
an average of 10 grams and thus possessed an average weight of 76.6 gm when
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transferred to an ocean netpen. The actual smolt transfer to seawater
occurring on
26 June 2000 was notable for the unusual vigor of the smolt that would have
normally been problematic since this time is well past the normal window for
ocean
placement of smolt. The ocean temperature at the time of Process I smolt
netpen
placement was 15.1 C. In contrast to the counterpart S1 smolts subjected to
standard
industry practices described above, Process I smolts fed vigorously within 48
hours
of ocean placement and continued to increase their consumption of food during
the
immediate post-transfer period. The mortality of Process I smolts was
comparable
to that of smolts placed earlier in the summer (6.1%) during initial 50 days
after
ocean netpen placement and two thirds of those mortalities were directly
attributable
to scale loss and other physical damage incurred during the transfer process
itself.
In contrast, corresponding control fish (held under identical conditions
without
Process I treatment) did not fare well during transfer to the netpen (17%
transfer
mortality) and did not feed vigorously at any time during the first 20 days
after ocean
netpen placement. This smaller number of control fish (176) were held in a
smaller
(1.5mX1.5m*1.5m) netpen floating within the larger netpen containing Process I
smolts. Their mortality post-ocean netpen placement was very high at 63%
within
the 51 day interval.
Both Process I and control smolts were fed on a daily basis in a manner
identical
to that experienced by the Industry Standard Fish shown on Table 6. Process I
fish
were sampled 51 days after their seawater placement and compared to the
Industry
Standard smolts shown on Table 6. As shown in Table 7, comparison of their
characteristics reveals dramatic differences between Industry Standard smolts
vs
Process I.
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Table 7: Comparison of the characteristics of St. John Si Process I Smolts
subjected
to Process I treatment and then placed in ocean netpens vs corresponding
industry
standard smolts.
Process I Smolts Averaged Industry Standard
Data from Table 6 in this
Example
Total Fish 10,600 150,577
Mean Date of Seawater 6/26/00 5/7/00
Transfer
Average Size at Transfer 76.6 95.8
(grams)
Mortalities after 30 days 648; 6.1% 5,016; 3.3%
(# and %)
Time to achieve full 2 days 56 days
Feeding after transfer
Interval between netpen 51 115
placement and analysis
Average size at Analysis
Weight (gram) 175.48 50 327.2 97
Length (cm) 26.2 32 30.7
Condition Factor (k) 0.95 0.9 1.14
SGR 1.80 1.09
In summary, notable differences between Process I, Control smolt and Industry
Standard smolt include:
1. The mortalities observed after ocean netpen placement were low in Process I
(6.1%) vs Control (63%) despite the that fact these fish were transferred to
seawater
1.5 months after the smolt window and into a very high (15.1 C) ocean water
temperature. The mortality of Process I was comparable to that of the accepted
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Industry Standard smolt (3-10%) transferred to cooler (10 C) seawater during
the
smolt window. This characteristic of Process I provides for a greater
flexibility in
freshwater hatchery operations since placement of Process I smolts are not
rigidly
confined the conventional "smolt window" currently used in industry practice.
2. The Process I fish were in peak condition during and immediately after
seawater transfer. Unlike industry standard smolt that required 56 days to
reach fall
feeding, the Process I smolts fed vigorously within 2 days. Moreover, the
initial
growth rate (SGR 1.8) demonstrated by Process I smolts are significantly
greater
than published data for standard smolt during their initial 50 days after
seawater
placement (published values (Stradmeyer, L. Is feeding nonstarters a waste of
time.
Fish Fanner 3:12-13, 1991; Usher, ML, C Talbot and FB Eddy. Effects of
transfer to
seawater on growth and feeding in Atlantic salmon smolts (Salmo salar L.)
Aquaculture 94:309-326, 1991) for SGR's range between 0.2-0.8). In fact, the
growth rates of Process I smolts are significantly larger as compared to
Industry
standard smolts placed into seawater on the same site despite that industry
standard
smolt were both larger at the time of seawater placement as well as that their
growth
was measured 120 days after seawater placement. These data provide evidence
that
the Process I smolts were not subjected to significant osrnoregulatory stress
which
would prevent them from feeding immediately.
3. The rapid growth of Process I smolts immediately upon ocean netpen
placement provides for compounding increases in the size of salmon as seawater
growout proceeds. Thus, it is anticipated that if Industry Standard Smolts
weighing
112.5 gram '(gm) were subjected to Process I treatment, placed in ocean
netpens and
examined at 120 days after ocean netpen placement their size would be average
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.
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
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period. By comparison, the industry standard smolts consumed little or no feed
within the first week after transfer, Figure 27A compares the weekly feed =
consumption on a per fish basis between Process I treated smolts and industry
standard smolts. As shown, Process I treated smolts consumed approximately
twice
as much feed per fish during their FIRST WEEK as compared to the industry
standard smolts after 30 days. Since smolts treated with Process I fed
significantly
more as compared to Industry standard smolts, the Process I treated smolts
grew
faster.
Figure 278 provides data on the characteristics of Process I smolts after
seawater transfer. These experiments were carried out for over 185 days.
Application of the Process Ito Atlantic Salmon pre-adult Fish that are Smaller
than
the Industry Standard "Critical Size" Smolt.
A total of 1,400 Landcatch/St John strain fingerlings possessing an average
weight of 20.5 gram were purchased from Atlantic Salmon of Maine Inc., Quossic
Hatchery, Quossic, Maine, USA on 1 August 2000. These fingerlings were derived
from an egg hatching in January 2000 and considered rapidly growing fish. They
were transported to the treatment facility using standard conventional truck
transport. After their arrival, these fingerlings were first placed in typical
freshwater
growout conditions for 14 days. These fingerlings were then subjected to
Process I
for a total of 29 days while being exposed to a continuous photoperiod. The
Process
I were then vaccinated with the Lipogen Forte product (Aquahealth LTD.) and
transported to ocean netpens by conventional truck transport and placed into
seawater (15.6 C) in either a research ocean netpen possessing both a predator
net as
well as net openings small enough (0.25 inch) to prevent loss of these smaller
Process I smolts. Alternatively, Process I smolts were placed in circular
tanks within
the laboratory. Forty eight hours after sea water transfer, Process I smolts
were
begun on standard moist (38% moisture) smolt feed (Connors Bros.) that had
been
re-pelletized due to the necessity to provide for smaller size feed for
smaller Process
I smolts, as compared to normal industry salmon. In a manner identical to that
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described for 70 grain smolts above, the mortality, feed consumption, growth
and
overall health of these 30 gram Process I smolts were monitored closely.
Figure 28 displays the characteristics of a representative sample of a larger
group of 1,209 Process I smolts immediately prior to their transfer to
seawater.
These parameters included an average weight of 26.6+8.6 gram, length of
13.1+1.54
cm and condition factor of 1.12+0.06. After seawater transfer, Process I
smolts
exhibited a low initial mortality despite the fact that their average body
weight is
26-38% of industry standard 70-100 gram SO-S1 smolts. As shown in Table 8,
Process I smolts mortality within the initial 72 hr after seawater placement
was
1/140 or 0.07% for the laboratory tank. Ocean netpen mortalities after
placement of
Process I smolts were 143/1069 or 13.4%. Figure 28 shows representative
Landcatch/St John strain Process I smolts possessing a range of body sizes
that were
transferred to seawater either in ocean netpens or corresponding laboratory
seawater
tanks. Process I smolts possess a wide range of sizes (e.g., from about 5.6
grams to
about 46.8 grams body weight) with an average body weight of 26.6 gram.
Experiments with these data were carried out for 84 days after the transfer of
fish to
seawater tanks, and the data from these experiment's are described in co-
pending
application No: 09/975,553, Attorney Docket No: 2213.1004-001.
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Table 8: Characteristics and survival of Landcatch/St. John Process I fish
after their
placement into seawater in either a laboratory tank or ocean netpen.
Laboratory Tank Ocean Netpen
Total Fish 140 1,069
Date of Seawater 9/5/00 (40); 9/12/00 9/12/00
Transfer (100)
Average Size at Transfer 26.6 26.6
(gram)
Total mortalities after 4 1; 0.7% 143;13.4%
days (# and % total)
% mortality of fish 0; 0.0% 4; 0.4%
weighing 25 gm and
above
Time to achieve feeding 48 hrs 72 lirs
Figure 29 shows a comparison of the distributions of body characteristics for
total group of Landcatch/St John Process I smolts vs. mortalities 72 hr after
seawater
ocean netpen placement. Length and body weight data obtained from the 143
mortalities ocpurring after seawater placement of 1,069 Process I smolts were
plotted on data obtained froth a 100 fish sampling as shown previously in
Figure 28.
Note that the mortalities are exclusively distributed among the smaller fish
within
the larger Process I netpen population.
Length and weight measurements for all mortalities collected from the bottom
of the ocean netpen were compared to the distribution of Process I smolt body
characteristics obtained from analysis of a representative sample prior shown
in
Figure 29. The data show that the mortalities occurred selectively amongst
Process I
smolts possessing small body sizes such that the mean body weight of
mortalities
was 54% of the mean body weight of the total transfer population (14.7/27 gram
or
54%). Thus, the actual mortality rates of Process I smolts weighing 25-30 gram
is
0.4% (4/1069) and those weighing 18-30 gram is 2.9% (31/1069).
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Application of Process I to Trout pre-adult Fish that are Smaller than the
Industry
Standard "Critical Size" Smolt.
Table 9 displays data on the use of the Process I on small (3-5 gram) rainbow
trout. Juvenile trout are much less tolerant of abrupt transfers from
freshwater to
seawater as compared to juvenile Atlantic salmon. As a result, many commercial
seawater trout producers transfer their fish to brackish water sites located
in estuaries
or fresh water lenses or construct "drinking water" systems to provide fresh
water for
trout instead of the full strength seawater present in standard ocean netpens.
After a
prolonged interval of osmotic adaptation, trout are then transferred to more
standard
ocean netpen sites to complete their growout cycle. In general, trout are
transferred
to these ocean sites for growout at body weights of approximately 70-90 or 90-
120
gram.
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Table 9: Comparison of the Survival of Rainbow Trout (3-5 gram) in Seawater
After
Various Treatments.
Percent Survival of Fish'
Hours Post Control Constant 14 Constant 14 Constant 23
Seawater Freshwater day day day
Transfer Photoperiod Photoperiod
Photoperiod +
Process I Process I
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
Fish Per 10 20 30 80
Experiment
1Survival percentages expressed as rounded whole numbers
A total of 140 trout from a single pool of fish less than 1 year old were
divided
into groups and maintained at a water temperature of 9-13 C and pH 7.8-8.3 for
the
duration of the experiment described below. When control freshwater rainbow
trout
are transferred directly into seawater, there is 100% mortality within 24 hr
(Control
Freshwater). Exposure of the trout to a constant photoperiod for 14 days
results in a
slight improvement in survival after their transfer to seawater. In contrast,
exposure
of trout to Process I for either 14 days or 23 days results in significant
reductions in
mortalties after transfer to seawater such that 30% and 46% of the fish
respectively
have survived after a 5 day interval in seawater. These data demonstrate that
application of the Process I increases in the survival of pre-adult trout that
are less
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than 7% of the size of standard "critical size" trout produced by present day
industry
standard techniques.
Application of the Process Ito Arctic Char pre-adult Fish that are Smaller
than the
Industry Standard "Critical Size" Smolt.
Although arctic char are salmonids and anadromous fish, their tolerance to
seawater transfer is far less as compared to either salmon or trout. Figure 30
shows
the results of exposure of smaller char (3-5 gram) to the Process I for a
total of 14
and 30 days. All fish shown in Figure 30 were exposed to a continuous
photoperiod.
Transfer of char to seawater directly from freshwater results in the death of
all fish
within 24 hr. In contrast, treatment of char with the Process I for 14 and 30
days
produces an increase in survival such that 33% (3/9) or 73% (22/30)
respectively are
still alive after a 3 day exposure. These data demonstrate that the
enhancement of
survival of arctic char that are less than 10% of the critical size as defined
by
industry standard methods after their exposure to the Process I followed by
transfer
to seawater.
Figure 30 shows a comparison of survival of arctic char after various
treatments.
A single group of arctic char (3-5 gram were obtained from Pierce hatcheries
(Buxton, ME) and either maintained in freshwater or treated with the Process I
prior
to transfer- to seawater.
SECTION The Use of the Process If to Peanit Successful Transfer of 10-30 gram
Smolt into Seawater Netpens and Tanks.
The Process II protocol is utilized to treat pre-adult anadromous fish for
placement into seawater at an average size of 25-30 gram or less. This method
differs from the Process I protocol by the inclusion of L-tryptophan in the
diet of
pre-adult anadromous fish prior to their transfer to seawater. Process II
further
improves the osmoregulatory capabilities of pre-adult anadromous fish and
provides
for still further reductions in the "critical size" for Atlantic salmon smolt
transfers.
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In summary, Process II reduces the "critical size" for successful seawater
transfer to
less than one fifth the size of the present day industry standard SO smolt.
Application of Process II to Atlantic Salmon Fingerlings:
St John/St John strain pre-adult fingerlings derived from a January 2000 egg
hatching and possessing an average weight of 0.8 gram were purchased from
Atlantic Salmon of Maine Inc. Kennebec Hatchery, Kennebec Maine on 27 April
2000. These fish were transported to the treatment facility using standard
conventional truck transport. After their arrival, these parr were first grown
in
conventional flow through freshwater growout conditions that included a water
temperature of 9.6 C and a standard freshwater parr diet (Moore-Clark Feeds).
On
17 July 2000, fingerlings were begun on Process II for a total of 49 days
while being
exposed to a continuous photoperiod. Process II smolts were then vaccinated
with
the Lipogen Forte product (Aquahealth LTD.) on Day 28 (14 August 2000) of
Process II treatment. Process II smolts were size graded prior to initiating
Process II
as well as immediately prior to transfer to seawater. St John/St John Process
If
smolts were transported to ocean netpens by conventional truck transport and
placed
into seawater (15.2 C) in either a single ocean netpen identical to that
described for
placement of Process I smolts or into laboratory tanks (15.6 C) within the
research
facility.
Figure 31 shows representative St. John/St John strain Process II smolts
possessing a range of body sizes were transferred to seawater either in ocean
netpens
or corresponding laboratory seawater tanks. Note that these Process II smolts
possess a wide range of body weights (3.95-28 gram) that comprised an average
body weight of 11.5 gram. Figure 31 shows the characteristics of St. John/St
John
Process II smolts. The average measurements of these St. John/St. John Process
II
smolts included a body weight of 11.50+/-5.6 gram, length of 9.6+1-1.5 cm and
condition factor of 1.19+1-0.09. The data displayed in Table 10 shows the
outcomes
for two groups of Process II smolts derived from a single production pool of
fish
after their seawater transfer into either laboratory tanks or ocean netpens.
Although
important variables such as the water temperatures and transportation of fish
to the
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site of seawater transfer were identical, these 2 groups of Process II smolts
experienced differential post seawater transfer mortalities after 5 days into
laboratory
tanks (10% mortality) and ocean netpens (37.7% mortality).
The probable explanation for this discrepancy in mortalities between seawater
laboratory tanks (10% mortality) and ocean netpens (37.7% mortality) is
exposure of
these fish to different photoperiod regimens after seawater placement.
Exposure of
juvenile Atlantic salmon to a constant photoperiod after seawater placement
reduced
their post-seawater transfer mortality from approximately 34% to 6%. Fish
transferred to ocean netpens experienced natural photoperiod that was not
continuous and thus suffered an approximate 4-fold increase in mortality. As
shown
in Table 10, a separate seawater transfer of St John/St John juvenile Atlantic
salmon
possessing an average weight of 21 gins exhibited only 0.2% mortality after a
six
week treatment with Process II and underwater lights. These fish were exposed
to a
continuous photoperiod by underwater halogen lights for an interval of 30
days.
Table 10: Characterization and survival of St. John/St. John Process II fish
after their
placement into seawater in ocean netpens containing underwater lights.
Total Fish 15,000
Seawater Transfer Date 8/9/01
Water Temperature (oC) 12.6
Size at Transfer (gram) 21+/-4.5
Total Mortalities after 30 days (# and % total) 250 1.7%
% Mortalities weighing15 grams or greater 30 0.2%
Time to achieve feeding after transfer 48 hr
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Table 12: Characteristics and survival of St. John/St. John Process II fish
after their
placement into seawater in either a laboratory tank or ocean netpen.
Laboratory Tank Ocean Netpen
Total Fish 100 1,316
Seawater Transfer Date 8/31/00 9/5/00
Water Temperature ( C) 15.6 15.6
Size at Transfer (gram) 11.5 11.5
Total Mortalities after 5 10; 10% 496; 37.7%
days (# and % total)
% mortalities weighing 0; 0% 1; 0.08%
_ 13 grams or-greater
Time to achieve feeding 48 hrs 48 hrs
after transfer
No apparent problems were observed with the smaller (10-30 gram) Process II
smolts negotiating the conditions that exist within the confines of their
ocean
netpen. This included the lack of apparent problems including the ability to
school
freely as well as the ability to swim normally against the significant ocean
currents
that are continuously present in the commercial Blue Hill Bay salmon
aquaculture
site. While these observations are (-still ongoing, these data do not suggest
that the
placement and subsequent growth of Process II smolts in ocean netpens will be
comprised because of lack of ability of these pre-adult anadromous fish to
swim
against existing ocean currents and therefore be unable to feed or develop
properly.
Figure 32 compares characteristics of survivors and mortalities of Process II
smolts after seawater transfer to either laboratory tanks (Figure 32A) or
ocean
netpens (Figure 32B) . Figure 32A data are derived from analyses of 100
Process II
smolts transferred to seawater tank where all fish were killed and analyzed on
Day 5.
In contrast, Figure 32B displays only mortality data from ocean netpen. In
both
cases, only smaller Process II smolts experienced mortality. Note differences
in Y
axis scales of Figures 32A-B.
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Comparison of the average body size of those Process II smolts that survived
seawater transfer vs. those Process II smolts that died shows that
unsuccessful
Process II smolts possessed significantly smaller body weights as compared to
average body size of whole Process II smolt transfer group. Thus, the average
weight of mortalities in laboratory tank (5.10+/-2.2 gram) and ocean netpen
(6.46+1-
1.5 gram) are 44% and 56% respectively the value of the average body weight
possessed by the entire transfer cohort (11.5 gram). In contrast, the
mortalities of
Process 1I smolts with body weights greater than 13 gram is 0/100 in the
laboratory
tank and 1/1316 or 0.076% for ocean netpens. Together, these data demonstrate
that
Process II is able to redefine the "critical size" of Atlantic salmon smolts
from
70-100 gram to approximately 13 gram.
Quantitation of Feeding and Growth of Process I and II smolts after Seawater
Transfer:
Landcatch/St John Process I smolts were offered food beginning 48 hr after
their
seawater transfer to either laboratory tanks or ocean netpens. While these
Process I
smolts that were transferred to laboratory tanks began to feed after 48 hr,
those fish
transferred to ocean netpens were not observed to feed substantially until 7
days. To
validate these observations, the inventors performed direct visual inspection
of the
gut contents from a representative sample of 49 Process I smolts 4 days after
their
seawater transfer to laboratory tanks. A total of 21/49 or 42.9% possessed
food
within their gut contents at that time.
The St John/St John Process II smolts fed vigorously when first offered food
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.
