Báo cáo khoa học: Globins and hypoxia adaptation in the goldfish, Carassius auratus potx

In recent years, hypoxia has received much attention in biomedical research [8,15] and, because of global Keywords cytoglobin; goldfish; hemoglobin; myoglobin; neuroglobin Correspondence

Carassius auratus

Anja Roesner1, Stephanie A Mitz1,2, Thomas Hankeln3and Thorsten Burmester2

1 Institute of Zoology, University of Mainz, Germany

2 Institute of Zoology, University of Hamburg, Germany

3 Institute of Molecular Genetics, University of Mainz, Germany

Many freshwater environments are characterized by

spatial, temporal or seasonal fluctuations in oxygen

availability Therefore, various fish species have

evolved physiological, anatomical and behavioral

mechanisms for coping with extended periods of

hypoxia [1–8] These strategies include O2 saving by

reduction of the metabolic rate, improved O2 uptake

by enhanced ventilation, aquatic surface respiration,

expansion of the gill surface and the increased O2

affinity of hemoglobin [9,10] Cyprinid fishes of the

genus Carassius (the crucian carp Carassius carassius

and its domestic Asian form, the goldfish C auratus) routinely experience hypoxia and even anoxic phases

in their environment of isolated ponds Carassius dis-plays remarkable tolerance against O2 deprivation This tolerance is conveyed by high glycogen stores in brain and liver, increased buffering capacities, meta-bolic rate depression and the ability to convert the lactate produced by anaerobic glycolysis into ethanol, which is excreted via the gills [2,3,8,11–14]

In recent years, hypoxia has received much attention

in biomedical research [8,15] and, because of global

Keywords

cytoglobin; goldfish; hemoglobin; myoglobin;

neuroglobin

Correspondence

T Burmester, Institute of Zoology,

University of Hamburg, Biozentrum Grindel,

Martin-Luther-King-Platz 3, D-20146

Hamburg, Germany

Fax: +49 40 42838 3937

Tel: +49 40 42838 3913

E-mail: thorsten.burmester@uni-hamburg.de

Database

The nucleotide sequences have been

sub-mitted to the GenBank database ⁄ EMBL

Data Bank under accession numbers

AM933143 (Hba), AM933144 (Hbb),

AM747267 (Mb1), AM747268 (Mb2),

AM933145 (Ngb) and AM933146 (Cygb1)

(Received 6 March 2008, revised 6 May

2008, accepted 15 May 2008)

doi:10.1111/j.1742-4658.2008.06508.x

Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures We investigated the contribution of respiratory proteins to hypoxia tolerance in C auratus We determined the complete coding sequence of hemoglobin a and b and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin Like the common carp (Cypri-nus carpio), C auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage Myoglobin is also expressed in non-muscle tissues By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at

Po2 6.7 kPa, 8 h at Po2 1.7 kPa, 16 h at Po2 6.7 kPa) at 20 C We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase Hypoxia significantly enhanced only the expression of myoglobin However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels These observations suggest that both myoglobin and neuroglobin may con-tribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin)

Abbreviations

ARP, acidic ribosomal phosphoprotein P0; Cygb, cytoglobin; GbX, globin X; Hb, hemoglobin; LDH-A, lactate dehydrogenase A; Mb,

myoglobin; Ngb, neuroglobin; SOD, superoxide dismutase.

warming, has become an important environmental

concern [16,17] Fish have become a prime model to

investigate hypoxia tolerance strategies at the organism

level Alternative metabolic pathways, as well as other

physiological responses are associated with major

changes in gene expression patterns For example,

genes encoding enzymes of the glycolytic pathway and

fermentation are expressed more strongly after

long-term hypoxia in some fish species [9,18,19] By

con-trast, genes required for oxidative energy production in

the tricarboxylic acid cycle or the respiratory chain, or

for the highly energy-consuming translation process

were found to be repressed Hypoxia may even cause

developmental arrest, which is reflected by the

repres-sion of growth or cell cycle-associated genes [19,20]

