From the National Research Institute of Fisheries Science, Fukuura 2-12-4, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan
Received for publication, March 6, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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Marine fish belonging to the genus Coryphaenoides known as
rattails or grenadiers have been studied extensively as an excellent model in
which to elucidate adaptation to the deep sea
(512),
because of their widespread bathymetric distribution up to a depth of about
6000 m (13). An important
parameter to study in the context of pressure is the change in volume that
accompanies such events as protein-ligand interactions and protein-protein
interactions, for the sign and magnitude of the reaction volume change
determine the reaction's sensitivity to pressure. Swezey and Somero
(14) have investigated the
volume change (V) that is associated with the polymerization
of G-actin to F-actin
-skeletal actin from C. armatus (abyssal
species) and C. acrolepis (non-abyssal species). The
V of actin from C. armatus was much smaller, which is
advantageous for a deep-sea habitat, than that from C. acrolepis
(14).
Actin is the main component of the microfilament system in all eukaryotic
cells and plays a central role in maintaining the cytoskeletal structure, cell
motility, cell division, intracellular movements, and contractile processes
(15,
16). It is one of the most
conserved proteins in eukaryotic cells, for example, -skeletal actin
proteins in carp and rat share 99.4% homology at the amino acid sequence level
(17,
18). It is therefore
surprising that differences in the
V of this highly conserved
protein have been found between two species of Coryphaenoides that
inhabit different niches.
In this study, we have cloned and sequenced the -skeletal actin
cDNAs from two abyssal Coryphaenoides, C. armatus and C.
yaquinae, of which C. yaquinae inhabits greater depths. These
actins contain three unique amino acid substitutions compared with the
previously sequenced
-skeletal actin from two non-abyssal
Coryphaenoides, C. acrolepis and C. cinereus
(19). Biochemical analyses of
the
-actin molecules purified from the skeletal muscles of C.
armatus, C. yaquinae, C. acrolepis, carp, and chicken show that these
amino acid substitutions are responsible for the adaptation of
-actin
to high pressures in abyssal species. Here we describe, for the first time,
the mechanism of adaptation of deep-sea fishes to high pressures at the amino
acid sequence level.
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EXPERIMENTAL PROCEDURES |
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Actin ProteinActin was isolated from the skeletal muscle of
each species according to Spudich and Watt
(20) and purified by
gel-filtration chromatography over Sephadex G-200 in G buffer (0.2
mM ATP, 0.2 mM CaCl2, 0.5 mM
-mercaptoethanol, Tris-HCl, pH 7.8). Actins were converted into the
Mg2+-G form as described previously
(21,
22).
Mg2+-G-actin was used immediately after its conversion
from Ca2+-G-actin. Actins were stored in G buffer at 4
°C after purification and used within 3 days. The concentration of G-actin
was determined spectrophotometrically using an absorption coefficient of 0.63
ml/mg at 290 nm.
Polymerization, Critical Concentration, and V
AssemblyPolymerization of Mg2+-actin was
initiated by adding KCl and MgCl2 to final concentrations of 50 and
2 mM, respectively. Polymerization was monitored by light
scattering, with excitation and emission wavelengths both set at 400 nm at 4
°C in a high pressure cell with a high pressure pump (PCI-400 cell and
TP-500 pump Teramecs Co. Ltd., Kyoto, Japan). The change in light scattering
was recorded as a function of time. The critical concentration was determined
as described previously (23,
24). The volume change
(
V) in assembly of G-actin into F-actin was calculated by the
method of Swezey and Somero
(14).
Isolation of -Skeletal Actin cDNAs from C. armatus and
C. yaquinaeC. armatus and C. yaquinae muscle cDNA
libraries were constructed, and
-skeletal actin cDNAs from each library
were cloned as described previously
(19). Structures of actin were
prepared using the program MOLSCRIPT
(25) with data from the
Protein Data Bank (accession number 1ATN
[PDB]
).