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The growth rates of identical fish treated with either Process I or II within
laboratory seawater tanks has been quantified. As shown in Table 12, both
Atlantic
salmon treated with Process I or II grow rapidly during the initial interval
(21 days)
after transfer to seawater. In contrast to industry standard smolt weighing 70-
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.
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Table 12: Comparison of Growth Rates of Pre-adult Atlantic Salmon Exposed to
either Process I or Process II and Placed in Laboratory Tanks During Initial
Interval
After Seawater Transfer
Process I Process II
Number of Fish 140 437
Weight at Placement into 26.6 11.50
Seawater
Days in Seawater 22 21
Placement Weight 26.6* 13.15*
Corrected for Mortalities
_ Weight after Interval in 30.3 15.2
Seawater
Weight Gained in 3.75 2.05
Seawater
SGR (% body 0.60 0.68
weight/day)
FCR 1.27 2.04
* Weight gain corrected for selective mortalities amongst smaller fish (4/140
or
2.9%' Process I; 103/437 or 23.6% Process II)
EXAMPLE 8. EXPOSURE OF SALMON SMOLTS TO CA2+ AND MG2+
INCREASES EXPRESSION OF PVCR IN CERTAIN TISSUES.
In smolts that were exposed to 10 mM Ca2+ and 5.2 mM me+, the expression of
PVCR was found to increase in a manner similar to that in smolts that are
untreated,
but are transferred directly to seawater.
Tissues were taken from either Atlantic salmon or rainbow trout, after
anesthesitizing the animal with MS-222. Samples of tissues were then obtained
by
dissection, fixed by immersion in 3% parafomialdehyde, washing in Ringers then
frozen in an embedding compound, e.g., 0.C.T.Tm (Miles, Inc., Elkahart,
Indiana,
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USA) using methylbutane cooled on dry ice. After cutting 8 micron thick tissue
sections with a cryostat, individual sections were subjected to various
staining
protocols. Briefly, sections mounted on glass slides were: 1) blocked with
goat
serum or serum obtained from the same species of fish, 2) incubated with
rabbit
anti-CaR antiserum, and 3) washed and incubated with peroxidase-conjugated
affinity-purified goat antirabbit antiserum. The locations of the bound
peroxidase-conjugated goat anti-rabbit antiserum were visualized by
development of
a rose-colored aminoethylcarbazole reaction product. Individual sections were
mounted, viewed and photographed by standard light microscopy techniques. The
methods used to produce anti-PVCR antiserum are described below.
The results are shown in Figs. 33A - 33G, which are a set of seven
photomicrographs showing immunocytochemistry of epithelia of the proximal
intestine of Atlantic salmon smolts using anti-PVCR antiserum, and in Fig. 34,
-
which is a Western blot of intestine of a salmon smolt exposed to Ca2+- and
Mg2+-treated freshwater, then transferred to seawater. The antiserum was
prepared
by immunization of rabbits with a 16-mer peptide containing the protein
sequence
encoded by the carboxyl terminal domain of the dogfish shark PVCR ("SKCaR")
(Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A). Specific binding of
the
anti-PVCR antibody is indicated by aminoethylcarbazole (AEC) reaction product.
Figs. 33A and 33B show stained intestinal epithelia from smolts that were
maintained in freshwater then transferred to seawater and held for an interval
of 3 -
days. Abundant PVCR immunostaining is apparent in cells that line the luminal
surface of the intestine. The higher magnification (1440X) shown in Fig. 33B
displays PVCR protein localized to the apical (luminal-facing) membrane of
intestinal epithelial cells. The pattern of PVCR staining is localized to the
apical
membrane of epithelial cells (small arrowheads) as well as membranes in
globular
round cells (arrows). Fig. 33C shows stained intestinal epithelia from a
representative smolt that was exposed Process I and maintained in freshwater
containing 10 mM Ca2+ and 5.2 mM Mg2+ for 50 days. Note that the pattern of
PVCR staining resembles the pattern exhibited by epithelial cells displayed in
Figures 33A and 33B including apical membrane staining (small arrowheads) as
well as larger globular round cells (arrows). Fig. 33D shows a 1900X
magnification
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of PVCR-stained intestinal epithelia from another representative fish that was
exposed to the Process I and maintained in freshwater containing 10 mI\4 Ca2+
and
5.2 mM Mg2+ for 50 days and fed 1% NaC1 in the diet. Again, small arrowhead
and
arrows denote PVCR staining of the apical membrane and globular cells
respectively. In contrast to the prominent PVCR staining shown in Figures 33A-
D,
Figs. 33E (1440X) and 13F (1900X) show staining of intestinal epithelia from
two
representative smolt that were maintained in freshwater alone without
supplementation of Ca2+ and Mg2+ or dietary NaCl. Both 13E and 13F display a
marked lack of significant PVCR staining. Fig. 33G (1440X) shows the lack of
any
apparent PVCR staining upon the substitution of preimmune serum on a section
corresponding to that shown in Figure 33A where anti-PVCR antiserum identified
the PVCR protein. The lack of any PVCR staining with preinunune antiserum is a
control to demonstrate the specificity of the anti-PVCR antiserum under these-
imimmocytochemistry conditions.
The relative amount of PVCR protein present in intestinal epithelial cells of
freshwater smolts (Figs. 33E and 33F) was negligible as shown by the faint
staining
of selected intestinal epithelial cells. In contrast, the PVCR protein content
of the
corresponding intestinal epithelial cells was significantly increased upon the
transfer
of these smolts to seawater (Figs. 33A and 33B). Importantly, the PVCR protein
content was also significantly increased in the intestinal epithelial cells of
smolts
maintained in freshwater supplemented with Ca2+ and Mg2+ (Fig. 33C and 33D).
The AEC staining was specific for the presence of the anti-PVCk antiserum,
since
substitution of the immune antiserum by the preimmune eliminated all reaction
product from intestinal epithelial cell sections (Fig. 33G).
Disclosure of localization of PVCR protein(s) in additional areas of
osmoregulatory
organs of Atlantic Salmon using paraffin sections. Demonstration that PVCR
proteins are localized to both the apical and basolateral membranes of
intestinal
epithelial cells.
Using the methods described herein, immunolocalization data from paraffin
sections of various osmoregulatory organs of seawater-adapted juvenile
Atlantic
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salmon smolt were obtained. PVCR proteins, as determined by the binding of a
specific anti-PVCR antibody, were present in the following organs. These
organs
are important in various osmoregulatory functions. These organs include
specific
kidney tubules and urinary bladder responsible for processing of urine, and
selected
cells of the skin, nasal lamellae and gill each of which are bathed by the
water
surrounding the fish. The PVCR was also seen in various portions of the G.I.
tract
including stomach, pyloric caeca, proximal intestine and distal intestine that
process
seawater ingested by fish. These tissues were analyzed after treatment with
Processes I and II, and after their transfer from freshwater to seawater. In
addition, it
is believed that the PVCR protein can also act as a nutrient receptor for
various
amino acids that are reported to be present in stomach, proximal intestine,
pyloric
caeca.
In particular, higher magnification views of PVCR immunolocalizations in
selected cells of the stomach, proximal intestine and pyloric caeca were
obtained.
The PVCR protein is not only present on both the apical (luminally facing) and
basolateral (blood-facing) membranes of stomach epithelial cells localized at
the
base of the crypts of the stomach, but also is present in neuroendocrine cells
that are
located in the submucosal area of the stomach. From its location on
neuroendocrine
cells of the G.I. tract, the PVCR protein is able to sense the local
environment
immediately adjacent to intestinal epithelial cells and modulate the secretion
and
.synthesis of important G.I. tract hoiniones (e.g., 5-hydroxytryptamine (5-
HT),
serotonin, or colecystokinin (CCK)). Importantly, it is believed that the
constituents of Process II effect G.I. neuroendocrine cells by at least two
means. The
first way that constituents of Process II remodel the G.I endocrine system is
through
alterations in the expression and/or sensitivity of PVCRs expressed by these
cells.
The second way is to supply large quantities of precursor compounds, for
example,
tryptophan that is converted into 5-HT and serotonin by G.I. metabolic
enzymes.
In a similar manner, PVCR protein is localized to both the apical and
basolateral
membranes of epithelial cells lining the proximal intestine. From their
respective
locations, PVCR proteins can sense both the luminal and blood contents of
divalent
cations, NaC1 and specific amino acids and thereby integrate the multiple
nutrient
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and ion absorptive-secretory functions of the intestinal epithelial cells.
Epithelial
cells of pyloric caeca also possess abundant apical PVCR protein.
To further demonstrate the specificity of the anti-CaR antiserum to recognize
salmon smolt PVCRs, Fig. 34 shows a Western blot of intestinal protein from
salmon smolt maintained in 10 mM Ca2+, 5 mM Mg2+ and fed 1% NaC1 in the diet.
Portions of the proximal and distal intestine were homogenized and dissolved
in
SDS-containing buffer, subjected to SDS-PAGE using standard techniques,
transferred to nitrocellulose, and equal amounts of homogenate proteins as
determined by both protein assay (Pierce Chem. Co, Rocford, IL) as well as
Coomassie Blue staining were probed for presence of PVCR using standard
western
blotting techniques. The results are shown in the left lane, labeled "CaR",
and shows
a broad band of about 140-160 kDa and several higher molecular weight
complexes.
The pattern of PVCR bands is similar to that previously reported for
shark.kidney
(Nearing, J. et al., 1997, J. Am. Soc. Nephrol. 8:40A) and rat kidney inner
medullary
collecting duct (Sands, J.M. et al., 1997, J. Clin. Invest. 99:1399-1405). The
lane on
the right was treated with the preimmune anti-PVCR serum used in Fig. 33G, and
shows a complete lack of bands. Taken together with immunocytochemistry data
shown in Figure 33, this immtmoblot demonstrates that the antisenun used is
specific for detecting the PVCR protein in salmon.
EXAMPLE 9: IMMUNOLOCALIZATION OF POLYVALENT CATION
RECEPTOR (PVCR) IN MUCOUS CELLS OF EPIDERMIS OF SALMON.
The skin surface of salmonids is extremely important as a barrier to prevent
water gain or loss depending whether the fish is located in fresh or seawater.
Thus,
the presence of PVCR proteins in selected cells of the fish's epidermal layer
would
be able to "sense" the salinity of the surrounding water as it flowed past and
provide
for the opportunity for continuous remodeling of the salmonid's skin based on
the
composition of the water where it is located.
Methods: Samples of the skin from juvenile Atlantic Salmon resident in
seawater for over 12 days were fixed in 3% parafonnaldehyde dissolved in
buffer
(0.1M NaPO4, 0.15M NaC1, 0.3M sucrose pH 7.4), manually descaled, rinsed in
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buffer and frozen at -80 C for cryosectioning. Ten micron sections were either
utilized for immunolocalization of PVCR using anti-shark PVCR antiserum or
stained directly with 1% Alcian Blue dye to localize cells containing acidic
glycoprotein components of mucous.
Results and Discussion: Figure 35A shows that salmon epidennis contains
multiple Alcian Blue staining cells present in the various skin layers. Note
that only
a portion of some larger cells (that containing acidic mucins) stains with
Alcian Blue
(denoted by the open arrowheads). For purposes of orientation, note that
scales have
been removed so asterisks denote surface that was previously bathed in
seawater.
Figure 35B shows immunolocalization of salmon skin PVCR protein that is
localized to multiple cells (indicated by arrowheads) within the epidermal
layers of
the skin. Note that anti-PVCR staining shows the whole cell body, which is
larger
than its corresponding apical portion that stains with Alcian Blue as shown in
Figure
35A. The presence of bound anti-CaR antibody was indicated by the rose color
reaction product. Although formal quantitation has not yet been performed on
these
sections, it appears that the number of PVCR cells is less than the total
number of
Alcian Blue positive cells. These data indicate that only a subset of Alcian
Blue
positive cells contain abundant PVCR protein. Figure 35C shows the Control
Preimmune section where the primary anti-PVCR antiserum was omitted from the
staining reaction. Note the absence of rose colored reaction product in the
absence
of primary antibody.
These data demonstrate the presence of PVCR protein in discrete epithelial
cells
(probably mucocytes) localized in the epidermis of juvenile Atlantic salmon.
From
this location, the PVCR protein could "sense" the salinity of the surrounding
water
and modulate mucous production via changes in the secretion of mucous or
proliferation of mucous cells within the skin itself. The PVCR agonists (Ca2+,
Mg2+) present in the surrounding water activate these epidermal PVCR proteins
during the interval when smolts are being exposed to the process of the
present
invention. This treatment of Atlantic salmon smolts by the process of the
present
invention is important to increased survival of smolts after their transfer to
seawater.
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EXAMPLE 10: DEMONSTRATION OF THE USE OF SOLID PHASE ENZYME-
LINKED ASSAY FOR DETECTION OF PVCRS IN VARIOUS TISSUES OF
INDIVIDUAL ATLANTIC SALMON USING ANTI-PVCR POLYCLONAL
ANTISERUM.
The PVCR content of various tissues of fish can be quantified using an ELISA
96 well plate assay system. The data, described herein, demonstrate the
utility of a
96 well ELISA assay to quantify the tissue content of PVCR protein using a
rabbit
polyclonal anti-PVCR antibody utilized to perfouu immunocytochemistry and
western blotting. These data form the basis for development of commercial
assay
kits that would monitor the expression levels of PVCR proteins in various
tissues of
juvenile anadromous fish undergoing the processes of the present invention, as
described herein. The sensitivity of this ELISA is demonstrated by measurement
of
the relative PV-CR content of 14 tissues from a single juvenile Atlantic
salmon, as
shown in Figure 36.
Description of Experimental Protocol:
Homogenates were prepared by placing various tissues of juvenile Atlantic
salmon (St. John/St. John strain average weight 15-20 gm) into a buffer (10 mM
HEPES, 1.5 mM MgC12, 10 mM KC1, 1mM Phenylmethylsulfonyl fluoride (PMSF),
0.5 dithiothreitol (DTT) and linM benzamidine pH 8.8) and using a standard
glass
Potter-Elvenhiem homogenizer with a rotary pestle. After centrifugation at
2,550Xg
forr 20 min. at 4 C to remove larger debris, the supernatant was either used
directly or
frozen at -80 C until further use. Homogenate protein concentrations were
determined using the BCA assay kit (Pierce Chem. Co.). Aliquots of individual
tissue homogenates were diluted into a constant aliquot size of 100
microliters and
each was transferred to a 96 well plate (Costar Plastic Plates) and allowed to
dry in
room air for 15hr. After blocking of nonspecific binding with a solution of 5%
nonfat milk powder + 0.5% Tween 20 in TBS (25 mM Tris 137 mM sodium
chloride, 2.7 mM KC1 pH 8.0), primary antiserum (either rabbit anti-PVCR
immune
or corresponding rabbit pre-irnmune antiserum) at a 1:1500 dilution was added.
After a lhr incubation, individual wells were rinsed 3 times with 500
microliters of
TBS, an 1:3000 horseradish peroxidase conjugated goat anti-rabbit (Gibco-BRL )
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were added and allowed to incubate for 1 hr. Individual wells were then rinsed
and
bound complex of primary-secondary antibody detected with Sigma A3219 2,2'
Azino-bis(3-ethylbenzthiazidine-6-sulfonic acid) color reagent after 15 min of
incubation using a Molecular Devices 96 well plate reader (Molecular Devices,
VMAX) at 405nm. Relative amounts of tissue PVCR content were determined after
corrections for minimal background and nonspecific antibody binding as
measured
by binding of preimmune antiserum.
Results and Data Interpretation:
Figure 36 shows the data obtained from a representative single ELISA
determination of PVCR protein content of 14 tissues of a single juvenile
Atlantic
salmon. Under the conditions specified in the Experimental Protocol as
outlined
above, nonspecific binding of both primary and secondary antibodies were -
minimized. While these quantitative values are measured relative to each other
and
not in absolute amounts, they provide data that parallels extensive
immunocytochemistry examination of each of the tissues. Note that the PVCR
content of various organs reflects their importance in osmoregulation of
Atlantic
salmon. Immunocytochemistry data described herein shows that tissues such as
intestine (proximal and distal segments), gill, urinary bladder and kidney
contained
PVCR protein. In each case, epithelial cells that contact fluids that bathe
the
surfaces of these tissues express PVCR. In contrast, other organs-including
liver, ,
heart and muscle contain minimal PVCR protein. Note that the highest PVCR
content of any tissue tested is the olfactory lamellae where salmon possess
the ability
to "smell" alterations in calcium concentration in water. The olfactory bulb
containing neurons that innervate the olfactory lamellae also possess abundant
PVCR. Taken together, these data demonstrate the utility of ELISA kits to
measure
tissue content of PVCR proteins and form the basis for development of
commercial
assay kits that would monitor the expression levels of PVCR proteins in
various
tissues of juvenile anadromous fish undergoing the processes of the present
invention. Alterations in PVCR tissue content measured in either relative
changes in
tissue PVCR content or absolute quantity of PVCR per tissue mass could, in
turn, be
utilized as correlative assays to determine the readiness ofjuvenile
anadromous fish
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for sea water transfer or initiation of feeding. These data demonstrate the
ability to
perform such assays on individual juvenile Atlantic salmon in the range of
body
sizes that would be utilized to transfer fish from fresh to seawater after
treatment
with the methods of the present invention.
EXAMPLE 11: ANTIBODMS MADE FROM THE CARBOXYL TERMINAL
PORTION OF AN ATLANTIC SALMON PVCR PROTEIN ARE EFFECTIVE IN
IMMLTNOCYTOCHEMISTRY AND IIVIMUNOBLOTTING ASSAYS TO
DETERMINE THE PRESENCE, ABSENCE OR AMOUNT OF THE PVCR
PROTEIN
Degenerate primers, dSK-F3 (SEQ ID NO: 15) and dSK-R4 (SEQ ID NO: 16),
described herein were constructed specifically from the SKCaR DNA sequence.
These primers have proved to be useful reagents for amplification of portions
of
PVCR sequences from both genomic DNA as well as cDNA.
To obtain more cDNA sequence from anadromous fish PVCRs, in particular the
putative amino acid sequence of the carboxyl terminal domain of PVCRs that are
targets for generation of specific peptides and, as a result, specific anti-
Atlantic
Salmon PVCR antisera, an unamplified cDNA library from Atlantic salmon
intestine
was constructed. Phage plaques originating from this cDNA library were
screened
under high stringency usineP-labeled 653 bp genomic Atlantic Salmon PCR
product. From-this cDNA library screening effort, a 2,021 bp cDNA clone was
isolated and contained a single open reading fi-aine for a putative amino acid
sequence corresponding to approximately one half of a complete cDNA sequence
from an intestinal PVCR protein. This putative amino acid sequence corresponds
exactly to the sequence encoded by the corresponding genomic probe as well as
the
putative amino acid sequence corresponding to the carboxyl terminal domain of
the
PVCR.
On the basis of the knowledge of this putative amino acid sequence, a peptide,
shown below, was synthesized and corresponded to a separate region of the
putative
carboxyl terminal PVCR amino acid sequence:
The peptide sequence for antibody production is as follows:
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Peptide #1: Ac-CTNDNDSPSGQQRIHK-amide (SEQ ID NO.:17)
producing rabbit antiserum SAL-1
The peptide was derivatized to carrier proteins and utilized to raise peptide
specific antiserum in two rabbits using methods for making a polyclonal
antibody.
