cloning of the hsp70 gene in barnacle larvae and its expression under hypoxic conditions

Cloning of the HSP70 gene in barnacle larvae and itsexpression under hypoxic conditions

S.H. Cheng *, C.H. So, P.K. Chan, C.W. Cheng, R.S.S. Wu

Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong

Dissolved oxygen (DO) is one of the most important

factors in coastal marine ecosystems (Diaz and Rosen-

berg, 1995). Hypoxia is generally defined as DO levels

between 0 mg l�1 (anoxia) and 2.8 mg l�1 in the marineenvironment. Hypoxia increases in frequency and se-

verity when coupled with meteorological and hydrody-namic events. Some marine environments, such as the

bottom water of the northern Gulf of Mexico, are ex-

posed to hypoxia/anoxic events regularly. The extent of

hypoxic zones could be expanding due to increased CO2arising from the introduction of anthropogenic nutrients

(Justic et al., 1997).

Animals employ different strategies to cope with a

decline in oxygen levels. Some escape hypoxic stress bybehavioral changes. For example, demersal fishes and

crustaceans migrated from an area with DO level < 2

mg l�1 and changed their diet (Pihl et al., 1991, 1992;Pihl, 1994). The brown shrimp Penaeus aztecus migrated

when the DO decline to below 1.96 mg l�1 (Renauld,1986). Benthic macrofauna such as Amphiura filiformis

and Pectinaria koreni left their position in the sediment

in moderate and severe hypoxia (Rosenberg et al., 1991;Nilsson and Rosenberg, 1994). Fishes disappeared and

lobsters emerged from burrows when oxygen saturation

declined below 40% in the Swedish west coast (Baden

et al., 1990a).

During severe hypoxia, a reversed sex ratio was ob-

served in the Norway lobster Nephrops norvegicus

(Baden et al., 1990b). Other physiological responses to

hypoxia included ventilatory and circulatory adapta-tions (Hagerman and Baden, 1998). Manganese con-

centrations in the gills of the Norway lobsters N.

norvegicus taken from areas of repeated hypoxia were

over 20 times higher than animals taken from other

Swedish locations (Eriksson and Baden, 1998).

Other animals may adjust their metabolism during

hypoxia. Our recent work has shown that the common

carp Cyprinus carpio responded to short term and longterm hypoxia exposures by switching to alternative

metabolic pathways (Zhou et al., 2000). A significant

reduction of the intraerythrocytic levels of adenosine

and guanosine was observed in hypoxic rainbow trout

(Oncorhynchus mykiss) (Val et al., 1995). Based on two

cell types of different hypoxia tolerance isolated from an

aquatic turtle, it was proposed that the first line of

defence was a suppression of ATP-demand and ATP-

supply pathways leading to inhibition of protein syn-

thesis (Hochachka et al., 1996, 1997). The second line of

defence, which was a ‘‘rescue’’ of protein synthesis me-diated by preferential expression of certain genes, oc-

curred mainly in hypoxia tolerant cells.

Despite the fact that significant progress has been

made toward elucidating components of the mecha-

nisms regulating physiological and cellular responses to

hypoxia, it is clear that all of the regulatory elements

have not been identified nor characterized. In particular,

genes and genes products that are expressed in a char-acteristic ‘‘on-off’’ manner following changes in the level

of oxygen in coastal waters have not been fully identified

in marine coastal organisms.

Barnacles are one of the most important functional

groups in intertidal zones (Wu and Levings, 1978).

Successful recruitment and settlement of barnacle larvae

affects the population dynamics of the barnacle popu-

lations. Barnacle larvae, therefore, have been used ex-tensively as a biomonitoring species for the assessment

of a wide range of toxicants including environmental

oestrogens, antifouling agents and water-soluble com-

ponents of petroleum oils (Billinghurst et al., 1998;

Vetere et al., 1997; Donahue et al., 1977). Previous

studies have shown that exposure to water and sediment

pollution affect the abundance of larvae significantly

(Silina and Ovsyannikova, 2000). Exposure to seasonalhypoxia on the Louisiana Continental Shelf resulted in a

reduction of benthic larval flux to the seabed and re-

duced settlement of barnacle cyprids under the pycno-

cline (Powers, 1995).