Hypoxia also affects the O2-binding respiratory

proteins, which are represented in vertebrates by the

globin superfamily [21–23] Globins are small globular

proteins that bind to O2 by virtue of a heme-bound

Fe2+ ion To date, five types of globins have been

identified in fish Hemoglobin (Hb) is included in the

red blood cells and serves for the transport of O2

within the circulatory system Hb is a heterotetramer

that consists of two a- and two b-chains Monomeric

myoglobin (Mb) supplies O2within the striated muscle

and heart of most vertebrates [24] Whereas Mb is a

single copy gene in most species, the common carp

(Cyprinus carpio) possesses two paralogous Mb genes

(Mb1 and Mb2) [25] Surprisingly, Mb1 is ubiquitously

expressed in various tissues, whereas Mb2 is restricted

to the brain Neuroglobin (Ngb) is located in the

cen-tral and peripheral nervous systems [26], the retina [27]

and some endocrine tissues [28] The exact role of Ngb

remains uncertain; it may supply O2 to metabolically

active neurons, although other functions such as the

detoxification of noxious reactive oxygen or nitrogen

species are conceivable [29–31] Cytoglobin (Cygb) is

located in the fibroblast cell lineage as well as in

dis-tinct populations of neurons [32,33] The function of

Cygb may be related to reactive oxygen species

detoxi-fication or the supply of O2 to particular enzymatic

reactions [30,33] Fish possess two paralogous Cygb

genes, which show divergent expression in neurons and

non-neuronal tissues [34] The most recently identified

vertebrate globin, which has been referred to as

glo-bin X (GbX), is restricted to fish and amphibians

[35,36] The physiological role of GbX, which is

expressed at low levels in a broad range of tissues, is

currently unknown

Because hypoxia reduces the availability of O2 to

mitochondria and respiratory proteins, it can be

assumed that low O2 levels change the expression of

globins Previously, we investigated the effect of

hypoxia on globin mRNA and protein levels in the zebrafish Danio rerio [23] Zebrafish exhibit moderate tolerance to hypoxia, surviving extended periods at

Po2 4 kPa It might be expected that increased expression of respiratory proteins should be advanta-geous at low O2 partial pressures, however, we found different globin responses Whereas Hb mRNA levels decreased under hypoxia, Mb and Ngb protein and mRNA levels increased significantly The data suggest that these globins are involved in conveying hypoxia tolerance to zebrafish Here we investigate the response

of globin levels in the extremely hypoxia-tolerant goldfish

Results

Cloning and analyses of goldfish globins Partial and complete cDNA sequences of goldfish Hba, Hbb, Mb1, Mb2, Ngb and Cygb1 were obtained

by RT-PCR from RNA extracted from various tissues (Fig 1 ) The complete coding sequences, including the 5¢- and 3¢-ends, of Hba, Hbb and Mb1 were then obtained from a mixed tissue cDNA library (supple-mentary Figs S1–S3) The cDNA of Mb2 was obtained

by RT-PCR (supplementary Fig S4) The 3¢-end of the coding sequence of Ngb was missing, and we obtained only the middle part of Cygb1 (supplemen-tary Figs S5 and S6) Cygb2 could not be obtained by RT-PCR Because our study was mainly aimed at investigating the regulation of globin expression, which requires only fragments of globin cDNA, we ignored the missing parts of the coding sequences The coding sequence of GbX had been obtained in a previous study [36]

We first compared goldfish globin sequences with their zebrafish orthologs (Fig 1) Both Hb cDNAs represent adult chains, whereas embryonic Hbs were not considered in this study The Hba chain we obtained by screening the cDNA library is 97% identi-cal at the nucleotide level to the goldfish Hba cDNA sequence available in the databases (accession number AF528157), suggesting allelic variation or the presence

of multiple isoforms in the C auratus genome Gold-fish and zebraGold-fish Hba proteins are 87.4% identical (99.3% similar; considering isofunctional replace-ments) The Hbb chains of these two fish species are 92.6% identical⁄ 98.0% similar; within the overlapping regions, the Cygb1 proteins of the two species are 79.7% identical⁄ 95.8% similar and Ngb is 92.4% iden-tical⁄ 98.1% similar GbX proteins are highly con-served, with scores of 98.0% identity and 99.0% similarity