The resulting peptide specific antiserum was then tested using both
immunoblotting and immunocytochemistry techniques to determine whether the
antibody bound to protein bands corresponding to PVCR proteins or yielded
staining
patterns similar to those produced using other anti-PVCR antiserum. A
photograph
of an immun.oblot was taken showing protein bands that were recognized by
antisera
raised against peptides containing either SAL-1 (SEQ JD NO.: 17) or SKCaR (SEQ
ID NO:2). As expected, antiserum raised to the peptide identified protein
bands that
co-electrophorese with PVCR proteins that are recognized by antisera raised to
SKCaR (SEQ ID NO:2). Immunostaining of juvenile Atlantic salmon kidney
sections with 3 different anti-PVCR antisera (anti-Sail, anti-4641, and anti-
SKCaR)
produces similar localizations of PVCR protein within the tubules of salmon
kidney.
Staining produced by anti-SKCaR antiserum is identical to that produced by
anti-4641 antiserum, an anti-peptide antisera corresponding to extracellular
domain
_
of mammalian PVCRs that is very similar to SKCaR (SEQ ID NO: 2). These PVCR
protein patterns stained identically to that produced by SAL-1 antiserum. Anti-
Sal-1
antisenun also exhibits a similar staining pattern for the distribution of
intestinal
PVCR protein, as compared to anti-SKCaR. Thus, this new antiserum is specific
for
a PVCR in Atlantic Salmon tissues. This antiserum can be used to determine the
presence, absence or amount of PVCR in various tissues of fish, using the
methods
described herein.
The Sal I antiserum is also useful in localization of SalmoKCaR proteins in
larval Atlantic salmon (See Figure 40B). The Sal I antiserum localizes
SalmoKCaR
proteins in the developing nasal lamellae of anadromous fish, including
Atlantic
salmon and trout, skin, myosepta, otolith and sensory epithelium. The
myoseptae
are collagenous sheets that separate the various muscle bundles in the fish.
Myosepta are important in both the development of muscle in larval fish as
well as
its function for muscle force generation in adult fish. Myosepta are also of
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significant commercial importance since they are one of the principal
determinants
of texture of smoked Atlantic salmon fillets.
The otolith is also of considerable importance to Atlantic salmon. It is a
calcified structure located in the inner ear of salmon where it is closely
associated
with epithelial cells responsible for sensing sound and direction. It is
likely that the
SalmoKCaRs associated with the otolith participate in the calcification of the
otolith
structure that consists of proteins and calcium precipitate.
A second peptide sequence was used for antibody production:
Peptide #2: CSDDEYGRPGTFKFEKEM (SEQ ID NO: 29).
This peptide was synthesized, derivatized in a manner identical to that
described
for Peptide #1 and antiserum was raised in rabbits as described above. As
expected,
this antiserum (Salmo ADD) produced a pattern of immonostaining on sections of
juvenile Atlantic salmon that is identical to that exhibited by Sal L (See
Figure
40C). Since both SalmoKCaR #1, #2 and #4, but not SalmoKCaR #3 possess the
carboxyl terminal sequence recognized by the Sal I antibody, the antibody-
staining
pattern displayed by Sal I show the distribution of SalmoKCaR proteins #1, #2
and
#4 but not #3 within the kidney of Atlantic salmon.
In contrast, the Salmo ADD antibody binds to a peptide sequence present in the
extracellular domain of all 4 SalmoKCaR proteins. Thus, any cells that possess
no
staining of Sal I but staining with Salmo ADD likely express either SalmoKCaR
#3
or some similar SalmoKCaR protein.
EXAMPLE 12: USE OF REVERSE TRANSCRIPTASE POLYMERASE CHAIN
REACTION (RT-PCR) TO DETECT EXPRESSION OF PVCRS IN VARIOUS
TISSUES
In Example 4, 2 degenerate primers, dSK-F3 (SEQ ID NO: 15) and dSK-R4
(SEQ ID NO: 16), are disclosed. These two primers were used to amplify genomic
DNA and obtain the sequence of a portion of the genomic DNA sequences of
PVCRs from various anadromous fish. These same primers can also be used to
amplify a portion of corresponding PVCR mRNA transcripts in various tissues.
DNA sequence analyses of amplified cDNAs from specific Atlantic salmon tissues
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(olfactory lamellae, kidney, urinary bladder) verifies these are all identical
to certain
genomic PVCR sequences described herein. These data show that:
1. PVCR mRNA transcripts are actually expressed in specific tissues of
anadromous fish. These data reinforce the data regarding PVCR protein
expression as detected by anti-PVCR antisera.
2. RT-PCR methods can be used to detect and quantify the degree of
PVCR expression in various tissues, as a means to predict the readiness of
anadromous fish for transfer to seawater.
3. cDNA probes can be generated from specific tissues of anadromous
-fish for use as- specific DNA probes to either detect PVCR expression using
solution or solid phase DNA-DNA or DNA-RNA nucleic acid hybridization or
obtain putative PVCR protein sequences used for generation of specific
anti-PVCR antisera.
RT-PCR Method:
Total RNA was purified from selected tissues using Teltest B reagent
(Friendswood, TX) and accompanying standard protocol. A total of 5 micrograms
of total-RNA was reversc transcribed with oligo dT primers using Invitrogen's
cDNA Cycle Kit (Invitrogen Inc, Madisbn, WI). The resulting cDNA product was
denatured and a second round of purification was perfouned. Two microliters of
the
resulting reaction mixture was amplified in a PCR reaction (30 cycles of 1
min. @
94 C, 2 min. @ 57 C, 3 min. @72 C) using degenerate primers dSK-F3 (SEQ ED
NO: 15) and dSK-R4 (SEQ ID NO: 16). The resulting products were
electrophoresed on a 2% (w/v) agarose gel using TAE buffer containing ethidium
bromide for detection of amplified cDNA products. Gels were photographed using
standard laboratory methods.
DNA sequencing of RT-PCR products were performed as follows:
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A total of 15 microliters of Atlantic Salmon urinary bladder, kidney and nasal
lamellae RT-PCR reactions were diluted in 40 microliters of water and purified
by
size exclusion on Amersham's MicroSpin S-400 FIR spin columns (Amersham Inc,
Piscataway, NJ). Purified DNA was sequenced using degenerate PVCR primers
(SEQ ID NO.: 15 and 16) as sequencing primers. Automated sequencing was
performed using an Applied Biosystems Inc. Model 373A Automated DNA
Sequencer (University of Maine, Orono, Maine). The resulting DNA sequences
were aligned using MacVector (GCG) and LaserGene (DNA STAR) sequence
analysis software.
Detection of Amplified RT-PCR cDNA products by Southern Blotting:
Alternatively, the presence of amplified PVCR products was detected by
Southern blotting analyses of gel fractionated RT-PCR products using a 'P.-
labeled
653 bp Atlantic salmon amplified genomic PCR product. A total of 10
microliters
of each PCR reaction was electrophoresed on a 2% agarose gel using TAE buffer
then blotted onto Magnagraph membrane (Osmonics, Westboro, MA). After
crosslinking of the DNA, blots were prehybridized and then probed overnight
(68 C
_
in 6X SSC, 5X Denhardt's Reagent, 0.5% SDS, 10Oug/m1 calf thymus DNA) with
the 653 bp Atlantic salmon PCR product (labeled with RadPrime DNA Labeling
System, Gibco Life Sciences). Blots were then washed with 0.1x SSC, 0.1% SDS
55 C and subjected to autoradiography under standard conditions.
Figure 37 shows the results of RT-PCR amplification of a partial PVCR mRNA
transcript from various tissues of juvenile Atlantic salmon. RT-PCR reactions
were
separated by gel electrophoresis and either stained in ethidium bromide(EtBr)
or
transferred to a membrane and Southern blotted using a 32P-labeled 653 bp
genomic
DNA fragment from the Atlantic salmon PVCR gene. Figure 37 shows the detection
of the PVCR in several tissue types of Atlantic Salmon using the RT-PCR
method,
as described herein. The types of tissue are gill, nasal lamellae, urinary
bladder,
kidney, intestine, stomach, liver, and brain.
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EXAMPLE 13: PRESENCE AND FUNCTION OF PVCR PROTEIN IN NASAL
LAMELLAE AND OLFACTORY BULB AS WELL AS GI TRACT OF FISH.
The data described herein described the roles of PVCR proteins in the
olfactory
organs (nasal lamellae and olfactory bulb) of fish as it relates to the
ability of fish to
sense or "smell" both alterations in the water salinity and/or ionic
composition as
well as specific amino acids. These data are particularly applicable to
anadromous
fish (salmon, trout and char) that are either transferred from freshwater
directly to
seawater or exposed to Process I or Process II in freshwater and then
transferred to
seawater.
These data described herein were derived from a combination of sources
including immunocytochemistry using anti-PVCR antisera, RT-PCR amplification
of PVCRs from nasal lamellae tissue, studies of the function of recombinant
aquatic
PVCR proteins expressed in cultured cells-where these proteins "sense"
specific ions
or amino acids as well as electrophysiological recordings of nerve cell
electrical
activity from olfactory nerves or bulb of freshwater salmon.
The combination of immunocytochemistry and RT-PCR data, described herein,
reveal the presence of PVCR proteins in both major families of fish
(elasmobranch-
shark; teleost-salmon) in both larval, juvenile and adult life stages.
Immunocytochemistry analyses reveal that one or more PVCR proteins are present
both on portions of olfactory receptor cells located in the nasal lamellae of
fish
(where they are bathed in water from the surrounding environmpt) as well as on
nerve cells that compose olfactory glomeruli present in the olfactory bulb of
fish
brain (where these cells are exposed to the internal ionic environment of the
fish's
body). Thus, from these locations fish are able to compare the ionic
composition of
the surrounding water with reference to their own internal ionic composition.
Alterations in the expression and/or sensitivity of PVCR proteins provides the
means
to enable fish to determine on a continuous basis whether the water
composition
they encounter is different from that they have been adapted to or exposed to
previously. This system is likely to be integral to both the control of the
homing of
salmon from freshwater to seawater as smolt and their return to freshwater
from
seawater as adults. Thus, fish have the ability to "smell" changes in water
salinity
directly via PVCR proteins and respond appropriately to regulate remain in
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environments that are best for their survival in nature.
One feature of this biological system is alteration in the sensitivity of the
PVCR protein for divalent cations such as Ca 2+ and Mg2+ by changes in the
NaC1
concentration of the water. Thus, PVCRs in fish olfactory organs have
different
apparent sensitivity to Ca2+ in either the presence or absence of NaCl. These
data
presented here are the first direct evidence for these functions via PVCR
proteins
present in the olfactory apparatus of fish.
Another feature of PVCR protein function in the olfactory apparatus of fish
is to modulate responses of olfactory cells to specific odorants (attractants
or
repellants). Transduction of cellular signals resulting from the binding of
specific
odorants to olfactory cells occurs via changes in standing ionic gradients
across the
plasma membranes of these cells. The binding of specific odorants to olfactory
cells
results in-electrical nerve conduction signals_that can be recorded using
standardized
electrophysiological electrodes and equipment. Using this apparatus, the
olfactory
apparatus of freshwater adapted salmon:
1. responded to PVCR agonists in a concentration-dependent manner
similar to that shown previously for other fish tissues including that
shown for winter flounder urinary bladder. These data provide the
functional evidence of the presence of a PVCR protein; and
2. that the presence of a PVCR agonist reduces or ablates the signal
resulting from odorants including both attractants or repellants. Thus,
PVCRs in the orfactory apparatus of salmon possess the capacity of
modulating responses to various odorants.
Another feature of PVCR proteins is their ability to "sense" specific amino
acids present in surrounding environment. Using the full-length recombinant
SKCaR cDNA, functional SKCaR protein was expressed in HEK cells and shown to
respond in a concentration-dependent manner to both single and mixtures of L-
amino acids. Since PVCR agonists including amino acids as well as polyamines
(putrescine, spermine and spemndine) are attractants to marine organisms
including
fish and crustaceans, these data provide for another means by which PVCR
proteins
would serve not only as modulators of olfaction in fish but also as sensors of
amino
acids and polyamines themselves. PVCR proteins in other organs of fish
including
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G.I. tract and endocrine organs of fish also function to sense specific
concentrations
of amino acids providing for integration of a wide variety of cellular
processes in
epithelial cells (amino acid transport, growth, ion transport, motility and
growth)
with digestion and utilization of nutrients in fish.
Description of Experimental Results and Data Interpretation:
PVCR protein and mRNA are localized to the olfactory lamellae, olfactory
nerve and olfactory bulb of freshwater adapted larval, juvenile and adult
Atlantic
salmon as well as the olfactory lamellae of dogfish shark:
Figure 38 show representative immunocytochemistry photographs of PVCR
protein localization in olfactory bulb and nerve as well as olfactory lamellae
in
juvenile Atlantic salmon. The specificity of staining for PVCR protein is
verified by
the use of 2 distinct antisera each directed to a different region of the PVCR
protein. _
Thus, antiserum anti-4641 (recognizing an extracellular domain PVCR region)
and
antiserum anti-SKCaR (recognizing an intracellular domain PVCR region) exhibit
similar staining patterns that include various glomeruli on serial sections of
olfactory
bulb. Using anti-SKCaR antiserum, specific staining of PVCR proteins is
observed
in discrete regions of the olfactory nerve as well as epithelial cells in the
nasal
lamellae that are exposed to the external ionic environment.
The presence of PVCR protein in both nasal lamellae cells as well as
olfactory bulb and nerve shows that these respective PVCR proteins would be
able
to sense both the internal and external ionic environments of the salmon. For
this
purpose, cells containing internally-exposed PVCRs are connected to externally-
exposed PVCRs via electrical connections within the nervous system. As shown
schematically in Figure 39, these data suggest that externally and internally-
exposed
PVCRs function together to provide for the ability to sense the ionic
concentrations
of the surrounding ionic environment using as a reference the ionic
concentration of
the salmon's body fluids. Changes in the expression and/or sensitivity of the
external set of PVCRs vs internal PVCRs would then provide a long term
"memory"
of the adaptational state of the fish as it travels through ionic environments
of
different composition. Figure 40 shows immunocytochemistry using anti-SKCaR
antiserum that reveals the presence of PVCR protein in both the developing
nasal
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lamellae cells and olfactory bulb of larval Atlantic salmon only days after
hatching
(yolk sac stage). As described herein, imprinting of salmon early in
development as
well as during smoltification have been shown to be key intervals in the
successful
return of wild salmon to their natal stream. The Sal I antiserum also
localizes
SalmoKCaR proteins in a variety of tissues in larval Atlantic salmon (Figure
40B).
These tissues include the developing nasal lamellae of salmon and trout, their
skin,
myosepta, otolith and sensory epithelium. Myosepta are important in both the
development of muscle in larval fish since they separate and define the muscle
bundles of the salmon. Myosepta are also of significant commercial importance
since they are one of the principal determinants of texture for smoked
Atlantic
salmon fillets. Sah-nol(CaR proteins are also present in the otolith which is
a
calcified structure located in the inner ear of the salmon where it is closely
associated with epithelial cells responsible for sensing sound and direction.
The
presence of PVCR proteins at these developmental stages of salmon lifecycle
indicate that PVCRs participate in this process.
Data obtained from using anti-SKCaR antiserum from other fish species
including elasmobranchs display similar staining of PVCR protein in cells
(marked
A) their nasal lamellae (Figure 41). Use of other methodology including RT-PCR
_
using specific degenerate primers (Figure 42) and ELISA methods (Figure 43)
detects the presence of PVCR proteins and mRNA in nasal lamellae of fish.
While
neither of these 2 techniques provide quantitative measurements as described,
both
sets of data are consistent and show abundant PVCR protein present in this
tissue.
Measurement of extracellular electrical potentials (EEG's) from olfactory
nerve
from freshwater adapted Atlantic salmon reveals the presence of functional
PVCR
proteins:
Figure 44 displays representative recordings obtained from 6 freshwater
adapted juvenile Atlantic salmon (approximately 300-400gm) using methods
similar
to those described in Bodznick, D. J Calcium ion: an odorant for natural water
discriminations and the migratory behavior of sockeye salmon, Comp. Physiol. A
127:157-166 (1975), and Hubbard, PC, et al., Olfactory sensitivity to changes
in
environmental Ca2+ in the marine teleost Sparus Aurata, Exp. Biol. 203:3821-
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3829 (2000). After anaesthetizing the fish, it was placed in V-clamp apparatus
where its gills were irrigated continuously with aerated seawater and its
nasal
lamellae bathed continuously by a stream of distilled water via a tube held in
position in the inhalant olfactory opening. The olfactory nerves of the fish
were
exposed by removal of overlying bony structures. Stimuli were delivered as
boluses
to the olfactory epithelium via a 3 way valve where 1 cc of water containing
the
stimulus was rapidly injected into the tube containing a continuously stream
of
distilled water. Extracellular recordings were obtained using high resistance
tungsten electrodes where the resultant amplified analog signals (Grass
Amplifier
Apparatus) were digitized, displayed and analyzed by computer using MacScope
software. Using this experimental approach, stable and reproducible recordings
could be obtained for up to 6 hr after the initial surgery on the fish.
As shown in Figure 44, irrigation of salmon olfactory epithelium with
distilled water produces minimal generation of large signals in olfactory
nerve. The
data in Figure 44 are displayed as both raw recordings (left column) and the
corresponding integrated signals for each raw recording shown in the right
column.
Exposure of the olfactory epithelium to 500 micromolar L-alanine ( a well
known
amino acid attractant for fish) produces large increases in both the firing
frequency
and amplitude in the olfactory nerve lasting approximately 2 seconds in
duration.
Similarly, application of either 1mM Ca" or 250 inM NaC1 also produce
responses
in EEG activity. To test for the presence of furictional PVCR protein, the
olfactory
epithelium was exposed to 50 micromolar gadolinium (Ge-a PVCR agonist) and
also obtained a response. Figure 45 shows dose response data from multiple
fish to
various PVCR agonists or modulators where the relative magnitudes of
individual
olfactory nerve response were noimalized relative to the response produced by
the
exposure of the olfactory epithelium to 10 mM Ca'. As shown in Figure 45, the
olfactory epithelium of freshwater adapted juvenile salmon is very sensitive
to Ca'
where the half maximal excitatory response (EC50) is approximately 1-10
micromolar. Similarly, exposure of olfactory epithelium to the PVCR agonist
Geproduces responses of a similar magnitude to those evoked by Ca" in a
concentration range of 1-10 micromolar. In contrast, olfactory epithelium
responses
to Mg" do not occur until 10-100 micromolar solutions are applied. These dose
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response curves (EC50 Ge<Ca2<Mg2+) are similar to those obtained for PVCR
modulated responses in other fish epithelium (flounder urinary bladder NaC1-
mediated water transport-see SKCaR application).
In contrast, analysis of the olfactory epithelium responses to NaC1 exposure
shows that it is unresponsive until a concentration of 250 millimolar NaC1 is
applied.
Since NaCl does not directly activate PVCRs in a manner such as Gd' Ca' or Mg'
but rather reduces the sensitivity of PVCRs to these agonists, these data are
also
consistent with the presence of an olfactory epithelium PVCR. The response
evoked
by exposure of the epithelium to significant concentrations of NaC1 likely
occurs via
other PVCR independent mechanisms.
These data suggest that PVCR proteins present in olfactory epithelium are
capable of sensing and generating corresponding olfactory nerve signals in
response
to PVCR agonists at appropriate concentrations in distilled water.
