TSINGHUA SCIENCE AND TECHNOLOGY

ISSN 1007-0214 08/20 pp405-412

Volume 12, Number 4, August 2007

Cloning, Characterization, and Distribution of an mRNA

Encoding a H+-ATPase Subunit in the Mantle
of Pearl Oyster, Pinctada fucata*
CHEN Lei ( )1, SUN Xude ()2, BAI Hongwei ()3, YIN Shande ()4,
XIE Liping ()1, XIONG Xunhao ()1, ZHANG Rongqing ()1, **
1. Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology,
Tsinghua University, Beijing 100084, China;
2. Department of Anaesthesology, Tangdu Hospital, the Fourth Military Medical University, Xian 710032, China;
3. Department of Hepatobiliary Surgery, the Naval General Hospital, Beijing 100037, China;
4. Department of Gynaecology and Obstetrics, the Naval General Hospital, Beijing 100037, China
Abstract: Mitochondrial ATP synthase is responsible for the production of the majority of the cellular ATP,
which is composed of two major units: F0 and F1. Although much is known about the active complex (5 subunits ()), the role of the subunit in the catalytic mechanism remains unclear, particularly in bivalve
animals. This study first cloned and identified the full-length sequence of the mitochondrial H+-ATP synthase
subunit cDNA gene in Pinctada fucata using the reverse transcriptase polymerase chain reaction (RT-PCR)
technique. The Pinctada fucata mitochondrial H+-ATP synthase subunit contains 1991 nucleotides, with
the translation start site at nt 48 (ATG) and the stop codon at nt 1660 (TAA), encoding a polypeptide 553
amino acids in length, which shares high similarity to that of other animals (81% identity to fruit fly, 82% to
carp, and 83% to humans). Alignment analysis of the well-conserved amino acid domains in the ATPase
subunit, the / signal transduction domain, showed that two residues (Asp

358

and Asn359) differ from any

other ATP synthase subunit. In situ hybridization analysis was used to reveal the wide-spread distribution
+
of mitochondrial H -ATP synthase in various tissues in Pinctada fucata. This work will help further research

on pearl energy metabolism to increase the output and quality of pearls to more efficiently utilize our rich
pearl oyster resources.
+
Key words: mitochondrial H -ATP synthase; Pinctada fucata; gene cloning

Introduction
Mitochondrial H+-ATP synthase (F1F0 ATPase) is responsible for the production of the majority of the
Received: 2006-09-08

Supported by the National High-Tech Research and Development

(863) Program of China (No. 2003AA603430) and the National
Natural Science Foundation of China (No. 30371092)

To whom correspondence should be addressed.

E-mail: rqzhang@mail.tsinghua.edu.cn; Tel/Fax: 86-10-62772899

cellular ATP required by eukaryotic organisms to meet

their energy needs. Its function is to couple the electrochemical proton gradient to the ATP synthesis. The
ATP synthase is composed of two major units: F0,
which is embedded within the inner membrane, and F1,
which lies in the matrix region. F0 is a hydrophobic
complex involved in proton translocation; and F1 is
composed of five subunits (33) which has catalytic domain[1,2]. Both the and subunits have
AT(D)P binding sites, but none of them alone has

406

ATPase activity[3,4]. ATP synthesis occurs on F1 with

ATP released from the tight binding site on F1 at the
expense of the energy present in the electrochemical
proton gradient[5]. The subunit, the catalytic center,
was recently investigated in detail[6]. Although known
to be necessary for the formation of an active complex,
the role of the subunit in this catalytic mechanism
remains unclear, particularly, in mollusk.
This paper reports the isolation and characterization
of a gene encoding an ATP synthase -subunit in a bivalve animal Pinctada fucata (P. fucata), which is an
important oyster used in mariculture of pearls in China.