In this study, we used the technique of RNA arbi-

trarily primed-polymerase chain reaction (RAP-PCR) to

obtain RNA fingerprinting of barnacle larvae exposed

to normoxic and hypoxic conditions. RAP-PCR onlyneeds minute amounts of RNA as templates for ampli-

fication and represents the ideal methodology in studies

involving nucleic acid fingerprinting of invertebrate

larvae (Welsh et al., 1992). We report here that RAP-

PCR can identify differential expression of genes in

barnacle larvae under hypoxic conditions. Sequence

analysis of the differentially expressed genes showed that

one of the clones contained a nucleotide sequence re-sembling the heat shock protein 70 (HSP70). Using

*Corresponding author. Tel.: +852-2788-9027; fax: +852-2788-

7406.

E-mail address: [email protected] (S.H. Cheng).

Baseline / Marine Pollution Bulletin 46 (2003) 659–676 665

rapid amplification of cDNA ends (RACE), we were

able to obtain the full length cDNA of the barnacle

HSP70 gene. The expression of this gene in barnacle

adults exposed to hypoxic conditions was investigatedby Northern blot analysis.

Barnacles (Balanus amphitrite Darwin) were collected

from the intertidal to sublittoral fringe at the Sai Kung

Ferry Pier in the New Territories of Hong Kong. Active

swimming stage II naupliar larvae showing strong

phototactic behaviour were collected from barnacle

brood sacs for subsequent experiments. Three thousand

nauplii were put into chambers incubated in fish tankscontaining 30 l of filtered seawater with continuous in-

flux of mixed oxygen (5%)/nitrogen (95%) gas. The level

of DO was maintained at 1.0 mgml�1. In the controlgroup, the larvae were incubated in a similar set up

except the gas mixture was replaced by natural air. Both

groups of larvae were incubated for 24 h with a sub-

merged probe for monitoring DO levels. Adult barna-

cles were also exposed to the same treatment in separatefish tanks.

Total RNA was extracted from 3000 larvae using 1

ml TriZol reagent (Life Technologies, USA), according

to manufacturer�s instructions. 100 ng of total RNA wasreverse-transcribed by Superscripte II Reverse Tran-

scriptase (Gibco BRL, USA) following the manufac-

turer�s instructions, using 400 ng of random hexamers asprimers. Differentially expressed genes were amplified byPCR using 46 arbitrary primers in thermal profile con-

taining low stringency steps and high stringency steps. A

negative control containing no cDNA template was in-

cluded. Amplified products showing differential patterns

of expression were purified from agarose gel using a

Qiaex II Gel Extraction Kit (Qiagen, Germany) ac-

cording to manufacturer�s instructions, and were thencloned into a pUC18 vector using a SureClone LigationKit (Amersham Pharmacia, USA) for sequencing reac-

tion using a dRhodamine Terminator Automated Se-

quencing Kite (PE Biosystems, England) and for probe

synthesis in Northern hybridization analyses. To con-

firm the differential expression of cloned genes under

hypoxia by northern hybridization, cloned products

were hybridized to total RNA isolated from adults ex-

posed to hypoxia.Full length cDNAs were synthesized by RACE re-

action from total barnacle larvae RNA isolated by

PolyAtract mRNA Isolation System III (Promega,

USA). RACE reactions were preformed by Marathon

cDNA amplification kit (ClonTech, USA) according to

the manufacturer�s instruction. Gene specific primerswere designed based on the sequences of the differen-

tially expressed genes identified.Sequences were searched for homology using the

program ‘‘BLAST’’ provided by National Center for

Biotechnical Institute (NCBI) (http://www.ncbi.nlm.

nih.gov/). The deduced amino acid sequences were ob-

tained by the ‘‘ORF Finder’’ program available at the

same NCBI site. Deduced amino acid sequences were

compared to known genes in the NCBI databases.