Addition of PVCR agonists such as Ca2+ or Gd3+ to distilled water containing
well
known salmon repellants reversibly ablates the response of the olfactory
epithelium
to these stimuli:
Figure 46 shows representative data obtained from a single continuous
_
recording where the olfactory epithelium was first exposed to a well-known
repellant, mammalian finger rinse. Finger rinse is obtained by simply rinsing
human
fingers of adherent oils and fatty acids using distilled water and has been
shown
previously to be a powerful repellant stimulus both in EEG recordings as well
as
behavioral avoidance assays (Royce-Malmgren and W.H Watson J. Chem. Ecology
13:533-546 (1987)). Note however that inclusion of the PVCR agonists 5mM Ca'
or 50 micromolar Gd3+ reversibly ablated the response by the olfactory
epithelium to
mammalian finger rinse. These data show that PVCR agonists modulated the
response of the olfactory epithelium to an odorant such as mam_malian finger
rinse.
The ablation of responses to both the PVCR agonists as shown in Figure 45 as
well
as mammalian finger rinse indicate that there are some complex interactions
between PVCR proteins and other odorant receptors. It is also extremely
unlikely
that inclusion of PVCR agonists removed all the stimulatory components of
mammalian finger rinse from solution such that they were not able to stimulate
the
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epithelium.
Addition of PVCR agonists such as Ca2+ or Gd3+ but not NaC1 to distilled
water containing the well known salmon attractant L-alanine reversibly ablates
the
response of the olfactory epithelium to these stimuli:
Figure 47 shows a time series of stimuli (2 mm between each stimulus in a
single fish) similar to that displayed on Figure 47 except that 500 micromolar
L-
Alanine (a salmon attractant) was used to produce a signal in the olfactory
nerve.
Note that the addition of either 5 inM Ca2+ (recording #2) or 50 micromolar
(recording #7) to 500 micromolar L-alanine resulted in the complete loss of
the
corresponding response from the olfactory nerve after injection of this
mixture. In
both cases, this was not due to a permanent alteration of the olfactory
epithelium by
either of these PVCR agonists because a subsequent identical stimulus without
the
PVCR agonist (recordings #3 and #8) caused a return of the signal. It is
noteworthy
that in the case of Ge addition, the magnitude of the subsequent L-alanine
signal
was decreased as compared to control (compare recordings #6 vs #8) indicating
that
the olfactory epithelium prefers an interval of recovery from its exposure to
this
potent PVCR agonist. However, the alteration of response to the L-Alanine
stimulus
is not permanent or nonspecific since combining the same dose of L-Alanine
with
_
250 mM NaC1 resulted initially in a similar response (recordings #4 and #9)
followed by an enhanced response to L-Alanine alone (recordings #5 and #10).
In summary, the data displayed in Flores 46 and 47 show that inclusion of a
PVCR agonist in solutions containing either a repellant (finger rinse) or
attractant
(L-alanine) causes a dramatic reduction in the response of the olfactory
epithelium to
those odorants. For both repellants and attractants, some form of complex
interactions occur within olfactory epithelial cells since mixing of PVCR
agonists
and odorants renders the epithelia temporary unresponsive to either stimulus.
While
the nature of such interactions are not known at the present time, such
interactions
do not occur at the level of the PVCR molecule itself as shown by data from
experiments using recombinant PVCR protein SKCaR. As further described herein,
inclusion of amino acids in the presence of Ca' enhances the response of SKCaR
to
ambient Ca"- concentrations. Regardless of their nature, these negative
modulatory
effects of PVCR agonists including Ca' is likely to produce major effects on
how
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freshwater salmon smell objects in their environment after transfer from a low
calcium to a high calcium environment. Use of this assay system would permit
the
identification and analyses of both specific classes of PVCR agonists and
antagonists
as well as the specific effects of each PVCR modulator on specific odorants
including both repellants and attractants.
Recombinant PVCR protein SKCaR possesses the capability to sense
concentrations
of amino acids after its expression in human embryonic kidney (HEK) cells:
Full length recombinant dogfish (Squalus acanthias) shark kidney calcium
receptor (SKCaR) was expressed in human embryonic kidney cells using methods
described herein. The ability of SKCaR to respond to individual amino acids as
well
as various mixtures was quantified using FURA-2 ratio imaging fluorescence.
Figure 48 shows a comparison of fluorescence tracings of FURA2-loaded
cells stably expressing SKCaR that were bathed in physiological saline (125 mM
NaC1, 4rnM KC1, 0.5 mM CaC12, 0.5 MgC12, 20 mM HEPES (NaOH), 0.1% D-
glucose pH 7.4) in the presence or absence of 10 mM L-Isoleucine (L-Ile)
before
being placed into the fluorimeter. Baseline extracellular Ca' concentration
was 0.5
mM. Aliquots of Ca' were added to produce final extracellular concentrations
of
2.5 mM, 5mM, 7.5mM, 10mM and 20 mM Ca' with changes in the fluorescence
recorded. Note that increases in cell fluorescence were greater in the
presence of
= 20 10mM Phe for extracellular Ca" concentrations less than 10 mM.
Figure 49 shows data plotted from multiple experiments as described in
Figure 48 where the effects of 10 in_M Phe, 10mM Ile or an amino acid mixture
(AA
Mixture) containing all L-isomers in the following concentrations in
micromoles/liter: 50 Phe, 50 Trp, 80 His, 60 Tyr, 30 Cys, 300 Ala, 200 Thr, 50
Asn,
600 Gin, 125 Ser, 30 Glu, 250 Gly, 180 Pro, 250 Val, 30 Met, 10 Asp, 200 Lys,
100
Arg, 75 Ile, 150 Leu. Note that both 10mM Phe and 10 mM Ile as well as the
mixture of amino acids increase SKCaR's response to a given Ca' concentration.
Thus, these data show that presence of amino acids either alone or in
combination
increase the apparent sensitivity to Ca'pennitting SKCaR to "sense"amino acids
in
the presence of physiological concentrations of Ca". These data obtained for
SKCaR are comparable to those obtained for the human CaR.
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The significance of these data for aquatic organisms stand in marked contrast
to the roles of human CaRs amino acid sensing capabilities. Figure 48 shows
that
SKCaR's maximal capability to sense amino acids is confined to a range of
Ca'that
is present both in aquatic external environments as well as the body fluids of
various
fish. The following physiological processes occur: 1) Sensing of amino acids
in the
proximal intestine and pyloric caeca of fish: The PVCR present on the apical
surface of intestinal epithelial cells is capable of responding to amino acids
such as
tryptophan as part of the Process II. Inclusion of tryptophan in the feed of
fish
interacts with the intestinal PVCR to improve the development of juvenile
anadromous fish to tolerate seawater transfer. 2) In both adult, juvenile and
larval
fish, PVCR localized to the apical membrane of stomach and intestinal
epithelial
cells could "sense" the presence of amino acids produced by the proteolysis of
proteins into amino acids. This mechanism could be used to infoun both
epithelial
and neuroendocrine cells of the intestine of the presence of nutrients
(proteins) and
trigger a multitude of responses including growth and differentiation of
intestinal
epithelia as well as their accompanying transport proteins, secretion or
reabsorption
of ions such as gastric acid. The apical PVCR also regulates the secretion of
intestinal hormones such as cholecystokin (CCK) and others. 3) PVCR proteins
_
present in cells of the nasal lamellae of fish "smell" both water salinity
(via Ca',
Mg' and NaC1) and amino acids which is an example of an attractant. At the
present time, it is unclear whether the amino acid sensing capabilities of
PVCRs are
utilized by the olfactory epithelium to enable fish to smell various amino
acid
attractants.
These data show that PVCR sensing of amino acids occurs in a range of
extracellular calcium that is present in various concentrations of seawater
present in
estuaries and fish migration routes as well as various compartments of a
fish's body
including serum and body cavities including intestine, pyloric caeca and
kidney
where transepithelial amino acid absorption occurs. These data constitute the
first
report showing the amino acid sensitivity of a PVCR in fish.
Companion Patent Application No. 10/125,778, filed April 18, 2002; U.S.
Application No. 10/125,772, filed April 18, 2002; and U.S. Application No.
10/125,792, filed April 18, 2002; U.S. Application No. 10/121,441, filed April
11,
CA 02481827 2012-01-19
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2002 all entitled "Polyvalent Cation-sensing Receptor in Atlantic Salmon;"
Patent
No. 6,463,882, issued on October 15, 2002, entitled "Growing Marine Fish in
Fresh
Water;" PCT Application No.: PCT/US01/31625, entitled "Growing Marine Fish in
Fresh Water," filed October 11, 2001; Patent No. 6,463,883, issued October 15,
20002, entitled "Methods for Raising Pre-adult Anadromous Fish;" Patent Na.
6,481,379, issued November 19, 2002, entitled "Methods for Raising Pre-adult
Anadromous Fish;" Patent No. 6,475,792, issued November 5, 2002, entitled
"Methods for Raising Pre-Adult Anadromous Fish;" Patent Application No.
09/975,
553, entitled "Methods for Raising Pre-adult Anadromous Fish," filed on
October
11, 2001; International PCT Application No. PCT/US01/31562, entitled, "Methods
for Raising Pre-adult Anadromous Fish," filed on October 11, 2001; Provisional
Patent Application No. 60/382, 464, "Methods for Growing and Imprinting Fish
Using an Odorant," filed October 11, 2001; and Patent Application No.
10/268,051,
"Methods for Growing and Imprinting Fish Using an Odorant," filed
October 8, 2002.
Additionally, Patent No 6, 334, 391, issued on January 8, 2002, International
PCT application No. PCT/US97/05031, filed on March 27, 1997, and Application
No. 08/622,738 filed March 27, 1996, all entitled, " Polycation Sensing
Receptor in
Aquatic Species and Methods of Use Thereof'. Additionally, EPO Application
No.: 97937017.8, filed July 24, 1997, which describes the SkCaR receptor.
CA 02481827 2005-10-06
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SEQUENCE LISTING
<110> MariCal, Inc.
<120> Polyvalent Cation-Sensing Receptor in Atlantic Salmon
<130> 08901536CA
<140> 2,481,827
<141> 2003-04-09
<150> 10/125,778
<151> 2002-04-18
<150> 10/125,772
<151> 2002-04-18
<150> 10/125,792
<151> 2002-04-18
<150> 10/121,441
<151> 2002-04-11
<150> 60/240,392
<151> 2000-10-12
<150> 60/240,003
<151> 2000-10-12
<160> 40
<170> PatentIn version 3.2
<210> 1
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<212> DNA
<213> Squalus acanthias
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!
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ggcggcagca aggagcgccc cacaactatg gaggaaacct aatccaactc ctccatcaac 3540
cccaagaaca tcctccacgg cagcaccgtc gacaactgac atcaactcct aaccggtggc 3600
tgcccaacct ctcccctctc cggcactttg cgttttgctg aagattgcag catctgcagt 3660
tccttttatc cctgattttc tgacttggat atttactagt gtgcgatgga atatcacaac 3720
ataatgagtt gcacaattag gtgagcagag ttgtgtcaaa gtatctgaac tatctgaagt 3780
atctgaacta ctttattctc tcgaattgta ttacaaacat ttgaagtatt tttagtgaca 3840
ttatgttcta acattgtcaa gataatttgt tacaacatat aaggtaccac ctgaagcagt 3900
gactgagatt gccactgtga tgacagaact gttttataac atttatcatt gaaacctgga 3960
ttgcaacagg aatataatga ctgtaacaaa aaaattgttg attatcttaa aaatgcaaat 4020
tgtaatcaga tgtgtaaaat tggtaattac ttctgtacat taaatgcata tttcttgata 4080
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcgg cccgacagca acgg 4134
<210> 2
<211> 1027
<212> PRT
<213> Squalus acanthias
<400> 2
Met Ala Gin Leu His Cys Gin Leu Leu Phe Leu Gly Phe Thr Leu Leu
1 5 10 15
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Gin Ser Tyr Asn Val Ser Gly Tyr Gly Pro Asn Gin Arg Ala Gin 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 Gin 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 Gin 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 Gin 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 Gin 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 Gin Gin Ala Thr Ala Met Ala Glu Ile Ile Glu His Phe
195 200 205
Gin 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 Gin Tyr Tyr Thr Gin Lys Gin Leu
245 250 255
Glu Phe Ile Ala Asp Val Ile Gin Asn Ser Ser Ala Lys Val Ile Val
260 265 270
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Val Phe Ser Asn Gly Pro Asp Leu Glu Pro Leu Ile Gin 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 Gin Leu Lys Asn Ser Lys Val Pro Ser His Gly Pro Ala Ala
370 375 380
Gin 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 Gin 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 Gin Val Leu Asn His Leu Leu His Leu Lys Phe Thr Asn Ser
465 470 475 480
Met Gly Glu Gin Val Asp Phe Asp Asp Gin Gly Asp Leu Lys Gly Asn
485 490 495
Tyr Thr Ile Ile Asn Trp Gin 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
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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
Gin 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 Gin Phe Leu Leu Val
725 730 735
Phe Leu Cys Ile Leu Val Gin 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
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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 Gin Arg Cys Ser Thr Gin 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 Gin Gly Pro Pro Gin Lys Cys
980 985 990
Glu Pro Gin 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
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Met Glu Glu Thr
1025
<210> 3
<211> 594
<212> DNA
<213> Salmo salar
<400> 3
cttggcatta tgctctgtgc tgggggtatt cttgacagca ttcgtgatgg gagtgtttat 60
caaatttcgc aacaccccaa ttgttaaggc cacaaacaga gagctatcct acctcctcct 120
gttctcactc atctgctgtt tctccagttc cctcatcttc attggtgaac cccaggactg 180
gacatgccgt ctacgccagc ctgcattcgg gataagtttt gttctctgca tctcctgcat 240
cctggtaaaa actaaccgag tacttctagt gttcgaagcc aagatcccca ccagtctcca 300
tcgtaagtgg tgggggctaa acttgcagtt cctgttagtg ttcctgttca catttgtgca 360
agtgatgata tgtgtggtct ggctttacaa tgctcctccg gcgagctaca ggaaccatga 420
cattgatgag ataattttca ttacatgcaa tgagggctct atgatggcgc ttggcttcct 480
aattgggtac acatgcctgc tggcagccat atrcttcttc tttgcattta aatcacgaaa 540
actgccagag aactttactg aggctaagtt catcaccttc agcatgctca tctt 594
<210> 4
<211> 197
<212> PRT
<213> Salmo salar
<220>
<221> misc_feature
<222> (171)..(171)
<223> Xaa = Any Amino Acid
<400> 4
Leu Ala Leu Cys Ser Val Leu Gly Val Phe Leu Thr Ala Phe Val Met
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 Gin 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
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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 Gin Phe Leu Leu
100 105 110
Val Phe Leu Phe Thr Phe Val Gin 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
<210> 5
<211> 2021
<212> DNA
<213> Salmo salar
<400> 5