Recently, Fraser et al [25] found two distinct Mb

sequences in the common carp (Cy carpio) Although

Mb1 could be readily obtained by screening the cDNA

library, Mb2 was identified by RT-PCR using

oligonu-cleotide primers designed according to the C carpio

Mb2 sequence Goldfish and carp Mb1 proteins are

93.9% identical and 98.0% similar; the Mb2 proteins

are 88.4% identical and 96.6% similar (Fig 2 ) The

paralogs are  78% identical and  90% similar

When compared with zebrafish Mb, the scores for

goldfish Mb1 are 81.6% identical⁄ 91.8% similar, and

for Mb2 78.2% identical⁄ 88.4% similar Using

zebra-fish anti-Mb serum, we examined the presence of Mb

protein in goldfish organs (Fig 3 ) We detected Mb

protein in all investigated tissues (brain, gills, heart, liver, kidney and swimbladder) As expected, the Mb signal was strongest in heart (note the different amounts of total proteins applied per lane), but we also observed apparently high Mb concentration in the goldfish gills

Changes in gene expression in hypoxic goldfish

In previous studies with zebrafish, we employed acidic ribosomal phosphoprotein P0 (ARP) as the nonregu-lated reference gene [23] Fragments of ARP were amplified from goldfish RNA using primers that had been designed according to known zebrafish sequences

Fig 1 Comparison of zebrafish (Dre) and goldfish (Cau) globins The amino acid sequences from Hba (HbA), Hbb (HbB), Mb, Ngb, Cygb and GbX were aligned The secondary structure of human neuroglobin is superimposed in the upper row, with alpha-helices designated A–H, the globin consensus numbering is given below the sequences Strictly conserved amino acids are shaded in gray Invariable (B12.2 and G7.0) and variable (E10.2 and H10.0) intron positions in vertebrate globin genes are indicated by arrows in the upper row.

Fig 2 Comparison of zebrafish (Dre) myoglobin and the paralogous carp (Cca) and goldfish (Cau) Mb1 and Mb2 The predicted secondary structure of zebrafish Mb is superimposed in the upper row, with alpha-helices designated A–H, the globin consensus numbering is given below the sequences Strictly conserved amino acids are shaded in gray, functionally important residues (PheCD1, HisE7 and HisF8) are white on a black background.

ARP mRNA levels remained constant in most tissues;

however, in brain and eye we found significant

upregu-lation of ARP mRNA by a factor of  1.7

(P < 0.01) Therefore, all expression levels were

subse-quently normalized according to total RNA content

However, none of the conclusions presented here was

affected if expression levels were normalized according

to ARP (data not shown)

We applied mixed chronic and acute hypoxia

regimes that lasted 3 days First, mild chronic hypoxia

was induced by a reduction in Po2 to  6.7 kPa for

48 h Acute hypoxia was achieved at Po2 1.7 kPa

for 8 h, followed by Po2  6.7 kPa for an additional

16 h before RNA extraction Reduction of Po2 to

close anoxia (< 0.5 kPa) led to the death of all

experi-mental animals within 16 h Normoxic controls were

kept at Po2 18.4 kPa The expression levels of

lac-tate dehydrogenase A (LDH-A), Hba, Hbb, Cygb1

and Mb1 were first analyzed in goldfish body

(car-casses without heart, brain and eyes) (Fig 4A ) Thus

the majority of tissue represents skeletal muscle, but

also includes blood vessels We observed a mild

( 25%) downregulation of Hba, Hbb and Cygb1

mRNA under hypoxia, which was, however, not

signif-icant LDH-A levels were unchanged, whereas Mb1

mRNA was found to be heavily upregulated (

18-fold; P < 0.05) In heart, Mb1 mRNA levels were

essentially unaffected (Fig 4B) In brain, we observed

a twofold increase in Mb2 mRNA (P < 0.01),

although Mb1, Ngb, LDH-A and superoxide

dismu-tase (SOD)-1 mRNA levels remained essentially

con-stant (Fig 4C) No significant changes in mRNA

levels of the investigated genes were observed in total

eye (Fig 4D)

Quantitative western blotting

To compare the protein levels of Ngb and Mb from

goldfish and zebrafish, we performed quantitative

wes-tern blotting We used specific antibodies that had

Brain Gill Hear

t Liver Kidne

y Swimb ladder

18 kDa

Fig 3 Myoglobin expression in goldfish tissues Protein extracts

from selected goldfish tissues were analyzed by western blotting

employing a zebrafish anti-Mb serum Protein extracts (100 lg)

were applied on each lane for brain, gills, liver and kidney; 50 lg

was loaded for heart and swimbladder plus associated tissues The

position of the 18 kDa molecular mass marker is indicated on the

right side.

