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thesis entitled

Isolation and Characterization of Atlantic Salmon
Salmo salar, Olfactory Receptor Genes

presented by

Jill Elizabeth Thorpe

has been accepted towards fulfillment
of the requirements for

M.S. degree in Fish. & Wildl.

 

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ISOLATION AND CHARACTERIZATION OF
ATLANTIC SALMON, SALMO SALAR, OLFACTORY RECEPTOR GENES

By

Jill Elizabeth Thorpe

A THESIS

Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of

MASTER'S OF SCIENCE

DEPARTMENT OF FISHERIES AND WEDLIFE

2000

ABSTRACT

ISOLATION AND CHARACTERIZATION OF
ATLANTIC SALMON, SALMO SALAR, OLFACTORY RECEPTOR GENES

By

JILL ELIZABETH THORPE

Olfactory cues mediate Atlantic salmon behavior during different life history stages. To
obtain an understanding of mechanisms influencing salmon behavior, we have addressed
olfactory discrimination at the molecular level by cloning and characterizing 8 olfactory
receptor gene fragments fiom the olfactory epithelium of the Atlantic salmon, Salmo
salar. Gene fragments were extracted from salmon representing two life stages, 4 genes
from mature parr and 4 genes from smolt. Gene sequence and structural analysis reveals
shared motifs with published putative odorant and pheromone receptors of other
vertebrates. Reverse Transcriptase-Polymerase Chain Reaction performed on the
olfactory tissues of mature pan' and smolt yielded gene products. No products were
amplified from non-olfactory tissues. Phylogenetic and Southern blot analysis suggest the
8 salmon gene fragments represent four distinct subfamilies. Northern blot assays
indicate that gene fragments from smolt are expressed solely in the olfactory organ of
smolts, and that mature parr OR genes are primarily expressed in the tissues of mature
parr. The findings represent the first identification of olfactory receptor genes in the
Salmonidae family and demonstrate differences in OR gene expression between the
mature parr and smolt. The results provide a foundation for understanding olfaction and

olfactory mediated behaviors in the mature parr and smolt characteristic life histories.

DEDICATION

To Ken, my family, and Christine for all of their love and support.

iii

ACKNOWLEDGMENTS

I am grateful to Scot Libants for guidance and mentorship through the molecular
biological experiments. I would like to express gratitude and appreciation to Dr. Weiming
Li for continuous support, assistance with the current work, and the opportunity to pursue
a graduate degree. I thank Dr. Kim Scribner and Dr. Tim Zacharewski for expert
technical assistance. I want to thank Dr. Steve McCormick and staff at the USGS Conti
Anadromous Fish Lab, Turners F ails, MA for assistance in obtaining wild fish, and Roger
Greil at Lake Superior State University for providing hatchery salmon. I wish to thank
Kenneth Oswald for assistance with phylogenetic analysis. The work was supported by a
grant from the United States Geological Survey (U SGS) administered through the USGS

Conti Anadromous Fish Lab and Monell Chemical Senses Center.

iv

TABLE OF CONTENTS

CHAPTER ONE: A Brief Review of Olfactory Receptor Genes .................................. 1
CHAPTER TWO: Isolation and Characterization of Atlantic Salmon, Salmo salar, ..... 7
Olfactory Receptor Genes.
INTRODUCTION .............................................................................................. 7
MATERIALS AND METHODS ....................................................................... 10

Isolation of Nucleic Acids
First Strand cDNA Synthesis
PCR and RT -PCR

Cloning and sequencing
Sequence Analysis

RACE experiments

dsDNA Probe Synthesis
Southern Blotting

Northern Blotting

RESULTS ........................................................................................................... 16
DISCUSSION ..................................................................................................... l 9

REFERENCES ................................................................................................................ 34

LIST OF TABLES

Table 1. Pearson Distance Matrix

vi

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5:
Figure 6:

Figure 7:

LIST OF FIGURES

Electrophoresis Analysis of PCR products using VNO primers

Electrophoresis Analysis of S'Rapid Amplification of cDNA Ends (RACE)
using cDNA fi'om smolt olfactory organ RNA

Gene Sequence Alignments

Comparison of Deduced Amino Acid Sequences of Atlantic salmon,
Goldfish, and Fugu cDNA's

Phylogenetic Tree of Gene Sequences
Southern Blot Analysis with two Atlantic Salmon cDNA's

Expression of Olfactory Receptor RNAs in Atlantic salmon tissues

vii

CHAPTER ONE: A Brief Review of Olfactory Receptor Genes

Olfactory systems of vertebrates detect and differentiate a large variety of
odorants. The molecular mechanisms leading to the versatility and specificity of
olfactory function and discrimination are poorly understood. Odor recognition begins
when chemical cues interact with specialized, peripheral receptors expressed within the
sensory epithelia (Lancet 1986). This primary discrimination mechanism may be
elucidated using molecular cloning techniques to characterize olfactory receptor (OR)
genes. The intronless, olfactory receptor genes represent the largest family of 7
transmembrane domain (TMD), G-Protein coupled receptors (Buck and Axel 1991).
Excellent reviews on the recent developments of OR genes are provided by Mombaerts
(1999), Hildebrand and Shepard (1997), and Buck (1996). Although significant advances
in understanding the transduction mechanisms, characterization, and genetic composition
of olfactory receptors have emerged (Bargrnann et al. 1993; Barth et a1. 1997; Ranting et
a1. 1993), questions regarding olfactory evolution, odorant recognition, and olfactory
impact on physiology and behavior have yet to be answered.

Molecular research presents an approach to deciphering olfactory function.
Identification of olfactory receptor genes provides insight into the evolution of stimulus
discrimination, reinforces the understanding of inter-species relationships, and confirms
the olfactory receptor 7 transmembrane domain characteristic (F reitag et a1. 1998; Buck
and Axel 1991; Ngai et a1. 1993; Byrd et a1. 1996). In addition, characterization of OR
genes allows the identification of functional domains, offers the opportunity to associate
a particular gene with a specific odorant molecule, and finthers an understanding of

olfactory mediated behaviors.