gtgatcacaa aggtaagaaa gacagtgaaa aatctgaact accccattat ataatctgtt 60
gctatttcat atgtttctat caataataca aacactactt ctctattcct gcagatgcca 120
gtgtttgtac caagtgtccc aatgactcat ggtctaatga gaaccacaca tcttgtttcc 180
tgaaggagat agagtttctg tcttggacag agccctttgg gatcgccttg gcattatgct 240
ctgtgctggg ggtattcttg acagcattcg tgatgggagt gtttatcaaa tttcgcaaca 300
ccccaattgt taaggccaca aacagagagc tatcctacct cctcctgttc tcactcatct 360
gctgtttctc cagttccctc atcttcattg gtgaacccca ggactggaca tgccgtctac 420
gccagcctgc attcgggata agttttgttc tctgcatctc ctgcatcctg gtaaaaacta 480
accgagtact tctagtgttc gaagccaaga tccccaccag tctccatcgt aagtggtggg 540
ggctaaactt gcagttcctg ttagtgttcc tgttcacatt tgtgcaagtg atgatatgtg 600
tggtctggct ttacaatgct cctccggcga gctacaggaa ccatgacatt gatgagataa 660
ttttcattac atgcaatgag ggctctatga tggcgcttgg cttcctaatt gggtacacat 720
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gcctgctggc agccatatgc ttcttctttg catttaaatc acgaaaactg ccagagaact 780
ttactgaggc taagttcatc accttcagca tgctcatctt cttcatcgtc tggatctctt 840
tcatccctgc ctacttcagc acttacggaa agtttgtgtc ggctgtggag gtcatcgcca 900
tactagcctc cagctttggc ctgctggcct gtattttctt caataaagtc tacatcatcc 960
tcttcaaacc gtccaggaac actatagagg aggttcgctg tagcactgcg gcccattctt 1020
tcaaagtggc agccaaggcc actctgagac acagctcagc ctccaggaag aggtccagca 1080
gtgtgggggg atcctgtgcc tcaactccct cctcatccat cagcctcaag accaatgaca 1140
atgactcccc atcaggtcag cagagaatcc ataagccaag agtaagcttt ggaagtggaa 1200
cagttactct gtccttgagc tttgaggagt ccagaaagaa ttctatgaag tagggaagtg 1260
tcttttggtg ggccgagagc cttgtcaaaa cctgagttgg tgttgcattc tttgttggct 1320
gggtagttgg agcagaaatt atgatattaa aagctttgat gtattcagaa tggtgacaca 1380
gcataggtgg ccaagattcc attatattac aataatctgt gttgttcatt atgaggacat 1440
ttcaaaatgc tgaaaatcat caaatacata atttactgag ttttcttgat aatcttgaga 1500
atagaatagc ctattcaagt catcgttgag cagacattaa ttaacaatga tgtaatactt 1560
tccataccta ttttctttaa caatagattc acattgttaa agttcaacta tgacctgtaa 1620
aatacatgag gtataacagg agacaataaa actatgcata tcctagcttc tgggcctgag 1680
tagcaggcag tttactctgg gcacgctttt catccaaact tccgaatgct gcccccaatc 1740
ctagtgaggt taaaggccca gtgcagtcat atcttttctc taggcacgct tttcatccaa 1800
acttccgaat gcggctatat cagtctcttt cctactgtct ttttcattag gccagtgttt 1860
aacaaccctg gtccttaagt acacacaaca gaacacattt ttgttgtagc cctggacaat 1920
cactcctcac tcagctcatt gagggcctga tgattagttg acaagttgaa tcaggtgtgc 1980
ttgtccaggg ttacaataca aatgtgtact gttgggggta c 2021
<210> 6
<211> 388
<212> PRT
<213> Salmo salar
<400> 6
Tyr Lys His Tyr Phe Ser Ile Pro Ala Asp Ala Ser Val Cys Thr Lys
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
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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 Gin Asp Trp Thr Cys Arg Leu Arg
100 105 110
Gin 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 Gin Phe Leu Leu Val
145 150 155 160
Phe Leu Phe Thr Phe Val Gin 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
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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 Gin Gin 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
<210> 7
<211> 3941
<212> DNA
<213> Salmo salar
<400> 7
ttccaacagc atatttttgt tgtatttgct ttggtttgtc tgaaatcaag cattatcaag 60
atcaagaaca gcatgagtca gaaacaaggc gacagccaga gtcactggag gggacaagac 120
tgaggttaac tctgaagtct aatgtgctga gaggacaagg ccctcctgag agctgaacga 180
tgagatttta cctgtattac ctggtgcttt tgggcttcag ttctgtcatc tccacctatg 240
ggcctcatca gagagcacag aagactgggg atattctgct gggcgggctg tttccaatgc 300
actttggtgt tacctccaaa gaccaagacc tggcagcgcg gccagaatcc acagagtgtg 360
ttaggtacaa tttccgggga ttccgttggc ttcaggccat gatttttgca atagaggaga 420
tcaacaacag cagtactctc ctgcccaaca tcacactggg ctacaggatc tttgacacct 480
gcaacaccgt gtccaaggcc ctggaggcta ccctcagttt cgtagcacag aataagattg 540
actctctgaa cttggatgaa ttctgtaact gcactgatca catcccatcg actatagcag 600
tggtgggggc ttctgggtca gcggtctcca ctgctgttgc caatctgttg ggccttttct 660
acatcccaca gatcagctat gcctcttcca gtcgcctact aagcaacaag aaccagttca 720
aatccttcat gaggaccatt cccacagatg agcaccaggc cactgccatg gcagatatca 780
tcgactactt ccaatggaat tgggtcattg cagttgcgtc tgatgatgag tatggacgtc 840
cggggattga aaaatttgag aaagagatgg aagaacgaga catttgtatc catctgagtg 900
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,
,
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
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agaggtccag cagtgtgggg ggatcctgtg cctcaactcc ctcctcatcc atcagcctca 2880
agaccaatga caatgactcc ccatcaggtc agcagagaat ccataagcca agagtaagct 2940
ttggaagtgg aacagttact ctgtccttga gctttgagga gtccagaaag aattctatga 3000
agtagggaag tgtcttttgg tgggccgaga gccttgtcaa aacctgagtt ggtgttgcat 3060
tctttgttgg ctgggtagtt ggagcagaaa ttatgatatt aaaagctttg atgtattcag 3120
aatggtgaca cagcataggt ggccaagatt ccattatatt acaataatct gtgttgttca 3180
ttatgaggac atttcaaaat gctgaaaatc atcaaataca taatttactg agttttcttg 3240
ataatcttga gaatagaata gcctattcaa gtcatcgttg agcagacatt aattaacaat 3300
gatgtaatac tttccatacc tattttcttt aacaatagat tcacattgtt aaagttcaac 3360
tatgacctgt aaaatacatg aggtataaca ggagacaata aaactatgca tatcctagct 3420
tctgggcctg agtagcaggc agtttactct gggcacgctt ttcatccaaa cttccgaatg 3480
ctgcccccaa tcctagtgag gttaaaggcc cagtgcagtc atatcttttc tctaggcacg 3540
cttttcatcc aaacttccga atgcggctat atcagtctct ttcctactgt ctttttcatt 3600
aggccagtgt ttaacaaccc tggtccttaa gtacacacaa cagagcacat ttttgttgtg 3660
gccctggaca atcactcctc actcagctca ttgagggcct gatgattagt tgacaagttg 3720
agtcgggtgt gcttgtccgg ggttgcaata cagatgtgta ctgttggggg tactcgagga 3780
ccaggattgg gaaacattac attaggacta ctgtaggttc ttcaatatgg tgtcatacgg 3840
tcatatggtg tcatatggtg tctggttgtt ttctgcatat gtgtatttca ccaagttact 3900
gcacatgtta gacctataca ctggaataaa catttttttt c 3941
<210> 8
<211> 941
<212> PRT
<213> Salmo salar
<400> 8
Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val
1 5 10 15
Ile Ser Thr Tyr Gly Pro His Gin 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
Gin Asp Leu Ala Ala Arg Pro Glu Ser Thr Glu Cys Val Arg Tyr Asn
50 55 60
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Phe Arg Gly Phe Arg Trp Leu Gin 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 Gin 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 Gin Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
Lys Asn Gin Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His
180 185 190
Gin Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gin 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 Gin Tyr Phe Glu Glu Trp Gin Ile Gin 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
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Ala Leu Arg Ala Gly Glu Ile Pro Gly Phe Lys Asp Phe Leu Gin 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 Gin 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 Gin Ala Leu
405 410 415
Gin 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 Gin Val Leu Lys Gin
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 Gin 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
CA 02481827 2005-10-06
129/17
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 Gin Asp Trp Thr Cys Arg Leu Arg Gin 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 Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin
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
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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 Gin Gin
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
<210> 9
<211> 4031
<212> DNA
<213> Salmo salar
<400> 9
gttccaacag catatttttg ttgtatttgc tttggtttgt ctgaaatcaa gcattatcaa 60
ggattgagca agacaactga gttgtcagac taagaatata cacatttcca gttctctctt 120
taatggactt ctcacactga tgttcttcag atcaagaaca gcatgagtca gaaacaaggc 180
gacagccaga gtcactggag gggacaagac tgaggttaac tctgaagtct aatgtgctga 240
gaggacaagg ccctcctgag agctgaacga tgagatttta cctgtattac ctggtgcttt 300
tgggcttcag ttctgtcatc tccacctatg ggcctcatca gagagcacag aagactgggg 360
atattctgct gggcgggctg tttccaatgc actttggtgt tacctccaaa gaccaagacc 420
tggcagcgcg gccagaatcc acagagtgtg ttaggtacaa tttccgggga ttccgttggc 480
ttcaggccat gatttttgca atagaggaga tcaacaacag cagtactctc ctgcccaaca 540
tcacactggg ctacaggatc tttgacacct gcaacaccgt gtccaaggcc ctggaggcta 600
ccctcagttt cgtagcacag aataagattg actctctgaa cttggatgaa ttctgtaact 660
gcactgatca catcccatcg actatagcag tggtgggggc ttctgggtca gcggtctcca 720
ctgctgttgc caatctgttg ggccttttct acatcccaca gatcagctat gcctcttcca 780
CA 02481827 2005-10-06
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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
CA 02481827 2005-10-06
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,
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
<210> 10
<211> 941
<212> PRT
<213> Salmo salar
<400> 10
Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val
1 5 10 15
Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gin Lys Thr Gly Asp Ile
20 25 30
CA 02481827 2005-10-06
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Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp
35 40 45
Gin 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 Gin 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 Gin 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 Gin Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
Lys Asn Gin Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His
180 185 190
Gin Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gin 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 Gin Tyr Phe Glu Glu Trp Gin Ile Gin 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
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129/22
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 Gin 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 Gin 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 Gin Ala Leu
405 410 415
Gin 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 Gin Val Leu Lys Gin
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 Gin 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
CA 02481827 2005-10-06
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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 lie Gly Glu
645 650 655
Pro Gin Asp Trp Thr Cys Arg Leu Arg Gin 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 Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin
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
CA 02481827 2005-10-06
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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 Gin Gin
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
<210> 11
<211> 3824
<212> DNA
<213> Salmo salar
<400> 11
gttccaacag catatttttg ttgtatttgc tttggtttgt ctgaaatcaa gcattatcaa 60
gatcaagaac agcatgagtc agaaacaagg cgacagccag agtcactgga ggggacaaga 120
ctgaggttaa ctctgaagtc taatgtgctg agaggacaag gccctcctga gagctgaacg 180
atgagatttt acctgtatta cctggtgctt ttgggcttca gttctgtcat ctccacctat 240
gggcctcatc agagagcaca gaagactggg gatattctgc tgggcgggct gtttccaatg 300
cactttggtg ttacctccaa agaccaagac ctggcagcgc ggccagaatc cacagagtgt 360
gttaggtaca atttccgggg attccgttgg cttcaggcca tgatttttgc aatagaggag 420
atcaacaaca gcagtactct cctgcccaac atcacactgg gctacaggat ctttgacacc 480
tgcaacaccg tgtccaaggc cctggaggct accctcagtt tcgtagcaca gaataagatt 540
CA 02481827 2005-10-06
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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
CA 02481827 2005-10-06
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attgatgaga taattttcat tacatgcaat gagggctcta tgatggcgct tggcttccta 2460
attgggtaca catgcctgct ggcagccata tgcttcttct ttgcatttaa atcacgaaaa 2520
ctgccagaga actttactga ggctaagttc atcaccttca gcatgctcat cttcttcatc 2580
gtctggatct ctttcatccc tgcctacttc agcacttacg gaaagtttgt gtcggctgtg 2640
gaggtcatcg ccatactagc ctccagcttt ggcctgctgg cctgtatttt cttcaataaa 2700
gtctacatca tccatcagcc tcaagaccaa tgacaatgac tccccatcag gtcagcagag 2760
aatccataag ccaagagtaa gctttggaag tggaacagtt actctgtcct tgagctttga 2820
ggagtccaga aagaattcta tgaagtaggg aagtgtcttt tggtgggccg agagccttgt 2880
caaaacctga gttggtgttg cattctttgt tggctgggta gttggagcag aaattatgat 2940
attaaaagct ttgatgtatt cagaatggtg acacagcata ggtggccaag attccattat 3000
attacaataa tctgtgttgt tcattatgag gacatttcaa aatgctgaaa atcatcaaat 3060
acataattta ctgagttttc ttgataatct tgagaataga atagcctatt caagtcatcg 3120
ttgagcagac attaattaac aatgatgtaa tactttccat acctattttc tttaacaata 3180
gattcacatt gttaaagttc aactatgacc tgtaaaatac atgaggtata acaggagaca 3240
ataaaactat gcatatccta gcttctgggc ctgagtagca ggcagtttac tctgggcacg 3300
cttttcatcc aaacttccga atgctgcccc caatcctagt gaggttaaag gcccagtgca 3360
gtcatatctt ttctctaggc acgcttttca tccaaacttc cgaatgcggc tatatcagtc 3420
tctttcctac tgtctttttc attaggccag tgtttaacaa ccctggtcct tgagtacaca 3480
caacagggca catttttgtt gtagccctgg acaatcactc ctcactcagc tcattgaggg 3540
cctgatgatt agttgacaag ttgggtcagg tgtgcttgtc cagggttaca atacagatgt 3600
gtgctgttgg gggtactcga ggaccaggat tgggaaacat tacattagga ctactgtagg 3660
ttcttcaata tggtgtcata cggtcatatg gtgtcatatg gtgtctggtt gttttctgca 3720
tatgtgtatt tcaccaagtt actgcacatg ttagacctat acactggaat aaacattttt 3780
tttcacaatg catccaatga caataaaatc accatatgcc aatg 3824
<210> 12
<211> 850
<212> PRT
<213> Salmo salar
<400> 12
Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val
1 5 10 15
Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gin Lys Thr Gly Asp Ile
20 25 30
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Leu Leu Gly Gly Leu Phe Pro Met His Phe Gly Val Thr Ser Lys Asp
35 40 45
Gin 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 Gin 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 Gin 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 Gin Ile Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
Lys Asn Gin Phe Lys Ser Phe Met Arg Thr Ile Pro Thr Asp Glu His
180 185 190
Gin Ala Thr Ala Met Ala Asp Ile Ile Asp Tyr Phe Gin 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 Gin Tyr Phe Glu Glu Trp Gin Ile Gin 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
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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
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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 Gin Asp Trp Thr Cys Arg Leu Arg Gin 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 Gin 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
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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 Gin Pro Gln
835 840 845
Asp Gin
850
<210> 13
<211> 3988
<212> DNA
<213> Salmo salar
<400> 13
gttccaacag catatttttg ttgtatttgc tttggtttgt ctgaaatcaa gcattatcaa 60
gatcaagaac agcatgagtc agaaacaagg cgacagccag agtcactgga ggggacaaga 120
ctgaggttaa ctctgaagtc taatgtgctg agaggacaag gccctcctga gagctgaacg 180
atgagatttt acctgtatta cctggtgctt ttgggcttca gttctgtcat ctccacctat 240
gggcctcatc agagagcaca gaagactggg gatattctgc tgggcgggct gtttccaatg 300
cactttggtg ttacctccaa agaccaagac ctggcagcgc ggccagaatc cacagagtgt 360
gttaggtaca atttccgggg attccgttgg cttcaggcca tgatttttgc aatagaggag 420
atcaacaaca gcagtactct cctgcccaac atcacactgg gctacaggat ctttgacacc 480
tgcaacaccg tgtccaaggc cctggaggct accctcagtt tcgtagcaca gaataagatt 540
gactctctga acttggatga attctgtaac tgcactgatc acatcccatc gactatagca 600
gtggtggggg cttctgggtc agcggtctcc actgctgttg ccaatctgtt gggccttttc 660
