水研センター研報，第21号，47−87，平成19年
Bull. Fish. Res. Agen. No. 21, 47‑87, 2007

博士号論文

Studies on the regulatory mechanisms involved in the＊1expression of heat-shock protein genes from rainbow trout
Nobuhiko OJIMA＊2

Abstract The present study was undertaken to examine the gene structure, the mRNA expression profiles and the mechanism of transcriptional regulation of fish heat-shock proteins（HSPs）,using the rainbow trout
as a model system.
Published studies using teleost cultured cells clearly demonstrate that teleosts can mounta stress response very similar to that observed with other vertebrates. However, HSP genes
have only been cloned from a modest number of different fish species, and molecular studiesof the fish HSPs are still in their early descriptive phase.
Heat-shock protein 70（Hsp70）is the major stress-inducible protein in vertebrates andhighly conserved throughout evolution. Rainbow trout have evolved by tetraploidization froma diploid ancestor, raising the possibility that multiples exist in the genome. To obtain full-length cDNA clones encoding Hsp70, a plasmid cDNA library was constructed froma heat-shocked juvenile of rainbow trout and screened. Consequently, twosand, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes
in the genome. Northern blot analysis showed that each ofandexpressed
two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells.
To comprehensively analyze the mRNA expression profile ofs multiple
s wereisolated from RTG-2 cells and quantitatively compared for their mRNA levels between
unstressed and heat-shocked cells using real-time quantitative RT-PCR analysis. To clone
multiple HSP genes at once, 200 cDNAs were arbitrarily isolated from the cDNA library
constructed from heat-shocked RTG-2 cells and sequenced. Consequently, nine cDNAs encoding HSPs were identified, namely,
β
β ,
, and
homolog. By Northern blot analysis, single bands were detected for
all genes examined except
s where two mRNA species having different sizes weredetected in heat-shocked cells irrespective ofandQuantitative RT-PCR
analysis showed that the accumulation levels of Hsp70a and Hsp70b mRNAs were dramatically increased after heat shock, and the mRNA levels in heat-shocked（28℃, 3 h）cells were480- and 510-fold of those in controls, respectively. The increased levels of Hsc70a, Hsc70b
and Hsp47 mRNAs were 1.3-, 2.8- and 1.6-fold, respectively, after heat shock. There were nodifferences in mRNA levels of the other four HSP genes between control and heat-shocked
cells with a significance level of 0.05.
To demonstrate the existence of the heat-shock transcription factor（HSF）in rainbow
trout, genes encoding HSF1 were cloned from RTG-2 cells. Consequently, two distincts, named
andwere identified. Southern blot analysis showed that each
HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were coexpressed in unstressed
RTG-2 cells and in various tissues. In electrophoretic mobility shift assay, eachtrans2007年 月 日受理（Received on March 9, 2007）*1 東京大学審査学位論文（掲載に際し投稿規定に沿って一部修正した）*2 中央水産研究所 〒236-8648 横浜市金沢区福浦2-12-4（National Research Institute of Fisheries Science, 2-12-4, Fukuura, Kanazawa, Yokohama
236-8648, Japan）

Nobuhiko OJIMA

lated HSF1 bound to a cis-acting DNA element, the heat-shock element. Chemical cross-linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers aswell as homotrimers.
The present study demonstrated that, in rainbow trout, at least someandhave
duplicate genes attributed to ancestral tetraploidization. This finding suggests that the comprehensive identification of duplicate genes is prerequisite to accurately examining gene
expression profiles of rainbow trout cells. Since the mRNA expression profiles of HSPs may
differ among stressors, it is interesting to compare the differences of the profiles for utilizings as a biomarker of various environmental stresses. Further, the present study suggeststhat a unique molecular mechanism, which functions through two distinct HSF1 isoforms,
underlies the stress response in tetraploid and/or cold-water fish species such as rainbow
trout. Since the lower activation temperature of rainbow trout HSF1 is a unique feature
among vertebrate counterparts, a detailed comparison of rainbow trout and other vertebrate
HSF1s will lead to further insight into the activation mechanism of the transcription factor.
Key words: gene expression profiling, gene expression regulation, heat-shock proteins, heatshock response,.

Contents

Section 3. Construction of HSF1
expression vectors

Abbreviations

Section 4. DNA-binding ability of HSF1

Introduction

Section 5. Oligomeric state of HSF1

Chapter I. Cloning and mRNA expression profile ofSection 6. Discussion

Hsp70

Summary and general discussion

Section 1. Isolation and sequence analysis

Acknowledgmentsof cDNAs encoding HSP70

References

Section 2. mRNA expression profiling ofAbbreviations

─ Temperature shift
experiment
Section 3. mRNA expression profilingof─ Time course

experiment
Section 4. Discussion
Chapter II. Cloning and mRNA expression profiles of

ANOVA, analysis of variance
CCT, chaperonin containing TCP1
cDNA, complementary DNA
DBD, DNA-binding domain
DDBJ, DNA Data Bank of Japan

HSP family members

DIG, digoxigenin

Section 1. Cloning of HSP cDNAs expressed

DNA, deoxyribonucleic acidin heat-shocked RTG-2 cells
Section 2. Accumulation of HSP mRNAs

EDTA, ethylenediaminetetraacetic acid
EGS, ethylene glycol bis（succinimidylsuccinate）

in unstressed and heat-shocked

EMSA, electrophoretic mobility shift assay

RTG-2 cells

ER, endoplasmic reticulum

Section 3. Discussion
Chapter III. Cloning and characterization of HSF1
Section 1. cDNA cloning and genomic
organization of HSF1
Section 2. mRNA expression pattern of
HSF1 in various tissues

EST, expressed sequence tag
GRP, glucose-regulated protein
HA, hemagglutinin
HEPES,-（ 2 - h y d r o x y e t h y l ）p i p e r a z i n e --2-ethanesulfonic acidHR, hydrophobic repeat

Regulation of expression of HSP genes in rainbow trout
HSC, heat-shock cognateof proteins called the heat-shock proteins（HSPs）

HSE, heat-shock element（Lindquist and Craig, 1988）. The response is the

HSF, heat-shock transcription factor

most highly conserved genetic system known,

HSP, heat-shock protein

existing in every organism in which it has beenIg, immunoglobulin

sought, from archaebacteria to eubacteria, from

IOD, integrated optical density

plants to animals（Lindquist and Craig, 1988）. HSPs

mRNA, messenger RNA

are traditionally classified by molecular weights into

NCBI, National Center for Biotechnology Information

several families（Sonna

NLS, nuclear localization signal

a 70-kDa heat-shock protein belongs to the HSP70

ORF, open reading frame

family. HSP family members possess three principal

PCR, polymerase chain reaction

biochemical functions: molecular chaperone activity,

PVDF, poly
（vinylidene difluoride）

regulation of cellular redox state, and regulation of

RACE, rapid amplification of cDNA ends

protein turnover（Sonna

RNA, ribonucleic acid
RT-PCR, reverse transcription polymerase chain
reaction

2002）. For example,

2002）.

The inducible HSP expression is regulated by theheat-shock transcription factors（HSFs）（Pirkkala2001）. In response to various inducers such as

SD, standard deviation

heat stress, most HSFs acquire DNA binding activity

SDS, sodium dodecyl sulfate

to the heat-shock element（HSE）, thereby mediating

SDS-PAGE, sodium dodecyl sulfate-polyacrylamidetranscription of the heat-shock genes, which results

gel electrophoresis

in accumulation of HSPs（Pirkkala2001）asSSC, standard saline citrateshown in Fig. 1. In vertebrates, several members of

TCP1, t-complex polypeptide 1

the HSF family, HSF1‑4, have been found（Pirkkala

TPR, tetratricopeptide repeat2001）. Among the family members, HSF1 is

UTR, untranslated region

functionally analogous to yeast andHSF

as the principal stress-induced transcription factor
Introduction（Morimoto, 1998）.
Published studies using teleost cultured cells

Poikilotherms like fish are directly affected byclearly demonstrate that teleosts can mount a stressambient temperature, which is among the most

response very similar to that observed with otherpervasive of environmental factors（Hawkins, 1996）.

vertebrates（Hightower and Renfro, 1988）.In regard

Temperature influences their metabolism, activity

to HSPs, most studies in fish have been performed

levels, spawning, development and growth; andat the protein level, and these results suggest that

because of selective pressures associated with these

the cellular stress response is likely to be playing

processes, temperature is an ecological resource,some role in enhancing the survival and health of

influencing the proportion of potential habitat

the stressed fish（Basuthat is suitable for a species（Hawkins, 1996）. In

genes have only been cloned from a modest number

this manner, adaptive responses to ambient waterof different fish species, and molecular studies of the2002）. However, HSP

temperature are essential for the survival of aquatic

fish HSPs are still in their early descriptive phaseectotherms. In particular, adaptation to heat stress is（Basu2002）.

a critical factor for the viability of cold-adapted fishThus, the present study was undertaken tosuch as salmonids because they receive heat stressexamine the gene structure, the mRNA expression

at moderately low temperatures. For instance, theprofiles and the mechanism of transcriptionalultimate incipient lethal temperature for rainbow

regulation of fish HSPs. For this purpose, rainbow

trout, at which half of the fish tested die within 1

trout was chosen as an experimental model fish

week, is about 26.2℃（Kaya, 1978）.

because of the following reasons. First, temperature

At a cellular level, all organisms respond totolerance is an important trait from both anheat stress by inducing the synthesis of a groupeconomic and an evolutionary perspective in fishes,Nobuhiko OJIMA

Fig. 1. Model of the regulatory mechanisms underlying the expression ofheat-shock protein genes in mammalian cells. HSF1 is present in the cytosol
as inactive monomers under normal conditions. The inactivation of HSF1 isassociated with the binding of HSPs to the protein. When unfolded proteins
accumulate in the cytosol by heat shock, HSPs dissociate from HSF1 to function
as molecular chaperones. Subsequently, HSF1 acquires DNA binding ability
through trimerization and translocates to the nucleus. HSF1 binds to HSEs in the
upstream region of HSP genes and activates their transcription.

particularly among cold-water salmonids such as

and the associated structural and regulatory generainbow trout（Somorjai

changes（Thorgaard

2003）. Second, since2002）.

rainbow trout is one of the most intensively studied

In rainbow trout, the major stress-inducible

fishes in a wide range of research areas（Thorgaard

protein in the cells has been reported to be Hsp70

2002）, considerable information is available（Kothary and Candido, 1982）, and partial cDNAs

about the physiology and biology of the fish as

encoding Hsp70 have been isolated （Kotharya background for molecular studies. Third, since

1984b）. Based on these data, the present

rainbow trout have evolved by tetraploidization fromstudy began with isolation of a full-length cDNA

a diploid ancestor（Ohnoencoding rainbow trout Hsp70. Chapter I describes1968）, wider-scale

DNA sequence information for the fish will provide

the isolation ofand its expression profile inan excellent and distinctive system for studying

rainbow trout RTG-2 cells exposed to heat stress.

the aftermath of a genome-wide duplication event

Next, to comprehensively explore the expression

Regulation of expression of HSP genes in rainbow trout
profile of HSP genes, isolation of genes encoding

has been identified in rainbow trout（Zafarullah

the other HSP family members was undertaken.