In 1991, Buck and Axel's study on rat olfactory receptor genes gave great impetus
to the genetic basis of olfactory research (reference thereafter). The investigators
reported the presence of a large, multigene family encoding hundreds of putative
olfactory receptor proteins. The findings were based on three assumptions. First, OR's
have the characteristic of seven transmembrane domain, G-protein coupled receptors.
Secondly, olfactory receptor genes may represent a multi-gene family due to the olfactory
system's ability to discriminate a wide range of chemical structures. Finally, OR gene
expression should be localized to sensory epithelium. Using degenerate primers prepared
from transmembrane domains I], III, and VII, cDNA from rat olfactory epithelia was
subjected to the polymerase chain reaction (PCR). Amplified PCR products,
approximately 600-1300 base pairs in length, were cloned and sequenced. These DNA
fragments were treated with restriction enzymes. The restriction digest revealed that the
sum of the fi'agment lengths exceeded that of the original PCR product suggesting the
presence of a gene family. Sequence analysis of the cDNA clones implied introns were
not contained within the OR gene coding region. Intronless genes consist only of coding
regions and can not be differentially spliced. The deduced amino acid residues consisted
of seven hydrophobic domains, supporting the 7 transmembrane domain characteristic.
Unlike other 7 TMD proteins that exhibit 80% sequence conservation, olfactory proteins
show considerable sequence divergence within the III, IV, and V domains. These variant
domains may reflect binding sites for different odorant structures. Northern blot assays
indicated that the members of the multi-gene family are expressed solely in the olfactory
epithelia of the rat. Lastly, Buck and Axel (1991) estimated that the rat genome contains

500-1000 OR genes, corresponding to 0.8-1.6% of the 60,000 or so mammalian genes.

Recent research indicates that the OR gene family consists of smaller sub-families
as suggested by sequence and Southern blot analysis (N gai et al. 1993, Ressler et a1.
1993; Berghard and Dryer 1998). OR genes contain variant sequences and share
sequence conservation with a sub group of related genes. Therefore, subfamilies exhibit a
greater sequence similarity amongst themselves than with other gene subfamilies.
Subfamily definition may be dependent upon» researcher discretion. Buck and Axel
considered related gene sequences that share 91% identity to be of the same subfamily,
whereas Barth, et a1. (1997) set the criterion at 60%. Southern blot assays provide
additional support of the subfamily characteristic. Southern analysis of an organism's OR
genes reveal a number of hybridization bands that do not appear to cross hybridize. The
degree of cross hybridization may be related to the degree of gene similarity. Thus,
variations in Southern assay hybridization patterns may suggest the presence of
subfamilies. The significance of subfamilies may be inherent to their structural and
functional properties. It has been proposed that genes possessing similar structural
properties bind compounds with a specific structural motif (Kobilka et a1. 1988).

Since the discovery of OR genes, researchers have employed similar approaches
to identify receptor genes that may detect pheromones (Ryba et a1. 1997; Cao et al. 1998).
It is hypothesized some vertebrates maintain a vomeronasal organ (VNO), an accessory
olfactory organ, to detect pheromones and odorants involved in foraging and reproductive
behaviors (Eisthen 1997). Tetrapods appear to possess the VNO (Doving and Trotier
1998), while fish do not. The presence and function of the VNO is still under
investigation. Therefore, for simplicity purposes, all receptors capable of detecting

chemical stimuli will be referred to as olfactory receptors.

OR genes have been cloned fiom various vertebrates, including rats, mice, dogs,
cows, pigs, chicken, river lamprey, chimpanzees, orangutans, goldfish, zebrafish, catfish,
and fi'ogs (Cf. Buck and Axel 1991; Ngai et a1. 1993; Byrd et a1. 1996; Blache et a1.
1998; Mombaerts 1999). The cloning strategies of Buck and Axel have continued to be
used, but the primer sequences have been refined to reflect changes in similarity and
degeneracy across species. Confirmation of OR genes is supported by sequence
similarity to known OR genes, Northern blot analysis, and in situ hybridization
experiments. 1

Subsequent research has provided information on spatial expression patterns of
OR genes. In situ hybridization experiments on rats and mice reveal specific OR genes
correspond to distinct zones in the olfactory epithelia (Ressler et a1. 1993; Vassar et a1.
1993; Barth et al. 1997). Topographical patterns appear to consist of three (Ressler et a1.
1993) or four (Vassar et al. 1993) expression zones. A distinctive pattern of odorant
receptor expression on olfactory sensory neurons has yet to be firmly established in fish
(Byrd et a1. 1996; Ngai et al. 1993a; Weth et a1. 1996). Researchers suggest zones may
correspond to distinct subfamilies involved in the detection of particular odorants.

In addition, olfactory receptor genes are not isolated to the olfactory epithelium.
OR gene expression has been documented in the testis of rat, mouse, human, and dog
(Parrnentier et a1. 1992; Mombaerts 1999). The presence of OR's in reproductive organs
implies a link between olfaction and reproduction. Ultimately, isolating olfactory
receptor genes will be an important component in comprehending the impact of olfaction

on an organism’s behavior and reproductive physiology.

Our approach to comprehending olfactory stimulus discrimination involves the
molecular characterization of OR genes and their expression in the olfactory organ of the
Atlantic salmon, Salmo salar. Atlantic salmon offer an efficient model for studying
olfactory function at the molecular level. In comparison to tetrapods, fish possess a
smaller repertoire of OR genes (N gai et a1. 1996; Barth et al.1997). Additionally,
information on salmon behavior, olfactory anatomy, and physiology is readily available
(Hasler and Scholz 1978; Hara 1994). The olfactory sense is vital to the survival and
behavior of salmon. In addition to migratory behavior and spawning stream selection
(Hasler and Scholz 1978), chemical cues are critical to sexual maturation, reproductive
behavior, and food recognition (Moore and Scott 1991; Moore and Waring 1996;
Sorensen et a1. 1988; Meams 1985). Therefore, investigating salmon olfactory function
at the molecular level presents an opportunity to understand the role of chemical cues in
mediating salmonid behaviors.

Olfaction plays a critical role of sensory discrimination in migratory behavior.
Salmon, an anadromous fish, spend early life stages, as alevin, fry, and parr, in fi'esh
water. After spending one to two years in the natal stream, some fish undergo
smoltification in the spring and migrate to sea in order to feed and grow. When the fish
are sexually mature and capable of reproducing, the salmon undergo upstream migration
to spawn. The salmon are directed to their natal streams by chemical cues (Sutterfin et a1.
1973; Hasler and Scholz 1978). Understanding the role of olfactory receptors in this
process may lead to ascertaining the specific odorants that guide a fish to its place of

birth. In addition to being the first documentation of odorant receptor gene sequences in

a sahnonid species, identifying salmon OR genes will provide a basis for determining
stimuli that mediate salmon migration.