Materials and Methods

1.1

Animal and total RNA extraction

Adult P. fucata were obtained from the Guofa Pearl

Farm in Beihai, Guangxi Province, China. Tissues including mantle, digestive gland, muscle, gill, and gonad were dissected and kept in RNAlater (Ambion
Austin, TX, USA). Total RNA was isolated from the
tissues with RNAzol RNA Isolation Kit (Biotech,
Houston, TX, USA) according to the manufacturers
instructions. The RNA integrity was examined by fractionation on 1.0% formaldehyde-denatured agarose gel
followed by staining with ethidium bromide. The quantity of RNA was determined by measuring the OD260 nm
with an Ultraspec 3000 UV/Visible Spectrophotometer (Amersham, Piscataway, NJ, USA).
1.2

Reverse transcriptase polymerase chain

reaction (RT-PCR)

A pair of degenerate oligonucleiotide primers was designed based on the conserved amino acid segments of
the ATP subunit. The primers were ATPF1 [5ATGAAG(T/C)T(G/C)GA(G/A)TT(G/A)GCICAGT-3]
corresponding to (MKLELAQY) and ATPR1 [5-AC
(A/T)(G/C)(C/T)(A/T/G/C)ACTTGTTCTTCAAT-3]
corresponding to the antisense strand of the sequence
encoding (MAIEEQV). Altogether, 2.5 g of mantle
total RNA was used as a template for the RT reaction
with Superscript II and oligio (dT) primers (Invitrogen,
California, USA). The RT reaction and the following
PCR reaction were performed according to the manufacturers instructions on Tgradient Thermocylcer
(Biometra, Gottingen, Germany). PCR cycles were
performed with denaturation at 95 for 5 min,

Tsinghua Science and Technology, August 2007, 12(4): 405-412

followed by 30 cycles at 95 for 45 s, 57 for 30 s,

and 72 for 45 s. The final extension was carried out
at 72 for 7 min after the cycles. The PCR products of
the expected size (164 bp) were excised and purified
with the Wizard PCR prep DNA Purification System
(Promega, Madison, WI, USA). The purified PCR
products were subcloned into pMD 18-T Vector (TaKaRa, Dalian, China) and sequenced. The resulting sequence information was used to design gene-specific
primers for 5- and 3- rapid amplification of cDNA
end (RACE).
1.3

Isolation and sequencing of the full-length

pfATPase subunit cDNA

Single-stranded cDNA for all RACE reactions were

prepared from the mantle total RNA using PowerScriptTM (Clontech, Plao Alto, CA). 5-RACE was
conducted according to the manufacturers instructions,
using the SMART RACE cDNA Amplification Kit
(Clontech, Plao Alto, CA) and the Advantage 2 cDNA
Polymerase Mix (Clontech Plao Alto, CA). The genespecific primer 5ATP was 5-AGTAACTCGGTC
AGACGAACTCCCCT-3. The 5-RACE PCR cycles
were performed using touchdown PCR with 5 cycles at
94for 5 s and 72 for 3 min, then 5 cycles at 94
for 5 s, 62 for 10 s, and 72 for 3 min, followed by
25 cycles at 94 for 5 s, 60 for 10 s, and 72 for
3 min.
The 3-RACE was performed using the SMART
RACE cDNA Amplification Kit (Clontech, Plao Alto,
CA) and the Advantage 2 cDNA Polymerase Mix
(Clontech, Plao Alto, CA). The gene-specific primer
3ATP was 5-ACAGGGGAGTTCGTCTGACCGAGT
TAC-3. The 3-RACE PCR cycles were performed using touchdown PCR with 5 cycles at 94 for 5 s and
72 for 3 min, then 5 cycles at 94 for 5 s, 62 for
10 s and 72 for 1 min, followed by 25 cycles at 94
for 5 s, 60 for 10 s, and 72 for 1 min. All amplified products were cloned into pMD 18-T Vector
(TaKaRa, Dalian, China) for sequencing.
1.4

In situ hybridization

The tissues were removed from the adult P. fucata and

immediately fixed in 4% paraformaldehyde containing
0.1% diethypyrocarbonnate (DEPC, Sigma, USA)
overnight. The digoxigenin-labeled probe was generated from the initially-cloned 164 bp fragment using

CHEN Lei ( ) et alCloning, Characterization, and Distribution of an

the DIG Labeling Kit (Roche, Hong Kong, China).