Exposing stage II naupliar larvae to hypoxic condi-tions provides a means of characterising induction and

inhibition of hypoxia responsive genes. In order to

identify differentially expressed genes in these barnacle

larvae, we used the procedure of RAP-PCR. Screening

with 46 arbitrary primers revealed at least 39 gene

products that exhibited elevated or reduced expression

between normoxic and hypoxic treated larvae. The

likelihood of other differentially expressed genes presentin these two conditions, other than those described here,

is very high since this study only used one fifth of the

usual number of primers normally engaged in a RAP-

PCR. Sizes of the gene products ranged from 110 bp to

1.1 kb. Among these 39 gene products, 16 were up-

regulated under hypoxia and 23 were down-regulated

under hypoxia. One of the RAP-PCR experiments is

shown in Fig. 1. In this experiment, nine sets of arbitraryprimers were used. Six differentially expressed gene

products, with sizes ranging from 300 bp to 1 kb, were

obtained using three of the primers sets.

These 39 gene products were then re-amplified by

PCR to obtain sufficient amounts for subcloning into

plasmid vectors. Insert containing colonies were selected

and plasmid DNA was then extracted for nucleotide

sequencing. Of the 39 gene products, 4 showed similarityto cloned genes following nucleotide analysis. The sizes

and similarities to cloned genes of these 4 clones are

listed in Table 1.

Optimal conditions for 50 and 30 RACE of the cloneG512 (Table 1) were obtained after 9 experiments (data

not shown). A 1.2 kb product was obtained from the 50

RACE and a 1.7 kb product was obtained from the 30

RACE. The nucleotide sequences from clone G512and its RACE products were aligned to obtain the full

length nucleotide sequence (Fig. 2; accession number:

AY150182). The full length nucleotide sequence was

2250 bp and sequences such as start codon and stop

codon could be identified. The total number of amino

acids expected is 650.

The predicted amino acid sequence of barnacle

HSP70 was compared and aligned to cloned HSP genesfrom other species (Fig. 3). Most of the HSP70 genes

showing the highest percentage of identity were cloned

from terrestrial and marine invertebrates (Table 2).

Total RNA, extracted from adult barnacles exposed

to normoxic and hypoxic conditions, was separated in a

formaldehyde gel. Using the RT-PCR product of HSP70

as a probe, a signal was obtained in both control and

hypoxic treated barnacles (Fig. 4). The size of the signalwas found to be 2.3 kb according to the molecular

weight markers. The house keeping gene b-actin wasused as an internal probe. The intensity of the hybridi-

sation signals was compared by image analysis. It was

666 Baseline / Marine Pollution Bulletin 46 (2003) 659–676

found that HSP70 expression was 2.5-fold stronger in

the hypoxic treated barnacles than the controls.

We have used the RAP-PCR method to clone dif-

ferentially expressed genes in barnacle larvae exposed tohypoxic conditions. The RAP-PCR was first used by

Welsh et al. (1992) to distinguish differentially expressed

genes in different murine tissues. We used 46 different

arbitrary primers in six RAP-PCR experiments and

obtained 39 differentially expressed gene products. This

study is one of the first reports on differentially ex-

pressed genes in barnacle larvae. Four out of the 39 gene

products shared similarity to cloned genes (Table 1)while the remaining 35 appeared to be novel sequences.

The quest for finding differentially expressed genes

has extended to different methodologies including the

RAP-PCR, differential display and more recently,

macro- and micro-cDNA arrays. Molecular character-

ization and cloning of differentially expressed genes have

been carried out in various aquatic species. Up-regula-

tion of a novel secretogranin-II mRNA was identified byRAP-PCR in the goldfish pituitary (Blazquez et al.,

1998). A novel cadherin was cloned and characterized

from the colonial invertebrate Botryllus schlosseri using

differential display (Levi et al., 1997). Systematic isola-

tion of peptide signals regulating developmental proce-

dures of the hydra was achieved by differential display

(Takahishi et al., 1997). The postmolt stage in the

decapod crustacean Penaeus japonicus was analysed bydifferential display and novel gene products associ-

ated with this physiological stage have been identified

(Watanabe et al., 2000).