A

B

C

D

Fig 4 Expression of goldfish globins at different oxygen levels mRNA quantities were determined by quantitative real-time RT-PCR The white columns represent mRNA levels from goldfish kept

at normoxia (P O 2  18.4 kPa), gray columns are mRNA levels from goldfish kept at hypoxia (48 h P O2 6.7 kPa, 8 h P O2 1.7 kPa,

16 h at P O2 6.7 kPa) RNA was extracted from carcasses (A), heart (B), brain (C) or eye (D) Bars represent SD The significance

of the data was estimated with a Student’s t-test, with n = 4.

**P < 0.01 Gene abbreviations: Cygb, cytoglobin; HbA, hemo-globin a; HbB, hemohemo-globin b; LDH-A, lactate dehydrogenase A; Mb1, myoglobin 1; Mb2, myoglobin 2; Ngb, neuroglobin; SOD-1, superoxide dismutase-1.

been raised against recombinant zebrafish Ngb or Mb.

First, we evaluated Ngb and Mb protein levels in

normoxic and hypoxic goldfish No discernable

changes of Ngb in protein extracts from brain and eye,

or Mb in extracts from brain, heart and liver were

observed (supplementary Fig S7) For interspecific

comparisons, total proteins were extracted from brains,

total eyes and hearts of individual goldfish and

zebra-fish specimens that had been kept under normoxic

con-ditions We applied a constant amount of total protein

extracts to SDS⁄ PAGE (100 lg per lane for brain and

eye; 15 lg for heart) and western blotting (Fig 5 ) To

avoid variations due to different treatments, samples

from both species were applied to the same gel and

transferred to a single membrane, which was then

incubated with the antibodies We observed an

approx-imately fivefold higher Ngb protein level in the

gold-fish brain compared with zebragold-fish brain (Fig 5A)

The difference was highly significant, as estimated by a

Student’s t-test (P < 0.001) In protein extracts from

goldfish eyes, Ngb protein levels were 3.2-fold higher

than in zebrafish eyes (P < 0.05) We observed no

dif-ferences in Mb levels in goldfish and zebrafish, either

in brain or in heart (Fig 5B)

Discussion

Many fish species have evolved strategies that allow

them to survive phases of acute and chronic hypoxia

[2,5–8] Carassius species are particularly hypoxia

toler-ant, with various mechanisms that help them to better

survive hypoxia and even anoxia [2,11,37] We recently

analyzed the expression regulation and putative roles

of the various globins in another, less hypoxia-tolerant species of the Cypriniformes, the zebrafish D rerio [23] Comparison of these data with those obtained from C auratus will help to delineate the contribution

of globin expression regulation to hypoxia tolerance

In particular, we focused on Mb and Ngb; although a respiratory role for Mb is well documented, our results provide better understanding of the physiological role

of the recently discovered Ngb

Two paralogous Mb genes in Cyprininae

Mb is an intracellular respiratory protein that mainly facilitates the diffusion of O2 from the capillaries to the mitochondria and stores O2 [37] Until recently, it had been commonly assumed that there is only a single

Mb gene in vertebrates and that Mb expression is con-fined to muscle tissue Mb protein is present at high concentrations in the skeletal and heart muscles of most vertebrates [37], but has also been identified in smooth muscle [38,39] However, Fraser et al [25] demonstrated that the common carp possesses two paralogous Mb genes, of which one (Mb1) is ubiqui-tously present also in nonmuscle tissues; Mb2 expres-sion is restricted to the carp’s brain We confirmed these results in goldfish, which also possesses two dis-tinct Mb genes (Fig 2) and exhibits ubiquitous expres-sion of Mb (Fig 3) This suggests that the duplication

of Mb genes and the altered expression patterns occurred before the divergence of the genera Cyprinus and Carassius (both belonging to the subfamily Cyprininae), which separated  11 million years ago [40] As revealed by database searches, the zebrafish

Fig 5 Comparison of neuroglobin (A) and

myoglobin (B) protein levels in goldfish and

zebrafish Protein levels were estimated by

quantitative western blotting (cf Fig S8).

Three individual zebrafish specimens and

four goldfishes were used for the

experi-ments The units of the y-axis are arbitrary,

with zebrafish brain = 1 Note that the

rela-tive levels of Mb in brain and heart cannot

be compared ***P < 0.001; *P < 0.05

(t-test), n.s., not significant.