Furthermore, salmon exhibit high sensitivity to odorants during specific months
of the year and particular periods of physiological development (Moore & Scott 1991;
Dittrnan et a1. 1997). By screening tissues of sexually mature and immature salmon for
OR genes, differences in patterns of gene expression between two stages of maturity can
be deciphered. The relationship and interaction between the reproductive and olfactory
sensory systems may be linked as implied by OR gene expression in the sex organs of
vertebrates. Overall, cloning salmon olfactory receptor genes will help advance
knowledge of olfactory reception and provide a basis for further studies in olfactory

function.

CHAPTER TWO: Isolation and Characterization of Atlantic salmon, Salmo salar,
Olfactory Receptor Genes

 

INTRODUCTION

Olfaction plays a primary role in the survival and life history of Atlantic salmon,
Salmo salar. Chemical cues are critical to migratory behavior, spawning stream
selection, sexual maturation, reproductive behavior, and food recognition (Hasler et al.
1978; Moore and Scott 1991; Moore and Waring, 1996; Meams 1985). Although the
electrical properties and signal transduction cascades of Pacific salmon olfactory receptor
cells have been elucidated (N evitt and Moody 1992; Nevitt 1987; Dittrnan et al. 1997),
knowledge of the genetic sequence of Pacific and Atlantic salmon olfactory receptors is
minimal. Furthermore, the molecular mechanisms whereby odorants mediate
characteristic behavior of salmon life histories have yet to be established.

Atlantic salmon provide an efficient model for investigating olfactory firnction at
the molecular level. In comparison to tetrapods, fish possess a smaller repertoire of OR
genes (N gai et al. 1996; Barth et al. 1997). Furthermore, due to its large role in
aquaculture industry (Anderson 1997), commercial and recreational importance (Myers
1984), and suitability as a model in behavioral and ecological studies, the Atlantic salmon
species is intensely studied. For these reasons, information on salmon behavior,
physiology, and olfactory anatomy is readily available. Moreover, studying Atlantic
salmon sensory discrimination presents the opportunity to ascertain the role of olfaction

in mature parr and smolt life history behaviors.

Among vertebrates, Atlantic salmon maintain unique life history strategies.
Anadromous salmon, hatched in streams, spend 20-50% of their life in fiesh water
(Hutchings and Jones 1998). Depending on growth opportunities, genetic variables, and
physiological drives, a subset of male fish, the mature parr, mature as yearlings in the
autumn and reproduce with adult females (Thorpe et al. 1992). Another subset, sexually
immature parr, undergo smoltification in spring and migrate to seawater as smolts. When
smolts become sexually mature and capable of reproduction, the fish, now termed
salmon, guided by chemical cues, enter their natal stream to spawn (Sutterlin et al. 1973;
Hasler and Scholz 1978). Mature parr may smoltify after they have completed their
reproductive cycle. Understanding the variation in mature parr and smolt life history
behaviors involves identifying distinctive characteristics in their physiology, behavior,
and anatomy (Thorpe et al. 1992; Lundqvist et a1. 1986; Saunders et al. 1982; Jakobsson
et al. 1997). Differences of olfactory sensitivity in Atlantic salmon life stages have been
established (Moore and Scott, 1991). Therefore, mature parr and smolt behaviors may be
distinguished further by variations in sensory discrimination.

The first step in transduction of odorants is stimulus interaction with olfactory
receptors. One approach to comprehend the mechanism of Atlantic sahnon odorant
discrimination is the molecular characterization of olfactory receptor genes. Buck and
Axel (1991) discovered a family of genes they proposed to encode putative olfactory
receptors in the rat olfactory epithelium. The intronless genes represent the largest family
of 7 trans-membrane, G-protein coupled receptors. Identification of OR genes provides
insight into the evolution of stimulus discrimination, reinforces the understanding of

inter-species relationships (F reitag et al. 1998), and confirms the olfactory receptor 7

transmembrane domain (TMD) characteristic (Cf. Buck and Axel 1991; Ngai et al. 1993;
Byrd et al. 1996). In addition, OR gene characterization allows identification of
functional domains (Buck and Axel 1991), offers the opportunity to associate a particular
gene with a specific odorant (Rarning et a1. 1993; Krautwurst et al. 1998), and provides a
foundation for understanding olfactory mediated behaviors. Since the findings of Buck
and Axel (1991), olfactory receptor genes have been cloned in over 25 species, including
mammalian, fish, amphibian, avian, and invertebrate organisms (Cf. Buck and Axel
1991; Ngai et al. 1993; Freitag et al. 1995; Byrd et a1. 1996; Barth et al. 1996; Blache
1998; Berghard and Dryer 1998; Mombaerts 1999; Bargrnann et a1. 1993; ORBD 1999).
Although significant advances in the understanding of transduction mechanisms,
characterization, and genetic composition of olfactory receptors have emerged (Goulding
et al. 1992; Bargrnann et al. 1993; Barth et a1. 1997; Rarning et a1. 1993), questions
regarding the molecular strategy by which OR function mediates the distinctive behavior
of the Atlantic salmon have yet to be established.

This study aims to isolate and identify OR genes in the Atlantic salmon. In
addition to illustrating olfactory receptor gene expression in various salmon tissues,
differential expression of OR genes at two critical life stages, mature parr and smolt, was
investigated. The findings represent the first documentation of OR gene expression in

two salmon life history stages.

 

MATERIALS AND METHODS

Isolation of Nucleic Acids

Atlantic sahnon, Salmo salar, were obtained from the USGS Conti Anadromous
Fish Lab, Turners Falls, MA and Lake Superior State University (LSSU) Hatchery,
Saulte Ste. Marie, Michigan. 6 and 10 mature parr were obtained through electro-fishing
in the Sawmill (Fall, 1998) and Four Mile Brook (Fall, 1999) rivers, respectively. 10 and
15 hatchery smolts were obtained fi'om the USGS Conti Anadromous Fish Lab during the
fall of 1998 and 1999, respectively. 30 hatchery smolts were obtained from LSSU during
the spring of 1999. Tissue samples fiom all mature parr and smolt salmon were collected
and preserved on dry ice or in RNAlater (Ambion, Austin, TX) for total RNA extraction.
In the fall of 1999, muscle tissue from 2 mature parr and 2 smolt salmon was preserved in
95% ethanol for genomic DNA extraction. Sexual maturity of fish was determined by the
developmental stage of the gonads. Mature parr had sexually mature testes, while smolts
did not (Saunders et al. 1982). RNA was prepared from the olfactory organ, gills, brain,
liver, muscle, and gonads of all mature parr and smolts according to the Trizol protocol
(Gibco, Rockville, MD) and quantified by spectrophotometry (Ausubel et a1. 1995). Due
to the small olfactory organ size and amount of RNA required for molecular applications,
olfactory organs fiom three like fish were combined into one sample (mature parr and
smolts were pooled separately). Genomic DNA was extracted in accordance with the
PureGene protocol (Gentra, Minneapolis, Minnesota) and quantified by fluorimetry

(Hoefer: DNA Quant 200).