1992）. Hsc70 is a member of the HSP70 family

Chapter II describes the isolation of HSP genes andthat is constitutively expressed in unstressed

their expression profiles in RTG-2 cells in response

cells（Hightower and Leung, 1997）. Nevertheless,to heat stress. Finally, to investigate the molecular

only the partial, not full-length, cDNA sequences

mechanism of transcriptional regulation of thementioned above have been used to detect rainbow

HSP genes, isolation of a gene encoding HSF wastrout Hsp70 transcripts. Therefore, an unambiguous

undertaken. Chapter III describes the isolation andinterpretation on mRNA expression profiles ofmRNA expression ofand the activation of thetranscription factor by heat stress.

and following evaluation of heat stress on
rainbow trout were likely impossible. To this end,

According to the nomenclature used in themuch more detailed information is required on theprevious publication （Gething, 1997a）, fully

possible existence of the multiple

capitalized names were used to denote the protein

sequence variations along with differences froms and theirfamily（e.g. HSP90, HSP70, HSP60 etc.）and initial. The resulting information is prerequisite

capital letter was used for specific family members

to utilization of（e.g. Hsp90, Hsc70, Cct8 etc.）in this thesis.

molecular biomarker for stressed state of fish.

expression profiles as a

Furthermore, it is interesting to explore different
Chapter I. Cloning and mRNA expression profile offunctional properties of the possible multiple Hsp70s

Hsp70

of fish in terms of evolutional interpretation.
Thus, the purpose of the study was to isolate

Hsp70 is the major stress-inducible protein in

full-length cDNA clones encoding rainbow trout

vertebrates and highly conserved throughout

Hsp70 and to investigate their mRNA expression

evolution. The transcripts of Hsp70 are induced

profiles during heat stress. This chapter describes theunder various physiological stresses and theidentification of two distinct heat-inducible

resulting translation products act as the molecularrainbow trout and their different mRNA accumulations inchaperone that mediates the correct assembly andlevels in cultured cells exposed to heat stress.

localization of intracellular proteins（Miao
1997）. In fish,s have been cloned from anumber of species; however, most studies of Hsp70

Section 1. Isolation and sequence analysis of
cDNAs encoding HSP70

have been performed at the protein level（Basu2002）. Hence, relatively little is known about theexpression profiles of Hsp70 transcripts in fish cells
exposed to a variety of stresses.

s attributed to ancestral tetraploidization.
Furthermore, since Hsp70 is a member of the highly

Until now, two partial cDNA sequences encoding
Hsp70 have been isolated from rainbow trout（Kothary

Rainbow trout is likely to have duplicate

1984b）. Their sequences have beenconserved HSP70 family, homologous genes such as
are generally present in vertebrate genomes.
Thus, to specifically analyze the mRNA expression

used as probes to detect Hsp70 mRNA in rainbow

profile of rainbow trout, it is essential totrout cells exposed to stresses such as heat shock

identify the number ofs in rainbow trout（Currie

1984a; Lund

genome and to determine their full-length DNA

1999）, sodium arsenite

sequences. This section describes the isolation of

1999; Kothary

2000; Samples（Kothary

1984a）, and hypoxia（Currie1999）. However, rainbow trout have evolved

full-length cDNAs encoding Hsp70 and their genestructures.

by tetraploidization from a diploid ancestor（Ohno
1968）, raising the possibility that multiple

Materials and Methodss exist in the genome, as inferred fromSouthern blot analysis （Kothary

1984b）.

Furthermore, a heat-shock cognate 70（Hsc70）gene

Construction of cDNA library
Rainbow trout

s wereNobuhiko OJIMA
obtained from the Nikko Station of the National

Phylogenetic analysis

Research Institute of Fisheries Science（Tochigi

Amino acid sequences of vertebrate Hsp70s andPrefecture, Japan）and reared on a commercial diet

Hsc70s were retrieved from the DNA Data Bankat 10℃. One individual of rainbow trout（3 monthsof Japan（DDBJ; http://www.ddbj.nig.ac.jp/Welcoold）was heat-shocked for 30 min in an aquarium

me-e.html）. A phylogenetic tree was constructed

at 25℃, and the whole body was homogenized infrom the multiple sequence alignment by using the

TRIZOL Reagent（Invitrogen, Carlsbad, CA, USA）

neighbor-joining method（Saitou and Nei, 1987）, as

using a power homogenizer（Polytron; Kinematica,

implemented in CLUSTAL W（Thompson

Lucerne, Switzerland）. The total RNA was isolated1994）. A graphical output of the tree was visualizedaccording to the manufacturer s instructions. Polyby the program TreeView（Page, 1996）.

（A）RNA was purified from the total RNA using
an Oligotex-MAG mRNA Purification kit（Takara

Southern blot analysis

Bio, Otsu, Japan）. A directional cDNA library

Genomic DNA was isolated from RTG-2 cells

was constructed from poly（A） RNA using aby using a DNeasy Tissue kit （Qiagen, Hilden,

SuperScript Plasmid System for cDNA Synthesis

G e r m a n y ） a c c o r d i n g t o t h e m a n u f a c t u r e r ʼs

and Plasmid Cloning kit（Invitrogen）according to

instructions. Each 10-μg DNA was digested

the manufacturer s instructions.

with restriction enzymes

cDNA cloning

capillary-transferred to a nylon membrane（Hybond

HI,

RI, anddIII, electrophoresed on a 1% agarose gel, andScreening of the cDNA library was performed

N+; Amersham Biosciences）with 10 × SSC（1 ×using a G ENE T RAPPER cDNA Positive Selection

SSC is 0.15 M NaCl plus 0.015 M sodium citrate）.

System （Invitrogen）. An oligonucleotide probe

DNA was UV-crosslinked to the membranes, which

for a cDNA capture hybridization was designed

were subsequently hybridized for 1 h in PerfectHyb

based on the sequence of a rainbow trout

hybridization solution（Toyobo, Osaka, Japan）withgenomic clone isolated in a preliminary study

digoxigenin （DIG）-labeled DNA probes. Probes（GenBank accession no. AB062281）. The nucleotide

specific forsequence of the probe was 5 -TGGTCCTGGTGAA

in their 3 -untranslated regions（UTRs）, namely,

GATGAGG-3 . Biotinylation of the probe and cDNA

nucleotides 2561‑2773 and 2029‑2191, respectively.

capture hybridization were performed according

The GenBank accession numbers of the twoto the manufacturer s instructions. The captured

are as follows:

single-stranded DNA was repaired and transfected

AB176855. The probes were labeled with a PCR

into E LECTRO MAX DH10B cells （Invitrogen） by

DIG Probes Synthesis kit （Roche Diagnostics,

using an electroporation apparatus（MicroPulser;

Mannheim, Germany）. The hybridized membranes

Bio-Rad Laboratories, Hercules, CA, USA）. The

were washed twice with 2 × SSC plus 0.1% SDS

electroporation was carried out at 1.8 kV using 20-

at room temperature for 5 min, and then twice

μl cells in a 0.1-cm-gap cuvette. White colonies

with 2 × SSC plus 0.1% SDS at 68℃ for 15 min.

were selected after transformation, and whether

The chemiluminescent detection of the probes wasandwere designedsAB176854 andthey were truly positive were analyzed by PCR

performed with a DIG Luminescent Detection kit

using the above-mentioned probe sequence as a

（Roche Diagnostics）according to the manufacturerʼssense primer and a SP6 promoter primer（5 -GTG

instructions. The positive signals were detected

ACACTATAGAAGAGCTATGACGTC-3 ）as an

by exposure on Hyperfilm-ECL （Amersham

antisense primer. Subsequently, the positive clones

Biosciences）.

were sequenced with an automated DNA sequencer（Gene Rapid; Amersham Biosciences, Piscataway,

Results

NJ, USA）after labeling with a Thermo Sequenase
Cy5.5 Dye Terminator Sequencing kit（Amersham
Biosciences）.

To obtain full-length cDNA clones encoding
Hsp70, a plasmid cDNA library containing 1.1 ×

Regulation of expression of HSP genes in rainbow trout
10 6 independent clones was constructed from a

encoding Hsp70 were identified in rainbow trout,heat-shocked juvenile of rainbow trout and screened.

and the nucleotide sequences of their 3 -UTR

After cDNA capture hybridization andwere found to have low homology with each other.

transformation, 14 out of 93 colonies were

Indeed, each

was specifically detected in

PCR-positive. Each positive clone was partially

Southern blot analysis by using the part of their 3

sequenced（approximately 400 bases）from the 5-UTR sequences as DNA probes. By applying theend and, consequently, 12 out of 14 clones showedsame probes to Northern blot analysis, this section

sequences highly homologous to those of Hsp70

describes the mRNA expression profiling of

cDNAs previously reported for other eukaryotic

in RTG-2 cells exposed to different temperatures.

sspecies. Moreover, the clones were divided into two
Materials and Methods

groups based on the nucleotide sequences, and eachgroup was comprised of six identical clones. The
clones having the longest size from each group were

Cell cultures and exposure to heat stressselected, named C1 and C11, and their complete

RTG-2 cells, immortalized rainbow trout gonadal

cDNA sequences were determined. Consequently,

fibroblasts（Wolf and Quimby, 1962）, were cultured

clones C1 and C11 were 2804 and 2229 bp in length,

at 20℃ in Leibovitz s L-15 medium（Invitrogen）

respectively. Hereafter, these are referred to as

supplemented with 5% fetal bovine serum andandrespectively.

antibiotic-antimycotic mixture（100 U/ml penicillin

The deduced amino acid sequences ofand

G , 1 0 0 μg / m l s t r e p t o m y c i n , a n d 0 . 2 5 μg / m lboth contain 644 residues with calculated

amphotericin B; Invitrogen）. The culture dishes

molecular weights of 71 015 and 70 988, respectively

were sealed with Parafilm and immersed into a（Fig. 2）. The two proteins shared 98.1% identity withwater bath at 24, 28, and 32℃ for 1 h each.

variations in 12 amino acid residues. Furthermore,
rainbow trout Hsp70s shared many identical amino

Northern blot analysis

acid residues with those of other vertebrates（Fig. 2）.

Total RNA was isolated from RTG-2 cells withFor example, Hsp70a and Hsp70b showed 84.5% and

TRI ZOL Reagent （Invitrogen） according to the

84.2% identity with human Hsp70.1, respectively.

manufacturer s instructions. For Northern blotting,Fig. 3 shows a phylogenetic tree constructed with

5μg of the total RNA was separated on a 1.2%

cytosolic HSP70 family members in vertebrates.

agarose-formaldehyde gel and capillary transferred

The tree was composed of the three major groups:

to a Hybond N+ nylon membrane with 10 ×mammalian Hsp70, fish Hsp70, and vertebrate Hsc70.

SSC. Hybridization and detection of target genes

Among the three groups in the tree, rainbow trout

were performed through Southern blot analysis

Hsp70s belonged to the fish Hsp70 group, confirming

as described in Section 1 of this chapter. A probe

the isolated cDNA clones to be those of

specific fors.

Fig. 4 shows the Southern blots of rainbow trout
andTo accurately distinguishbetween the two genes, the DNA probes were

was designed in its 3 -UTR,

namely, nucleotides 6188‑6408, based on the genomic
DNA sequence（GenBank accession no. S85730）
previously reported（Zafarullah1992）.

designed to hybridize to 3 -UTR specific torespective genes. Consequently, the band patterns

Quantification and statistical analysis of mRNAdiffered between

accumulation levels

andsuggestingthat the two rainbow trout Hsp70s were encoded by
distinct genes in the genome.

The fluorograms of Northern blots were
scanned in 8-bit grayscale into Adobe Photoshop（Adobe Systems, San Jose, CA, USA）at 300 dpi

Section 2. mRNA expression profiling of─

Temperature shift experimentusing a flatbed scanner（GT-8700F; Seiko Epson,Tokyo, Japan）and accompanying software. The
integrated optical density（IOD）of positive bands

In the preceding section, two distinct genes

was quantified using Gel-Pro Analyzer softwareNobuhiko OJIMA

Fig. 2. Comparison of the amino acid sequences of rainbow trout Hsp70a and Hsp70b with those of othervertebrate Hsp70s. Dots represent amino acid residues that are identical to those of the rainbow trout
Hsp70a sequence, and dashes indicate gaps set to maximize the alignment. The box represents thelocation of the sequence used for designing a probe to screen rainbow trouts. GenBank accession
numbers for cited genes are presented in Fig. 3.

Regulation of expression of HSP genes in rainbow trout

Fig. 3. Phylogenetic analysis of cytosolic HSP70 family members in vertebrates.
Arrowheads indicate the position of rainbow trout Hsp70a and Hsp70b. The threegroups of HSP70 family members are boxed.
Hsp70B was used as an
outgroup. Numbers at the nodes represent the percentage of bootstrap values（1000replicates）. The scale bar represents a phylogenetic distance of 0.1 amino acidsubstitutions per site. GenBank or Swiss-Prot accession numbers for cited proteins areindicated in parentheses.

Nobuhiko OJIMA

Fig. 4. Genomic Southern blot analysis of rainbow trout（left panel）
and（right panel）. RTG-2 genomic DNA digested with
HI,
RI,
and
dIII was separated by agarose gel electrophoresis, blotted onto anylon membrane, and hybridized with DIG-labeled probes. The probes arecomplementary to sequences in 3 -UTR of the two genes. λDNAs digested withdIII were used as size markers and are indicated on the left.