tacatcccac agatcagcta tgcctcttcc agtcgcctac taagcaacaa gaaccagttc 720
aaatccttca tgaggaccat tcccacagat gagcaccagg ccactgccat ggcagatatc 780
atcgactact tccaatggaa ttgggtcatt gcagttgcgt ctgatgatga gtatggacgt 840
ccggggattg aaaaatttga gaaagagatg gaagaacgag acatttgtat ccatctgagt 900
gagctgatct ctcagtactt tgaggagtgg cagatccaag gattggttga ccgtattgag 960
aactcctcag ctaaagttat agtcgttttc gccagtgggc ctgacattga gcctcttatt 1020
aaagagatgg tcagacggaa catcaccgac cgcatctggt tggccagcga ggcttgggca 1080
accacctccc tcatcgccaa accagagtac cttgatgttg tagttgggac cattggcttt 1140
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gctctcagag caggcgaaat acctggcttc aaggacttct tacaagaggt cacaccaaag 1200
aaatccagcc acaatgaatt tgtcagggag ttttgggagg agacttttaa ctgctatctg 1260
gaagacagcc agagactgag agacagtgag aatgggagca ccagtttcag accattgtgt 1320
actggcgagg aggacattat gggtgcagag accccatatc tggattacac tcatcttcgt 1380
atttcctata atgtgtatgt tgcagttcac tccattgcac aggccctaca ggacattctc 1440
acctgcattc ctggacgggg tcttttttcc aacaactcat gtgcagatat aaagaaaata 1500
gaagcatggc aggttctcaa gcagctcaga catttaaact tctcaaacag tatgggagaa 1560
aaggtacatt ttgatgagaa tgctgatccg tcaggaaact acaccattat caattggcac 1620
cggtctcctg aggatggttc tgttgtgttt gaagaggtcg gtttctacaa catgcgagct 1680
aagagaggag tacaactttt cattgataac acaaagattc tatggaatgg atataatact 1740
gaggttccat tctctaactg tagtgaagat tgtgaaccag gcaccagaaa ggggatcata 1800
gaaagcatgc caacgtgttg ctttgaatgt acagaatgct cagaaggaga gtatagtgat 1860
cacaaagatg ccagtgtttg taccaagtgt cccaatgact catggtctaa tgagaaccac 1920
acatcttgtt tcctgaagga gatagagttt ctgtcttgga cagagccctt tgggatcgcc 1980
ttggcattat gctctgtgct gggggtattc ttgacagcat tcgtgatggg agtgtttatc 2040
aaatttcgca acaccccaat tgttaaggcc acaaacagag agctatccta cctcctcctg 2100
ttctcactca tctgctgttt ctccagttcc ctcatcttca ttggtgaacc ccaggactgg 2160
acatgccgtc tacgccagcc tgcattcggg ataagttttg ttctctgcat ctcctgcatc 2220
ctggtaaaaa ctaaccgagt acttctagtg ttcgaagcca agatccccac cagtctccat 2280
cgtaagtggt gggggctaaa cttgcagttc ctgttagtgt tcctgttcac atttgtgcaa 2340
gtgatgatat gtgtggtctg gctttacaat gctcctccgg cgagctacag gaaccatgac 2400
attgatgaga taattttcat tacatgcaat gagggctcta tgatggcgct tggcttccta 2460
attgggtaca catgcctgct ggcagccata tgcttcttct ttgcatttaa atcacgaaaa 2520
ctgccagaga actttactga ggctaagttc atcaccttca gcatgctcat cttcttcatc 2580
gtctggatct ctttcatccc tgcctacttc agcacttacg gaaagtttgt gtcggctgtg 2640
gaggtcatcg ccatactagc ctccagcttt ggcctgctgg cctgtatttt cttcaataaa 2700
gtctacatca tcctcttcaa accgtccagg aacactatag aggaggttcg ctgtagcact 2760
gcggcccatt ctttcaaagt ggcagccaag gccactctga gacacagctc agcctccagg 2820
aagaggtcca gcagtgtggg gggatcctgt gcctcaactc cctcctcatc catcagcctc 2880
aagaccaatg acaatgactc cccatcaggt cagcagagaa tccataagcc aagagtaagc 2940
tttggaagtg gaacagttac tctgtccttg agctttgagg agtccagaaa gaattctatg 3000
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aagtagggaa gtgtcttttg gtgggccgag agccttgtca aaacctgagt tggtgttgca 3060
ttctttgttg gctgggtagt tggagcagaa attatgatat taaaagcttt gatgtattca 3120
gaatggtgac acagcatagg tggccaagat tccattatat tacaataatc tgtgttgttc 3180
attatgagga catttcaaaa tgctgaaaat catcaaatac ataatttact gagttttctt 3240
gataatcttg agaatagaat agcctattca agtcatcgtt gagcagacat taattaacaa 3300
tgatgtaata ctttccatac ctattttctt taacaataga ttcacattgt taaagttcaa 3360
ctatgacctg taaaatacat gaggtataac aggagacaat aaaactatgc atatcctagc 3420
ttctgggcct gagtagcagg cagtttactc tgggcacgct tttcatccaa acttccgaat 3480
gctgccccca atcctagtga ggttaaaggc ccagtgcagt catatctttt ctctaggcac 3540
gcttttcatc caaacttccg aatgcggcta tatcagtctc tttcctactg tctttttcat 3600
taggccagtg tttaacaacc ctggtcctta agtgcacaca acagggcaca tttttgttgt 3660
ggccctggac aatcactcct cactcagctc attgagggcc tgatgattag ttgacaagtt 3720
gagtcaggtg tgcttgtccg gggttgcaat acagatgtgt actgttgggg gtgctcgagg 3780
accaggattg ggaaacatta cattaggact actgtaggtt cttcaatatg gtgtcatacg 3840
gtcatatggt gtcatatggt gtctggttgt tttctgcata tgtgtatttc accaagttac 3900
tgcacatgtt agacctatac actggaataa acattttttt tcacaatgca tccaatgaca 3960
ataaaatcac catatgccaa tgaaaaaa 3988
<210> 14
<211> 941
<212> PRT
<213> Salmo salar
<400> 14
Met Arg Phe Tyr Leu Tyr Tyr Leu Val Leu Leu Gly Phe Ser Ser Val
1 5 10 15
Ile Ser Thr Tyr Gly Pro His Gin Arg Ala Gin 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
Gin 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 Gin 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
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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
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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
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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 Gin Asp Trp Thr Cys Arg Leu Arg Gin 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 Gin Phe Leu Leu Val Phe Leu Phe Thr Phe Val Gin
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
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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 Gin Gin
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
<210> 15
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 15
tgtcktggac ggagccctty ggratcgc 28
<210> 16
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 16
ggckggratg aargakatcc aracratgaa g 31
<210> 17
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide for Sal-1 antibody production
<400> 17
Cys Thr Asn Asp Asn Asp Ser Pro Ser Gly Gin Gin Arg Ile His Lys
1 5 10 15
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<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 18
caagcattat caagatcaag 20
<210> 19
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 19
ctcagagtgg ccttggc 17
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 20
cagttctctc tttaatggac 20
<210> 21
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 21
ctcagagtgg ccttggc 17
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 22
agtctacatc atccatcagc c 21
<210> 23
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<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 23
gattttattg tcattggatg c 21
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 24
tggaagatga aatcgccgc 19
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 25
gtggtggtga aactgtaacc gc 22
<210> 26
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide for 4641 antibody production
<400> 26
Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu Lys Phe Arg Glu Glu
1 5 10 15
Ala Glu Glu Arg Asp Ile Cys
<210> 27
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide for 4641 antibody production
<400> 27
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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
<210> 28
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide for SKCaR antibody production
<400> 28
Ala Arg Ser Arg Asn Ser Ala Asp Gly Arg Ser Gly Asp Asp Leu Pro
1 5 10 15
Cys
<210> 29
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> peptide for Sal-ADD antibody production
<400> 29
Cys Ser Asp Asp Glu Tyr Gly Arg Pro Gly Ile Glu Lys Phe Glu Lys
1 5 10 15
Glu Met
<210> 30
<211> 1078
<212> PRT
<213> Homo sapiens
<400> 30
Met Ala Phe Tyr Ser Cys Cys Tip Val Leu Leu Ala Leu Thr Tip His
1 5 10 15
Thr Ser Ala Tyr Ser Pro Ser Gin Pro Ala Gin 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
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Gin 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 Gin 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 Gin 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 Gin Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn
165 170 175
Lys Asn Gin 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 Gin Tyr Ser Asp Glu Glu Glu Ile Gin Met Val Val
245 250 255
Glu Val Ile Gin 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
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Ile Ala Met Pro Gin Tyr Phe His Val Val Gly Gly Thr Ile Gly Phe
305 310 315 320
Ala Leu Lys Ala Gly Gin 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 Gin 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 Gin
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 Gin Val Leu Lys His Leu
450 455 460
Arg Asn Leu Asn Phe Thr Asn Asn Met Gly Glu Gin 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
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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
Gin Asp Trp Thr Cys Arg Leu Arg Gin 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 Gin Phe Leu Leu Val Phe Leu Cys Thr Phe Asn Gin Ile
725 730 735
Val Ile Cys Val Ile Trp Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg
740 745 750
Asn Gin 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
CA 02481827 2005-10-06
129/43
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 Gin Lys Gin Gin Gin Pro Leu Ala Leu Thr Gin Gin Glu Gin Gin
930 935 940
Gin Gin Pro Leu Thr Leu Pro Gin Gin Gin Arg Ser Gin Gin Gin Pro
945 950 955 960
Arg Cys Lys Gin Lys Val Ile Phe Gly Ser Gly Thr Val Thr Phe Ser
965 970 975
Leu Ser Phe Asp Glu Pro Gin Lys Asn Ala Met Ala Asn Arg Asn Ser
980 985 990
Thr Asn Gin Asn Ser Leu Glu Ala Gin Lys Ser Ser Asp Thr Leu Thr
995 1000 1005
Ala Asn Gin Pro Leu Leu Pro Leu Gin Cys Gly Glu Thr Asp Leu
1010 1015 1020
Asp Leu Thr Val Gin Glu Thr Gly Leu Gin Gly Pro Val Gly Gly
1025 1030 1035
Asp Gin Arg Pro Glu Val Glu Asp Pro Glu Glu Leu Ser Pro Ala
1040 1045 1050
CA 02481827 2005-10-06
129/44
Leu Val Val Ser Ser Ser Gin Ser Phe Val Ile Ser Gly Gly Gly
1055 1060 1065
Ser Thr Val Thr Glu Asn Val Val Asn Ser
1070 1075
<210> 31
<211> 3606
<212> DNA
<213> Fugu rubripes
<400> 31
taatctcagg agagtttcgt ttccagagga aaccgaactt cttaaacgca ggtaggttgc 60
aagcgtcata tttcacagtg gaatcaaagg caagaaaacg ggtcatttca agagaaattt 120
gtcctccttt ctcccagtcg cagcaggaaa aacatgagtc tcctgaaaaa atgcagcagg 180
cagactgatg ggacaagata agagggaccg tatcacctta aaaaaattat ttttccaaaa 240
aaatccatta gaaagggcag gatggtgcga ctggtgctgc attatctcat acttctgggg 300
tccggttatg tcatttcaac atatgggcct aatcagagag cacagatgac tggcgatatt 360
ctgctgggag gacttttccc catacacttt ggcatttctt ccaaagacga aaacctcgct 420
gcccgaccgg aatccacaaa gtgtgtcagg taaaactcag cacttcatat gtaactgcat 480
gtaaatgctt aaatcacgtg tcagcactgt tgagcacttt caaaccttct ttccgcaggt 540
tcaatttccg tggtttccgc tggctgcagg ccatggtttt cgccatcgag gagatcaaca 600
acagcagcag cctcctgccc aacatcaccc tcggctacag gatcttcgac acgtgcaaca 660
cggtttcaaa agccctggag gctacgctga gcttcgtggc ccaaaacaag atcgactccc 720
tgaatttgga tgaattttgc aactgcacgg atcacatccc ggcaaccatc gcggtggtgg 780
gagcggcagg gtctgcggtc tctacggcag tcgcaaacct gctgagtctc ttttatattc 840
ctcaggtgag tgacggaatt atctttacac tccagttaaa accttaaagt caccgcaatc 900
ctctatcaca ccagatcagc tatgcgtcct ccagccgctt gctgagcaac aagaatcaat 960
acaagtcctt catgagaacc atccctacag atgagcacca ggccacagcc atggccgacg 1020
tcatcgagta cttccagtgg aactgggtca ttgccgtggc ctctgatgac gattatgggc 1080
ggcctgggat agaaaagttt gagaaggaga tggaagagcg agacatttgc atccatctga 1140
acgaactcat ctctcagtac ttcgaggact gtgaaatcaa agctctcgtg gacagaattg 1200
aaaactccac agccaaagtc atcgttgtgt ttgccagcgg ccctgatatc gagcctctga 1260
tcaaagaaat ggtgaggagg aacatcaccg atcggatctg gttagccagt gaagcgtggg 1320
caagctcctc cctcatcgct aaaccagaat atctcgatgt tgtggagggc acaatcggct 1380
ttgttttgaa agcagggaac atacctggtt tcagggagtt cttacagcag gtccagccaa 1440
agagaggcag ccacaacgaa tttgtcaggg agttttggga ggaaaccttc aactgttacc 1500
CA 02481827 2005-10-06
129/45
tggaggacag cccgagactg caagaaagtg agaatggtag cgacagtttc aggccgcttt 1560
gcaccagcga ggaagacatc acgagtgtgg agaccccgta cctcgaccac acacacctcc 1620
gtatctccta caatgtctat gttgcagttt actccatcgc tcacgccctc caggacatac 1680
tctcctgcac tcctggacat ggactctttg ctaacaactc ttgcgcggat ataaagaaaa 1740
tggaagcatg gcaggtaaaa actgctcttt tgcccacaat gttctacttt gtcagcctca 1800
aagggtcaaa tgtgtttgca ggtcctgaag cagctcagac atttgaacta caccaacagc 1860
atgggggaaa aggttcattt tgatgagaac gcagacatgg aagcaaacta caccatcata 1920
aactggcacc ggtctgctga ggacggctca gtggcgttca gggaggtggg ctactaccac 1980
atgcacgcca ggcggggagc caaactgctc attgataaca caaagatgat gtggaatgca 2040
tacagttcag aggtcagtca tcttcagggc aatttcctgt ttgtcctgat gtacagtgtc 2100
tctgttcctg acatcattta cgtggcaggg acaccaaatg tggtgtcatg tgagcatcta 2160
agggtttgca tctgttgtcc tttaaggtgc cgttttctaa ctgcagcgag gactgcgaac 2220
ctgggacgag gaagggcatc attgacagca tgcccacgtg ctgctttgaa tgtaccgagt 2280
gctcagacgg cgagtacagc gatcataaag gtagtagcgt ttgtttactg cttcctgtta 2340
ctgtccacga acactcaggt aatcctcaca tgcatgtttc agacgccagc atttgcacca 2400
agtgtccaaa caactcctgg tccagtggga accacacctt ctgctttctg aaggaaattg 2460
agttcctggc ctggtcagaa ccctttggga tcgctttggc catctgcgcg gtcctggggg 2520
ttctcctgac ggcatttgtg atgggagtct ttgtcagatt tcgcaacact ccgatcgtga 2580
aggcctcaaa ccgagaactg tcctacgtcc tcttgttgtc actcatctgc tgtttttcca 2640
gctcactcat cttcatcggg gagccccaag actggacctg ccgtttacgc cagcctgcct 2700
tcgggatcag ttttgtcctt tgcatctcct gcatcctcgt gaaaaccaac agggttcttt 2760
tggtgtttga agccaagatc cccaccagca tccatcgtaa atggtggggc ctgaacctgc 2820
agttcctcct ggtgtttctg tgcaccttcg ttcaggtcat gatatgtgtg gtctggcttt 2880
acaacgcccc cccctccagc tacaggaatc atgacatcga tgagatcatt ttcatcacct 2940
gtaatgaggg atccgtgatg gcgctcgggt ttctcattgg ctacacgtgc ttactagcgg 3000
ccatatgctt cttctttgcg tttaagtcaa ggaaacttcc cgaaaacttc acggaggcca 3060
agttcatcac tttctgcatg ctcatcttct ttattgtttg gatttctttt atccctgcgt 3120
acttcagtac ttacggcaag tttgtttcag cggtagaggc cattgccatc ctggcatcca 3180
gctacgggat gctagcttgt attttcttta ataaggtcta catcatcctg ttcaaaccct 3240
gtaggaacac catagaagag gtcaggtgca gcaccgcggc ccacgctttc agagtggccg 3300
ccaaagctac attaaaacac agaaccactg tgaggaaaaa gtcgaacagc atcggctcta 3360
CA 02481827 2005-10-06
129/46
ccgcctccac tccatcatca tcaattagcc tgaagaccaa cagcaacgac tgcgactccg 3420
cctcagggag gcataggccg agggtgagct tcggcagcgg aactgtcatg ctgtccttga 3480
gctttgagga gtccaggagg agctctctga tgtgacacac aaaccagttt catccacccg 3540
tcctttcagc ctctaatcca tctgtccacc cttctaatta ccttgattat taacctgctt 3600
aataaa 3606
<210> 32
<211> 4856
<212> DNA
<213> Fugu rubripes
<400> 32
cttgtggctg gggtcagtag aagaacctag actagcggta catgaaccgt atcaaaagtg 60
taacggtgaa acattagcac aactgtgtgg gggaggaaat agagtcattg catagtttct 120
taaaccagtt gtgcagtacc tgagtgacaa actttgtttt gtccatttta tttttgcatt 180
aaacaaaagc gattttattg gaaattgcct cgatagatgt caataaatct cacatgatgt 240
tatcagcact tcattacagg tatcataaag atataatagg aacaactgta acttgtattt 300
gttctaaagg ttataaccct tggggaacag ccagataagg accacgtaat gttttgtctg 360
actgattaat aataataagc tgacatgagc agaaccaaca cctcagtcta taaaagcatt 420
tgtgcagaat gctccgagac atttttagga tggagatctc tgttatcttc ctttgcttca 480
tttctctgtt tgatttaaac tcagctggtg acctgaaggc tcctgagaac agtctgaaac 540
aacaggttgg accgagggag gacacaaccg gtgccgcagc tccgtctgac atatgtcgcc 600
tccagggttc tgctcgccta cctgctttct caaaggatgg agactttgtc atcggggggg 660
ttttctccat acaccgctac acagtgacgg tgaaccacaa ctacaccacc atgcctgagc 720
catttcgctg cagagtgtgc ggagaagaaa atgtttgatt gtttcatttc tggagtatat 780
atgatgttca cattatgatg cattttttat ttatacctac acttgtgaat gtttgttttc 840
tgtcatgatg tgcagcatcg accatcatga attgcaactg tctcatgcaa tggtcttcgc 900
catcgaggag atcaacaaca gcacggagct gcttcctgga atcaaactcg gttaccagat 960
ccacgactcg tgcgcagccg tgcccatcgc agtaaatgtg gccttccaac tgttaaacac 1020
tctggaccct gtgtttgtca caggtgacaa ctgttcacag tccggtatgg tgatggctgt 1080
cgtcggcgag tctggttcca ctccgtccat cagcatatcc cgcgtcatcg gctccttcga 1140