10

First Strand cDNA Synthesis

Total RNA from the olfactory organ, gills, brain, liver, muscle and gonads of
mature parr and smolts was used to generate cDNA. RNA samples were treated with
DNase I (0.1 U per ug of RNA). First strand cDNA synthesis was carried out in 20 pl
reaction volumes. 1 ug of total RNA was incubated with 1 ul oligo dT 18 bases (500
rig/ml) at 70°C for 10 minutes. The RNA, oligo dT, 0.1 M DTT, 10 mM each dATP,
dGTP, dCTP, and d'I‘TP, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgC12 were
incubated at 37°C with 200 units of M-MLV enzyme (Gibco, Rockville, MD) for 50
minutes. Inactivation of the reaction was accomplished by heating at 70°C for 15
minutes.
PCR and RT-PCR

cDNA fi'om mature parr and smolt olfactory organ, gills, brain, liver, muscle, and
gonads was used as a template for amplification in the Polymerase Chain Reaction (PCR)
(Mullis 1990). Degenerate primers, designed from conserved regions of transmembrane
domains H, 111, and VH of published vertebrate OR and vomeronasal (VNO) gene
sequences, were utilized (Buck and Axel 1991; Ngai et al. 1993; Byrd et al. 1996; Cao et
al. 1998). Primers were chosen at random. PCR reactions of 50 pl total volume contained
100 ng of template, 20 mM Tris—HCI (pH 8.4), 50 mM KCl, 2.0 mM MgC12, 0.2 mM
dNTP's, 1U Taq Polymerase (Gibco, Rockville, MD), and 100 pmol each primer.
Thermal cycling parameters (MJ Research FTC-200) for the goldfish VNO primer pair, 5'
primer: 5'-ACNCCNATHGTNAARGCNAAYAA—3' and the 3' primer: 5'—

YTTNGCYTCRTTRAANGYRTC-3', (Cao et al. 1998), were as follows: 1 cycle- 94° C

11

for 2 min.; 30 cycles- 94° C for 1 min., 38° C for 1 min., 72° C for 2 min.; 1 cycle- 72° C
for 10 min. Aliquots of PCR products were analyzed by agarose gel electrophoresis.
Cloning and sequencing

PCR products were excised from a 1.2 % agarose gel, purified using the Wizard
Purification System (Promega, Madison, WI), ligated into the pGEM Easy Vector System
(Promega, Madison, WI), transformed into heat shock JM109 competent cells (Promega,
Madison, WI), and cultured with SOC media (Sambrook et al. 1989). After incubation of
plates for 24 hours, colonies were screened for vector inserts. Prospective colonies were
picked and grown in 4 m1 of LB media with 30 pl of ampicillin. Plasmid DNA was
subjected to PCR to amplify the gene insert and obtain clone size using T7 and SP6
primers. PCR reactions of 50 pl total volume contained 100 ng of DNA, 20 mM Tris-
HCl (pH 8.4), 50 mM KCl, 2.0 mM MgC12, 0.2 mM dNTP's, 1 U Taq Polymerase, and
50 pmol each primer. Thermal cycling (MJ Research PTC-ZOO) parameters were: 1
cycle- 94° C for 4 min; 30 cycles- 94° C for 45 sec., 50° C for 45 sec., 72° C for 2 min; 1
cycle- 72° C for 10 min. Plasmid DNA was prepared and purified according to the 5'-3'
Mini-Prep kit protocol (5Prime-3Prime, Boulder, Co).

Cloned DNA was sequenced using Hex labeled T7 and SP6 primers from
Integrated DNA Technologies, Inc. (Coralville, IA). Sequencing reactions followed the
Epicentre Biotechnologies cycle sequencing protocol (Epicentre Technologies, Madison,
WI). Sequencing reactions of 6 pl contained 50 ng of plasmid DNA, 2.5 pmol of primer,
2 pl of dNTPs/ddNTPs termination mix (G, A, T, C), 1.8 p1 SequiTherm EXCEL II
sequencing buffer, and .25 pl SequiTherm EXCEL H DNA Polymerase (5 U/pl).

Thermal cycling parameters: 1 cycle- 95° C for 2 min; 30 cycles- 95° C for 45 sec., 55° C

12

for 45 sec., 72° C for 2 min. Reaction products were separated on a 6% polyacrylamide
gel, scanned on a Hitachi FMBio H Multi-view System, read using FMBiolOZ Read
Image v1.1 Software, and scored by FMBio Analysis 8.0 Software. Gene specific
primers were designed to walk across the gene to obtain the complete sequence of insert
DNA. Complementary strands of the insert were sequenced.
Sequence Analysis

The cloned fragments were compared to sequences in the National Center for
Biotechnology Information (NCBI) database using BLAST software in order to receive
homologous gene sequences (see: http://www.ncbi.nlm.nih.gov/). Nucleotide and
deduced amino acid sequences were aligned using the CLUSTAL—W algorithm (The
Baylor College of Medicine Search Launcher). Representatives of goldfish, Carassius
auratus, (Acc: AF 083081) and pufferfish, F ugu rubripes, (Acc: AB009040) odorant
receptor gene sequences were included for comparison. The goldfish and pufferfish OR
genes were chosen randomly. A phylogeny was created using PAUP 4.0 (Swofford 1998)
with salmon gene fragments for the ingroup and representatives of goldfish (Acc:
N233788; AJ233787), pufferfish (Acc: AB009032; AB009034), zebrafish (Acc:
U72691; U72692), catfish (Acc: L09219; L09218), medaka (Acc: AB029476;
ABOZ9478), frog (Acc: AJ011429; A101 1430), rat (Acc: AF 016178; AF 053992), river
lamprey (Acc: AF 069553; AF069554), and mouse (Acc: NM_013618; NM__013619) OR
genes as outgroups. Two OR genes fi‘om each outgroup species were chosen randomly
and utilized in the phylogeny. Each gene spanned TMD III-V and was aligned with the

Atlantic salmon clones prior to phylogenetic analysis to ensure gene homology.