In this study, we identified 16 up-regulated gene

products and 23 down-regulated gene products in bar-

nacle larvae exposed to hypoxia. Out of these 39 gene

products, 4 shared similarity to genes with known

functions (Table 1). Identification of hypoxia responsivegenes in mammalian cell lines and tissues using the dif-

ferential display methodology have been reported. In

HeLa cells exposed to 1% oxygen, novel genes and genes

with known functions, such as the glucose transporter 3

and adenylate kinase isoenzyme 3, were identified

(O�Rourke et al., 1996). Rat lung fibroblasts exposed to5 Pa pO2 were subjected to differential display analysis

(Takahashi et al., 1998) and induction of the phospho-glycerate mutase B gene, which might contribute to the

regulation of glycolytic flux under reduced O2 tension,

was found. Over-expression of genes such as the tissue

inhibitor of metalloproteinase-1, prostate tumour in-

Fig. 1. RAP-PCR amplification of total RNA of barnacle larvae in different experimental treatments. Barnacle larvae were treated in (1) hypoxia 1.0

mg O2 l�1; (2) normoxia. RAP-PCR using different arbitrary primers is indicated by the following: M: 1 k plus DNA marker (Gibco BRL), C:

negative control without cDNA template.

Table 1

Clones sharing similarities to known genes

Clone designation Size (bp) Shared similarity to known genes (accession number)

E121 110 79% similar to Xenopus laevis protein kinase (Z17205)

G512 600 81% similar to Crassostrea gigas HSP70 (AF144646)

D521 410 56% similar to human p76 (NM_00480)

D221 260 82% similar to human cell division factor (XM_009947)


ducing factor-1, enolase-alpha and prothymosin-alpha

were observed in differential display experiments on

hypoxia treated human microvascular endothelial cells

(Roland et al., 2000).

One of our differentially expressed gene products,

clone G512, shared 81% similarity to the Pacific oyster

HSP70 gene (Table 1). Using RACE, the full length

cDNA of the barnacle HSP70 was cloned and the nu-cleotide sequence shared high homology to invertebrates

and mammals alike (Table 2). The HSP family of pro-

teins, also known as the stress proteins, function as

molecular chaperones (see Buchner, 1996). They sup-

press the irreversible folding reactions of other proteins

and hence enhance survival of the organisms. They are

induced by many environmental stresses including ex-

posure to toxicants, changes in temperature and osmo-

larity, and hypoxia/anoxia (see Lewis et al., 1999).

Among the HSP family, HSP70 is the most conserved

member and has been identified in marine algae, inver-

tebrates and chordates. This stress protein has been used

as a biomarker in these aquatic species.

In our investigation of HSP70 expression in barnacleadults exposed to hypoxic conditions (Fig. 4), it ap-

peared that 24 h treatment at 1.0 O2 mg l�1 could induce

a 2.5-fold increase in RNA levels. Our data supported

the hypothesis that induction of HSP70 transcription

occurred following hypoxia exposure. Altered expression

of HSP70 under hypoxia conditions has been investi-

Fig. 2. Full length nucleotide sequence of Balanus heat shock protein 70 (accession number: AY150182).


gated in other organisms. Induction of HSP70 expression

in ischaemia and hypoxia in the mammalian brain and

myocardium has been well documented (see Sharp and

Sagar, 1994; Gray et al., 1999). In HSP70-overexpressing

Fig. 3. Conserved sequences among Balanus HSP70 protein and other species. Multiple alignment of Balanus HSP70 predicted amino acid sequences

(accession number: AY150182) with HSP70 gene products cloned from other species (accession numbers are: AAA74394, AAC23392, AAD31042,

AAF09496, AAB06239, P19120, AAK17898). Identical amino acid sequences are shaded.


rat hearts subjected to global ischaemia, the number

of apoptotic cells has been observed to be less than that

of the control-transfected hearts (Suzuki et al., 2000).