The in situ hybridization procedures were performed mainly as described previously with some
modifications[7].

Results and Discussion

2.1

Cloning and sequence analyses of P. fucata

ATPase subunit cDNA

PCR was performed with mantle of P. fucata cDNA as

template using two degenerate oligonucleotides
(ATPF1 and ATPR1) derived from the conserved
amino acid segments of the ATPase subunit. A 164bp PCR product of appropriate size was subcloned and
sequenced. Sequence analysis of the PCR fragment revealed that it most likely encoded a ATPase subunit
homologue. The full-length cDNA of the pfATPase
subunit was obtained using gene-specific primers for
5-RACE and 3-RACE designed based on the sequence derived from the initial fragment (Fig. 1). The
touchdown PCR with 5-RACE yielded the amino terminus of the open reading frame (ORF) and a 47-bp 5untranslated regions (UTR), while the touchdown PCR
with 3-RACE yielded a 282-bp fragment and 3-UTR.
The 3-UTR contained a canonical poly-adenylation
signal and a poly (dA) tail. In summary, the full-length
cDNA was 1991 bp in size, with the translation start
site at nt 48 (ATG) and the stop codon at nt 1660
(TAA), encoding a polypeptide of 553 amino acid in
length (Fig. 1). The deduced amino acid sequence contained a perfect match to the peptide sequence of the
ATPase subunit (Fig. 1).
2.2

Sequence, phylogenetic, and predicted

structure analysis

The nucleotide sequence was submitted to the GeneBank with Accession No. DQ986328. From an evolutionary standpoint, the ATPase subunit is a remarkably well conserved protein. Multiple protein sequence
alignment analysis showed that the pfATPase subunit sequence shared high similarity with those of
ATPase subunits from carp, Xenopus laevis (X. la),
fruit fly, and mammalian (81% identity to fruit fly,
82% to carp, and 83% to human) (Fig. 2a). The

407

well-conserved amino acid domains in the ATPase

subunits, the / signal transduction domain, which is
an essential residue for cooperative catalysis between
and subunits[8-10], were found in the deduced amino
acid sequence. Alignment analysis of this domain
showed two residues (Asp358 and Asn359) that differ
from any other ATPase subunit (Fig. 3). This can be
considered to be a characteristic residue in oyster.
To investigate the evolutionary relationships between the P. fucata ATPase subunit and known ATPase subunit isoforms, a phylogenetic tree was constructed using the ClustalX program[11] with the
neighbor joining (NJ) algorithm of Saitou and Nei[12]
(Fig. 2b). The phylogenetic tree revealed that the P. fucata ATPase subunit is closely related to the fruit fly
ATPase subunit. Within the ATPase subunit, the
pfATPase was fairly close to that of fruit fly and carp.
However, the pfATPase diverged from that of mouse
and rat.
2.3

Distribution and expression of pfATPase

subunit mRNA in tissues

The distribution of the hybridization signal of pfATPase

subunit mRNA was studied using in situ hybridization. This mRNA is widely expressed in the mantle,
gill, spermatogenic epithelia, digestion gland epithelia,
and muscle cells in P. fucata. The pfATPase subunit
mRNA hybridization signals were dark-brown and the
background was not stained. As shown in Figs. 4a-4e,
the positive signals were only distributed in the cytoplasm with hybridization negative nuclei detected in all
five tissues. Strong pfATPase subunit mRNA hybridization signals were observed in the mantle gland,
mantle epithelia cells, and gill epithelia cells, while
weak signals were observed in the digestive gland and
gonad. Positive signals were not present in the muscle.
The pfATPase mRNA hybridized signal was not detected on the negative control. F1F0 ATP synthase plays
a major role in cellular energy production. Strong hybridization signals in the mantle and gill epithelia cells,
which are involved in periostracum, shell formation[13],
and calcium metabolism, indicate that the energy
metabolism of these two organs is very active. This
is consistent with previous results[14,15].