（Media Cybernetics, Silver Spring, MD, USA）. The

5A）. A single mRNA species was detected at 24

relative amounts of Hsp70 mRNAs were calculated℃ for bothby dividing IOD of

To

mRNA differed from that of Hsp70b mRNA. The

by that ofconfirm the validity of usings, where the length of Hsp70a

as an internal

apparent sizes of the bands were consistent withstandard, possible changes in the amounts of Hsc70

the calculated ones based on the cloned cDNAs

mRNAs themselves during heat stress were tested

described in the preceding section. Besides thewith one-way analysis of variance（ANOVA）at thesingle mRNA species at 24℃, longer mRNA species

significance level ofwere detected at 28 and 32℃ for each

<0.05.

（Fig.

5A）. Hereafter, for convenience, the shorter and
Results

longer mRNA species are referred to as Hsp70-shortand Hsp70-long mRNAs, respectively.

Fig. 5 shows the expression profiles of rainbow

To compare the expression levels of different

trout Hsp70 mRNAs in RTG-2 cells exposed tomRNA species, the amounts of thedifferent heat shock temperatures for 1 h. Neithertranscripts were determined by measuring IOD

Hsp70a nor Hsp70b mRNAs were detected at theof the bands. IOD of Hsc70 mRNA was used as ancontrol temperature（20℃）, whereas both mRNAs

internal standard because there was no statisticallywere apparently induced at 24℃ and above（Fig.

significant difference in the level of Hsc70 mRNA at

5A）. In contrast, Hsc70 mRNA was constitutively

different temperatures（ >0.05, one-way ANOVA）.

expressed at all temperatures examined （Fig.

In this regard, however, IOD at 32℃ was excluded

Regulation of expression of HSP genes in rainbow trout

Fig. 5. Expression profiles of Hsp70 mRNAs in RTG-2 cells exposed to different temperatures.（A）Northern blot analysis ofand. The probes are complementary to sequencesin 3 -UTR of respective genes. The positions of 28S and 18S rRNAs are indicated on the left as size
markers. The blots are representatives of three independent experiments.（B）Quantification of
Northern blot analysis. The amounts of Hsp70 mRNAs normalized to those of Hsc70 mRNAs areshown. The values were calculated by dividing IOD of Hsp70 mRNA by that of Hsc70 mRNA, andare presented as means ± SD of three independent experiments.

Nobuhiko OJIMA
from the ANOVA because of its large variance.

there was no statistically significant changes in the

Therefore, the normalized mRNA levels of Hsp70 at

level of Hsc70 mRNA during heat stress（ >0.05,

32℃ were calculated for reference. As shown in Fig.

one-way ANOVA）, IOD of Hsc70 mRNA was again

5B, Hsp70b-short and Hsp70b-long mRNAs displayed

used as an internal standard. In this experiment,

higher expression levels than Hsp70a counterparts

Hsp70 mRNA levels reached the maximum after 3

at any heat shock temperatures tested. For example,

h at 28℃ and then decreased（Fig. 6B）. Hsp70a-longthe mean value of Hsp70b-short mRNA was 3.4-foldand Hsp70b-long mRNAs disappeared by 12 h,higher than that of Hsp70a-short mRNA at 28℃. In

whereas Hsp70a-short and Hsp70b-short mRNAs

addition, the expression level of Hsp70a-short mRNA

remained detectable after 24 h. Although

was almost as high as that of Hsc70 mRNA at 24

andand 28℃, whereas the maximum expression level oflevels of Hsp70b-short and Hsp70b-long mRNAs

Hsp70b-short mRNA was 2.6-fold higher than that ofwere consistently higher than Hsp70a counterparts（Fig. 5B）.

had similar expression profiles, theduring heat stress（Fig. 6B）.

Section 3. mRNA expression profiling of─Section 4. Discussion

Time course experiment
In this chapter, two distinct

s wereIn the preceding section, a 1 h heat shock at 28℃

identified in rainbow trout, and their mRNA

gave rise to the highest accumulation levels of Hsp70

expression profiles were separately investigatedmRNAs. When heat shock is prolonged, how do theusing RTG-2 cells exposed to heat stress.

mRNA levels change in RTG-2 cells? To answer

Considering the possibility that multiplesthis question, this section describes the mRNA

are expressed in rainbow trout cells, a screening

expression profiling of

to identify the cDNAs was carefully designed.

s during prolonged

heat stress.

First, to eliminate individual variations, the cDNA
library was constructed using poly（A） RNA
Materials and Methods

prepared from one individual of juvenile rainbow
trout. Second, 12 full-length cDNA clones encoding

Cell cultures and exposure to heat stress
RTG-2 cells were cultured at 20℃ as described in

Hsp70 were isolated and correctly classified by
DNA sequencing. Consequently, it was found that

Section 2 of this chapter. The culture dishes weretwosealed with Parafilm and immersed into a waterin rainbow trout. Until now, two cDNA clones haves,bath at 28℃ for 1.5, 3, 6, 12, and 24 h.

been isolated as family members of
rainbow trout:

Northern blot analysis

and, were expressedands from（Kothary

1984b）; however, both clones were only partiallyNorthern blot analysis was performed as describedin Section 2 of this chapter.

sequenced. Comparing the partial cDNAs with thecomplete cDNA sequences isolated in the present
study,

Quantification and statistical analysis of mRNA
accumulation levels

was found to correspond to. On the other hand, it could not be identified

with certainty thatis eitheror

Quantification and statistical analysis of mRNA

becausewas the fragment of theexpression levels were performed as described incoding region ofthat is highly conserved. In

Section 2 of this chapter.

addition, a genomic clone of rainbow trout
was previously isolated（GenBank accession no.

Results

AB062281）, which corresponds toand hasan intronless open reading frame.
Fig. 6 shows the expression profiles of Hsp70

Rainbow trout Hsp70s were highly homologous tomRNAs during prolonged heat stress at 28℃. Since

those from other vertebrates（Fig. 2）. Phylogenetic

Regulation of expression of HSP genes in rainbow trout

Fig. 6. Expression profiles of Hsp70 mRNAs in RTG-2 cells exposed to prolonged heat stress at
28℃ .（A）Northern blot analysis of, and. The probes are complementary
to sequences in 3 -UTR of respective genes. The positions of 28S and 18S rRNAs are indicatedon the left as size markers. The blots are representatives of three independent experiments.（B）Quantification of Northern blot analysis. The amounts of Hsp70 mRNAs normalized to those of
Hsc70 mRNAs are shown. The values were calculated by dividing IOD of Hsp70 mRNA by thatof Hsc70 mRNA, and are presented as means ± SD of three independent experiments.

Nobuhiko OJIMA
analysis of cytosolic Hsp70 family members indicatesin the cell morphology after 1 h at 24 and 28℃（datathat there is a fish Hsp70 group containing rainbow

not shown）. Thus, temperatures exceeding 28℃

trout Hsp70s（Fig. 3）. Moreover, the phylogenetic

would be too stressful for RTG-2 cells.

tree showed the other two groups: mammalian

A new finding is that longer mRNA species

Hsp70 and vertebrate Hsc70. Even if other Hsp70

were induced in rainbow trout cells at above 28

family members, glucose-regulated protein 75℃ for both

（Grp75） and 78 （Grp78）, were included in thethe expression of multiple mRNA species has been

analysis, the clustering of the three groups was notreported for several heat-shock genes（Dellavalle

altered（data not shown）. Somes belonging

and1994; Takechi（Fig. 5）. Although, 1994）, two different

to the fish Hsp70 group are heat-inducible; e.g.,mRNA species from a single

Japanese flounderbeen detected. Since the longer mRNA species were

（Yokoyama

1998）, platyfish

have not yet

detected only under severe heat stress conditions,and zebrafishsthey may be useful as a biomarker for the high2004）. Therefore, rainbow trout

degrees of heat stress. However, whether such

Hsp70s are suggested to be heat-inducible forms,

mRNAs are induced at the individual level remains

and indeed, their transcription was induced by heatto be clarified. Now, what mechanisms underlie

stress（Figs. 5 and 6）.

the generation of different Hsp70 mRNA species（Yamashita

Southern blot analysis indicated that rainbow trout

under severe heat stress condition? There are

Hsp70a and Hsp70b are encoded by distinct genes

three possible mechanisms:（i）polyadenylation andin the genome（Fig. 4）, which would be common

deadenylation［e.g.,among salmonid fishes because, as suggested by
Ohno1994）］,（ii）defective splicing［e.g.,（1968）, most of them have evolved bytetraploidization from a diploid ancestor. In fact, Rise
（2004）have reported the presence of distinct
forms of several genes in Atlantic salmon（Dellavalle（Dellavalle1994）］, and（iii）alternative

splicing［e.g., mouse（Takechi1994）］.

Among them, it is most likely that the mRNA
species are generated by alternative splicing.

by preliminary comparisons of its expressed

One reason is that the lengths of the longer

sequence tag（EST）clusters. Thus, rainbow trout

mRNA species appear to be too long if they areandare suggested to be paralogous

genes.

（Figs. 5A and 6A）. For example, a polyadenylated

Northern blot analysis showed that bothandpolyadenylated forms of the shorter mRNA species
form ofwere induced by heat stress in RTG-2

Hsp70 mRNAs has poly（A）tailsof about 120 to 160 nucleotides（Dellavalle

cells（Figs. 5 and 6）. In this analysis, Hsp70a and1994）. Another reason is that rainbow trout Hsc70

Hsp70b mRNAs could be clearly distinguishedmRNA, which contains introns in the genomic DNA

by using probes specific to respective genes. As（Zafarullah

shown in Fig. 5, the mRNA levels of

1992）,showed no change in lengthandat above 28℃（Fig 5A）. This indicates that splicing

decreased at 32℃ and the variance of data

is not defective after severe heat stress. Taken

at this temperature was large, suggesting that a

together, the longer mRNA species of the rainbow

temperature of 32℃ was too severe for rainbow

trout

trout cells. For instance, the synthesis of Hsps

spliced products. However, the possibility thatwas not observed at 32℃ in RTG-2 cells, and thepolyadenylation and deadenylation may be involved

temperature range in which RTG-2 cells couldin the event cannot be excluded because thesurvive for at least 7 days was 0‑28℃（Mosser

sequences of poly（A）tails from the longer mRNA., 1986）. Furthermore, Mosser（1986）

reported that RTG-2 cells became shriveled and took

s are thought to be alternatively

species have not yet been determined.
Another finding is that the accumulated mRNAon a threadlike appearance after 1 h at 32℃. The

levels ofwere higher than that ofobservation in the present study was consistent withduring heat stress（Figs. 5 and 6）. This difference

these results, and there was no observable change

seems not due to the difference of labelingRegulation of expression of HSP genes in rainbow trout
efficiency between the two DIG-labeled probes.

have been performed at the protein level, and HSP

The efficiency for both probes were evaluated bygenes have only been cloned from a modest number

gel electrophoresis, confirming that there was noof different species（Basusignificant difference between the signal intensities

since the structure and the mRNA expression ofof the two bands （data not shown）. However,

HSP genes have been analyzed individually andquantitative PCR analysis will be needed to discuss

separately, a comprehensive mRNA expression

more accurately the difference of the mRNA levels.

profile of HSP genes has not been established in

On the other hand, the mRNA expression profiles

cells from a single fish species. Additionally, HSPs

of the two genes were similar to each other inare good examples of molecular level biomarkers

both temperature shift（Fig. 5）and time course

to perceive sublethal cellular damages as the result

experiments（Fig. 6）. In the time course experiment,of an environmental stress（Ryan and Hightower,

prolonged heat stress at 28℃ for longer than 3 h1996）. However, the use of HSPs as indicators forcaused a decrease in Hsp70 mRNA levels（Fig. 6B）.

stress states of fish in general is premature（Iwama

These results generally agreed with the induction2004）, in part because little information ispattern of Hsp70 mRNA previously reported

available about a comprehensive stress response（Kothary

profile of HSP genes in fish cells.

1984a）, in which, however, multiple

Hsp70 mRNA species were examined together
without separation.

2002）. Furthermore,Thus, this chapter describes the isolation ofmultiple HSP genes from rainbow trout and analysis

At the protein level, the synthesis of Hsp70 hasof their expression profiles at the mRNA levels. In

been reported to attain a peak at 2 h and maintainthis regard, the mRNA levels between unstressedhigh levels between 2 and 6 h after treatment ofand heat-shocked cells were quantitatively compared

RTG-2 cells at 28℃（Mosser

using real-time RT-PCR analysis.