These authors suggested that HSP70 could be associated

with a reduction in myocardial apoptosis following is-

chaemia. In the over-wintering common frogs, Rana

temporaria, a significant increase in the HSP70 was

found after 1 month of hypoxic submergence (Currie andBoutilier, 2001). It was suggested that the HSP70 might

contribute to the heart�s remarkable hypoxia and anoxiatolerance and might act to defend metabolism during the

over-wintering months.

Down-regulation of HSP70, however, was observed

in human microvascular endothelial cells (Oehler et al.,

2000). Rainbow trout exposed to hypoxia did not show

any increase in HSP70 in their nucleated red blood cells,hepatocytes, gill epithelial cells and myocardial cells

(Currie et al., 1999; Airaksinen et al., 1998; Gamperl

et al., 1998).

We have cloned the HSP70 in barnacle larvae and

demonstrated an induction of HSP70 expression fol-

lowing exposure to hypoxia. Relatively few genes have

been cloned in the genome of the barnacle. Our report

not only adds to the understanding of the barnacle ge-

nome by cloning of the HSP70 gene, it also provides a

protocol for measuring the induction of this gene in this

species. In addition, our data also support the use of

HSP70 as a biomarker of exposure and expand the ap-plication of barnacles as a model organisms for bio-

monitoring in the marine environment.

Acknowledgements

We thank Prof. David Randall and Prof. Nora Tam

for their critical comments. This project is supportedsubstantially by a research grant awarded by the Re-

search Center for Coastal Pollution and Conservation,

City University of Hong Kong.

References

Airaksinen, S., Rabergh, C.M.I., Sistonen, L., Nikinmaa, M., 1998.

Effects of heat shock and hypoxia on protein synthesis in rainbow

trout (Oncorhynchus mykiss) cells. J. Exp. Biol. 201, 2543–2551.

Baden, S.P., Loo, L.O., Pihl, L., Rosenberg, R., 1990a. Effects of

eutrophication on benthic communities including fish: Swedish

west coast. Ambio 19, 113–122.

Baden, S.P., Pihl, L., Rosenberg, R., 1990b. Effects of oxygen

depletion on the ecology, blood physiology and fishery of the

Norway lobster Nephrops norvegicus. Mar. Ecol. Prog. Ser. 67,

141–155.

Billinghurst, Z., Clare, A.S., Fileman, T., Mcevoy, J., Readman, J.,

Depledge, M.H., 1998. Inhibition of barnacle settlement by the

environmental oestrogen 4-nonylphenol and the natural oestrogen

17 beta oestradiol. Mar. Pollut. Bull. 36, 833–839.

Blazquez, M., Bosma, P.T., Chang, J.P., Docheety, K., Trudeau, V.L.,

1998. Gamma-amionbutyric acid up-regulates the expression of a

novel secretogranin-II messenger ribonucleic acid in the Goldfish

pituitary. Endocrinology 139, 4870–4880.

Buchner, J., 1996. Supervising the fold: functional principles of

molecular chaperones. FASEB J. 10, 10–19.

Currie, S., Boutilier, R.G., 2001. Strategies of hypoxia and anoxia

tolerance in cardiomyocytes from the overwintering common frog,

Rana temporaria. Physiol. Biochem. Zool. 74, 420–428.

Currie, S., Tufts, B.L., Moyes, C.D., 1999. Influence of bioenergetic

stress on heat shock protein gene expression in nucleated red blood

cells of fish. Am. J. Physiol. 276, R990–R996.

Diaz, R.J., Rosenberg, R., 1995. Marine benthic hypoxia: a review

of its ecological effects and the behavioural responses of ben-

thic macrofauna. Oceanogr. Mar. Biol. Annu. Rev. 33, 245–

303.

Donahue, W.H., Wang, R.T., Welch, M., 1977. Effects of water-

soluble components of petroleum oils and aromatic hydrocarbons

on barnacle larvae. Environ. Pollut. 13, 187–202.