D rerio genome harbors only a single Mb gene It

may be assumed that the emergence of an additional

Mb gene is linked to a genome duplication event,

which occurred in the Cyprininae 12–16 million years

ago [40] We observed Mb protein expression in

goldfish as well as in zebrafish brain, suggesting that

nonmuscle expression of Mb emerged before the

diver-gence of D rerio and Cyprininae within the lineage of

the Cypriniformes As proposed by Fraser et al [25],

the occurrence of Mb in tissues other than muscle, its

hypoxia-inducible expression, as well as the occurrence

of the brain-specific isoform Mb2 may be part of the

strategy of the Cyprininae for better survival during

prolonged periods of hypoxia

Expression-regulation of globins and their

function in hypoxia

As in zebrafish [23], we observed a mild

downregula-tion of Hba and Hbb mRNA levels at hypoxia

com-pared with normoxic controls (Fig 4A) The hypoxia

response of Hb has been investigated in various fish

species, with conflicting results For example,

Timmer-man and ChapTimmer-man [41] reported an increase in Hb

levels in the sailfin molly (Poecilia latipinna), whereas

Person Le Ruyet et al [42] observed no difference in

normoxic and hypoxic juvenile turbot

(Scophthal-mus maxi(Scophthal-mus) and seabream (Sparus aurata) In

zebra-fish, the Hb mRNA levels decrease under hypoxia

[23,41] The contribution of the transcription

regula-tion of Hb to hypoxia tolerance is species specific and

may also depend on the hypoxia regime [43]

Never-theless, Carassius Hb is 50% saturated even at

Po2 = 0.33 kPa, thereby contributing to hypoxia

tolerance [44] Most likely, the O2-affinity of fish Hb is

largely regulated on a post-translational level, i.e via

alteration of O2affinities by means of modulators such

as ATP and GTP [45] Therefore, alterations in Hb

mRNA levels are not necessarily required Mb1 and

Mb2 were the only globin-types in goldfish to show

hypoxia induction at the mRNA level It may be

assumed that enhanced expression of Mb is associated

with hypoxia tolerance in goldfish Additional Mb in

various tissues increases the availability of O2 to the

respiratory chain of the mitochondria, thereby

prom-oting the survival of the cells

Putative role of Ngb in preadaptation of the brain

to hypoxia

Altered expression levels of certain proteins may help to

improve the animal’s survival under unfavorable

condi-tions For example, interspecific variations in heat

shock proteins have been found in marine gastropods and have been attributed to the acclimatization to dif-ferent habitats [46,47] The high Mb content in the mus-cles of marine mammals such as whales and seals is considered an adaptation to long-term dives [48] Noth-ing is known about the more recently discovered Ngb Although the localization, expression, regulation and evolution of Ngb have been thoroughly investigated in recent years, its exact role in vertebrate neurons is not well understood [29,30] Whereas some studies point to

a Mb-like role of Ngb in supplying O2 [26,49], thereby enhancing the survival of neurons under hypoxia [50], other authors have proposed a function for Ngb in the detoxification of reactive nitrogen species and NO [51,52] or hypoxia-related signaling [53,54] Most stu-dies agree that in vivo hypoxia does not significantly enhance Ngb expression in the mammalian brain [21] This is not surprising, because under normal condi-tions most mammals never experience low O2 environ-ments during their adult life However, as already pointed out, many fish live in hypoxic environments

In fact, we previously observed an increase in Ngb levels of up to 5.7-fold in hypoxic zebrafish brain compared with normoxia controls, which suggests the involvement of this protein in hypoxia response in this fish species [23]

It is well established that the Carassius brain has evolved various strategies to survive very low oxygen levels [37] However, C auratus, which is actually more hypoxia-tolerant than zebrafish, does not show a hypoxia response in Ngb expression At first sight, this

is difficult to reconcile with the hypothesis that Ngb is involved in O2 supply for respiration or has any other

Po2-related function such as reactive oxygen species detoxification However, we did not observe an increase in the expression of the typically hypoxia-responsive gene LDH-A, or of the reactive oxygen species-defense gene SOD, although the fact that Mb1

is heavily upregulated shows that the hypoxia regime

we applied in this study actually induce changes in gene expression As shown in Fig 5, there is an appro-ximately fivefold higher level of Ngb protein in the goldfish brain compared with the related, less hypoxia-tolerant zebrafish This is the first time that higher Ngb concentrations could be correlated with hypoxia tolerance, which may be interpreted as a preadaptation

of the goldfish brain A similar observation has been made in the subterranean mole rat Spalax ehrenbergi,

a mammal that can survive extended periods of hypoxia without neuronal damage, and which has con-stitutively higher expression levels of Ngb compared with rats (A Avivi, F Gerlach, T Burmester, E Nevo