1986）. These

results are roughly consistent with the mRNA
expression profiles of Hsp70 shown in Fig. 6. In

Section 1. Cloning of HSP cDNAs expressed inaddition, Mosser

heat-shocked RTG-2 cells.（1987）reported that theinduction of HSP synthesis at 28℃ paralleled thedevelopment of thermotolerance in RTG-2 cells. On

To comprehensively analyze the mRNA expression

the other hand, rainbow trout Hsp70a and Hsp70b

profile ofhave not been clearly distinguished at the protein

each gene is prerequisite to the specific detection.

level as well as at the mRNA level. Nevertheless,However,

two proteins supposed to be Hsp70 have been

trout except forreported by Zafarullah（1992）ands described in the preceding

detected in heat-stressed RTG-2 cells（Airaksinen
1998; Mosser1986）. Their apparent

molecular sizes were 70 and 68 kDa（Mosser1986）, and 69 and 67 kDa（Airaksinens, DNA sequence information fors have not been cloned from rainbow

chapter. Thus, isolation of genes encoding HSP
family members is required, and it is desirable thats would be isolated as many as possible at once.

1998）. Considering the predicted molecular weights

This section describes an efficient isolation of cDNAs

described in Results of Section 1, Hsp70a and Hsp70b

encoding HSPs from RTG-2 cells and the features ofseem to be their equivalents.

their gene structures.

Chapter II. Cloning and mRNA expression profiles

Materials and Methodsof HSP family members
Cell Culture
To resolve the regulation and functionalsignificance of HSPs, fish are ideal organisms because

RTG-2 cells were cultured at 15℃ as described in
Section 2 of Chapter I.

they are ectothermic vertebrates and naturally
exposed to thermal stress in their environment（Basu2002）. However, most studies on HSPs in fish

Construction of directional cDNA library
Five 100-mm culture dishes with confluent

Nobuhiko OJIMA
RTG-2 cells were heat-shocked at 25℃ for 24 hRNAs using a

in an incubator. Cells were lysed with TRI ZOL

of a

I primer-adapter. After addition

Reagent（Invitrogen）, and total RNA was isolated

and size-fractionated by column chromatography

I adapter, cDNAs were digested withI

according to the manufacture s instructions. Poly

according to the manufacturer s instructions. The（A）RNA was isolated from the total RNA usingdigests were ligated into plasmid vector pSPORT1,

an Oligotex-MAG mRNA Purification kit（Takara

and the plasmids were introduced into ELECTROMAX

Bio）. A directional cDNA library was constructed

DH10B competent cells （Invitrogen） using anusing a SuperScript Plasmid System for cDNA

electroporation apparatus（MicroPulser; Bio-Rad

Synthesis and Plasmid Cloning kit（Invitrogen）.

Laboratories）. The electroporation was performed

cDNAs were constructed from 5μg of poly（A）

using a 0.1-cm-gap cuvette containing 1μl of theTable 1. List of cDNAs isolated from heat-shocked RTG-2 cells

Regulation of expression of HSP genes in rainbow trout
Table 2. Summary of cloned cDNAs encoding HSP family members

plasmid DNA and 20μl of the competent cells atare summarized in Table 1, where the BLAST hits

1.8 kV and one pulse. The cells were plated onto LB

with anagar plates containing 100-μg/ml ampicillin.

of 200 clones, 121 clones（60.5%）showed significant

value 10‑5 were defined as unknown. Out

similarities（
Sequencing and bioinformatic analysis

value<10‑5）with proteins in the NCBI

database, and 11 clones（5.5%）were putatively

Two hundred white colonies were arbitrarily

identified as HSP family members. Additionally, four

selected for isolation of plasmid DNA. The plasmid

clones（2.0%）were classified as chaperone-related

DNA was purified using a conventional alkaline/SDS

proteins , which was designated in the present

lysis method. The 5 end each of cDNA inserts

study, including proteins with chaperone or catalytic

was sequenced with a T7 promoter primer（5activity for protein folding.

-TAATACGACTCACTATAGG-3 ）using a Thermo

Next, the cDNA clones encoding HSP family

Sequenase II dye terminator cycle sequencing kit

members were completely sequenced, resulting（Amersham Biosciences）and an automated DNA

in identification of nine genes（Table 2）. These

sequencer（373A; Applied Biosystems, Foster City,

clones were designated as in Table 2, based on

CA, USA）. After removing the vector sequence,

their deduced protein homology with HSP family

the deduced amino acid sequences were comparedmembers of other vertebrates. Among them, twowith sequences in the nr protein database using

types having different sequences were found eachthe blastx program in the NCBI BLAST homepage

in（http://www.ncbi.nlm.nih.gov/BLAST/）
.

and
β and

They were designated as
β , andand,respectively. Both Hsp90βa and Hsp90βb cDNAs
Results

contained complete open reading frame（ORF）, andtheir deduced amino acid sequences shared 97.7%

To clone multiple HSP genes at once, 200 cDNAs

identity with each other. The two proteins contained

were arbitrarily isolated from heat-shocked RTG-2

the conserved C-terminal tetrapeptide motif, EEVD

cells and their 5 ends were sequenced. The readable（data not shown）, which plays an important rolesequence length was approximately 340 bp. The datain cofactor binding mediated by a tetratricopeptide

Nobuhiko OJIMA
repeat（TPR）domain（Scheufler

2000）. A（Munro and Pelham, 1987）. Besides those described

BLAST search indicated that both proteins had

above, cDNAs encoding chaperonin containing

the highest amino acid identity with Atlantic

TCP1 subunit 8（Cct8）, Hsp47 and DnaJ homolog

salmonHsp90β（Table 2）. Meanwhile,

were identified. Cct8 and Hsp47 cDNAs contained

Hsc70b cDNA contained an incomplete ORF, andcomplete ORFs, whereas DnaJ homolog cDNA wasits deduced protein lacked 121 amino acid residues

a partial fragment containing putative intron（s）.

from the N-terminus compared with that of Hsc70a

The deduced Hsp47 protein contained an RDEL

cDNA. The cDNA sequence of Hsc70a was identicaltetrapeptide motif at the C terminus （data notto that of the genomic clone reported previously

shown）that acts as an ER-retention signal（Satohas rainbow trout
For（Zafarullah1992）.

1996）.

one clone was isolated and identified aswhich was cloned from a juvenile rainbow

trout in the preceding chapter. The two Hsp70a

Section 2. Accumulation of HSP mRNAs in
unstressed and heat-shocked RTG-2 cells

proteins derived from different sources showed
only one amino acid variation, namely, T429I（datanot shown）. For

one incomplete cDNA

As a result of the preceding section, DNA
sequence information for severals becameclone, which lacked the 5 end, was isolated. The

available. Based on this information, it is possible

deduced protein possessed a C-terminal tetrapeptide

to design probes and primers specific to respective

KDEL（data not shown）, which is conserved ingenes. By using these probes and primers, this

BiP/Grp78 and required for retrieval of BiP/Grp78

section describes the mRNA expression profiles ofmolecules that leave the endoplasmic reticulum（ER）

s and the quantitative analysis of their mRNA

Table 3. Primers used for synthesis of DIG-labeled probes

Regulation of expression of HSP genes in rainbow trout
accumulation levels before and after heat stress.

Northern blot analysis
Five micrograms of total RNA were separated

Materials and Methods

on a 1% agarose-formaldehyde gel and capillarytransferred to Hybond N+ nylon membranes

RNA preparation（Amersham Biosciences）with 10 × SSC. RNAs

Control RTG-2 cells were cultured at 20℃ as

were UV-crosslinked to the membranes, which

described in Section 2 of Chapter I. The culture

were subsequently hybridized at 68℃ for 1 h indishes were sealed with Parafilm and immersed into aa PerfectHyb hybridization solution （Toyobo）

water bath at 28℃ for 3 h. This heat-shock condition

containing DIG-labeled DNA probes. The probes

gave rise to the highest accumulation levels of

were labeled with a PCR DIG Probes Synthesis

Hsp70 mRNAs in Chapter I. Total RNA was isolatedkit（Roche Diagnostics）using primers specific tofrom the control and heat-shocked RTG-2 cells

3 -UTR of each gene（Table 3）. The hybridized

with TRIZOL Reagent（Invitrogen）according to themembranes were washed twice with 2 × SSC plus

manufacturerʼs instruction. For real-time RT-PCR,

0.1% SDS at 68℃ for 5 min, and then twice with

20μg of each RNA was incubated at 37℃ for 20

2× SSC plus 0.1% SDS at 68℃ for 15 min. The

min with 10 U RNase-free DNase I（Takara Bio）

chemiluminescent detection of the probes wasin a 50-μl reaction volume containing 20 U RNase

performed with a DIG Luminescent Detection kit

inhibitor（Toyobo）. RNAs were extracted with phe-

（Roche Diagnostics）according to the manufacturerʼs

nol:chloroform:isoamyl alcohol（25:24:1 mixed, pH5.2;

instruction. Positive signals were detected by using

Nacalai Tesque, Kyoto, Japan）followed by ethanol

a luminescent image analyzer（LAS-1000 mini; Fuji

precipitation.

Photo Film, Tokyo, Japan）.

Table 4. Primers used for real-time RT-PCR

Nobuhiko OJIMA
Quantitative RT-PCR analysis

the PCR product, as displayed by a single peak

Quantitative RT-PCR was performed using a（data not shown）. The control, containing all the

QuantiTect SYBR Green RT-PCR kit （Qiagen）

reaction components except for the template, wasand ABI PRISM 7000 Sequence Detection System

included in all experiments. The amount of each（Applied Biosystems）. One-step RT-PCR was

HSP mRNA was then normalized to the abundance

performed in a 25-μl total reaction volume includingof a housekeeping gene, β12.5μl of 2 × QuantiTect SYBR Green RT-PCR

validity of using βmaster mix, 0.25μl QuantiTect RT mix, 50 ng RNA

possible changes in the amounts of β-actin mRNA

template, and 0.2μM each of target specific primers

were tested before and after heat shock by using

designed to amplify a part of 3 -UTR of each gene

λpoly（A）+ RNA-A（Takara Bio）as an external（Table 4）. To quantify each target transcript, astandard. The normalized values of each HSP

standard curve was constructed with serial dilutions

mRNA in heat-shocked cells were divided by thoseof total RNA extracted from heat-shocked（28℃,

in controls. Student s unpaired

3 h）RTG-2 cells for every set of primers. Reverse

group comparisons.

. To evaluate theas an internal standard,

test was used fortranscription was performed at 50℃ for 30 min,and thermal cycling conditions were as follows: 95

Results℃ for 15 min; and 40 cycles of 95℃ for 5 s and 60℃ for 31 s. After PCR, a melting curve analysis

To investigate whether the cloned HSP genes

was performed to demonstrate the specificity ofare actually transcribed in rainbow trout cells,

Fig. 7. mRNA accumulation levels ofs in control（C）and heat-shocked（HS）RTG-2 cells as
revealed by Northern blot analyses. The probes are complementary to sequences in 3 -UTR ofrespective genes. The positions of 28S and 18S rRNAs are indicated on the left as size markers. The
blots are representatives of three independent experiments.