Tsinghua Science and Technology, August 2007, 12(4): 405-412

408
A: RT-PCR
(164 bp)
5'-RACE (1458 bp)
3'-RACE (558 bp)
Full-length cDNA (1991 bp)
B:

(1344 - 1507 bp )
(1-1458 bp )
(434 -1991 bp )
(1-1991 bp )

ACGCGGGGATATTGGTCGAAACCGCACACATACGACAAGTAGACAAA
ATGCTTTCGGCCAGATTAGCAGCAAGTCTCGTAAGACAGCTTCCCAGGGCTGCACCAAAGGTTTGCCAACATGCC
M L S A R L A A S L V R Q L P R A A P K V C Q H A
CTGGGAGCTGGACTTATTTCTGCCAAAAAGTTTTCCACATCTACACATCACCATACTGCAGGAGCTAGTGCGGAA
L G A G L I S A K K F S T S T H H H T A G A S A E
GTGTCATCCATCTTGGAGGAGAGGATTTTGGGACAGACAACACAAGCTGGATTAGAGGAAACCGGTCGTGTACTA
V S S I L E E R I L G Q T T Q A G L E E T G R V L
TCCATCGGAGACGGTATTGCTCGTGTTTATGGTCTAAAGAATATTCAGGCAGAAGAAATGGTAGAATTCTCCAGT
S I G D G I A R V Y G L K N I Q A E E M V E F S S
GGTTTAAAGGGTATGGCCTTGAACTTGGAGAGAGATAACGTAGGAGTTGTCGTATTTGGTAACGATAAATTAATT
G L K G M A L N L E R D N V G V V V F G N D K L I
AAAGAAGGAGACATCGTAAAAAGAACAGGAGCTATTGTGGATGTCCCCGTAGGAAAGGAATTATTAGGAAGAGTT
K E G D I V K R T G A I V D V P V G K E L L G R V
GTTGATGCTTTAGGAAATCCTATTGATGGAAAGGGACCAATCACATCACCTGACAGACAGAGGGTAGGAGTTAAG
V D A L G N P I D G K G P I T S P D R Q R V G V K
GCCCCTGGTATCATCCCCCGTATCTCAGTAAAGGAACCTATGCAGACCGGTATTAAGACTGTAGATAGCTTAGTA
A P G I I P R I S V K E P M Q T G I K T V D S L V
CCTATTGGTAGAGGTCAGAGAGAACTCATCATCGGTGACAGGCAGACTGGCAAAACCGCTATTGCAATTGACACA
P I G R G Q R E L I I G D R Q T G K T A I A I D T
ATTATTAACCAGAAGAGGTTCAACGATGGAACAAATGAGAAGGCCAAGCTTTATTGTATCTATGTTGCTATTGGT
I I N Q K R F N D G T N E K A K L Y C I Y V A I G
CAGAAGAGATCCACAGTGGCTCAGATTGTAAAGAGGCTTACTGATGCAGATGCCATGAAGTACACTGTGATTGTC
Q K R S T V A Q I V K R L T D A D A M K Y T V I V
AGTGCTACAGCTTCTGATGCTGCTCCCTTACAGTATCTGGCTCCTTACTCGGGATGTGCCATGGGAGAATTCTTC
S A T A S D A A P L Q Y L A P Y S G C A M G E F F
AGAGATAACGGCATGCACGCCCTCATCATCTACGACGATCTGTCAAAACAGGCTGTAGCTTACCGTCAAATGTCA
R D N G M H A L I I Y D D L S K Q A V A Y R Q M S
CTACTTTTGAGACGTCCCCCCGGTCGTGAAGCCTACCCTGGTGATGTGTTCTACCTCCATTCACGTTTGCTGGAG
L L L R R P P G R E A Y P G D V F Y L H S R L L E
CGTGCTGCCAAGATGAATGACGATAACGGTGGTGGATCCCTCACTGCTCTCCCTGTCATCGAGACCCAGGCTGGT
R A A K M N D D N G G G S L T A L P V I E T Q A G
GATGTGTCAGCCTACATCCCCACTAACGTCATCTCTATCACAGACGGACAGATCTTCTTGGAGACTGAGTTGTTC
D V S A Y I P T N V I S I T D G Q I F L E T E L F
TACAAGGGTGTACGTCCAGCAATTAATGTAGGATTATCTGTCAGTAGGGTAGGATCGGCAGCCCAAACAAAGGCC
Y K G V R P A I N V G L S V S R V G S A A Q T K A
ATGAAACAGGTAGCAGGCTCCATGAAGTTGGAGTTGGCCCAGTACAGAGAAGTGGCAGCTTTCGCTCAGTTTGGA
M K Q V A G S M K L E L A Q Y R E V A A F A Q F G
TCTGATTTGGATCAGGCTACACAGAATTTACTGAACAGGGGAGTTCGTCTGACCGAGTTACTCAAACAGGCTCAG
S D L D Q A T Q N L L N R G V R L T E L L K Q A Q
TATGTACCAATGGCCATTGAAGAACAAGTAGCAGTTATCTATGCTGGTGTCAAGGGACGTTTAGACAAAGTTGAT
Y V P M A I E E Q V A V I Y A G V K G R L D K V D
CCATCTAGAATCACAGAATTTGAAGCAGCTTTTGTATCACACATCAGAGGAAGTCAGCAAGAATTATTAGGTCAG
P S R I T E F E A A F V S H I R G S Q Q E L L G Q
ATCAGGAAAGACGGTCAAATCACAGAAGCCTCCGACGCTAAATTAAAGGAGGTTGTCACACAGTTCTTGTCCACC
I R K D G Q I T E A S D A K L K E V V T Q F L S T
TTCGAAGCATAAATTCATGTGACATTAAACATTTTCTCAAACGAATGGTGATATAGACAGTGCGGTTTTCCTTGT
F E A *
GACACTTATTGACTTAGAGTCTATTTATATCAGTGCTAACTTTGAAAGTATGTTGAATTGATTTGTACATGTAAT
GTCCCTTGTTTAAAAGATCTTTATAAACATTACATGGCAGTGACTGTTTTACATTGTTTTTGATGAATTCAGACA
ACTGTTGATGTATAGATGACATTATTAAAGGGATATTATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