cattccacta gtgaatattt tcaactttta ccaaagcaca tgaacacgta atattaacca 1200
tcactgtaat gacacaatgt tcaacttcag gtgagccact ttgccacctg tgcatgcctg 1260
tctgataaac agaagtaccc aagtttcttc agaacgattc ccagtgacca gttccaggcc 1320
gacgctttgg tcaaactcat caaacacttt ggctggactt ggataggtgc tgtgtgctca 1380
CA 02481827 2005-10-06
129/47
gattcagact atggcaataa tggcatggca gcgttcctgc atgcagctca gaaggagggc 1440
atctgtgtgg agtactcgga atccttttat cgcacccacc cgcacagcag gatcaagaga 1500
gtggctgatg tcatccgcag gtgtatagat ctatttttgc ttcatgattc atgaaggtat 1560
ttgtgtttgt ttgaaatgac gcatatactt gtgagttttc tgcaggtcaa cagctgtggt 1620
ggttgtagcc tttacagcat ctacagaaat gatgatcctg ctcgaggaac tgtctcatga 1680
accctcacca cctcgccagt ggataggcag tgaatcctgg gtaactgacc cagacttgct 1740
gaggttcagc ttctgtgctg gaactattgg atttgccatt cagaggtctg tcatcccagg 1800
cctgagagac ttcctgctgg atctctcccc ctcgaaagtg gcttcatctc ctgtgctcac 1860
tgagttctgg gaggactcat tcaactgccg gttgggaaaa ggtgacagaa gttatacctc 1920
ttcttaattt actgtggata gattttaaat tagtgtccaa atctaataac aaaggctttc 1980
aacagatctc ctgtgttgtc agttgctgtt gcaggtgagc gcatgtgtga tggatctgaa 2040
gacataatga ctctccagag tccgtacact gacacttctg agctccgcat taccaacatg 2100
gtgtacaagg ctgtttatgc aatagctcat gccattcata atgcagtgtg tcaggacaca 2160
aatgctacca ctcggtgtag caaattcacc acgattaacc ccaaaaaggt caaacctaaa 2220
gaattgcctc tgtaactact aacatggttt attacaagtg tttcagctga agtctttcct 2280
ctgactctct gtttcattca attcaggttc tcactcagct gaagacagta aatttttcac 2340
aaaatggtta tgctgtatcc tttgatgcca acggggatcc tgtggcatca tatgagctgg 2400
taaactggaa aaagagtggg agcgggagca tcgaagttgt gccagtagga tactatgatg 2460
cttcactgcc agaaggccaa gagttccgta tcttcaggga catcacatgg gtggatggca 2520
gaaagcaagt acgacagcca gaataattaa taagacagta attcaggcat gaaaacacaa 2580
catctctgaa ttaaaggctt ttttgttcaa ttgtgactct atgtaaaggc accaacggca 2640
gttctgttaa aatctttttc aactggtggg tgtgtatgtc tcaggtgcca gtgtcagtgt 2700
gcagcgacag ctgtcctcaa ggaactcgta aggtactgca gaaaggaaaa cccatctgct 2760
gttatgactg tgtacaatgt ccagagggag agattagcaa tgttacaggt agttatctag 2820
atttcaaaga cagcagggct aaataaataa tttgtttaaa ccctacatga tttttgtttc 2880
ctagattctc ctgaatgcat cccttgtctt gatgacttct ggcctaaccc agagagaaat 2940
gcctgtttcc ccaaacctgt ggagtttctt tcctttaacg aggttctagg aatcatccta 3000
gctgtgttct cagtcggtgg tgcctgtctg gctgttatca cagcagctgt atttttccat 3060
cacaggacct ctcctattgt cagagccaac aactctgagt tgagcttcct gttgctcttc 3120
tctttgactc tgtgcttctt atgttcactc actttcattg gtgctccctc tcacctgtcc 3180
tgcatgctgc gccacacagc gtttgggatc acctttgtcc tttgcatctc atgtgtttta 3240
gggaaaactg tggtggtgtt gatggccttc agagcgactc tccccgggag taatgtcatg 3300
CA 02481827 2005-10-06
129/48
aagtggttcg gtccacctca gcaaaggatg actgttgtga ctttcacgtc tatccaggtt 3360
ttaatatgca ttgtttggtt ggttgtcaat cccccgtttc cagtgagaaa tctgaccacg 3420
tacaaggaga gaatcatcct ggagtgtgcg ttaggctcat ctgttggatt ctgggctgta 3480
ctcgggtaca tcggccttct ggcagctgtt tgtttagttc tagccgtcct cgcccgaaaa 3540
ctccccgata atttcaacga agccaaaatg atcaccttca gcatgctgat attctgtgcc 3600
gtctggatca ccttcatccc cgcttacgtc agctctcctg ggaaattcac cgtggccgtg 3660
gagatctttg ccattttggc ctccagtttt ggactcatcc tgtgcatttt tgctccaaaa 3720
tgttttatta ttttatttaa gccagagaaa aatagcaaaa aacacttgat gaacaagaaa 3780
taatcagaat gtctgcaata tgttgggtga gtttaacgac tggaagtatg ggtccgtaca 3840
gcagcaatag cccagtctgt agatgagtga gctgtgggtt tgagggtatc tggttcaagt 3900
tccatttggg aacaaagatt aggaaatgga cctgtagaag gagatgtgca gggacatatt 3960
aagaacccca ctatggtatc attaagcaaa ataccaaacc cccaaatgat aagattgcct 4020
ttatctctct tcatgattgc atgtgcatgt gtttattgta gcttcatgtt taaccgcttg 4080
taacttgtgt gtaacaaagt gtaaacacag acatttcccc tttgtgcttt taatgtatgc 4140
tttttttctt ctcttgtttt acactgtact gccattcctc ccaacgttat gtgacaggct 4200
agattaggct gcatgatgta tgagttttag aaaatctcac cctttacaac acaagtccgc 4260
tgatacagtt ttccatctct cctcccttat gtccaccata acctcttttc tattttctat 4320
gtgattgtta aacaattaca cttttcattg ggaatgttat tgataaatgc aagtaaaact 4380
actgtgatat ctgtgtagat tctcaatcat ccaggtcatg gtcatccaat gcagtttgct 4440
ttgtcaaatg gactgttttt cttctctttg aagttctgga gtgtgatgta ttcaaaacat 4500
cattaaagtt ataaagtggg aacgactgta acagatttgt tcaaaaggtt ataacccttg 4560
gggaacagcc agataaagac cacataattg cagaatgctc caagacattt ttaggatgga 4620
gatctgttct cttcctttgc ttcatttctc tgtgtgattt gaactcagct ggtgacctga 4680
aggttcctgg gaacagtctg aaacaacagc ttggactgag ggaggacaca accgctgccg 4740
cggctccgtc tgtcatatgt cggctccagg gttttgctcg cctacctgcc ttctcaaaga 4800
atggagactt tgtcatcggg gggggttttc tccatatacc actacaaaat gacagt 4856
<210> 33
<211> 4981
<212> DNA
<213> Fugu rubripes
<400> 33
gtgtatttgt ttatgcattc ataacgaagc aacaaactca tattctgcaa gttttgatgg 60
atttgctggc attattaata cttacaatta gattagatta gattagatta gattagatta 120
CA 02481827 2005-10-06
129/49
gataaaagaa attcaggaag ggaaatgcag acattatatt taccaatcct atgaactatc 180
caaatcttgc atagccttaa attaaacatg gtctagatga taaaagtaac atgtatgaag 240
tgaacgaatc tgacgtcatc ggtcacacct tgatgggtgg tgtcatcaga catggtgaag 300
acacaactct gcatataaaa tatgcagtag agatgaagag aacctgaggt ggaggaagga 360
ggaagagtga tggctgacat cactggcaca ttgggcctct tcttcaccct catcacattg 420
tttgtctcct cttccacctc cttcaatgct ccgacttgca agctatggag gaaatttcag 480
cttaatgaga tgcacgaacc gggcgatgtg cttctaggcg ggctgtttca agttcattac 540
agctctgttt ttcccgagtg gacgtttaca tctgaaccac atcagcctgt ctgcactagg 600
taagtttcat gcacaccaca aacaatgatt caagctcaga ttggtcctgg aagaaaaatg 660
tcaattaaat gtttgtgatg ggtgaatcca tccataaagg gtgtatctgg agccagtttc 720
accaggttct tgttaatggt tttggtattt tgcatgtcag gtttgacatt ctaggcttcc 780
gtcacgctat gaccatggcc ttcgctgttc aggagataaa caagaaccct gacctgcttc 840
ctaatctgac tctgggatac cgtctttatg acaactgcgg agcccttgtg gttggattca 900
gtggagcgtt agcactggca agtggtcagg aggaggcgtt tgctcttcag ggaggctgtg 960
ctgggagccc accggtcctg gggatcgtgg gtgattcact ctctacgttc actattgctt 1020
ctgccagtgt tttaggcttg tacaaaatac ccatggtcag tgattagtgg cactaaagtt 1080
tgtgttttgt atgtgaatta taaactttca tcttttgaat aatattgtaa aacactggaa 1140
gaaatgtgat tggttatgta gagaaatgga tgtcatctac tgaagacata taaataatta 1200
ttacattaat ttgatcaagt tcataaatta accctttttc aaatgaaaaa gtatttttta 1260
ccaaacaaaa actgcaaaat gaacttaaat cttagttaaa tttagtgtaa aagtgatact 1320
cacgcttcgg atcagagtac agacgcagac agctaatctt tggagaaaat ctaagtgcac 1380
tgagatttaa aatttttgtt taaacattca aatgtctttt tcttttcttt tctgttttta 1440
aaaacgttga tctctcttac acagttaaat ctgttaaaat aaaagaataa tttcatgttc 1500
atgctgagtg ctccggaaat ctttaatgct cgaaaacata tttaaaacat ttgggcttgt 1560
gctgtttaat ttcctatttt taggtaagct attttgctac atgttcctgt ctgaccaatc 1620
gtcagcgctt tccgtccttt tttagaacca tcccgagcga tgatttccag gtaaaatatt 1680
cctttcaagc tgagaatcta cagagattaa aaaaaactcc ccaccctcaa attcaaacaa 1740
atattactta gtatcagatt acaaactgtg ggaactaaaa cactcaattg tctggactga 1800
tcccccattc aggtacgtgc aatgatccag atcctgaaac actttggatg gacttgggtg 1860
ggactgctgg tcagtgatga cgattatggg ctccatgtgg cccgttcgtt ccagtctgac 1920
ctggtccagt cagggcaagg ttgtctggcc tacttggagg ttttgccctg ggacaattat 1980
CA 02481827 2005-10-06
129/50
ctgagtgaaa acaggaggat tgtgcacgtg ataaaggaat caacggctcg cgtgctgatg 2040
gtgtttgcac accagtccca catgattcat ctcatggagg aggtttgatg tgcacagaaa 2100
aaactgcagt gaacacgcta aagaatgcta tttctggtat taataatgat atgtacaggt 2160
ggtgagacag aaggtgacag gccttcagtg gttagcaagt gaagcttgga ctggaacgac 2220
cttcctccag acgccggact tcatgccgta cctgaatggc acgctgggca tcgccatccg 2280
tcgaggtgaa ataacaggtc tcagagattt cctcctacga atacggcctg gccagagtag 2340
caacaacact agttatgata tggtaattat ttcttactat tttaactccc agaggaactg 2400
ttcatttaaa aaataaatga tatattttaa tcttttttca ggttcaacag ttttgggaat 2460
actcattcca gtgtaaattt ggtgcgtcag gttcagcaga agcctgcaca ggagatgaaa 2520
atatccagca ggtggacgct gagtttttgg acgtgtctaa cctcagacca gagtataaca 2580
tttacaaagc tgtgtacgct ttggcgtacg cgcttgatga catgctgcag tgtgagccag 2640
ggagggggcc gttcagtgga ggcagctgcg cggatattca caaactggag ccctggcagg 2700
tgagacctga cactggaaaa taacaagcgc ttcccctttt agtgtgagcc tttgaaattt 2760
agattcaccc agtggtccct cagacctgat tactgtcaac ctccatcagc tgttcaaatt 2820
agagcaacag ggtccagaat taggattcga cctaataagc ctgatgtggt attgtgcatt 2880
tgcagttcgt gcattatcta caacatgtca atttcaccac aacgtttggc gatcaagtat 2940
catttgatga aaatggggac gtcctaccca tctatgacat cctcaactgg cagtggctcc 3000
ctgatggaag aactcaggtt cagaatgtgg gtgaggtcaa gaggtcaccg tccagaggtg 3060
aagaactcca gatccatgaa gacaagatct tctggaactt tgaatctaat aaggttattc 3120
ttgtgtgctc ctgttatctg ttaaaagaaa tgcatgaaaa tcagatgttg atttttcctc 3180
ctgcagcccc cacactcagt gtgcagtgaa agctgtcctc ctggaacccg catgtccagg 3240
aagaaaggac aacctgtctg ctgctttgac tgtcttcttt gttctgaggg gaaaatcagc 3300
aacacaacag gttggtattt gatttcaaac actattttgg gcacattaat ctcaaagctg 3360
cgtccatgtt ttgcttcaga ctccatggag tgcaccagtt gtcctgagga cttctggtcc 3420
agcccccagc gggaccactg cgttcccaag aaaactgagt tcctctccta ccatgaacct 3480
ctgggtatct gtctgacagc cgcctccttg ttgggaacag tgatcagcgt ggttgtgttg 3540
ggcatcttca tccatcatcg cagtacacct gtagtacggg ccaacaattc cgagctcagc 3600
ttcctgctcc tggtgtccct taagctgtgt ttcctctgtt ctctgctgtt cattggtcgt 3660
cccaggctgt ggacgtgcca gctgagacac gcagcattcg ggatcagctt tgtgctctgt 3720
gtctcgtgca tcctggtgaa aaccatggtg gtcctggctg tcttcagggc ctccaagcca 3780
ggagggggcg ccactctgaa gtggtttggt gccgtgcagc agagaggaac agttctgggc 3840
cttacttcaa tccaggcagc aatctgcttt gcctggctat tgtcgtcttc accaaagcca 3900
CA 02481827 2005-10-06
129/51
cataaaaaca ttcagtacca caaagacaag atcgtttttg agtgtgtcgt tgggtccaca 3960
gtgggtttcg ctgtattgct cagttacatc ggtttactgg ccatcctcag tttcctgtta 4020
gcatttcttg cacggaatct tcccgacaac ttcaatgagg ccaaactcat cactttcagc 4080
atgctgatct tttgtgctgt gtgggtggcc tttgtcccag cttacatcaa ctcaccgggc 4140
aaatatgcag atgccgtcga ggtctttgct atcctcacct ccagttttgg cctcttggtg 4200
gcgctgtttg gaccaaaatg ttatataatc ctgtttcgtc ctgagaggaa cacaaaaaga 4260
gcaataatgg cccgttgaaa tcagaaccag cacttccaaa tcacaataac aaacactcat 4320
tgaagcatca acagcaaaag tacaaatgca aaaaaaatac aattaaaatt aaagcaattc 4380
accccctagt ggttaaagat tgcactgcat tacacgtttt ccccatttcc ccacctttcc 4440
atgtcacatt aacaatgtgc actgctgcat gtattccaac atccacgggg taagataaga 4500
ccatataaga cagaatctca gtcttcattt ttgaatgtca tcaataactg attcttctat 4560
ttctccacac tttctgtttt gcttctcttc ccttctcact gtatttcttg tgctgttatg 4620
ttaaacttta ttatgatgat caatgagggc agttgagctg tataatatgc tcactcttgt 4680
tttattgtgg taaataaatt tatatttaaa tctgccacat gcttttggtc agtaaaggtt 4740
tgtgaacgtt ttttttgtcc tgcaatatag aaaaaataat atgtaataaa gatttcaagg 4800
gtcaaacaga aaggtatgac tccatctatc tgtgtggaat agaaaaataa gtaacattta 4860
agcagcaatt aaatacattt ttatcacact tcacatacgc atacaattta aaaaaacaaa 4920
acaaaattcc agcccacgtg gctgaagccc tcacgaggga tgaggaagcc tgagtgactg 4980
a 4981
<210> 34
<211> 4781
<212> DNA
<213> Fugu rubripes
<400> 34
cagccttcat aaacacaaat aggatatcct atagattggc atagtgaaat ttgaccactt 60
tcaacttgcc gtctggcatc tggagactag atccagacct gatcagtgcc aaggtcaaac 120
gctgtaacac agttaaccct tgaatagctg tctttcatgg tgggggaaac tcaaacttca 180
gtcaaacaga aaaagagaaa gttctaatca gactgatact cccttcatcc ctccactgca 240
acctggctta gcatccacag gtcaaacctt gtctttatgt tggggagggc acacacattc 300
catgcacaca gaagtacaat gtgctgattc tacatggaaa aaaattcagg aatctgacag 360
agattcagat tcagattcag attcagattc agattattga catgatgaaa aggtgataaa 420
tgttgttgtg ctaaactacc tatctatcat acctgtggcc acaaaacaat cactgcgctc 480
ttccaaatat catgacttaa agaaaactcc tcaagtatca cattttcatt tgaggtctga 540
CA 02481827 2005-10-06
129/52
catgacatga cagttctgag acattcttct cacacattca actactttca tctttaactt 600
ccaactaaac tcaatttaga ctatttacaa aacatgaact aataactcag tttgtttgtg 660
atagcggggt cattcattcc agtgcctgaa agctggctac cagttcatta attggtgacc 720
ctgaggagac aaattcagtt tatctaacca cttaatccta attaaggttt aaggagactg 780
aggcaggaaa aaaccctgga caggtcacct gtccaccagt aactgttcac tatcaactag 840
ggactattaa tcatcactga acctaatatg catgttttta tgctgtggaa gcaagggggg 900
acagagagaa cgcaaacttc gcgcagaaat gccagagtct ggagccctga atgctcctgc 960
tctgatattg gtgatcactg caccctcatg cagtctctac aatgacctcc agatgttttc 1020
aggggtttcc aaaaatattt ttttaatcca tctttatttt ctgcgatatt tacaaacaat 1080
gaatcatctc aacaaattct gcagtttttc ctcctgtagg atgaaggaaa atcttgctac 1140
ggcccaaagt tgtcccctgg gaaatgcagg atgcagagtt cttgatgcaa tagatgcaaa 1200
tctattagta agttgtcgtc atggtgatta tgtaatgaag tagaaaaagc aagttaaatg 1260
ttgaagagct gcaccctctg acatcagcaa tgagggcttt cttcttataa aaggatgtta 1320
aacattagga tggcctgcag gagtatataa ttcttctgtt ttcttctgtg agacattttt 1380
gcttacgatt gtgtgtgtca ggttccacca gatgcctgtc tgtgtctgtg taatgcttct 1440
gtttgcgctt ttccacggag cctttggagc cgaggacaat ctaaagtgta agatgttggg 1500
gagaccagag tttccactgt tatctcagga aggagacatc actattgggg gagccttcac 1560
tctccacagc caaatgtcaa aaccttctct ctcctttgaa gagacaccag aagatctcac 1620
atgctccagg taacatttat tttctactgt agtgctattg ttttaccttc agagaaccta 1680
aacctctgtt attttccttt caggattaat ttaagagaat ttcgctttgc ccaaacaatg 1740
atttttgcaa tagaggagat caacaacagc agctcgcttt tgccaaatat ttccattggt 1800
tataaagtat ttgacacgtg cggtttgacg ctgccttcta cacgagcagt aatggcctta 1860
atgaatggaa agacaagaac cccagaagga ggctgctcca gtcggacatc tgttcatgca 1920
attattggtg cttcggagtc gtcttcaacc attgtgatgc ttcaaatttc agggatattt 1980
caaataccgg tggtaataat gtgtcatatt tgtgattggt ttgcttttga ttctgtcttt 2040
ggtaaaaaaa atgaccagat tctgattcat gtattgcttt ccctcttgtt ttttcctctc 2100
tttttgttat cttttttgtt cagataagcc actttgccac ctgtgcatgc ctcagtaaca 2160
ggaaggagta cccctccttc ttcagaacca tccccagtga cttctatcag agcagagctc 2220
tggctaaact agtgaaacat ttcgggtgga cgtgggtcgg ggctgtgaaa agtgacaatg 2280
attatgggaa caatggcctg gcgacattta ttatggctgc tgagcaggaa ggggtctgtg 2340
ttgagtactc ggagggcttt tcgtggacag accccagcga gcagattgcg agggtggtca 2400
CA 02481827 2005-10-06
129/53
cagtcattaa aagtggcagc gcaagggttt tagtcgcctt ccttgcacag agtgagatgt 2460
ccgctctgct ggaggaggct gtaaagcaga acctgacggg gctgcagtgg gttgggagcg 2520
agtcctggat cacagcaggt cacctggctc ttaagaaata ctcagcaatc ctgacagggt 2580
ccctgggctt caccatcaga aagacaaaga taactggcct tcaggagttt cttttacagg 2640
ttaacccaag tcagaaccct cagaataatc ttctcaaaga attctgggag accacatttg 2700
gttgtagttt ccagtctgat gtgcacgggg ccacccagtg ctctggtgtt gagaagctaa 2760
aggacattca gaatcctttt acagatgtgt cagaacttag gatctccaac aatgtgtata 2820
aagctgtgta cgctgtggct catgctatgc atagcatgct aaaatgtggc caaagtggcg 2880
aggcagtgaa tcagtcatgt accactaaaa aggattttga gctaaagcag gtaagatgtt 2940
gtctctaaga attatgtcca gctgaatttc tttacacccc ccattgacct gttctttctg 3000
acaggttgta gagcacctgc aatcagtgaa cttcactctt cagtcaggtg aaagagtgta 3060
ttttgatgac tatggagacc cggcggctac ttatgagctg gtgaactggc agagaagccc 3120
agaaggaaat acagtatttg tggtcgttgg gaactatgac gcttcacaac caaacgggag 3180
gcagtttacc atgaacaaca tcaatataac atgggctgcc aggctgcaga aggtacgacg 3240
catgttaaaa agcattgatt tacacgctga tgtttataca ggcatgggtt aaatggcttc 3300
atctatgtat tttgtatttt cgtcttaaaa cagagaccgc tgtctgtgtg cagtcagagt 3360
tgcattccag gcttccgaca ggctgtgatt aaggggaaac ccatctgctg tttcacctgc 3420
gtcgcctgtg ctgcaggaga gatcagcaac tccagcagta agttcaagtc ttcagagcag 3480
gcaatattta tcctgaaaca atctttttta ctctcttctg cttcaagctt tattcatggc 3540
acctttgatt tttagactct gcagagtgtt tgcagtgccc actggagttc tggtcaaacg 3600
aagatcacag ccagtgcgtt ccaaaggtga tcgagttctt atcatttgaa gaaaccatgg 3660
gggcccttct tgccgccgtt tcactgtttg gggctgcgtt aacgtcgctg gtgttttgtg 3720
tcttttttcg gttccgtcac acacctcttg tcaaagccag taactccgag ctgagctttc 3780
ttcttctttt ctctctgact ctgtgcttcc tttgttccct cacattcatc gggaggccct 3840
caaggtggtc ctgcgtgctg cgacacacgg cgttcggcat cacctttgct ctctgcatgt 3900