Table 5. mRNA accumulation levels ofs relative to those in control

Regulation of expression of HSP genes in rainbow trout
Northern blot analysis was performed using DNA
Section 3. Discussion

probes specific to 3 -UTR of respective genes. In
this regard, the DnaJ homolog gene was excluded
from this analysis because its cDNA was incomplete

In this chapter, multiple genes encoding HSP

where 3 -UTR was uncertain. Meanwhile,family members were isolated, and their mRNA

isolated in Chapter I was included. Consequently,

accumulation levels were comprehensively comparedsingle bands were detected for all genes examined

in RTG-2 cells before and after heat shock. To

exceptaccurately compare the mRNA levels of these HSP

s where two mRNA species having

different sizes were detected in heat-shocked cells
irrespective ofandgenes, real-time RT-PCR was used.

as observed in

In preliminary experiments, it was found that

Chapter I（Fig. 7）. Therefore, it was concluded thatthe mRNA accumulation level of the β-actin genethe cloned genes are actually transcribed in RTG-2

was reduced in RTG-2 cells after 24-h exposure tocells.

heat shock at 25℃（data not shown）. Accordingly,

Although Hsp70a and Hsp70b transcripts wereit was considered that the ratios of mRNAs of theapparently induced by heat shock, changes in mRNA

HSP genes to those of the house-keeping geneaccumulation levels were ambiguous for the otherwould be relatively high in the heat-shocked cells,

HSP family members（Fig. 7）. Thus, to accurately

and utilization of the cells as a source of a cDNA

determine the changes, quantitative RT-PCR analysis

library was attempted to isolate HSP genes as manywas performed. In this regard, the β-actin gene was

as possible. As expected, multiple HSP genes wereused as an internal standard for normalizing theidentified by arbitrarily isolating cDNAs from themRNA accumulation levels of HSP genes, because

RTG-2 cells（Table 1）. Among the cloned genes, two

no significant differences were found in the β-actin

cDNAs having different sequences were found inmRNA levels between control and heat-shocked cells

each of（ =0.36, Student s

and. These were suggested totest, two-sided）, at least underbe paralogous genes as in the case of rainbow trout

the heat-shock conditions applied here（data nots in Chapter I. Although only one clone could

shown）. Consequently, significantly increased afterbe identified each for the other HSP family genes

heat shock were the mRNA accumulation levels ofin the present study, each may have a possible

five HSP genes, namely,

paralog because it has been inferred that most of, and（Table 5）. In particular, theaccumulation levels of Hsp70a and Hsp70b mRNAs

the rainbow trout genes are duplicated（Ohno
1968; Palti

2004）.

were dramatically increased after heat shock, andSequence analyses of the two Hsp90 cDNAs

the mean values in heat-shocked cells were 480- andshowed that both deduced proteins were highly

510-fold of those in controls, respectively（Tableh o m o l o g o u s t o A t l a n t i c s a l m o n H s p 9 0β. I n

5）. The increased levels of Hsc70a, Hsc70b andvertebrates, two major Hsp90 isoforms are present in

Hsp47 mRNAs were 1.3- to 2.8-fold on average after

the cytosol, namely, Hsp90α and Hsp90β（Sreedharheat shock and considerably lower than those of2004）. Hsp90α is highly heat-inducible,

Hsp70 mRNAs（Table 5）. Further, when

whereas Hsp90β is expressed constitutively at aandwere compared, the increased mRNA

high level at normal temperatures and its expression

was more remarkable than that ofis weakly dependent on heat shock（Scheibel andas suggested by a statistical analysis（Table

Buchner, 1997）.The two Hsp90 genes in the present

level of5）
. On the other hand, there were no differences instudy, designatedmRNA levels of the other four HSP genes between

constitutively expressed in RTG-2 cells irrespective

β and

β,

were

control and heat-shocked cells with a significanceof heat-shock treatment （Fig. 7 and Table 5）,level of 0.05（Table 5）.

suggesting that both genes actually encode a β
isoform of Hsp90. On the other hand, Sathiyaa（2001）isolated a partial fragment of rainbow trout
Hsp90 cDNA, showing that its transcripts were

Nobuhiko OJIMA
induced by heat shock in hepatocytes as revealed bytrout

Northern blot analysis. The reported partial

accumulation levels in heat-shocked cells differeds. Furthermore, since the mRNA

sequence is not identical to the Hsp90s in the

betweenpresent study, raising the possibility that there

have promoters with different activities.

and, the two genes may

exists at least one α isoform of Hsp90 besides theCct8, one of the subunits of the CCT complex

two β isoforms in rainbow trout. This possibility

abundant in eukaryotic cytosol, is a member of theis also supported by the result that an α isoform

chaperonin/HSP60 family（Kubota and Willison,of Hsp90 has been isolated from another salmonid,

1997）. Until now, the heat shock inducibility of

the Chinook salmon

CCT subunits has not been investigated in fish. In（Palmisanorainbow trout RTG-2 cells, quantitative RT-PCR

1999）
.

In contrast to the other HSP family members

analysis showed that there was no significant

examined, the accumulation levels of Hsp70 mRNAs

increase in the Cct8 mRNA levels after heat shock

were dramatically increased after heat shock（Table 5）. Likewise, it has been reported that no（Fig. 7 and Table 5）. In Northern blot analysis

significant increase of CCT subunits was detected（Fig. 7）, two mRNA species having different sizes

in response to heat shock at protein levels in bothwere detected in heat-shocked cells irrespective

HeLa and mouse BALB/3T3 cells（Kubotaof1999）. However, as discussed by Kubota

andas described in Chapter I.

,

Since nearly the same regions were detected for the（1999）, CCT may be induced depending on the typetwo genes in Northern blot and real-time RT-PCR

of stress, cells, organisms and other environmental

analyses, it is inferred that the accumulation levels

conditions.

of Hsp70 mRNAs in the latter analysis were those

In RTG-2 cells, the mRNA accumulation levels

of integrated values of the two mRNA speciesofdetected in the former analysis. In any case, given

shock（Table 5）. It is known thatthe magnitude of the changes in the mRNA levels,

is induced by heat shock at the transcriptional

it is strongly suggested thatlevel in zebrafish embryos（Pearson

s are the mostuseful biomarkers of heat stress among HSP family
members in fish, at least in rainbow trout.
The study in this chapter demonstrated that
there exist twos in rainbow trout cells likes in Chapter I. Zafarullah

were significantly increased after heat
expression
1996）

and chicken embryonic fibroblasts（Hirayoshi
1991）. In vertebrate cells, Hsp47 is located in
the lumen of ER（Nagata, 1997）, as is BiP/Grp78（Gething, 1997b）. In rainbow trout, however, the.（1992）isolated

cellular localizations of Hsp47 and Grp78 have nota genomic clone encoding Hsc70 from rainbow trout

been determined. Considering that rainbow trout

and designated it as, which corresponds

Hsp47 and Grp78 had ER retention signals（datain this chapter（Table 2）. Hsc70

not shown）, it is suggested that both proteins areis a member of the HSP70 family and exhibits

located in the ER lumen like their counterparts

constitutive expression （Hightower and Leung,from other vertebrates. In addition, consistent with1997）
. It has been reported that Hsc71 mRNA levels

mammalian cells（Gething, 1997b）, the results in thisdid not significantly increase after heat shock inchapter indicate that rainbow trout Grp78 mRNA

RTG-2 cells（Zafarullah

was constitutively expressed in unstressed cells（Fig.

to the1992）, and similar

results were obtained by Northern blot analysis in

7 and Table 5）.

Chapter I. However, by using quantitative RT-PCR

The enhanced synthesis has been observed withanalysis, it was demonstrated that the accumulation

polypeptides having 100, 87, 70, 68, 60, 39, and 27 kDalevels of both Hsc70a and Hsc70b mRNAs werein RTG-2 cells exposed to heat shock at 28℃（Mosser

significantly increased after heat shock in the1986）. Under the same heat-shock conditions,present study（Table 5）. Considering that several

the enhanced syntheses of a similar set of HSPs

putative HSEs have been found in the promoter

have also been found by another research group

region of

1992）, it is likely（Kothary and Candido, 1982）, although there werethat there exist functional HSEs for the rainbow

slight differences in the polypeptide size together（Zafarullah

Regulation of expression of HSP genes in rainbow trout
with an additional polypeptide having 32 kDa.

Thus, the purpose of the study was to isolate a

Judging from the molecular sizes, it is thought that

cDNA clone encoding rainbow trout HSF and to

the above-mentioned polypeptides belong to either

characterize its translation products. This chapterof the HSP100, HSP90, HSP70, HSP60, HSP40, anddescribes the existence of two distinct HSF1

HSP27 families. Among these, the genes encoding

isoforms in rainbow trout and the evidence for

the HSP90, HSP70, HSP60, and HSP40 families were

heterotrimer formation of these isoforms

cloned in this chapter. Unfortunately, however,cDNAs encoding HSP100 and HSP27 families could

Section 1. cDNA cloning and genomic organization

not be found in 200 cDNAs arbitrarily isolated in theof HSF1

present study.
As a result of the preceding chapters, it was
Chapter III. Cloning and characterization of HSF1

shown that some rainbow trouts, especiallys, are induced at the transcriptional level
The expression of HSP genes is regulated byby heat stress. This suggests that cells of rainbow

heat-shock transcription factors（HSFs）that bind totrout have an HSF as do other vertebrates. Thus,a specificto demonstrate the existence of the transcription-acting element, namely, HSE（Morimoto,

1998; Pirkkala

2001; Wu, 1995）. In vertebrates,

genes encoding four types of HSFs, HSF1‑4, havefactor, this section describes the cDNA cloning of
rainbow trout HSF and its gene structure.

been cloned. Among the HSF family members, HSF1
Materials and Methods

is the principal transcriptional factor activated byexposure to stresses such as heat shock, and thisprotein is known to form homotrimers that bindDNA（Morimoto, 1998; Pirkkala, 2001; Wu,

1995）
.

Cell culture and animals
RTG-2 cells were cultured at 15℃ as describedin Section 2 of Chapter I. Rainbow trout（3 months

During evolution, fish have adapted to live inold）, which were used to extract total RNA forvarious ambient temperatures. Reflecting such

RT-PCR, were obtained from the Nikko Station

adaptations, the threshold temperature for HSPof the National Research Institute of Fisheries

induction differs between cold- and warm-adapted

Science（Tochigi Prefecture, Japan）and reared on afish. For example, HSPs are induced in the 26‑30

commercial diet at 15℃ .

℃ range in rainbow trout RTG-2 cells（Mosser1986）, whereas HSP70 is induced in the 35‑37℃ range in zebrafish tissues （RåberghcDNA cloningA r a n d o m p r i m e d λZ A P I I c D N A l i b r a r y

2000）. However, little is known about the molecular

was constructed by using a λZapII predigested

mechanisms underlying the difference in HSP

RI/CIAP-treated vector kit（Stratagene, La Jolla,

induction temperatures among fish species. To

CA, USA）with RNA isolated from RTG-2 cells as

date, Råbergh（2000）reported the isolation

described below. Approximately 1.2 × 106 plaques

cDNA from zebrafish, which is awere screened at 2 × 105 plaques per 140 × 100-mm

warm-adapted fish. Although a cDNA fragment

plate by hybridization of duplicate nitrocellulose

encoding an HSF has also been cloned from bluegill

membranes with a 2.7-kb fragment of chicken HSF1

sunfishcDNA （Nakai and Morimoto, 1993） as a probe.

of one（Råbergh2000）, a full-length HSF1 cDNA clone has not been

The membranes were soaked in 2×SSC for 5 min,isolated from any fish other than zebrafish. Someprehybridized at 42℃ for 2 h with hybridization

authors（Airaksinen

buffer ［6×SSC, 1×Denhardtʼs solution, 0.15%

1998; Le Goff and Michel,1999） have reported the presence of a protein

SDS, and 100μg/ml denatured calf thymus DNA

that possesses HSF1-like activity in rainbow trout;（Invitrogen）］, and hybridized with a 32P-labeled

however, a gene encoding HSF1 itself has not been

DNA probe in the same buffer at 42℃ for 16 h. The

identified in this cold-adapted fish.

membranes were then rinsed twice with 2 × SSC

Nobuhiko OJIMA
plus 0.1% SDS at room temperature for 5 min perC-3 ）as antisense primers. The nested PCR was

rinse, washed twice in 2 × SSC plus 0.1% SDS at 50

performed with the SP6 promoter primer （5℃ for 20 min per wash, dried, and exposed to X-ray-ATTTAGGTGACACTATA-3 ）as a sense primer

film for 2 days. Positive clones were isolated throughandthree rounds of screening. Phagemid pBluescript SK

CTCCCCACTCTGCTGGAGA-3 ; for（−）was excised from purified plaques with helper

CCCACTCTGCTGGAGCTGGAGG-3 ）as antisense

phage according to the manufacturer s instructions.

primers. The amplified products were subcloned into

The 5 - and 3 - termini of rainbow trout HSF

pGEM-T Easy vector（Promega, Madison, WI, USA）.

-specific primers（for, 5 -CGGAC, 5 -TC

cDNAs were isolated by rapid amplification of
cDNA ends（RACE）. A directional cDNA library

Sequence determination

constructed in Section 1 of Chapter II was used as

Nucleotide sequences were determined fromthe PCR template. For 5 -RACE, the first PCR wasboth strands by a 373A DNA sequencer（Applied

performed with the M13 reverse primer（5 -AGCGG

Biosystems） and a Thermo Sequenase II Dye

ATAACAATTTCACACAGG-3 ）as a sense primer

Terminator Cycle Sequencing kit （Amersham

and a rainbow trout

Biosciences）.