13

RACE experiments

In order to identify additional characteristics of gene fragments, Rapid
Amplification of cDNA Ends (RACE) was performed according to the SMART RACE 5'
and 3' Kit (Clontech, Palo Alto, CA). Nested primers were generated from the sequences
of interest. The gene specific primer (GSP) for 5' RACE, the anti-sense primer, is 5'-
TCTGCATCTCTTGTGTTCTGGGG- 3'. The GSP for 3' RACE, the sense primer, is 5’-
GCTGAACCCACATCACACTCTAG— 3'. All procedures followed the protocol outlined
in the Clontech SMART RACE manual, except for thermal cycling parameters. The
appropriate adjustments for using the MJ Research PCR thermocycler were as follows: 5
cycles: 94° C for lOsec., 70° C for 30 sec., 72° C for 3 min; 10 cycles: 94° C for 203ec.,
68° C for 30 sec., 72° C for 3 min.; 20 cycles: 94° C for 205ec., 65° C for 30 sec., 72° C for
3 min. RACE products were analyzed by electrophoresis on 1.2 % agarose gels. cDNA
fiagments over 300 bases were excised fi'om the gel, purified using the Wizard
Purification kit (Promega, Madison, WI), and cloned according to the procedure outlined
above.
dsDNA Probe Synthesis

Biotinylated probes were generated according to the protocol provided in the
BrightStar Psoralen-Biotin non-isotopic labeling kit (Ambion, Austin, Texas). Based on
sequence identity, genes were grouped into subfamilies whereby members exhibit over
85% nucleic acid similarity. Assuming gene fragments with a high degree of similarity
would reveal similar hybridization patterns, only one representative clone of each
subfamily was generated into a probe to be used in blotting assays (ASOR16, ASORl 1,

ASOR17, and ASORSS). Each representative clone was chosen randomly. Double

14

stranded DNA was obtained from plasmid PCR using SP6 and T7 primers with
recombinant pGEM vectors as templates. The Plasmid PCR products were run on a 1.2%
low-melting temperature agarose gel. Target fragments were excised and subjected to
Phenol/Chloroform extraction. Gene products were exposed to a restriction digest using
Ndel and Ncol to eliminate flanking primer regions. This dsDNA (0.5 pg) template was
labeled according to the protocol outlined in the BrightStar Psoralen-Biotin instruction
manual. Probes ranged in size from 200 bases to 500 bases.
Southern Blotting

Atlantic salmon genomic DNA, isolated from muscle tissue from 2 hatchery
smolts obtained from LSSU, was digested with EcoRV and HindIII, electrophoresed on a
1% agarose gel, and blotted, through downward capillary transfer, onto a positively
charged nylon membrane (Ambion, Austin, Texas). Blots were hybridized with
Psoralen-Biotin labeled probes corresponding to gene fragments ASOR16, ASORI l,
ASOR17, and ASOR55. Hybridizations were carried out at 45° C. Low stringency
washes, using 0.1 standard saline citrate (SSC), were performed at 42° C. Labeled DNA
probes were detected using the BrightStar BioDetect Nonisotopic Detection Kit (Ambion,
Austin, Texas). Detection was carried out using chemoluminescence generated from
enzymatic reactions of conjugated biotinylated probes.

Northern Blotting

Probes representing ASOR16, ASORl l, ASOR17, and ASOR55 were used in
Northern Blotting experiments to characterize OR gene expression in olfactory and non-
olfactory tissues of smolt and mature parr. Total RNA extracted from the olfactory

organ, gills, liver, and testes of mature parr and smolts obtained fi'om Turners Falls and

15

LSSU was utilized in assays. Hybridization and washing conditions were as
follows: 45° C for hybridization; 45° C for washing. Detection of biotinylated probes

followed the chemoluminescent detection procedure employed in Southern Blot Analysis.

 

RESULTS

Isolation of Atlantic salmon odorant receptor genes was based upon their
expected homology to published OR genes of aquatic and terrestrial organisms (Buck and
Axel 1991; Cao 1998). Degenerate oligonucleotide primers, designed from conserved
regions of vomeronasal receptor gene sequences (Cao et al. 1998), yielded PCR products
of approximately 500 base pairs from olfactory tissue cDNA templates RNA (Figure 1).
PCR using cDNA templates synthesized from the mRN A of mature parr and smolt gill,
heart, liver, muscle, and gonadal tissues did not yield results.

Cloning and sequencing of PCR products generated six gene fragments that may
encode for olfactory receptors. Six plasmid PCR reactions contained gene fragments of
the expected length (samples 2, 5, 6, 16,17, and 18). Samples 2, 5, 6, 16, and 18 were
received from reactions using cDNA based on mature parr olfactory mRN A. Sample 17
was obtained from reactions using cDNA based on smolt olfactory mRN A. Plasmid
DNA was subjected to DNA sequencing. Five of the six DNA sequences (ASOR2,
ASORS, ASOR16, ASOR17, and ASOR18) share over 75% sequence similarity to
goldfish and Fugu odorant receptor genes at the HI, IV, and V TMD regions. Another
gene sequence, OR55, identified through additional PCR experiments using template

cDNA derived from smolt mRN A, is homologous to published OR genes.

16

RACE performed on cDNA derived from smolt olfactory mRN A yielded PCR
products of 200, 300, 400, and 1000 base pairs (Figure 2). According to calculations
based on the size of the cloned insert and OR gene sequences, the 5' end fragment was
expected to range from 350-500 base pairs. The 300 and 400 base pair 5' RACE PCR
products were cloned and sequenced. Two clones shared 80% sequence similarity with
goldfish and pufferfish OR gene families between transmembrane domains III and V.
The 3' cDNA end of the clones, ASOR6 and ASORl l, were aligned with the 5' cDNA
end of the previous six OR genes. Overlapping sequences were not apparent in either
gene. The two clones may represent partial OR gene sequences different fi'om the gene
fragments previously identified. The Gene Specific Primer (GSP) designed for the 5'
RACE may correspond to a highly conserved region in the OR gene family.

Nucleotide and amino acid alignments revealed regions of inter— and intra- species
identity (Figure 3 and Figure 4). The deduced amino acid sequences of salmon OR gene
fragments were approximately 200 residues long. Clones appear to represent TMD HI,
IV and V. Sequence comparisons exhibited similar OR motifs among the Atlantic
salmon, Goldfish, and Pufferfish: SELSFLL in leflII, LCISCVL at the interface of
intracellular loop 3 and TMIV, and NVMK in TMIV.