cctgcgtcct ggcaaaaact gtggctgttt tgttcgcatt tacagcaaaa aggccaggaa 3960
acacagtttt ttactgttct gttccacttc agagaacaag tgtttttgcc tgcatcactt 4020
tgcaggttat aatttgtgtg ctctggttga cgcttgcccc accacaccct cacaaaaata 4080
ccgctcatgc caaagagcgc attattttag agtgtaattt aggttcccca gtgtggtttt 4140
gggtggtatt ggggtatatc gggctcttgg ctgtgatttg ctttatcctt gcttttcttg 4200
ctagaaagct gcctgataat tttaacgaag ctaaattcat caccttcagc atgctgatat 4260
tttgtgcagt ttgggtcact tttatcccag cgtacgtcag ctctcctggg aagttcacag 4320
CA 02481827 2005-10-06
129/54
ttgccgtgga gatctttgct atcctggcct caagttttgg gttacttttc tgtatatttg 4380
ccccaaaatg ttatatatta atattgaagc cagaaaaaaa cactaaaaaa cacatgatgg 4440
ggagaaacca tcaaaattag gctgaactga gaattgtatc acttaaataa cagtaatttt 4500
gttctttaat atttaagtcc agaaagtgtt ttattaatac agagcattat gtaagtgcac 4560
atatataaaa tgcaccttat gtcagtttga ttttgggatg gaattgactc acatgttaat 4620
gtcatttatc gattaaagct acaataaagt taaactacgt ggacagttgt ttaatagttt 4680
ccctgtcaag tattttaaaa gcaacaattg catgtaaaag caacacagct gtaataggcc 4740
cactcgtctg tcactcacag cagtgaggtg atgccattgg a 4781
<210> 35
<211> 4846
<212> DNA
<213> Fugu rubripes
<400> 35
gatccaaact tgcgacaggg atttgtgcca gtatatcaac gccagaacgg cgtctggtat 60
ttgacagtta aggctgtgca gttgtgggag aaagtagatg ctttttaact gtttgctcta 120
gaatttgtgg acacttttag tcttaatgga tgtataatca atttcctaat ttctcttgaa 180
tctggcatat tgcaaacatt tgttgatcac cacaaacaca cacacacaca cacacgcaca 240
cacacacaca cacacacaca cacacataca cacacactac aaatctacga acttgagctg 300
cagcagtggg tgcacacgtt tgtattgggc tgtgagtctg ttcacagaat gagttggatg 360
ggatggattc cctcacaaag aggtgtacaa cttctctgcc ttctgtgctg catgataatt 420
cctgttgtca ttgccctcct ggatcagagc caacattgca gggtcatccc gggctccatg 480
tccctgccag tgctggagaa gaggggggac atcattttag gcggtctctt ctctcttcac 540
gacatggtgg tagagccaaa tctgcccttc acctctacac caccacccac tcagtgcact 600
aggtaaggta caataatccc ttctacactt ttattcatgt gtaaatttta agttttttgc 660
tgtaaatata gcagtatgca caattccttg tctcttactg acaagtcagc atcctgtttt 720
tcatttcttc ttcccagatt cagttttcgg acattccgtt ggatgcaaac catgatcttt 780
gccgtcgagg agatcaacag aaatgccgaa atccttccca atatcacact gggctacaag 840
atctacgact cgtgcagcac gccccatcag agtttgaagg ctgccatcga tttaatgggg 900
agtgagaaag attcccagtt tgagggaaag ttgcagaggg aaggctgcga tgggaacgtg 960
ccggccgtca tcggagacgg aggatctact cagtctctcg tcgtggcccg tttccttggg 1020
gtctttcatg taccgcaggt atctcgcatt gctgatgctt caaagggaac tcttctctca 1080
tgctagaaat catttgtttg ctagggttca cttaaaagca attatcactt atttttggtc 1140
gtgtcaggtc agttattttt ccagctgtgc ctgtctcagc gacaaaacgc agttcccagc 1200
CA 02481827 2005-10-06
129/55
ctttttaagg acgatgccca gcgatctctt tcaggtttga ggcagcagcc ttaaaacttt 1260
tttttttaaa tttaaagcta tgagagaacc ttcatctcct tcatttctag gtgggtgccc 1320
ttgtacagct cgtcaagtat ttcggctgga cttgggtcgg agtgattgca ggagacgacg 1380
cttacggccg tggcggagcg gccatattcg ctaatgaggt agtaatgaga acaaatggac 1440
atttcaatgc tttattgagc atcgtgcggg cactgaaaat taggattaat ggcatgtctt 1500
ctatctgggg taggtgagaa ggctcggggc ttgcattgct ctctatgaga tgatccccaa 1560
gacacaatca caggctgcca tttcatctat catttccaac atccgctctt ccggggctcg 1620
tgtagttctg gtgtttgctg ttgagcaaga cgtggccagg ttgttcgatg aggcagtcag 1680
gtatggcacc tgtgtatccg caaacgtaca aaaacaactc taagctattt agcttctcct 1740
aaatatagac agaagctgac tgggattcag tggttagcca gtgaggcgtg gagcacagct 1800
gccatcctct ccacccctaa aaggtaccac cacatcctgc aggggtctat gggatttgct 1860
attcggagag cggacatccc tggactgcaa gacttcctcc ttcgcttgca tccctcgagc 1920
gccgaggctg atgacgatcc tttcctgatc ccattctggg aggaggtgtt tcagtgtagc 1980
ctggatccgc acggccactc tgaggctaaa cgtccctgct ctgggacaga ggagcttagg 2040
agcgtgaaga acatctattc tgatgtatca cagcttagga tttcctacaa tgtctacaag 2100
gcggtttatg cccttgccta cgccatcaaa gccatgagaa gttgtgaaaa aggaagtggg 2160
ccattttctc aacaagcctg ccctgattta gacaatatcc acccgtggca ggtatgcaat 2220
taacaaaatt ccatgtgcag aatgacttgt gatgttggaa tcgaatcttc cgctcttttt 2280
tcgcagcttc accactacat aaaacaagtg aactacacga acagattcgg cgatgagatt 2340
aaatttgatg aaaacgggga tcccgccgcc atgtatgact tgattaactg gcagctgacg 2400
ccgggcgggg acatggactt cgtcaccgtt gggaaatttg atgacatagc cgggaccgga 2460
aggaagaacc tccacattga ggaagagaag atcgtgtgga atggcaacaa cacccaagta 2520
ggagctgtca ataaaatgct tcaacactca cgggtgactc agagatgtat ccttatgtga 2580
ccgtgtgctg tactctagtt tcctcttttg ctcaggttcc cttatcagta tgcagtagca 2640
tctgtccccc ggggacccgc aaggcaatcc gacctaatta ccctatctgc tgccatgact 2700
gtgtggtttg tacagcaggg gagattagca atcaaacagg tgagaagagg aggagaatgg 2760
tcacagtttt actcttccag acctctgtcc ataagccagt gaagtggatt tttgataaaa 2820
tgttccttta cgttttcctc tttttctgca gatgccatag aatgtgcccg ctgcctgccg 2880
gagttctggt ccaatgctga caggacagcc tgtgtcccca aacaagtgga gttcctctcc 2940
ttcggtgaca caataggcat tgctctgttg gttgtttccc tgatcggctc cttccttacc 3000
tgcgccgtgg ccctcgtatt cttctatcac aggacctccc ccatcgtcag agccaacaac 3060
CA 02481827 2005-10-06
129/56
tctgacctga gcttcctgct cctcttctct ctgactctgt gcttcctgtg ctctctgacc 3120
ttcatcagcc caccctccca gtggtcctgc atgctgcgac acacagcctt cgggatcacg 3180
tttgtcctct gcatctcgtg cattctgggg aaaacaattg tggtcttaat ggcgttcaga 3240
gccacacttc ctggtagtga tgtgatgaaa tggttcggac cagggaagca aaaggcaatt 3300
atcactttca gcacactggt ccaggtaagg gcttctttta agttgtctca aattgttacg 3360
gttgcatgta ttgaatgtaa catttctgat catttcctct tttgttgcag gttgttattt 3420
gcacggtgtg gctggttgtt gctcctccca ccccacgaca gtacatgcca cgtgaaagtg 3480
ctatcatcat tctcttatgt gacgaaggct caaccatagc cttctccctt gttttgggat 3540
acattggcgt tctggcctgt atgtgtttcc tcctagcctt cctggcgaga aaactgccag 3600
acaatttcaa cgaggccaga ctaatcgcct tcagcatgct cattttttgt gcagtctggg 3660
tggcctttgt cccagcttat atcagctctc cagggaaata ctccacgctc acggaaatct 3720
ttgccatctt ggcctccagt tacggactgc tgggctgcat ctttgcaccc aagtgctata 3780
taattctcat gaagtcagaa aagaacacaa ggaaacactt gatgtcaaaa agtgaaagat 3840
tttagagata tattattgct tgcgttgctg tgggttttga aatgaaatgg ggggacaact 3900
ttaaacgtcc gcgtcatgga tgaagtgtct cttttggcca ccagagggaa gtgttgtgct 3960
gctcatgcaa cactggttca ctgcttatac tggaaaaagc acaaaaagaa catgtattgt 4020
ttccacatac tacagtgtgt tcccgataaa acagtaaaat atatggttag cttataaatt 4080
catatagcaa tcatcccatt aatatattga agtgctcatt gtcattaaag tgcaatattc 4140
tcagtgacag tgtgaaggac agcttttttt ttatgacagt gggatttggc agcattttca 4200
gtgctgatat ggcttgaggt ggaatgaggc cctgaggtgt attctgtgga aatcccagct 4260
gttaaattaa atccatacac cccgacggcc ctttcacata aacataaggc cctggctgat 4320
tccctataat aacaaactaa aacgatatat ttcccgccct ccacagctgc tctcaggact 4380
gtatcgaccg ccttccgccg attgatctgt tgtccctccc gaagctgagg agggattgga 4440
aattgaaaga tggggagtca tctctgctgt ccatcaggaa atggcatatg agcacaatct 4500
atttacattc acttcttacc gtcattgttc ttaaatgtcc cactacagcc atctgtgccc 4560
tccccctctc cccatctcaa aagggattat gacaaagtga atttcccttc atagcatgtc 4620
catttcagga gtccttcagt ggctttgctg ttggtttgat tttaattaag gtgaacagga 4680
gagttcacct cagagggttt aatcatcgag tattagaaac acagtcaaag aaaagagtac 4740
aaaagagtca aatggctttc aaataacaat tcacaggaaa catatcagca gaaagtagag 4800
agttaaacaa aggccacgac ttttttagca cattaaaccc aataga 4846
<210> 36
<211> 4743
CA 02481827 2005-10-06
129/57
<212> DNA
<213> Fugu rubripes
<400> 36
ggacaggggt caacatgggc accctgactg gtctgtaact ggtccccatt acactgaaat 60
ctggcatcat gttgattatt acgatccagc tgtaaagcag ctgacggctc agaagatgaa 120
gtctgattgt tgatgtctga tgttccttct tagcccagtt tagctggtgt agtggtgaac 180
agatccctgt actccagggg aggggcccca tgtttctgga actggaccct gatcactgtg 240
actggtgggc tctatttttg cactaaataa taaatttaaa ggctgtatta cttttattta 300
ttattaaagt ttgatcttcc accatgatca taaaaaactg tatcacatga gatcagtgtg 360
aatttacaat gtggtttgaa aaacaattta atgaggcatc ttcataacga tcccatgatg 420
aaacaacaat gtgctgaggg gggcctggag gggccggagc ctgacaggcc ttctgggata 480
cgcctggtca ctgccctcca ggtgaacgct gcagtccctc tttcctctct ctctctctca 540
cacatgcaca cacatgaaca catgcacaca aatcactcag tcatttcatt attgtttttc 600
ttttgccttt gagagcggta atttgatgct gtgcagttct gtttactgcc acccctccct 660
gggctttaca gcagttataa atatcagcgc aggcctgctg ggcagtcata tgttcgctcc 720
gtgtcatgtg taaggagtga tgcgttgcag cgccgtcagg acatgcctgc gctccatact 780
ttgcctctgt cgtgatctgc tgtattggtt tgtttgagca aatgtaactt tccatttcca 840
ggctgttgtc tggtcattcc gtcggcgcga ggtgggctga acagcatcat ccatgtctca 900
gttatcacgg atcttcacct tgattgtggg cttcggtggc agggagctgg gactcggggg 960
ggtcttacag gtagttcagg ctctgacttg ttctcagtgg tccacaccaa ctgaacaggg 1020
cctcttccag gatgggcacg tggttgtcgg tgggctcttt aaccttcatt acacgcctcc 1080
agacacagcc aacaacttca ctcagcaatc gcattacaaa gcttgcaccg ggtacagctg 1140
caatctttca cgtcatttgt gtgaactgtt gtgacctttt cccctccaaa tccaatgttt 1200
acacttggat tctcctcatt cttggcttca ggctggaaaa cctccctttg cagtacattt 1260
acgccatggt gtttgcagtg gaggagatca atcacagcgc agcgctgctg ccaggcgtga 1320
agctcgggta ccacattcgc gatagctgcg ccctccaccc ctggaccact caggcggcgc 1380
tggcactggt cgcaggagac agtgccagct gtgaattggc aaccccagcg gactactctg 1440
cagagacgag tgaagaaaaa ggtactgatt aagccttgca atacctgatg aactcaaact 1500
tgatttaaaa ttgtaatttt taaaacaatt taggtgctgc ctccgttcct ttgattattg 1560
gtggcgcttc ttccaatgca gcaaaaatac tcctgggaac cctgagtcca ctatctgtac 1620
ctttagtgag ttttccgtcc tttcagttta tataaatcat atgacatgtt tttaaagcac 1680
acatgagcct gcggatgatg taattggatt ctatttataa tataataata ataataaagc 1740
tttaaggatc tttgtggttg ttcttccaga taagctacac agctagctgc ccttgcctga 1800
CA 02481827 2005-10-06
129/58
gtgacaggca ccgatatccc accttcttca ggaccatggc cagtgatatt taccaggctc 1860
aggctctcgc ccagcttgtg ttacgcttca actggacgtg gatcggggca gtggtggcaa 1920
acaatgatta cggtcatgtg gcagttaagg tgaaatattc tggctaagca tgcaagacat 1980
tctcctgtgg ataatcctac atgcacagct gcggtgtgtt tatctgtgta cgtcaggtgt 2040
ttcaagagca gactcagggg aaaggtgtgt gtctggcgtt tgtcgagact ctccagaggg 2100
agacgatcgt ggcagacgcc gtgcgtgcgg cgcgcacaat tcaggcctcg actgcgaggg 2160
tgattctggt ttttagctgg tacactgacg tagggcatct cttccgtcag ctgcagaaaa 2220
taaacgtgag aagttctgtg cagggatctg agtgtctaat cagtcagagg agttctgatc 2280
tttgaacgct gcctccgctg caggtgaccg acagacagtt tctggccagc gaggcctgga 2340
gcaccagtga ggttcttctc aaagatcctg acacttctac agtggcgagt ggagtcgtcg 2400
gcgtggccat cgcaagccaa cacatccctg ggtttgaccg tttcctcaga ggcctgaacc 2460
cgtctcttcg gcccagcgac aagtttttac aagaattctg ggaggaggaa tttggctgca 2520
gcccctcgcc tccttcttca gagacctctg gtgatttgaa cgcttcgctg cctccctgca 2580
gtggtgcaga gtctctggag ggagtgcagc atcccttcac cgatacctca cacctgaggg 2640
tgacgtataa cgtctacctg gctgtatatg ctgcggccaa cgcccttcac agccttctct 2700
cttgccccat tcataacagc ccttctggaa cttctcactg cacctccccg aagggcatta 2760
aaacaacaga ggtaaagaaa ataatacaca gatcaaataa acgcagtaaa atcacaaaat 2820
aataataagt ggcaacaccg tcgtgtttac agctgctgca acacttgagc aaagtgaatt 2880
tcaccacgcc gcagggcaaa cacttgtact tccgaggtgc agatattccg gcaatgtacg 2940
acctcattaa ctggcagagt ggcacagacg ggaccctcca gctcgtcctc atcggtgccg 3000
tggctggatt tgacctgcag ctcaatgagt cagaaattga gtggagcgcc aaatataatc 3060
aggtgattca gaggaagcat ctcaatcaca aaaatctgga tacatggaaa cgtatttaat 3120
atgctggggg tgtgcatgtc taggtgcctg tgtcagtgtg cagtgagagc tgcccccccg 3180
gcaccagaaa ggccaacagg aagggagaac ctctctgctg cttcgactgt atcccttgtg 3240
ctgacggcga gattagcaac acaagcggtg agggaaatat cacagcagct tctttctgtt 3300
ctgaactttg ggggtttgaa cgtctgactg tcactgttta ggttctcttc agtgtgaccg 3360
ttgccctcct gagttctggt ccaacgatgg acggactgct tgtgttcctc gacagctgga 3420
ttttctgtcc tttaatgaaa ccttgggcgt tgctctgacc gccgtggccg tgtccggcgc 3480
tgtggtgaca acagccgtgt ttgtggtgtt ccttcactat cgtcacacgc ccatggtgag 3540
aactcagagt cacgtcctgg aaaagaaacc actgaatctg tttaatcagt tcatcctaat 3600
ttccattgga aggttcgagc caacaactct gaattgagct tcctgctgct gctgtcactc 3660
CA 02481827 2005-10-06
129/59
aagctgtgtt tcctgtgctc gctggtgttc atcggtcgtc cgtccgtctg gtcctgtcgg 3720
ttccagcagg cggcttttgg gatcagcttc gtgctttgtg tttcctgcct ccaagtcaag 3780
actatagtgg ttctggcagc cttccgctcg gcccggccgg gcgcgggggc cctaatgaag 3840
tggttcggcc cgtcccaaca gagaggaagc gtttgcatct ttacttgtgt acaggcaaga 3900
gttacagatg gattaagaga aaacattgtt tttttaatgt cagtatgatt tatgtgatgt 3960
tatatgagtg tttgttttca cagtgttgag ctgatgttgc acaatgcttt ctttcatcaa 4020
ggttatcatc tgtattgttt ggctgtcact gagcccccca gtgccccaag ctgacttgga 4080
tgtgccgggc ttacaggtca ccctggagtg cgccatggcc tccgtggtgg gcttctctct 4140
ggtcctgggc tacatcggtc tgctggcctg cacctgcctc ctgttggcgt tcctggctcg 4200
gaaactcccc gacaacttca acgaggccaa gctgatcacc ttcagcatgc tgatcttctg 4260
cgccgtctgg gtggccttcg tccccgctta catcagctct cccggaaaat actcagttgc 4320
cgtggaaata tttgccatcc tggcttccag ttatggcctg ctcttctgta tctttgctcc 4380
aaagtgtttc atcatcctgt tgagacccga gaaaaacaca aagaaacacc tgatgatgcg 4440
atagcaaaca aaatatgaga tttatattcc ttttttagaa atatgcagtc ttaatatctg 4500
ctgctgtgtg ttatttatgc atctgcatca ataaaacgct gaaagaaatt gtaaaacaag 4560
taaaaacatt ctgattctta tttgctttca ttggctattt agaaaaaggt aatgcattat 4620
gggacattta aagcacaggg ttaataaaaa atatactata tcatgtgtct gttgtgtgta 4680
tattctaatg cagtttgatt atttattcat tatggattcc ttatcatgcc acctcaacag 4740
atc 4743
<210> 37
<211> 469
<212> DNA
<213> Fugu rubripes
<400> 37
catcatcctc gctggaatct tcctgggcta tatttgtccc ttcaccctca tcgcccgccc 60
cactgtagct tcctgctacc tccagaggct cctcgtgggg ctctccgctg ccatatgcta 120
ctctgccctt gtcaccaaaa ccaatcgcat cgctcgtatt ctggcaggca gcaaaaagaa 180
gatttgcacc aggaaaccga ggttcatgag cgcctgggct caggtggtca tcgcctttat 240
cctgatcagt ctacagctca gtctggaggt caccctcatc gtcctggagc ctcctgaacc 300
catcaaatcg caccccagca taagagaggt tttcctcatt tgcaacacca gcaacatggg 360
cgtcgtggcg ccgctcggat acaacggcct gcttataatg agctgcacct actacgcctt 420
caagacgcgc aatgtacctg ccaactttaa cgaagccaaa tacatcgcc 469
<210> 38
CA 02481827 2005-10-06
129/60
, 4
<211> 318
<212> DNA
<213> Fugu rubripes
<400> 38
gcccgcatct tcagcggcgt gaaagatgga gcccagcgac cgcgcttcat cagccccacc
60
tcccagttag ccatctgcgg cgctcttatc tcctgccagt tgctggtggc gctgatctgg
120
ttaatggtgg aggtgcccgg ggtccgcaag gaagtgagct cggagcggag gaacgtggtc
180
atcctcaagt gcaacagcaa ggacagcagc atgctgatgt cgctgaccta caactgcgtc
240
ctcatcatcc tctgcaccgt ctacgcgttc aagacgcgaa agtgccccga gaacttcaac
300
gaggccaagt tcatcggc
318
<210> 39
<211> 559
<212> DNA
<213> Fugu rubripes
<220>
<221> misc_feature
<222> (528)..(535)
<223> n is a, c, g, or t
<400> 39
cgtgttgctt acgggcattt tcctcatcta cctcatcacc ttcctcatga tcgccgagcc
60
aagtgtggct gtgtgtgcct tccgcaggct gtttctgggg ctcggcatgt gcatcagcta
120
ctcggccatg ctcaccaaga ccaaccggat ctaccggatc tttgagcagg gcaaaaagtc
180
agtcacgccc ccgaaattta tcagccccac ctcccagctg attatcacct tcatactcat
240
ctcagtgcag gtatacatgc gcattcacgt gcacgatctg ctgcgaagcc tctgctaacc
300
ctagcccccg tgctccttct cgcagcttct cggggtcttc atctggtttg gcgtgatgcc
360
tccgcacacc atcatcgact acgaagagca gaagcccccg aatccggagt ttgcccgcgg
420
agtcctgaag tgcgacatgt ctgacctctc cctcatctta tgtctgagct acagtctggt
480
gctgatgatc acctgcacgg tgtacgccat caagaacaga agggtccnnn nnnnnttcaa
540
cgaagccaag cccatcggc
559
<210> 40
<211> 499
<212> DNA
<213> Fugu rubripes
<220>
<221> misc_feature
<222> (85)..(85)
<223> n is a, c, g, or t
<400> 40
CA 02481827 2005-10-06
129/61
1
taccgcatct tcgagcaggg caagaggtcg gtgaccccgc ccaagttcat cagccccact 60
tcccagctca tcatcacctt cgtgntgatc tctgtgcagg tgagcctggt ggcctgctcc
120
cacttcctgc ccgcctgaag gggctttttt tgaccttcgg ctggtgtttg tgtgcttcag
180
gtcttcggcg tgttcgtgtg gtttgctgtg gtccctcctc acaccatcat cgattacgag
240
gaactgcgtc ctccccagcc caacctggcc cggggcatcc tgaagtgcga catgtccgac
300
ctgtccatca tctgctgcct cagctacagc atcgtgctca tggtaacaca gcgaccttca
360
gccacgtcaa ctcccgctct tgtgaacgat aacaggaaat aagcgtggcg tgtcggtgtg
420
ttgctaggtg acgtgtacgg tgtacgccgt gaagagccgc ggcgttccag agacgtttaa
480
cgaagccaag cccatcggg
499