-specific primer（5 -ATCTTTCTCTTCATCCCCAGGACT-3 ）as an antisense
primer. The nested PCR was performed with the T7
promoter primer（5 -TAATACGACTCACTATAG
GG-3 ）as a sense primer andPhylogenetic analysis
A phylogenetic tree was constructed as described-specific primers

in Section 1 of Chapter I. The setting parameters（for

5 -TGCCTTTTATGTTCTGCACGA-3

were as follows: MATRIX, BLOSUM; GAPOPEN,; for

5 -CCTCCCTCCACAGAGCTTCA-3

10.0; GAPEXT, 0.05; GAPDIST, 8; MAXDIV, 40;）as antisense primers. For 3 -RACE, the first PCR

ENDGAPS, off; NOPGAPS, off; NOHGAPS, off.

was performed with the M13 forward primer（5-CCCAGTCACGACGTTGTAAAACG-3 ）as a sense
primer and-specific primers（for5Southern blot analysis
Genomic DNA was isolated from RTG-2 cells by-GAAGCAGCTTGTCCAGTACACTAA-3 ; fora GenomicPrep Cells and Tissue DNA Isolation, 5 -GAAGCAGCTGGTCCAGTACACCT

kit （Amersham Biosciences） according to theTable 6. Comparison of rainbow trout HSF1s with other vertebrate
HSF1

Regulation of expression of HSP genes in rainbow trout
manufacturerʼs instructions. Ten micrograms ofshowed 55.3% and 56.4% identity to human HSF1,

genomic DNA was digested withrespectively（Table 6）.

HI,

RI, ordIII, resolved by electrophoresis on a 1% agarose

Next, the structural features of HSF1a andgel, and transferred to a nylon membrane（Hybond

HSF1b were examined in comparison to those of

N+; Amersham Biosciences）. The membranes wereother vertebrate HSF1s. HSF1 has been reported

hybridized for 1 h in PerfectHyb hybridization

to contain conserved regions referred to as thesolution（Toyobo）with DIG-labeled DNA probes.

DNA-binding domain（DBD）, and the amino-terminal

As the probes, the 3 -UTRs ofand carboxyl-terminal hydrophobic heptad repeats

andwere labeled by a PCR DIG Probes Synthesis kit（HR-A/B and HR-C, respectively）（Morimoto, 1998;（Roche Diagnostics）. The probed regions of

Pirkkala

andcorrespond to nucleotides 1651‑2027 andalignment demonstrated that both of the rainbow

1691‑2052, respectively. The hybridized membranes

trout HSF1s contained these conserved domain

were washed twice with 2 × SSC plus 0.1% SDS

structures（Fig. 8）, as shown schematically in Fig.

at room temperature for 5 min, and then twice10.

, 2001; Wu, 1995）. Multiple sequencewith 0.1 × SSC plus 0.1% SDS at 68℃ for 15 min.

Region I （DBD） of the rainbow trout HSF1s

The chemiluminescent detection of the probes wasshowed high similarity to the corresponding region

performed with a DIG luminescent detection kitof zebrafish, chicken, and human HSF1 （Table（Roche Diagnostics）according to the manufacturerʼs6）; for instance, DBD of rainbow trout HSF1b

instructions. The positive signals were detectedshared 90.7% identity with that of human HSF1.

by exposure on Hyperfilm-MP （Amersham

By contrast, regions II（HR-A/B）and IV（HR-C）

Biosciences）.

of the rainbow trout HSF1s showed less similarity
to the corresponding regions of other vertebrate
Results

HSF1s （Table 6）. However, the actual heptad
repeats of hydrophobic amino acids are conserved

By screening an RTG-2 cDNA library using aacross the whole HSF1 family（Fig. 8）. In addition,

chicken HSF1 cDNA probe, two positive clones,

two KRK tripeptides, which are conserved among

named C1 and C2, were isolated. Sequence analysis

characterized HSF1 family members, were identified

revealed that these two clones encoded distinct

in both of the rainbow trout HSF1s（Fig. 8）. In

isoforms of HSF. Clone C1 was a partial cDNA

contrast to the highly conserved regions describedcontaining an insert of 983 nucleotides encoding

above, regions III and V of rainbow trout HSF1s

the DNA-binding domain of HSF, whereas clone C2

showed low similarity to the counterparts of othercontained an insert of 2771 nucleotides including

vertebrate HSF1s （Table 6）. Notably, region V

introns and an ORF encoding 513 amino acids.

showed low similarity across the HSF1 family, even

By using 5 - and 3 -RACE, the full-length
cDNAs of clones C1 and C2 without introns were

between rainbow trout HSF1a and HSF1b（78.8%
identity; Table 6）.

determined to be 2083 bp and 2142 bp, respectively.

The genomic organization of the rainbow trout

Clones C1 and C2 were predicted to encode proteinss were examined by Southern blot analysisof 501 and 513 amino acids, respectively（Fig. 8）.

using the 3 -UTRs of

Phylogenetic analysis indicated that the two proteins

These two probes showed different hybridized

andas probes.

belong to the HSF1 cluster（Fig. 9）. Accordingly,

patterns（Fig. 11）, demonstrating that each HSF1 isclones C1 and C2 are referred to hereafter as

encoded by a distinct gene in the genome.

rainbow trout HSF1a and HSF1b, respectively.
The sequence identity between the two predicted
proteins was 86.4% （Table 6）. By contrast, theSection 2. mRNA expression pattern of HSF1 invarious tissues

whole ORF of the rainbow trout HSF1s showed
low homology to those of other vertebrate HSF1s.

In the preceding section, it was shown that

For example, rainbow trout HSF1a and HSF1b

two distinct genes encoding HSF1 are present inNobuhiko OJIMA

Fig. 8. Comparison of the predicted amino acid sequences of rainbow trout（rt）HSF1aand HSF1b with the sequences of zebrafish（z）, chicken（c）, mouse（m）and human（h）HSF1. The three domain structures, DBD, HR-A/B and HR-C, are boxed. Open andfilled diamonds indicate the repeats of hydrophobic amino acids. The underlined KRK
tripeptides are putative nuclear localization signals（NLSs）. The numbers on the left
indicate the amino acid positions of each protein.

Regulation of expression of HSP genes in rainbow trout

Fig. 9. Phylogenetic tree of the vertebrate HSF family based on the amino acidsequences. The tree was calculated by neighbor joining, with
HSF
used as an outgroup. Arrowheads indicate the position of rainbow trout HSF1aand HSF1b. Numbers at the nodes indicate the percentage of bootstrap values（1000 replicates）. The scale bar refers to a phylogenetic distance of 0.1 amino acidsubstitutions per site. GenBank accession numbers for the sequences are as follows:
human HSF1（M64673）, HSF2（M65217）, HSF4（D87673）; mouse HSF1（X61753）,
HSF2（X61754）, HSF4（AB029350）; chicken HSF1（L06098）, HSF2（L06125）, HSF3
（L06126）;
HSF1（L36924）; zebrafish HSF1（AB062117）; rainbow trout
HSF1a（AB062548）, HSF1b（AB062549）;
HSF（M60070）.

Fig. 10. Schematic representation of HSF1 domain structures. The three regions ofidentity are denoted region I, corresponding to DBD; region II, corresponding
to the amino-terminal hydrophobic heptad repeat（HR-A/B）; and region IV,
corresponding to the carboxyl-terminal hydrophobic heptad repeat（HR-C）.
Regions III and V roughly correspond to domains of mammalian HSF1, namely, theregulatory and transactivation domains, respectively.

Nobuhiko OJIMA

Fig. 11. Genomic Southern blot analysis of rainbow trouts. In theleft panel, hybrization was carried out with a DIG-labeledprobe（a377-base fragment of the 3 -UTR ofcDNA）, whereas in the rightpanel, hybrization was carried out with a DIG-labeledprobe（a362-base fragment of the 3 -UTR ofcDNA）. λDNAs digested withdIII were used as molecular markers and are indicated on the left.

rainbow trout cells. This raises the question that the

RT-PCR kit（Invitrogen）. After reverse transcription,

two genes may have distinct expression patterns in

DNase I digestion was performed to eliminate

various tissues. To answer this question, this section

residual genomic DNA from the RNA samples. PCR

describes the expression of HSF1 mRNAs in various

was carried out in a total volume of 50μl with 0.5μtissues of rainbow trout.

l of cDNA synthesis mixture containing HotStarTaq
DNA Polymerase（Qiagen）in an automated thermal

Materials and Methods

cycler（model 2400; PerkinElmer, Wellesley, MA,
USA）. PCR consisted of one initiation cycle of 15

Isolation of RNA and RT-PCR analysis

min at 95℃, amplification cycles of 0.5 min at 94

Total RNA was isolated from RTG-2 cells℃, 0.5 min at 50℃ and 0.5 min at 72℃, and one

and rainbow trout （6 months old） tissues withtermination cycle of 1 min at 72℃, with 35 cycles in

TRI ZOL Reagent （Invitrogen） according to thetotal formanufacturer s instructions. Single-stranded cDNA

cDNA was amplified as a positive control, because

was synthesized from 5μg of total RNA by using

Hsc70 mRNA is constitutively expressed in different

a SuperScript First-Strand Synthesis System for

rainbow trout tissues（Zafarullah

andand 30 for. Hsc70., 1992）andRegulation of expression of HSP genes in rainbow trout

Fig. 12. RT-PCR analysis of theandgenes in rainbow
trout RTG-2 cells and tissues. Rainbow trout
was subjected
to RT-PCR analysis as a positive control. Molecular size markers areindicated on the right（in base pairs）.

in RTG-2 cells as shown in the preceding chapters.

transcription（data not shown）. Taken together,

The oligonucleotide primers were as follows:these results demonstrate that theforward, 5 -GAAGCAGCTTGTCCAGTACACCAA-3 ;reverse, 5 -TTCCAAGAGCTGAACAAA

CCATTG-3 ;andmRNAs are coexpressed in unstressed
rainbow trout cells without tissue specificity.

forward, 5 -GAAGCAGCTGGT

CCAGTACACCTC-3 ;reverse, 5 -GGCTGAATAAACCATGCCAGTAGC-3 ;Section 3. Construction of HSF1 expression vectors

forward, 5-ACATCAGCGACAACAAGAGG-3 ;reverse,In the preceding section, it was shown that two

5 -AGCAGGTCCTGGACATTCTC-3 . The amplified

rainbow trout

products were visualized by ethidium bromide

examined. To further characterize their function, thes are coexpressed in all tissues

staining.

proteins themselves are required. To this end, thissection describes the construction of plasmid vectors
Results

that express epitope-tagged HSF1s, and confirmationof the protein expression.

To determine whether the two

s cloned
Materials and Methods

from RTG-2 cells are actually transcribed in rainbow
trout, RT-PCR was used to analyze total RNA
isolated from unstressed RTG-2 cells and rainbow

Plasmids

trout tissues. As a positive control, rainbow trout

To distinguish between HSF1a and HSF1b in the

Hsc70 cDNA was analyzed. The PCR products were

following experiments, hemagglutinin（HA）tagged

predicted to be 423 bp for

HSF1a（HSF1a-HA）and Protein C tagged HSF1b

and 421 bp for439 bp for, and bands corresponding tothese sizes were amplified（Fig. 12）.

andtranscripts were both detected in all rainbow
trout tissues examined, as well as in RTG-2 cells.