Alignment data demonstrated Atlantic salmon genes possess sequence and
structural properties unique to the Atlantic salmon species. Clones share short motifs,
HWPA and VSP, that are not shared by goldfish or pufferfish OR genes (Cao et al. 1998;
Naito et al. 1998). Pairwise comparisons were carried out using PAUP 4.0 to predict the
percent identity amongst salmon nucleic acid sequences (Swofford 1998). Percent

nucleic acid identity is represented in Table 1. Four clones (ASOR2, ASORS, ASOR16,

17

and ASOR18) exhibit over 90% nucleic acid identity. ASOR6 and ASOR 11 share 95%
sequence identity, but only 45% nucleic acid identity with other gene fragments.
ASOR17 and ASORSS possess distinct sequences, exhibiting less than 60% identity with
the other Atlantic salmon clones.

A phylogeny was created using PAUP 4.0 (Swofford 1998) with Atlantic salmon
clones for the ingroup and representatives of teleost, mammalian, and amphibian OR
genes as outgroups. Bootstrap analysis supports the presence of distinct Atlantic salmon
gene families (Figure 5). Moreover, partitioning of the total genetic variance revealed the
majority of genetic variation resides across subfamilies rather than within.

Southern blot analysis revealed separate hybridization patterns for each Atlantic
salmon OR gene. Three cDNA clones, ASOR55, ASOR] l, and ASOR17, displayed 3-6
hybridization bands in genomic blots (Figure 6). Probe 16 revealed one hybridization
band in the HindIII digested DNA and two hybridization bands in the EcoRV digestion.
Patterns of hybridization for each probe appear to be different.

Northern blot analysis demonstrated that OR genes are expressed in salmon
tissues. The expected size of teleost olfactory receptor mRN A ranges from 800 bp- 5.0
kb (see: http://www.ncbi.nlm.nih.gov/). Assays were performed on smolt and mature
parr olfactory and non-olfactory tRNA samples. Four probes, ASOR16, ASOR] 1,
ASOR17, and ASOR55, were utilized individually in all experiments. Northern Analysis
performed with ASOR] 1, ASOR17, and ASORSS revealed hybridization to
approximately 1.3 kb, 2.2 kb, and 1.8 kb, respectively, in RNA from smolt olfactory
tissue (Figure 7), but not to RNA from mature parr olfactory organ. ASOR16 did not

appear to hybridize with RNA from mature parr gill and liver or RNA from smolt

l8

olfactory and non-olfactory tissue. Analysis with ASOR16 revealed one band at 1.0 kb in

mature parr testes.

 

DISCUSSION

Olfactory cues mediate Atlantic salmon behavior during different life history
stages. One approach to determine the significance of odorants in salmon behavior
involves identifying and characterizing olfactory receptor genes. We have cloned a novel
family of genes likely to encode for odorant receptors in Atlantic salmon, Salmo salar.
Our data signify the first record of olfactory receptor gene sequences in the
Salmoniforme order. Differential expression of olfactory receptor genes in tissues of the
mature parr and smolt was illustrated representing the first correlation between olfactory
receptor gene expression and salmonid life histories.

As a result of sequence and Northern analysis, we suggest that the Atlantic
salmon gene fragments encode for olfactory receptors, based on three lines of evidence.
First, gene products were observed exclusively in PCR's utilizing template cDNA from
the olfactory organ. These findings suggest genes are localized to olfactory tissue.
Secondly, deduced amino acid sequences of salmon cDNA's identify a family of proteins
that share structural and sequence properties with other seven trans-membrane domain,
olfactory receptors. BLAST analysis reveals sahnon gene sequences display over 75%
nucleic acid similarity to published goldfish and pufferfish OR genes. Finally, Northern
blot assays verified that gene fi'agments were expressed primarily in the olfactory organ.

Phylogenetic and amino acid alignment studies suggest that the Atlantic salmon

OR genes form a distinct ancestry in the olfactory receptor gene lineage. Phylogeny of

19

salmon sequences performed with representative teleost, mannnalian, and amphibian OR
genes as outgroups support the novel salmonid gene characteristic. Bootstrap analysis
supports the presence of distinct Atlantic salmon gene families. Additionally, salmon
gene fiagments exhibit motifs distinctive for Atlantic salmon. The salmon OR gene
lineage may have emerged as an adaptation to odorants mediating sahnon specific life
history behaviors. For example, spawning salmon migration is guided by chemical cues
particular to an individual's natal stream (Hasler et al. 1978).

Nucleic acid analysis and Southern blot results suggest salmon OR genes are
organized into subfamilies. Nucleic acid comparisons indicate that eight salmon gene
fragments represent four OR subfamilies in which members exhibit at least 85%
sequence similarity (Berghard and Dryer 1998; Ngai et al. 1993). Representative gene
fragments of the olfactory receptor subfamilies reveal distinct hybridization patterns in
Southern blot analysis. Southern blot analysis confirms the presence of separate OR gene
lineages. Probe hybridization pattern variations indicate genes may be clustered on
different loci of the genome. Therefore, results support the organization of genes into 4
subfamilies based on sequence similarity and hybridization patterns.

The existence of Atlantic salmon OR gene subfamilies may reflect an adaptation
of the olfactory system to various classes of odorants. In general, G-protein coupled
receptors possess structural domains that confer binding specificity onto the proteins
(Kobilka et a1. 1988). Therefore, OR gene subfarrrilies, sharing similar structural
properties within subfamilies, may detect a particular class of chemicals. Sequence
divergence between subfamilies may lead to variations in receptor/ligand relationships.

One distinguishing feature of odorant receptor proteins is the diversity exhibited in

20

several of the transmembrane domains. Although OR genes maintain maximal
conservation in the second intracellular and extracellular loops and within transmembrane
domains 11, VI, and VII (Buck and Axel 1991), hypervariable regions, associated with
transmembrane domains 1]], IV, and V, exist (N gai et al. 1993). The pronounced variable
regions may correspond to ligand binding domains, accounting for the olfactory systems
ability to discriminate a large variety of chemical structures. Our Atlantic salmon gene
fragments extend fi‘om TMDIII to TMDV, the most variable of domains in these G-
protein coupled receptors. Therefore, the variability in structural properties exhibited by
the salmon subfamilies may confer binding specificity upon each lineage of proteins.
Further testing of subfamily specificity requires functional data.