& T Hankeln, unpublished results) These data

provide additional support for an adaptive role of Ngb

in hypoxia tolerance of neurons, and for a possible

func-tion of Ngb in O2storage or facilitated O2diffusion

Distinct roles of Mb and Ngb in hypoxia

adaptation

Changing environmental oxygen concentrations have a

significant impact on the expression of intracellular

respiratory proteins, which increase the availability of

O2 to the tissues In mammals, the results on the

impact of hypoxia on Mb expression are variable

[55,56] Data on hypoxia-regulation of Ngb in

mam-malian systems are also not consistent Although Ngb

was found to be more highly expressed in hypoxic cell

and tissue culture systems, no changes in Ngb mRNA

levels were found in whole-animal experiments [21]

Here we have demonstrated that both Mb and Ngb

may contribute to the extreme hypoxia tolerance of

goldfish The increased expression of Mb under

hypoxia and the high intrinsic Ngb levels in goldfish

neuronal tissues agree with the proposed function of

these proteins in O2 supply However, neuronal tissues

require a consistent supply with sufficient O2 and any

shortage results in severe defects in the brains of most

vertebrates The function of the nervous system thus

requires an uninterrupted O2 supply The high intrinsic

concentration of Ngb may guarantee its immediate

availability upon the onset of hypoxia, and may be

part of the strategy that secures a constant flow of O2

to the highly energy-demanding neurons In contrast

to neurons, striated muscle cells can survive via

anaer-obic fermentation; therefore, a delayed increase in Mb

concentration (as reflected by enhanced Mb mRNA

levels) is sufficient to ensure the supply of O2 to the

muscle cells Together with the high-affinity Hb [44],

high levels of Mb and Ngb may contribute to the fact

that Carassius are able to maintain normal O2

con-sumption rates down to oxygen levels of 5–10% of air

saturation in water [57]

Experimental procedures

Experimental animals

Adult goldfish (C auratus L.) were purchased in a local pet

shop and kept for several months in a large tank Animals

used for the hypoxia or normoxia control experiments

(weighing around 4 g each) were directly transferred to a

100 L aquarium and kept at 14 h light⁄ 10 h dark cycle and

a temperature of 20C Water was filtered with a

thermofil-ter (Ekip 350; Hydor, Bassano del Grappa, Italy) Wathermofil-ter

quality was checked periodically (Multi Check; Amtra,

Rodgau, Germany) and partial water changes were carried out when necessary Animal handling and experiments were conducted according to a protocol that had been approved

by the county government office (Bezirksregierung Rhein-hessen-Pfalz, AZ 1.5 177-07⁄ 021-30)

Hypoxia treatment Groups of four goldfish were randomly assigned to hypoxia treatment or control groups The animals were not fed for

24 h before or during the experiments Hypoxia treatment was performed in a 40 L aquarium with loosely fitting covers Water was bubbled with gas mixtures (2% O2in N2

or 100% N2; Air Liquide, Du¨sseldorf, Germany) A ther-mopump (Ekip 350; Hydor) was used to ventilate the water and to keep the temperature constant at  20 C O2 par-tial pressure and temperature were measured every 15 min using an oxygen sensor (Oxi 340i, WTW, Weilheim, Germany) Hypoxia treatment was started by reducing the Po2 to  6.7 kPa ( 50 Torr) for 48 h, Po2 1.7 kPa ( 13 Torr) for 8 h, followed by Po2 6.7 kPa ( 50 Torr) for additional 16 h O2 partial pressure remained constant (± < 0.5 kPa) during experimental time Control animals were kept under the same conditions, but the water was gassed with room air (Po2 18.4 kPa,

 138 Torr) After the experiment, specimens were cooled

on ice and killed by decapitation Organs were removed, shock-frozen in liquid N2and stored at)80 C until use