-47
75
25
150
50
225
75
300
100
375
125
450
150
525
175
600
200
675
225
750
250
825
275
900
300
975
325
1050
350
1125
375
1200
400
1275
425
1350
450
1425
475
1500
500
1575
525
1650
550
1725
1800
1875
1944

Fig. 1 Oyster pfATPase subunit cDNA and deduced amino acid sequence. The conserved amino acid sequence residues used to deduce the degenerate primers are in bold and the corresponding nucleotides are shaded. The 3-RACE
primer is underlined. The 5-RACE primer is in the box. The stop codon is indicated with an asterisk.

409

CHEN Lei ( ) et alCloning, Characterization, and Distribution of an

Groups
P. fucata
Carp
Fruit fly

Sequence identities (%)

P. fucata

Carp

Fruit fly

Gallus

X. la

Cow

Homo

Mus

Rattus

100

82

81

82

83

83

83

83

83

80

88

90

90

91

91

90

100

79

81

81

80

80

80

100

Gallus

100

X. la
Cow

93

92

92

92

92

100

92

92

92

93

97

98

07

100

97

96

100

98

100

Homo
Mus
Rattus

100
(a) Matrix indicating the percentage identities of the aligned pfATPase

(b) Phylogenetic tree showing the evolutionary relationship of the oyster pfATPase subunit protein sequence. (The N-J tree was generated
using the method N-J of program Clustal X 1.81.)