Our data suggest mature parr and smolts maintain distinct olfactory receptor gene
sequences and expression patterns. Sequence analysis reveals mature parr genes retain
distinct sequences fiom smolt clones. The four clones obtained from mature parr tissue
represent one subfamily separate from the three subfamilies containing genes solely
extracted from molt tissue. Additionally, Northern assays suggest smolt genes hybridize
solely to RNA from the olfactory organ of smolt. No hybridization with smolt gene
probes was detected in olfactory or non-olfactory tissues of the mature parr. Sequence
variation and differential expression of olfactory receptor genes may reflect variations in
olfactory sensitivity of Atlantic salmon at different stages in their life history. Olfactory
receptor expression confers the ability upon an organism to detect stimuli (Amoore
197 7). Consequently, a fish at one stage of development, expressing a particular OR,
may detect chemicals not discernible by a fish, in a different life stage, which does not

appear to exhibit the same receptor gene. Olfactory sensitivity discrepancies are evident

21

in mature parr and smolt. For instance, Scott and Moore (1991) reported an increased
olfactory sensitivity to testosterone in mature parr compared to immature individuals.
Identification of OR genes provides a foundation for deciphering chemical cues driving
variations in sahnon behavior and offer a basis for understanding the impact of odorants
on salmon. Ultimately, these biochemical and molecular data assist not only in the
furthering of basic olfactory knowledge, but also offer important considerations for
natural variables influencing Atlantic salmon life histories and strategies.

Our findings have many implications for understanding and conserving the
threatened Atlantic salmon species. Through molecular characterization of odorant
receptor genes, we have provided a basis for comprehending olfactory homing, a
behavior essential for Atlantic salmon survival. Salmon rely on olfactory discrimination
to detect natal homing stimuli. If the chemical cues are not detected, salmon may not
locate adequate spawning ground for successful reproduction. Therefore, ensuring
species survival through olfactory homing requires a comprehensive understanding of
olfactory discrimination at the molecular level. Through identifying the genetic sequence
of olfactory receptors, information on olfactory discrimination is obtained. Moreover,
identification of Atlantic salmon OR genes will greatly assist management practices of
the salmonid species. Utilizing OR genes in functional assays may identify critical
odorants involved in olfactory imprinting, the process of forming olfactory memories for
homing. Understanding imprinting would provide for the development of techniques
used to detect its occurrence. Thus, it can be determined if hatchery raised fish have
been imprinted for their environment, thereby increasing the chances of successful

homing and reproduction. Additionally, it may be possible to imprint hatchery fish to

22

streams other than those utilized by wild salmon populations, preventing genetic
pollution of wild Atlantic salmon populations. Our results provide a basis for
comprehending molecular aspects of olfactory discrimination in salmon, in turn,

contributing to the efforts put forth to preserve this valuable resource.

Gene Accession Numbers
AF191680, ASOR2; AF191681, ASOR5; AF191682, ASOR16; AF191683, ASOR18;

AF 191684, ASOR17; AF 191685, ASOR6; AF 191686, ASOR] 1; AF206035, ASORSS

Acknowledgemenm

I am grateful to Scot Libants, Dr. Kim Scribner, Dr. Tim Zacharewski, and Kenneth
Oswald for expert technical assistance. I want to thank Dr. Steve McCormick and staff
at the USGS Conti Anadromous Fish Lab, Turners Falls, MA for assistance in obtaining
wild fish, and Roger Greil at Lake Superior State University for providing hatchery
salmon. The work was supported by a grant from the United States Geological Survey
(U SGS) administered through the USGS Conti Anadromous Fish Lab and Monell

Chemical Senses Center.

23

 

,3?A$0R16 0.5987
_ ASOR16 0.6022
.. iAsoms 0.5996

2,, a. ASOR2 0.5950
ASOR5 0.6280
3 ASOR17 0.6942

 

Table 1: Pearson Distance Matrix

Atlantic salmon sequences were analyzed using PAUP 4.0 (Swofford 1998).

0.1042
0.0926
0.3659
0.5555

0.0859

0.3561

0.5618 0.5684 0.6294

0.3643

Values represent the difference between Atlantic salmon sequences.

Example: 0.04 represents 4% difference between nucleic acid sequences.

24

 

lane 1 lane 2 lane 3

1000bp —'

500hn

 

Figure 1: Electrophoresis Analysis of PCR products using VNO primers
cDNA prepared from mature part and smolt olfactory organ RNA was subjected
to PCR amplification with degenerate oligonucleotide primers. PCR yielded
products of 500 base pairs using Atlantic salmon olfactory organ cDNA as
templates. Lane 1 contains a 100 base pair marker. Lane 2 contains a 500 base
pair PCR product amplified from cDNA fi'om smolt olfactory organ RNA. Lane 3
contains a PCR product of 500 base pairs amplified from cDNA of mature
parr olfactory organ RNA.

25

Lane 1 Lane 3 Lane 4

600bp

400bp

 

Figure 2: Electrophoresis Analysis of S'Rapid Amplification of cDNA Ends (RACE)
using cDNA from smolt olfactory organ RNA
Using the SMART RACE kit fi'om Clontech, 5' RACE ready cDNA prepared
from Atlantic sahnon olfactory organ RNA was subjected to PCR using Gene
specific primers. Lane 1 contains PCR products of 200, 300, 400, and 1000 base
pairs using smolt olfactory organ cDNA as a template. Lane 3 contains a PCR
negative control in which no template was utilized. Lane 4 contains a 100 base

pair marker.

26

 

 

PRPR GCCGCCTCC'I'I‘G‘I'PG mm W AW'ICCATCAT cocnmcaccrvm GTA '1'
CAOR ACTA'I'I'I‘CACI'IC'I'I‘ ocrocnrr'nnm Luann-mart ATA'I'I'I'I'I'CCGA‘I‘A‘!‘ MTACCCCMTA

 

Figure 3: Gene Sequence Alignments
The nucleic acid sequences of 8 Atlantic salmon cDNA's were aligned with
representatives of Goldfish (CAOR) and Pufferfish (FRPR) odorant receptor
genes (AF 083081 and AB009040, respectively) using the Clustal-W algorithm.
Inter-species sequence identity is indicated in the dark shaded region. Atlantic
salmon infra-species sequence identity is represented in the light shaded regions.