Eriksson, S.P., Baden, S.P., 1998. Manganese in the haemolymph and

tissues of the Norway lobster, Nephrops norvegicus (L.), along the

Swedish west coast, 1993–1995. Hydrobiologia, 375–376, 255–

264.

Gamperl, A.K., Vijayan, M.M., Pereira, C., Farrel, A.P., 1998. b-receptors and stress protein 70 expression in hypoxic myocardium

of rainbow trout and Chinook salmon. Am. J. Physiol. 274, R428–

R436.

Gray, C.C., Amrani, M., Yacoub, M.H., 1999. Heat stress proteins

and myocardial protection: experimental model or potential

clinical tool? Int. J. Biochem. Cell Biol. 31, 559–573.

Table 2

Comparison between predicted amino acid sequences of HSP70 cloned

in Balanus and in other species

Species Common name % similarity to

Balanus HSP70

Urechis caupo Marine worm 85

Ceratitis capitata Mediterranean Fruitfly 84

Crassostrea gigas Pacific oyster 86

Manduca sexta Tobacco hornworm 87

Trichoplusia ni Cabbage looper 87

Bos taurus Cow 85

Homo sapiens Human 85

Fig. 4. Hypoxia induces HSP70 expression. Northern blot hybridiza-

tion analysis of total RNA extracted from hypoxic treated and control

barnacles. HSP70 expression is up-regulated under hypoxia and the

internal control (b-actin) expression shows no difference.


Hagerman, L., Baden, S., 1998. Physiological flexibility: a necessity for

life in anoxic and sulphidic habitats. Hydrobiologia 375–376, 241–

254.

Hochachka, P.W., Buck, L.T., Doll, C.J., Land, S.C., 1996. Unifying

theory of hypoxia tolerance: molecular/metabolic defense and

rescue mechanisms for surviving oxygen lack. Proc. Natl. Acad.

Sci. USA 93, 9493–9498.

Hochachka, P.W., Land, S.C., Buck, L.T., 1997. Oxygen sensing and

signal transduction in metabolic defense against hypoxia: lessons

from vertebrate anaerobes. Comp. Biochem. Physiol. A 118A, 23–29.

Justic, D., Rabalais, N., Turner, R.E., 1997. Impacts of climate change

on net productivity of coastal waters: implications for carbon

budgets and hypoxia. Clim. Res. 8, 225–237.

Levi, L., Douek, J., Osman, M., Bosch, T.C.G., Rinkevich, B., 1997.

Cloning and characterization of BS-cadherin, a novel cadherin

from the colonial urochordate Botryllus schlosseri. Gene 200, 117–

123.

Lewis, S., Handy, R.D., Cordi, B., Billinghurst, Z., Depledge, M.H.,

1999. Stress proteins (HSP�s): methods of detection and their use asan environmental biomarker. Ecotoxicol. 8, 351–368.

Nilsson, H.C., Rosenberg, R., 1994. Hypoxic response of two benthic

communities. Mar. Ecol. Prog. Ser. 115, 209–217.

Oehler, R., Schmierer, B., Zellner, M., Prohaska, R., Roth, E., 2000.

Endothelial cells down regulates expression of the 70 kDa heat

shock protein during hypoxia. Biochem. Biophys. Res. Commun.

274, 542–547.

O�Rourke, J.F., Pugh, C.W., Barlett, S.M., Ratcliffe, P.J., 1996.Identification of hypoxically inducible mRNA in HeLa cells using

differential-display PCR. Role of hypoxia-inducible factor-1. Eur.

J. Biochem. 241, 403–410.

Pihl, L., 1994. Changes in the diet of demersal fish due to eutroph-

ication-induced hypoxia in the Kattegat, Sweden. Can. J. Fish.

Aquat. Sci. 51, 321–336.

Pihl, L., Baden, S.P., Diaz, R.J., 1991. Effects of periodic hypoxia on

distribution of dermersal fish and crustaceans. Mar. Biol. 108, 349–

360.

Pihl, L., Baden, S.P., Diaz, R.J., Schaffner, L.C., 1992. Hypoxia-

induced structural changes in the diet of bottom-feeding fish and

crustacea. Mar. Biol. 112, 349–361.