Fig. 2

Phylogenetic analysis of pfATPase subunit in Pincatada fucata and their homologies with other species

P. fucata
Carp
Cow
Fruit fly
Gallus
Homo
Mus
Rattus
X. la

--MLSARLAASLVRQLPRAA-PKVCQHALGAGLISAKKFSTSTHHHTAGASAEVSSILEERILGQTTQAGLEETGRVLSI
--MLSGRVAAALARTLPRRA-GFVSKNVAAAACVGAKNLHTARPWLQKTGTAEVSSILEEKILGADTSAALEETGRVLSI
--MLSVRVAAAVARALPRRA-GLVSKNALGSSFIAARNLHASNSRLQKTGTAEVSSILEERILGADTSVDLEETGRVLSI
MSIFSARLASSVARNLPKAANQVACKAAYPAASLAARKLHVAST--QR--SAEISNILEERILGVAPKADLEETGRVLSI
--MLSVRVAAVFARSLPRQA-GLVSRNALGAAFVATRNIHASKMRFQKTGTAEVSSILEERILGADTSAELEETGRVLSI
--MLSVRVAAAVVRALPRRA-GLVSRNALGSSFIAARNFHASNTHLQKTGTAEMSSILEERILGADTSVDLEETGRVLSI
--MLSVRVAAAVARALPRRA-GLVSKNALGSSFVGARNLHASNTRLQKTGTAEMSSILEERILGADTSVDLEETGRVLSI
--MLSVRIAAAVARALPRRA-GLVSKNALGSSFVGTRNLHASNTRLQKTGTAEMSSILEERILGADTSVDLEETGRVLSI
--MLSVRVAAALARALPRQS-GLVSKKALGAAFVATRNIHASGAWLQKSGTAEVSSILEERILGADISTDLEETGRVLSI

77
77
77
76
77
77
77
77
77

P. fucata
Carp
Cow
Fruit fly
Gallus
Homo
Mus
Rattus
X. la

GDGIARVYGLKNIQAEEMVEFSSGLKGMALNLERDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGKELLGRVVDALGNP
GDGIARVYGLRNVQAEEMVEFSSGLKGMSLKLEPENVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVYGLNNIQADEMVEFSSGLKGMALNLEPDNVGVVVFGNDKLIKQGDIVKRTGAIVDVPVGDELLGRVVDALGNA
GDGIARVYGLRNVQAEEMVEFFFGLKGMSLNLEPDNVGVVVFGNDRLIKEGDVVKRTGAIVDVPVGEELLGRVVVALGNP
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDVVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGDELLGRVVDALGNA
GDGIARVYGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGDELLGRVVDALGNA

157
157
157
156
157
157
157
157
157

P.fucata
Carp
Cow
Fruit fly
Gallus
Homo
Mus
Rattus
X. la

IDGKGPITSPDRQRVGVKAPGIIPRISVKEPMQTGIKTVDSLVPIGRGQRELIIGDRQTGKTAIAIDTIINQKRFNDGTN
IDGKGPLGSKQRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTAIAIDTIINQKRFNDGTE
IDGKGPIGSKARRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGAINTKDRFRVGIKAPGIIPRVSVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTALAIDTIINQKRFNEAQD
IDGKGPITSKTRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGPIGSKTRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGSD
IDGKGPIGSKTRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGPVGSKIRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGPLTSKIRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVSIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNEGTD

237
237
237
236
237
237
237
237
237

(a) N-terminal coupled domain (1-22 aa)