RNA extraction RNA samples from total goldfish or single organs were extracted using the RNeasy Mini Kit by Qiagen (Hilden, Germany) Tissues were weighed and homogenized in the required volume of RLT buffer (Qiagen) To avoid contami-nation with genomic DNA, a DNase digestion was per-formed on the Qiagen columns Quality and amount of RNA were checked photometrically and with gel electrophoresis

cDNA amplification, cloning and sequencing Total RNA was extracted from goldfish brain, liver, heart, skeletal muscle, spleen, eyes and gills as described above Partial or complete cDNA sequences of C auratus Hba, Hbb, Mb1, Mb2, Ngb, Cygb1, acidic ribosomal protein (ARP; also known as rplp0), LDH-A and Cu⁄ Zn-SOD-1were amplified via RT-PCR with the OneStep RT-PCR kit (Qiagen) using degenerated or specific oligonucleotide primers (supplementary Table S1) The cDNA fragments were cloned into the pCR4-TOPO-TA (Invitrogen, Karlsruhe, Germany) or the pGEMTeasy vector (Promega, Mannheim, Germany) Poly(A)+RNA was purified from total RNA using the PolyATractTM kit (Promega);

5 lg poly(A)+RNA were used for the construction of a

directionally cloned cDNA expression library applying the

Lambda ZAP-cDNA synthesis kit (Stratagene, Heidelberg,

Germany) according to the manufacturer’s instruction The

library was then screened with digoxigenin-labeled cDNA

fragments of the globins Positive phage clones were

con-verted to plasmid vectors using the material provided in the

cDNA synthesis kit cDNAs inserted in the pBK-CMV

vector were sequenced on both strands by a commercial

sequencing service (Genterprise, Mainz, Germany) In some

cases, the incomplete clones were extended by 5¢- and

3¢-RACE (Invitrogen) with a series of nested

oligonucleo-tide primers according to the manufacturer’s instructions

The sequences were obtained after the cloning of the

PCR products into pCR4-TOPO-TA (Invitrogen) or

pGEM-Teasy vector (Promega)

Quantitative real-time RT-PCR

RNA extractions and cDNA synthesis were carried out

from tissues of single specimens Quantitative real-time

RT-PCR was performed according to a two-step protocol

First, total RNA was converted into cDNA employing

Superscript II RNase H)Reverse Transcriptase (Invitrogen)

and an oligo(dT)16 primer according to the manufacturer’s

instructions The cDNA samples were diluted with the same

volume of DNase-free water Real-time RT-PCR

experi-ments were carried out on an ABI Prism 7000 SDS (Applied

Biosystems, Darmstadt, Germany) using the Power SYBR

Green PCR Master Mix (Applied Biosystems) Levels of

mRNA of ARP, LDH-A, SOD-1, Hba, Hbb, Mb1, Mb2,

Ngb and Cygb1 were evaluated To avoid amplification of

genomic DNA, all primer pairs included one

intron-span-ning oligonucleotide The oligonucleotide primers were

obtained from Sigma-Genosys (Hamburg, Germany)

(sup-plementary Table S2) Reactions were run in triplicate with

one or two repetitions, using 1 lL of diluted cDNA as

tem-plate in a reaction volume of 25 lL Primer concentrations

were 0.13 lm for each oligonucleotide The Taq DNA

poly-merase was activated for 15 min at 95C, followed by 40

cycles of a standard PCR protocol (15 s at 95C, 30 s at

60C, 30 s at 72 C) The efficiency of the reaction was

measured by the slope of a standard curve First evaluation

of results was performed in the ABI Prism 7000 sds

pro-gram; for normalization and calibration data were exported

to qBase (http://www.medgen.ugent.be/qbase/) Final data

analyses were carried out with the Microsoft excel2003

spreadsheet program (Microsoft, Redmond, WA, USA)