（HSF1b-Protein C）were constructed. The coding
regions of both HSF1 cDNAs were amplified withthe specific PCR primers possessing adIII orI restriction enzyme site. The primers were as

These bands were not due to contamination byfollows:

genomic DNA because no bands were amplified

TGGAGTTCCACGGTGG-3 ;forward, 5 -CCCAAGCTTGATAin the negative control reactions in which total-TATGCGGCCGCGAGGATAATTTGGGCTTGTCRNA was used as the template without reverse

TGG-3 ;

reverse, 5

forward, 5 -CCCAAGCTTGATA

Nobuhiko OJIMAATGGAGTTTCACGTTGG-3 ;reverse, 5/Translation System（Promega）according to the-TATGCGGCCGCGGATAGTTCGGGCTTGTCTG

manufacturer s instructions. For the reaction, 1μg

G-3 . PCR was carried out in a total volume of 50

each of the plasmids described above was used as a

μl with KOD-Plus-DNA Polymerase （Toyobo）

template in a 50-μl reaction mixture.

using 1μl of the plasmid RTG-2 cDNA library
described in Section 1 of Chapter II as the template.
PCR consisted of one initiation cycle of 2 min at 94

Western blot analysis
Ten microliters oftranslated products℃, amplification cycles of 0.25 min at 94℃, 0.5 min

were separated by SDS-PAGE on 10% gels andat 53.6℃ and 1.5 min at 68℃, and one terminationtransferred to poly（vinylidene difluoride）（PVDF）

cycle of 1 min at 68℃, with 37 cycles in total. The

membranes（Hybond-P; Amersham Biosciences）

C terminus of HSF1a was fused to an HA epitopeby electrophoretic transfer. The membranes were

tag in plasmid pMH（Roche Diagnostics）atdIII

blocked with Tris-buffered saline containing 5%

I restriction enzyme sites. Likewise, theskim milk for 1 h at room temperature. Antibodies

C terminus of HSF1b was fused to a Protein C

against HA or Protein C （Roche Diagnostics）

epitope tag in plasmid pMX（Roche Diagnostics）. In

were used to detect epitope-tagged proteins at acontrol experiments, pHMlacZ and pXMlacZ（Roche

working concentration of 1μg/ml each. Incubation

Diagnostics）, which contain the β-galactosidase

and washing procedures for these antibodies

gene cloned in-frame with an N-terminal tag of

were performed according to the manufacturerʼseither HA or Protein C, were used.

instructions. An ECL Western blotting analysis

andsystem（Amersham Biosciences）was used to detect
Coupled in vitro transcription and translation
Coupledtranscription/translation was

performed with a TNT Quick Coupled Transcription

the epitope-tagged proteins. Positive signals weredetected by exposure on Hyperfilm-ECL（Amersham
Biosciences）.

Fig. 13. Western blot analysis oftranslated expression vectors. Filled arrowhead
indicates the position of the epitope-tagged rainbow trout HSF1a and HSF1b（lanes 3
and 6, respectively）; open arrowhead indicates nonspecific bands（lanes 1‑3）. The TNT
Quick Master Mix（Promega）used fortranslations was analyzed as a negative
control（lanes 1 and 4）, and vectors encoding epitope-tagged β-galactosidase（HA-βgal
or Protein C-βgal）were translated
as positive controls（lanes 2 and 5）.

Regulation of expression of HSP genes in rainbow trout
inhibitor cocktail （Complete, Mini, EDTA-free;
Results

Roche Diagnostics）was added to the extraction
buffer at the concentration recommended by thetranscription/translation assay

manufacturer. The pellets were homogenized by five

was performed for the two HSF1s using cDNAs

A coupledfreeze-thaw cycles with liquid nitrogen and pipetting.

encoding epitope-tagged HSF1 （HSF1a-HA and

The homogenates were centrifuged at 10 000

HSF1b-Protein C）to check for protein expression.

℃ for 5 min. The supernatants were collected, andAs positive controls, cDNAs of epitope-tagged β

the protein concentrations were measured by a-galactosidase （HA- and Protein C-βgal） wereProtein Assay kit（Bio-Rad Laboratories）.

at 4

translated. The reaction mixtures were subjected toWestern blotting, and the translated products weredetected by antibodies against the epitope tags.

Electrophoretic mobility shift assay（EMSA）
The DNA-binding ability of HSF1 was analyzed

Specific translation products were detected in lanes

by EMSA as described previously（Mosser

containing those from the HSF1 expression vectors

1988）with the following modifications. The（Fig. 13, lanes 3 and 6）. From their migration on thetranslated products and the whole-cell extracts

gel, the apparent molecular masses of HSF1a-HA

from RTG-2 cells were used as the protein samples.

and HSF1b-Protein C were estimated to be 70 kDa

Binding reaction mixtures were incubated for 30

and 72 kDa, respectively（Fig. 13, lanes 3 and 6,

min on ice. Gels were run at 4℃ for 3 h at 150 V,

respectively）. These sizes were, however, larger

dried, and exposed on Hyperfilm-MP（Amersham

than the expected molecular masses of 57 200 Da

Biosciences）. A double-stranded synthetic HSE,

for HSF1a-HA and 58 590 Da for HSF1b-Protein C

which contains four inverted nGAAn repeats（5 -tcg

calculated from the predicted amino acid sequences.

actaGAAgcTTCtaGAAgcTTCtag-3 ）, was used as aprobe and a competitor. The probe was end-labeled

Section 4. DNA-binding ability of HSF1

with［32P］dCTP by the Klenow fragment of DNApolymerase I. For competition experiments, a 50-foldIn the preceding section, epitope-tagged HSF1s
were successfully synthesized. It is known

molar excess of unlabeled HSE oligonucleotides wasadded to the binding reaction mixtures.

that specific binding to HSE in the promoter ofs is a common feature of mammalian and avian

Results

HSF1. Do HSF1s of rainbow trout have HSE-binding
ability as in the case of other vertebrates? By usingthetranslated HSF1s, this section describes

The DNA-binding ability of each HSF1 was
examined by EMSA using thetranslated

the sequence-specific DNA binding ability of rainbow

proteins, and gel shift bands were observed in thetrout HSF1s.

lanes containing epitope-tagged HSF1（Fig. 14, lanes
5 and 8）. The bands were detected at a position
Materials and Methods

corresponding to the gel-shift band of heat-shocked
RTG-2 cell extract （Fig. 14, lane 2）, and werePreparation of whole cell extracts

abolished by the addition of excess unlabeled HSE

RTG-2 cells were cultured in a 100-mm dishprobe（Fig. 14, lanes 6 and 9）. Moreover, these（Asahi Techno Glass, Funabashi, Japan）at 15℃

bands were not detected in the lanes containing

as described in Section 2 of Chapter I. The dishes

epitope-tagged β-galactosidase（Fig. 14, lanes 4 andwere sealed with Parafilm and immersed in a water7）.This means that the gel-shift bands were not due

bath at 25℃ for 1 h for heat shock. The cells wereto a factor endogenous to theharvested, centrifuged, and rapidly frozen at ‑80

mixture or to the epitope tags. Taken together, these℃. The frozen pellets were suspended in extraction

results demonstrate thatbuffer［20 mM HEPES（pH 7.9）
, 0.2 mM EDTA, 0.1

and HSF1b, as well as endogenous HSF1 in RTG-2

M KCl, 1 mM dithiothreitol, 20% glycerol］. Proteasecells, bind specifically to HSE consensus sequences.

translationtranslated HSF1a

Nobuhiko OJIMA

Fig. 14. EMSA of endogenous rainbow trout HSF1 andtranslated HSF1a and HSF1b. Unlabeled HSE oligonucleotides wereused as a competitor and added to the binding reaction mixtures as
indicated by plus signs（+）. RTG-2 cells were cultured at 15℃（C）andheat shocked at 25℃ for 1 h（HS）.
translated HA-βgal andProtein C-βgal were used as negative controls.

（succinimidylsuccinate）（EGS; Pierce Biotechnology,
Section 5. Oligomeric state of HSF1

Rockford, IL, USA）as described previously（Sorger
and Nelson, 1989）with the following modifications.

In the preceding section, it was demonstrated

translated products containing 2 mMthat HSF1a and HSF1b both could bind to HSE

EGS were incubated at 22℃ for 30 min and thenconsensus sequence. It is known that mammalian

quenched by adding glycine to 50 mM at 22℃ for

and avian HSF1 binds to HSE as a homotrimer;

20 min. The samples were immunoprecipitated withhowever, rainbow trout has two HSF1 isoforms. Thisanti-HA or anti-Protein C Affinity Matrix（Rocheraises the question whether the two HSF1s form a

Diagnostics） according to the manufacturerʼsheterotrimer or not. Thus, this section describes the

instructions. The immunoprecipitates wereanalysis of oligomeric state of rainbow trout HSF1

separated by SDS-PAGE on 6% gels. The HSF1a-HA

by using the differentially epitope-tagged proteins.

and the HSF1b-Protein C were detected by Western
blot analysis using anti-HA and anti-Protein C Ig

Materials and Methods（Roche Diagnostics）, respectively, as described in
Section 3 of this chapter.

Chemical cross-linking and immunoprecipitationtranslated HSF1a and HSF1b werechemically cross-linked using ethylene glycol bis

Regulation of expression of HSP genes in rainbow trout

Fig. 15. Chemical cross-linking and immunoprecipitation.
translated products
containing either HSF1a or HSF1b, or both, were cross-linked using EGS andimmunoprecipitated with anti-HA or anti-Protein C Ig. The immunoprecipitates wereanalyzed by Western blotting using antibodies against HA（A）or Protein C（B）.
Molecular mass markers are indicated on the left（in kDa）. The asterisk indicates theband corresponding to a HSF1b dimer.

HSF1a-HA, HSF1b-Protein C, and a mixture of bothResults

HSF1a-HA and HSF1b-Protein C.
Fig. 15A shows the immunoprecipitated proteins

To investigate whether rainbow trout HSF1s

probed with anti-HA Ig. When the cross-linked

form oligomeric structures, chemical cross-linking

proteins were immunoprecipitated with anti-HA Ig,

with EGS, followed by immunoprecipitation withtwo bands were detected in the lanes containing

antibodies specific for the epitope tags, was

HSF1a-HA（Fig. 15A, lanes 1 and 3）. The apparent

performed. The immunoprecipitated proteins weremolecular masses of the bands wereanalyzed by Western blotting. In this experiment,

and

three

to the sizes of an HSF1 trimer and monomer,translated products were analyzed:

200 kDa

70 kDa. These molecular masses correspondNobuhiko OJIMA
respectively. This result therefore suggests that

（Table 6）, suggesting that both HSF1a and HSF1b

the 200- and 70-kDa products are cross-linked

bind specifically to the HSE consensus sequence.

trimers and monomers of HSF1a-HA, respectively.

As expected, both proteins did indeed bind to theMoreover, when the same cross-linked proteins were

HSE（Fig. 14）. HSF1a and HSF1b also possess otherimmunoprecipitated with anti-Protein C Ig, twodomains conserved in the HSF1 family, i.e., HR-A/B

similar bands were detected in the lane containing

and HR-C（Fig. 8）. HR-A/B has been reported to beboth HSF1a-HA and HSF1b-Protein C（Fig. 15A,

essential for forming HSF1 trimers through theirlane 6）. This suggests that the

α-helical coiled-coil structures（Clos200-kDa product is1990;

a cross-linked HSF1 trimer containing both HA andSorger and Nelson, 1989）. The second hydrophobic

Protein C epitope tags, i.e. an HSF1 heterotrimer.

repeat, HR-C, has been suggested to suppress trimer

Because theformation by interacting with HR-A/B under normal

70-kDa product is an HSF1a-HA

monomer that coimmunoprecipitated withconditions（RabindranHSF1b-Protein C, this provides evidence that thethe presence of these domain structures, the data

, 1993）. As predicted by

two isoforms interact with each other. By contrast,in this chapter demonstrate that both rainbow trout

no bands were observed in the lanes containing

HSF1s form trimers（Fig. 15）.

only HSF1a-HA or HSF1b-Protein C（Fig. 15A, lanes

Furthermore, it was found that an endogenous

4 and 5）, verifying that the epitope tags were notrainbow trout HSF1 is suppressed under normalinteracting with themselves. These results therefore

conditions but activated by heat shock in RTG-2

indicate that HSF1a and HSF1b interact with eachcells（Fig. 14, lanes 1 and 2）. This stress-inducible

other and form heterotrimers.

activation of HSF1 has been observed in rainbow

To confirm further the above results, the same

trout hepatocytes（Airaksinen1998）and in

immunoprecipitated proteins were probed withthe embryonic fibroblast cell line STE and maleanti-Protein C Ig by using a replica membrane

germ cells （Le Goff and Michel, 1999）. Taken

from the Western blotting（Fig. 15B）
. This analysis

together, the results in this chapter suggest

indicated that HSF1b also formed homotrimersthat rainbow trout HSF1s are activated to formand heterotrimers with HSF1a. In addition, a band

DNA-binding trimers by heat shock in a mannercorresponding to an HSF1b dimer was detected（Fig.

similar to the activation of other vertebrate HSF1s.

15B, lanes 5 and 6）. Taken together, these results

In addition to the conserved domain structures, bothdemonstrate that rainbow trout HSF1s form homo-rainbow trout HSF1s contain two KRK tripeptides,

and heterotrimers

which are also conserved among members of the.

HSF1 family （Fig. 8）. The cluster of the basic
Section 6. Discussion

residues preceding HR-A/B has been reported to be
the major NLS of human HSF1（Zuo

In this chapter, it was demonstrated that two1995）.