Tsinghua Science and Technology, August 2007, 12(4): 405-412

410
P. fucata

EKAKLYCIYVAIGQKRSTVAQIVKRLTDADAMKYTVIVSATASDAAPLQYLAPYSGCAMGEFFRDNGMHALIIYDDLSKQ 317

Carp

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGGYFRDNGKHALIIYDDLSKQ 317

Cow

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317

Fruit fly

ESKKLYCIYVAIGQKRSTVAQIVKRLTDSGAMGYSVIVSATASDAAPLQYLAPYSGCAMGEYFRDKGKHALIIYDDLSKQ 316

Gallus

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317

Homo

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317

Mus

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317

Rattus

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317

X. la

EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
(b) Nucleotides binding domain257-303 aa

P. fucata

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDDNGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

Carp

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDNFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

Cow

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDAFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

Fruit fly

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMSPAMGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 396

Gallus

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDSFGGGSLTALPAIETQAGDVSAYIPTNVISITDGQIFLET 397

Homo

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDAFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

Mus

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDSFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

Rattus

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDSFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

X. la

AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDHFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397

P. fucata

ELFYKGVRPAINVGLSVSRVGSAAQTKAMKQVAGSMKLELAQYREVAAFAQFGSDLDQATQNLLNRGVRLTELLKQAQYV 477

Carp

ELFYKGIRPAINVGLSVSRVGSAAQTRGMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYS 477

Cow

ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477

Fruit fly

ELFYKGIRPAINVGLSVSRVGSAAQTKAMKQVAGSMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYV 476

Gallus

ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYV 477

Homo

ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477

Mus

ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477

Rattus

ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477

X. la

ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYV 477

P. fucata

PMAIEEQVAVIYAGVKGRLDKVDPSRITEFEAAFVSHIRGSQQELLGQIRKDGQITEASDAKLKEVVTQFLSTFEA 553

Carp

PMAIEEQVAVINAGVRGHLDKMDPSKITKFEKAFLQHVISQQTDLFAAIRTDGKISEASDAKLKEVVLNFLASFE- 552

Cow

PMAIEEQVAVIYAGVRGYLDKLEPSKITKFENAFLSHVISQHQALLSKIRTDGKISEESDAKLKEIVTNFLAGFEA 553

Fruit fly

PMAIEDQVAVIYCGVRGHLDKMDPAKITKFEKEFLQHIKTSEQALLDTIAKDGAISEASDAKLKDIVAKFMSTFQG 552

Gallus

PMAIEEQVAVIYAGVKSHLDKLEPSKITKFESAFLAHVLSQHQALLSTIRTEGKISDQTEAKLKEIFTNFLSTFEA 553

Homo

PMAIEEQVAVIYAGVRGYLDKLEPSKITKFENDFLSHVVSQHQALLGTIRAEGKISEQSDAKLKEIVTNFLAGFEA 553

Mus

PMAIEEQVAVIYAGVRGYLDKLEPSKITKFENAFLSHVISQHQSLLGNIRSDGKISEQSDAKLKEIVTNFLAGFEP 553

Rattus

PMAIEEQVAVIYAGVRGYLDKLEPSKITKFESAFLSHVVSQHQSLLGNIRSDGKISEQSDAKLKEIVTNFLAGFEP 553

X. la

PMAIEEQVTVIYAGVRGHLDKMEPSKITKFESAFLAHVKSQHQELLATIRTDGKISEQADAKLKEIVLNFLSTFEA 553
(c) / signal transduction domain (322-388 aa)

Fig. 3 Alignment of the oyster pfATPase subunit sequence with other ATP synthase subunits from invertebrates to
mammalian. The amino acid domains are indicated by overlining. The 358th and 359th amino acids, which differ from
other species are Asp (D) and Asn (N), respectively.

CHEN Lei ( ) et alCloning, Characterization, and Distribution of an

411

Fig. 4 In situ hybridization of oyster pfATPase subunit mRNA in various tissues of P. fucata. Among these tissues, hybridization signals (arrows) were observed in the mantle epithelial cells (a), mantle gland (b), digestive gland (c), spermatogenic duct (d), and gill (e).