27

 

ASOR17
nsonss
nsoru
asons
nsorus
Asorua
Asorur - - . -e~‘~:é?3‘wyi

papa >33 --. CG‘I'I'I‘C'I'I‘A

A8031?
ASORSS
A5082
ASORS
ASORIG
ASORIO
ASOR6
ASORll

CAOR

ASOR17
ASORSS
ASORl
ASORS
ASORIG
ASORIB
ASOR6
ASORll

PRPR 'ACTNGCCTTTPHIE CGAGG CTT TCAGCNfi3KKHUTH'ITTGTGCAGTGTGOG
CAOR TGAAG TTT T TTIGTATGCTCATAT TTTGHGCTGTTTGGA

 

 

 

Figure 3 (continued): Gene Sequence Alignments

28

541 555 556 570 571 585 586 600 601 615 616 680

 

       

Asonss -------------------------------------------------------------------------- TI‘PIVKANNSE-AOL.
Asom7 -------------------------------------------------------------------------- IPIVKANNSELSFLLL
Asonz ------------------------------------------------------------------------------ DYPYCGLAEL“
ASOR5 --------------- - ------------------------- vmnoos
Asoms -------------------------------------------------------------------------- TPIVKANNSELSFLLL
ASOR18 -------------------------------------------------------------------------- TLSVKANNSELSFLLL
Asons ------------------------------------------------------------------------------------------
ASOR11 ..........................................................................................
crronao IPCAEGEISNETDSI NCKOCPGEYWPNAEK NKCVLKAVEFLSFTE IerLVFFSLFGVG LTVLVAILFYSKKNTPIVKANNSELSFLLL
annsa ............................................... GWLVFFTLLGFF LTLSVTALFVINKNTPLVKANNSELSFLLL
----------- TIDIV----—------
646 660 861 675 576 690 691 705 705 720
Asonss LWTCOHEACSI WVHWSPGILVEGO- WWVLAVFRTSKARGO COPEVVWCWATE—RN SPGSHLIPG-HCYL
ASOR17 wsccrnnan G ----- LASCSASP— ----- Aswsn--Psc CWLOGTVPVAFD-ET VRTFPANGHF-YCST
ASOR2 , SECACAHR FGSLLLtt‘t fauNW‘T-lfllfi W .
ASORS P srwsvnAsnsv wonrvrcrscv 9," -KTWCCFTTL-NOPV a

  
 

ASORIS Fsrncrrcsnria RPSE-WSCMLRHTAF er‘rrvrdrfspvimlrwwmrim -,

ASOR18 FS--LCFLCSLTFIA npsewscutaronw VHSSSASLVFWGNNv -SGVDGLOGLRFOPV n

ASOR6 --------------------- LCLLGRHGG AGIRFLHLLCSGEN- NSGVDGFRAT-LOPV
----------------------- ASRP

g
is?
1“
5E
32
j;
'%

CAOR80 FSLSLCFLCSLTFIG RPTE-WSCMLRHTAF qrmmrfibr 1‘ iM‘I’V‘WD .
annaa FSLTLCFLCSLTFIG nesc-wscnrnnmr ”minimise“ M . “1M1

--mv ------

736 750 788 780

721 735 751 765 781 795 796 010
mass usueFSLsusmKn AS---PYVRFVYG-- -vmoesencArs Amsussvrtm rrorpsrxrx --------------------
ASOR17 s-o E -VELLNSSP TK---P10vcnvenr HECLIAFHYFLG -------------------------------------------------

  
 

  

      

ASORZ ill- 1 #16th WWWIGLL-A 'VLcavLAFLxm LFNEAKN .......................
A3085 UL . MK-EKIILECDVAXL LV R----T LLSCLLLFWLE Hsrxmm .....................
11501110 SLROSHLLSLOEHED LG- 31;): 9mm“ -c NF YIGHP-G CLVXVLDFLWSRKLS LILSTKOK ......................
ASOR“ VNSLAS-ILOEHEN L 91.. = i: -L flmvtavt‘GEL-A LLWWSFLH ”1011st .......................

ASOR6 FL rosin ------------------------------------------------------------------------------
ASOR" er TRDAG ..............................................................................
CAORBO mrswvmmutv Im-Vfltt‘r‘tfilsrnsr WEWGLL-A vtdfl'llitmtmm ummrrrrsurrr CAWITFIPAYVSSP
anaa wtrrcpprnxumi pr-EkrrtedALesrt gemAvrcvrevr-A vrcErLAruirorpo nrumrrrrsuur CAVWITFIPAYVSSP

Figure 4: Comparison of Deduced Amino Acid Sequences of Atlantic salmon, Goldfish,
and F ugu cDNA's.
The deduced amino acid sequences of 8 Atlantic sahnon cDNA's were aligned
with representatives of goldfish (CAOR80) and firgu (FRPR380) olfactory
receptor gene sequences (AF 083080 and AB009038, respectively). Amino acid
residues were aligned with the Clustal- W aligorithm. Identical sequences of
Atlantic salmon, firgu, and goldfish are shown in the shaded, bold type region.
Identical salmon sequences are revealed in darker shaded regions with bold
typeface.

29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

___l 23.11
3 i——lN—-—-| As 6

Figure 5: Phylogenetic Tree of Gene Sequences

A phylogenetic tree was created with PAUP 4.0 (Swofl‘ord, 1998) using Atlantic Salmon
(AS), Goldfish (Gold), Zebrafish, (Zeb), Frog (Frog), Catfish (Cat), Medaka (Medaka),
Pufferfish (Fugu), River lamprey (LamP), Rat (Rat), and Mouse (Mouse) 0R gene
sequences. Accession numbers are provided in the Materials and Methods section and are
listed as gene 1 and 2, respectively. Values represent the proportion of 100 bootstrap
replicates supporting particular nodes. Values under 60 are not shown. Atlantic salmon
clones remain separate fiom other species.

1 .3.
44100—4 A818

 

 

 

 

30

EcoRV HIndIII EcoRV HindIII

    

4.0kb
4.0kb
3.5kb
3.0kb :
2.0kb
2.0kb
Probe 11 Probe 16

Figure 6: Southern Blot Analysis with Four Atlantic Salmon cDNA's

Atlantic salmon genomic DNA, isolated from muscle tissue, was digested with EcoRV
and HindIII, electrophoresed on a 1% agarose gel, and transferred to a positively charged
nylon membrane. The membrane was cut into four separate membranes and probed with
individual Psoralen Biotin labeled gene probes, ASORl 1, ASOR16, ASOR17, and
ASOR55.

31

EcoRV HIndIII

4.0kb
4.0kb —

3.51:5 3.51:1: —

3.01:1:
2.0kb

1.5kb

2.0kb

   

Probe 17 Probe 55

Figure 6 (continued): Southern Blot Analysis with Four Atlantic Salmon cDNA's

32

KB Smolt

i"

Olf. Smolt Gills

     

Figure 7: Expression of Olfactory Receptor RNAs in Atlantic salmon tissues

Psoralen Biotin labeled DNA probes were annealed to 30ug tRNA isolated fiom smolt
olfactory tissues and gills. Hybridization was performed with probe 1 1, representing
ASORll received from smolt tissue. Bands appear in smolt olfactory tissue at the 1.2 Kb
marker. No hybridization of this size is detected in tRNA from smolt gills.

33

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