The significance of the data was evaluated by Student’s

t-test

Recombinant protein expression and antibody

preparation

The complete coding sequences of D rerio Ngb was cloned

into the pET3a and Mb into pET15b expression vectors

(Novagen, Darmstadt, Germany) employing PCR-generated restriction sites Plasmids were transformed into Escherichia coli BL21(DE3)pLys and grown at 25C in TBY medium (0.5% NaCl, 1% tryptone, 0.5% yeast extract, pH 7.4) containing 100 lgÆmL)1 ampicillin, 30 lgÆmL)1 chloram-phenicol and 1 mmolÆL)1d-aminolevulinic acid The culture was induced at D600= 0.8 by isopropyl-b-d-thiogalacto-pyranoside (0.4 mmolÆL)1) After 16 h, bacteria were har-vested by centrifugation and resuspended in 50 mmolÆL)1 Tris⁄ HCl, pH 8.0, 1 mmolÆL)1 EDTA, 0.5 mmolÆL)1 di-thiothreitol, 8 lgÆmL)1 DNase and 4 lgÆmL)1 RNase supplemented with Complete proteinase inhibitor mixture (Roche Applied Science, Mannheim, Germany) and Pefab-loc (Roth, Karlsruhe, Germany) The cells were broken by freeze–thaw cycles in fluid nitrogen followed by ultrasonica-tion DNA and RNA were digested for 2 h at 37C Cell debris was removed by centrifugation (1 h at 4C at

10 000 g) Ngb was purified by ammonium sulfate precipi-tation, followed by DEAE ion-exchange column and size exclusion chromatography His-tagged Mb was purified by affinity chromatography (Protino Ni 2000 prepacked columns; Macherey and Nagel, Du¨ren, Germany) Final globin fractions were analyzed by gel electrophoresis, pooled, concentrated and stored frozen at )20 C Protein concentrations were determined using the Bradford [58] method Purified recombinant D rerio Ngb and Mb were used to raise a polyclonal antibody in rabbits Specific Ngb antibodies were affinity-purified from crude rabbit serum using recombinant D rerio Ngb coupled to a HiTrap NHS-activated HP column (Amersham Biosciences, Munich, Germany) according to the manufacturer’s instruc-tions The antibody was stored at )70 C in 50 mmolÆL)1 Tris, 100 mmolÆL)1 glycine, pH 7.4 or supplemented with 0.1% NaN3at 4C

Protein extraction and western blotting Tissues were removed from the animal (goldfish or zebra-fish) and immediately homogenized in 1· NaCl⁄ Pi (140 mmolÆL)1 NaCl, 2.7 mmolÆL)1 KCl, 8.1 mmolÆL)1 Na2HPO4, 1.5 mmolÆL)1 KH2PO4) by ultrasonication The debris was precipitated by centrifugation for 10 min at

13 000 g at 4C and the supernatant was stored at)20 C until use Protein concentrations in the samples were deter-mined according to Bradford [58] Protein extracts (100 lg) were diluted in sample buffer (31.25 mmolÆL)1 Tris⁄ HCl,

pH 6.8, 1% SDS, 2.5% b-mercaptoethanol, 5% glycerol) and heat-denatured for 5 min at 95C Samples were applied to a 15% SDS-polyacrylamide gel and run at 100–

120 V Proteins were transferred to a nitrocellulose mem-brane for 2 h at 0.8 mAÆcm)2 Nonspecific binding sites were blocked by incubating for 45 min with 2% BSA in NaCl⁄ Tris (10 mmolÆL)1 Tris, pH 7.4, 140 mmolÆL)1 NaCl) Membranes were then incubated for 2 h with anti-Ngb or anti-zebrafish-Mb serum, both diluted 1 : 500

in 2% BSA⁄ NaCl ⁄ Tris, and washed four times for 5 min

with NaCl⁄ Tris Membranes were incubated with the goat

anti-(rabbit IgG) coupled with alkaline phosphatase

(Dianova, Hamburg, Germany) for 1 h, diluted 1 : 10 000

in 2% BSA⁄ NaCl ⁄ Tris, and washed as described above

Detection was carried out with nitro blue tetrazolium

chloride and 5-bromo-4-chloro-3-indolyl-phosphate salt as

substrates The membranes were scanned at 1200 dpi and

the images were imported into the scion image program

(version Beta 4.0.3) Protein levels were estimated by

analy-ses of grey values Mean gray values of the background of

empty gel lanes were subtracted from the measurements of

Ngb or Mb protein levels Data were imported into

Micro-softexcel2003 spreadsheet program (Microsoft)

Statisti-cal analyses were performed by Student’s t-tests

Acknowledgements

We thank F Gerlach for his advice on the real-time

PCR experiments and critical reading of the

manu-script This work has been supported by grants of the

Deutsche Forschungsgemeinschaft (Bu956⁄ 5, Bu956 ⁄ 11

and Ha2103⁄ 3) and the Fonds der Chemischen

Industrie

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