Conclusions

In summary, the pfATPase subunit is unique among

those which belong to the ATPase subunit family
based on the following criteria. First, the deduced
amino acid sequence of pfATPase has not been reported for other bivalve mollusks. Secondly, the /
signal transduction domain, which may be involved in
cooperative catalysis, differs from any other ATPase
subunits. Thirdly, the analysis of the phylogenetic tree
also showed that the pfATPase subunit differs from
any other members of the ATPase subunit. Therefore,
the pfATPase subunit probably defines a new
ATPase subunit. The physiological function of this
pfATPase subunit needs further investigation,
particularly that of the / signal transduction domain.

[3]

Pedersen P L. Frontiers in ATP synthase research: Understanding the relationship between subunit movements and
ATP synthesis. J. Bioenerg. Biomembr., 1996, 28(5): 389395.

[4]

Weber J, Senior A E. Catalytic mechanism of F1-ATPase.

Biochim. Biophys. Acta, 1997, 1319(1): 19-58.

[5]

Cross R L. Molecular motors: Turning the ATP motor.

Nature, 2004, 427(6973): 407-408.

[6]

Senior A E, Nadanaciva S, Weber J. The molecular mechanism of ATP synthesis by F1F0-ATP synthase. Biochim.
Biophys. Acta, 2002, 1553(3): 188-211.

[7]

Huang W, Yao B, Sun L, Pu R, Wang L, Zhang R. Immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (GnRH) and its receptor in
rat digestive tract. Life Sci., 2001, 68(15): 1727-1734.

[8]

Jounouchi M, Maeda M, Futai M. The alpha subunit of

ATP synthase (F0F1): The Lys-175 and Thr-176 residues
in the conserved sequence (Gly-X-X-X-X-Gly-Lys-

References

Thr/Ser) are located in the domain required for stable sub[1]

Penefsky H S, Cross R L. Structure and mechanism of

unit-subunit interaction. J. Biochem. (Tokyo), 1993, 114(2):

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Relat. Areas Mol. Biol., 1991, 64: 173-214.
[2]

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[9]

Omote H, Le N P, Park M Y, Maeda M, Futai M. Beta sub-

Boyer P D. The binding change mechanism for ATP syn-

unit Glu-185 of Escherichia coli H(+)-ATPase (ATP syn-

thaseSome probabilities and possibilities. Biochim. Bio-

thase) is an essential residue for cooperative catalysis. J.

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Biol. Chem., 1995, 270(43): 25 656-25 660.

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412
[10] Yotov W V, St-Arnaud R. Cloning and functional expres-

[14] Chen L, Xie L, Xiong X, Dai Y, Fan W, Zhang R. Cloning

sion analysis of the alpha subunit of mouse ATP synthase.

and characterization of a novel G protein beta-subunit of

Biochem. Biophys. Res. Commun., 1993, 191(1): 142-148.

pearl oyster (Pinctada fucata), and its interaction sites with

[11] Thompson J D, Gibson T J, Plewniak F, et al. The

CLUSTAL_X windows interface: Flexible strategies for
multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res., 1997, 25(24): 4876-4882.
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405-416.

Symposium on Cell Signaling in Cancer, Development and

Stem Cells Held at Tsinghua University
A Symposium on Cell Signaling in Cancer, Development and Stem Cells was held at Tsinghua on June 13-16, 2007.
The symposium was co-organized by Chinas State Key Lab of Biomembrane and Membrane Biotechnology, the
US National Institute of Health, the University of Chicago, and the Beijing Society for Cell Biology.
Professor Michael Karin from the University of California at San Diego gave the keynote speech entitled How
inflammation affects cancer development and progression: the I kappaB Kinase (IKK) and beyond. About 36 scholars, including 24 foreign scientists from Japan, the USA, Israel, and the Netherlands, presented their latest research
results on cell signaling, differentiation, and modulation of stem cells and cancer cells, and embryo development
mechanisms at the three-day symposium. Nearly 200 people attended the symposium.
From http://news.tsinghua.edu.cn