Moreover, the basic peptide KRK has been reported

distinct isoforms of HSF1 exist in rainbow trout

to be a part of a bipartite type NLS in human HSF2

cells. In vertebrates,（Sheldon and Kingston, 1993）.

s have been alreadyisolated from human（Rabindran

1991）, mouseIn contrast to the highly conserved domains（Sarge1991）, chicken（Nakai and Morimoto,discussed above, other regions of the rainbow trout1993）,（Stump, 1995）, and zebrafish

HSF1s were poorly conserved in comparison with

2000）; however, the present study

other vertebrate HSF1s. These poorly conservedis the first report of the cloning of a gene encoding

regions are illustrated in Fig. 10 as regions III and

HSF1 from cold-water fish species.

V. Regions III and V roughly correspond to domains（Råbergh

Using multiple sequence alignment, domainof mammalian HSF1 that have been described

structures that are common to the HSF1 family wereby several authors（Green

identified in the rainbow trout HSF1s（Fig. 8）. The

1995; Zuo

1995; Shi,

DNA-binding domain in both rainbow trout HSF1s istransactivation domains, respectively. Greenhighly homologous to that of other vertebrate HSF1

（1995） have shown that the central regulatory, 1995）, namely, the regulatory andRegulation of expression of HSP genes in rainbow trout
domain of human HSF1 regulates the function of

It was assumed that thetranslated

the transactivation domain in a heat-shock inducible

HSF1s would be in the form of active trimers withmanner. Moreover, Newton

DNA-binding ability because the（1996） havetranslations

suggested that the regulatory domain of humanwere performed at 30℃, at which rainbow trout

HSF1 alone is sufficient to sense heat stress. Thus,

endogenous HSF1 is already activatedstructural differences in regions III and V between（Airaksinen

rainbow trout and other vertebrate HSF1s may

As predicted, thereflect differences in the activation temperatureindeed possess DNA-binding ability（Fig. 14, lanes 5of HSF1. For example, human, mouse, and chicken

and 8）.

HSF1 are activated at

1998; Le Goff and Michel, 1999）.
translated HSF1s did

42℃（Morimoto, 1998）,

Importantly, the chemical cross-linking andwhereas rainbow trout HSF1 is activated at 25℃

immunoprecipitation experiments in this chapter

in RTG-2 cells（Fig. 14）. Notably, regions III and V

showed that the two HSF1 isoforms have the abilityof rainbow trout HSF1s even share low similarity

to form heterotrimers

with the corresponding regions of zebrafish HSF1.

Fig. 15B, lane 3）. Given that the two HSF1 isoforms

Again, this may be related to differences in theform both homo- and heterotrimers, there are fourthreshold temperature for HSP induction between

potential assemblies of HSF1 trimer, namely, two

cold- and warm-adapted fishes, as discussed at thehomotrimers（a 3 and b 3）and two heterotrimers

beginning of this chapter. Moreover, because region（a2b1 and a1b2）. The existence of the four types of

V of rainbow trout HSF1a shows low similarity totrimer may be reflected in the broad band migratingthat of HSF1b（Table 6）, transactivation may differ

atbetween the two rainbow trout HSF1s.

corresponding to an HSF1b dimer was detected by（Fig. 15A, lane 6 and

200 kDa in Fig. 15. On the other hand, a band

It was demonstrated here that each rainbow

Western blot analysis with anti-Protein C Ig（Fig.

trout HSF1 is encoded by a separate gene（Fig.

15B, lanes 5 and 6）. It remains unclear whether

11）. To date, two isoforms of HSF1 generated bydimer formation is a feature only of HSF1b.

alternative splicing have been reported for mouse（Fiorenza

1995）and zebrafish（Råbergh

Although four HSFs, HSF1‑4, have been identified
in vertebrates, it has been previously stated that

2000）; however, among vertebrates, rainbow

HSF family proteins function as homotrimers. Sarge

trout HSF1 is the first case of having two genetically（1991）pointed out, however, that mouse

distinct isoforms. The HSF1a and HSF1b mRNAs

HSF1 and HSF2 are likely to co-oligomerize because

are coexpressed in rainbow trout tissues（Fig. 12）,they share highly homologous oligomerization

which suggests that both are involved in the heatdomains. Likewise, Sistonen

shock response of rainbow trout. However, since thethe possibility that human HSF1 and HSF2 may

existence of the proteins has not been checked inassociate to form heterotrimers for synergistic

the same cell, the actual protein abundance remains

induction of theto be elucidated.

concerning rainbow trout HSF1 raise the same

（1994）raised

The results of this chapter

To characterize rainbow trout HSF1 isoforms,

possibility of hetero-oligomerization. If hydrophobictranslated HSF1s having distinct epitope

interactions are the major stabilizing force of HSF

tags were used in this chapter. Although migration

trimerization, it is not surprising that HSF familyof the products was retarded in SDS-PAGE, thisproteins form heteromeric complexes because theyphenomenon may result from the poor binding of

possess similar heptad repeats of hydrophobic amino

SDS to the proteins because of their acidic isoelectricacids. However, since thepoint（HSF1a, 4.64; HSF1b, 4.63）. As pointed out

trout HSF1 has not been examined, it remains toby Sarge

be elucidated whether the HSF1 isoforms form

（1991）, such retarded migration of

HSF on SDS-PAGE seems to be characteristic ofstate of rainbow

heterotrimers

several HSFs that have been cloned to date. It was

Why are there two isoforms of HSF1 in rainbow

therefore concluded that the epitope-tagged HSF1s

trout? Although the existence of the two genes may

were successfully generated

be explained simply by ancestral tetraploidization

Nobuhiko OJIMAof salmonids, this does not rule out the possibility

the genome. Northern blot analysis showed thatthat the isoforms have acquired divergent functions

each ofduring evolution. One possibility is that the distinct

species having different sizes by heat stress in

HSF1 isoforms contribute to the tissue specificity

rainbow trout RTG-2 cells. The induction levels ofof the heat shock response. Airaksinen（1998）

total Hsp70b mRNAs were consistently higher thanreported that the induced expression of HSPs is

Hsp70a counterparts during heat stress, althoughboth cell type- and tissue-specific in rainbow trout.

the expression profiles of the two genes were similar

Furthermore, it has been reported that rainbow

to each other in temperature shift and time course

trout HSF1, as well as mouse HSF1（Goodson andexperiments. Interestingly, an mRNA species with

Sarge, 1995）, is activated at a lower temperaturea larger molecular size was expressed only underin male germ cells than in somatic cells（Le Goff

severe heat stress not less than 28℃ irrespective ofand Michel, 1999）. By contrast, the alternatively
spliced isoforms of HSF1 are suggested to regulate

and

and

expressed two mRNA.

In Chapter II, multiple

s were isolated fromthe tissue-specific gene expression of HSPs in

RTG-2 cells and quantitatively compared for theirzebrafish （Råbergh2000） and mouse

mRNA levels between unstressed and heat-shocked（Sarge, 1995）. In the present study, however, both

cells using real-time RT-PCR analysis. Consequently,

HSF1a and HSF1b mRNAs were coexpressednine cDNAs encoding HSPs were isolated fromin all rainbow trout tissues examined. Thus, theheat-shocked RTG-2 cells, namely, Hsp90βa, Hsp90

above-mentioned assemblies of HSF1 trimers, rather

βb, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47,

than transcriptional regulation ofand DnaJ homolog. Quantitative RT-PCR analyses,s themselves,

may regulate the tissue specificity of the heat shockin which

response in rainbow trout. Another possibility is thatshowed that the mRNA accumulation levels of

isolated previously was included,

the two homotrimers and/or two heterotrimers play

, and

were

the role of the other HSF family members, i.e., HSF2,

significantly increased after heat shock, and the

HSF3, and HSF4. For instance, the relationshipincreased levels of two

between rainbow trout HSF1a and HSF1b may be

were most conspicuous. In the case ofsimilar to that between chicken HSF1 and HSF3.

increased level ofTanabethat of（1998）reported that HSF3 has adominant role in regulating the heat shock response

s,

and

,
s, thewas more remarkable than.

In Chapter III, genes encoding HSF1 were cloned

and directly influences HSF1 activity in chickenfrom RTG-2 cells. Consequently, two distinct

cells. Unfortunately, in the present study, a cDNA

named

encoding HSF members other than HSF1 was notpredicted amino acid sequence offound. However, a cDNA encoding rainbow trout

86.4% identity to that of

HSF2 has been reported（Le Goff

2004）, thencontained the general structural motifs of HSF1,

the relationship between HSF1 and HSF2 in thisi.e. a DNA-binding domain, hydrophobic heptad

species is needed to be elucidated in future studies.

repeats and nuclear localization signals. Southern

and

s,

, were identified. The
showed. The two proteinsblot analysis showed that each HSF1 is encoded
Summary and general discussion

by a distinct gene. The two HSF1 mRNAs were
coexpressed in unstressed RTG-2 cells and in

In Chapter I, full-length cDNA clones encoding

various tissues. In EMSA, eachtranslated

Hsp70 were isolated from rainbow trout and

HSF1 bound to the HSE. Chemical cross-linking

investigated for their mRNA expression profiles

and immunoprecipitation analysis showed thatduring heat stress. Consequently, twos,

HSF1a and HSF1b form heterotrimers as well

, were identified and found toas homotrimers. Taken together, these results

have 98.1% identity in their deduced amino acidanddemonstrate that in rainbow trout cells there

sequences. Southern blot analysis indicated thatare two distinct HSF1 isoforms that can form

the two Hsp70s are encoded by distinct genes inheterotrimers, suggesting that a unique molecular

Regulation of expression of HSP genes in rainbow trout
mechanism underlies the stress response in
tetraploid and/or cold-water fish species.

I am grateful to Mrs. Misako Hojo and Mrs. Shiori

The present study demonstrated that, in rainbow
trout, at least someandadvice and discussion.

have duplicate

Fukumoto for their assistance with isolation andsequencing of cDNAs.

genes attributed to ancestral tetraploidization. The

I am grateful to Dr. Teruo Azuma, Freshwater

genes found to be duplicated in the present study

Fisheries Research Division, National Research

are summarized as follows:β,Institute of Fisheries Science, Fisheries Researchandmay have possible

Agency, for providing eyed eggs of rainbow trout.

. Since the otherparalogs as discussed in Section 3 of Chapter II, it

I am obliged to Dr. Masahito Yokoyama, Farming

is apparent that the comprehensive identification

Biology Division, National Research Institute ofof duplicate genes is prerequisite to accurately

Aquaculture, Fisheries Research Agency, and Dr.

examining gene expression profiles of rainbow trout

Ichiro Nakayama, Aquatic Genomics Researchcells. Meanwhile, HSPs are induced in fish cells byCenter, National Research Institute of Fisheries

a variety of stresses, including industrial effluents,

Science, Fisheries Research Agency, for theirpolycyclic aromatic hydrocarbons, several metals

appropriate assistance and encouragement.

such as copper, zinc and mercury, pesticides, andarsenite as well as heat（Iwama

I appreciate all members of Aquatic Genomics

2004）. Thus,Research Center, National Research Institute ofalthough the present study focused on heat stress, it

Fisheries Science, Fisheries Research Agency, foris likely that the mRNA expression profiles of HSPs

their cooperation and support.

differ among stressors. It is interesting to compare
the differences of the profiles for utilizing

Referencess as abiomarker of various environmental stresses.
Further, the present study demonstrated that two

Airaksinen S., Råbergh C. M. I., Sistonen L. anddistinct isoforms of HSF1 can form heterotrimers as

Nikinmaa M., 1998: Effects of heat shock andwell as homotrimers

hypoxia on protein synthesis in rainbow trout

This finding suggeststhat a unique molecular mechanism, which functions
through two distinct HSF1 isoforms, underlies the

（

201,

）cells.

2543‑2551.

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functional difference between HSF1a and

M. R., Nakano K., Schulte P. M. and Iwama G.

HSF1b remains to be elucidated. Since the lowerK., 2002: Heat shock protein genes and theiractivation temperature of rainbow trout HSF1 isfunctional significance in fish.

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Clos J., Westwood J. T., Becker P. B., Wilson S.,

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Lambert K. and Wu C., 1990: Molecular cloning

vertebrate HSF1s will lead to further insight intoand expression of a hexameric

the activation mechanism of the transcription factor.

shock factor subject to negative regulation.

heat

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I express my sincere gratitude to Professor Shugo
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Preferential deadenylation of Hsp70 mRNA

guidance and encouragement for the present study.

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I express my sincere gratitude to Dr. Michiaki
Yamashita, Biochemistry and Food Technology

in

14,

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