Powers, S.P., 1995. Effects of hypoxia on the vertical distribution and

settlement of invertebrate larvae. 23th Benthic Ecology Meeting,

17–19 March 1995.

Renauld, M.L., 1986. Detecting and avoiding oxygen deficient sea

water by brown shrimp Penaeus aztecus (Ives) and white shrimp

Penaeus setiferus (Linnaeus). J. Exp. Mar. Biol. Ecol. 98, 283–292.

Roland, I., Minet, E., Ernest, I., Pascal, T., Michel, G., Remacle, J.,

Michiels, C., 2000. Identification of hypoxia-responsive messengers

expressed in human microvascular endothelial cells using differen-

tial display RT-PCR. Eur. J. Biochem. 267, 3567–3574.

Rosenberg, R., Hellman, B., Johansson, B., 1991. Hypoxic tolerance of

marine benthic fauna. Mar. Ecol. Prog. Ser. 79, 127–131.

Sharp, F.R., Sagar, S.M., 1994. Alterations in gene expression as an

index of neuronal injury: heat shock and the immediate early gene

response. Neurotoxicology 15, 51–59.

Silina, A.V., Ovsyannikova, I.I., 2000. Long-term and seasonal

dynamics of the recruitment and growth of young Balanus rostratus

barnacles in Amurskii Bay, Sea of Japan. Russ. J. Mar. Biol. 26,

181–187.

Suzuki, K., Sawa, Y., Kagisaki, K., Taketani, S., Ichikawa, H.,

Kaneda, Y., Matsuda, H., 2000. Reduction in myocardial apop-

tosis associated with overexpression of heat shock protein 70. Basic

Res. Cardiol. 95, 397–403.

Takahishi, T., Muneoka, Y., Lohmann, J., Lopez De Haro, M.S.,

Solleder, G., Bosch, T.C.G., David, C.N., Bode, H.R., Koizumi,

O., Shimizu, H., Hatta, M., Fujisawa, T., Sugiyama, T., 1997.

Systematic isolation of peptide signal molecules regulating devel-

opment in hydra: Lwamide and PW families. Proc. Natl. Acad. Sci.

USA 94, 1241–1246.

Takahashi, Y., Takahashi, S., Yoshimi, T., Miura, T., 1998. Hypoxia-

induced expression of phosphoglycerate mutase B in fibroblasts.

Eur. J. Biochem. 254, 497–504.

Val, A.L., Lessard, J., Randall, D.J., 1995. Effects of hypoxia on

rainbow trout (Oncorhynchus mykiss): intraerythrocytic phos-

phates. J. Exp. Biol. 198, 305–310.

Vetere, V.F., Perez, M.C., Romagnoli, R., Stupak, M.E., Del-Amo, B.,

1997. Solubility and toxic effect of the cuprous thiocyanate

antifouling pigment on barnacle larvae. J. Coat. Technol. 69, 39–

45.

Watanabe, T., Persson, P., Endo, H., Kono, M., 2000. Molecular

analysis of two genes, DD9A and B, which are expressed during the

postmolt stage in the decapod crustacean Penaeus japaonicus.

Comp. Biochem. Physiol. B 125B, 127–136.

Welsh, J., Chada, K., Dala, S.S., Cheng, R., Ralph, D., McClelland,

M., 1992. Arbitrarily primed PCR fingerprinting of RNA. Nucleic

Acids Res. 20, 4965–4970.

Wu, R.S.S., Levings, C.D., 1978. An energy budget for individual

barnacles (Balanus glandula). Mar. Biol. 45, 225–235.

Zhou, B.S., Wu, R.S., Randall, D.J., Lam, P.K., Ip, Y.K., Chew, S.F.,

2000. Metabolic adjustments in the common carp during prolonged

hypoxia. J. Fish Biol. 57, 1160–1171.

0025-326X/03/$ - see front matter � 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0025-326X(03)00059-6


cloning of the hsp70 gene in barnacle larvae and its expression under hypoxic conditions

Documents