Phylogenetic AnalysisA molecular phylogenetic tree was
constructed from the sequences of the actin coding regions. The DNADIST and
NEIGHBOR programs in the PHYLIP version 3.5 program package
(26) were used for neighbor
joining (27). Bootstrap
analyses with 1000 replicates were performed to examine the confidence of
nodes within the resultant topology. Molecular phylogenetic analysis showed
that -actin genes from the Coryphaenoides species were
categorized into three types, actin 1, actin 2a, and actin 2b (see
"Results"). The GenBankTM accession numbers of actin
nucleotide sequences used in this study are give in parentheses: C.
armatus 2a (AB086240
[GenBank]
), C. armatus 2b (AB086241
[GenBank]
), C.
yaquinae 2a (AB086242
[GenBank]
), C. yaquinae 2b (AB086243
[GenBank]
), C.
acrolepis 1 (AB021649
[GenBank]
), C. acrolepis 2a (AB021650
[GenBank]
), C.
cinereus 1 (AB021651
[GenBank]
), C. cinereus 2a (AB021652
[GenBank]
); carp, C.
carpio (D50025
[GenBank]
); medaka, Oryzias latipes (D87740
[GenBank]
); fugu 1,
Fugu rubripes (U38850
[GenBank]
), fugu 2 (U38958
[GenBank]
); goldfish, Carassius
auratus (D50029
[GenBank]
); tilapia, Oreochromis mossambicus (AB037866
[GenBank]
);
zebrafish, Danio rerio (AF180887
[GenBank]
); chum salmon, Oncorhynchus
keta (AB032464
[GenBank]
); Atlantic salmon, Salmo salar (AF304406
[GenBank]
);
chicken, Gallus gallus (K02257); human, Homo sapiens
(M20543
[GenBank]
); mouse, Mus musculus (M12234); rat, Rattus
norvegicus (V01218
[GenBank]
); and bovine, Bos taurus (U02285
[GenBank]
). Medaka
-actin (D89627
[GenBank]
) was used as the outgroup gene.
Quantification of Actin IsoformsTo identify the ratio of
the -actin isoforms, quantitative polymerase chain reaction with
reverse transcription
(RT-PCR)1 and
two-dimensional electrophoresis were performed. These analyses were repeatedly
done four times using total RNAs, and actin proteins were isolated from four
individuals in each species. The conditions for RT-PCR were as described
previously (19), except for
the cycle number. The cycle number within a linear range of PCR amplification
was determined to be 25 on the basis of the signal intensities of RT-PCR
products with sequential cycles. The primer
5'-ATTGCTGACCGYATGCAGAA-3' and NOT-1 d(T)18 primer
amplified
480 bp and 680 bp, for actin 1 and actin 2b, respectively.
RT-PCR products were subjected to 1.5% agarose gel electrophoresis.
Two-dimensional electrophoresis was performed by Multiphor II electrophoresis
unit with a pH range of 4.07.0 or 5.06.0 gel (24 cm) and 12.5%
SDS-PAGE gel (Amersham Biosciences). The ratios of actin 2b to actin 2a, or
actin 1 to actin 2a, were quantified using a computerized image analysis
scanner STORM 860 (Amersham Bioscience).
Quin 2 AssayThe dissociation rate constant of
Ca2+ from actin was determined by assaying the
fluorescence intensity increase of Quin 2
(8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N, N,
N', N'-tetraacetic acid)
(28).
Ca2+-actin (5 µM) prepared by dialysis
against G-buffer (free CaCl2) was added to 100 µM
Quin 2 and 0.2 mM MgCl2, and the fluorescence was
measured at respective excitation and emission wavelengths of 340 and 500 nm
at 4 °C in a high pressure cell. For reaction rates, the relevant
thermodynamic equation is, ln k/
P =
V#/RT, where k is the rate
constant for the reaction, P is the pressure, V#
is activation volume, R is the gas constant, and T is the
absolute temperature (29). The
apparent binding rate constant and the apparent volume change
(
V#) of the Ca2+ dissociation
reaction were determined as described previously
(29,
30).
Nucleotide Exchange AssayThe apparent binding rate constant
of -ATP (1,N6-etheno-ATP) on actin was determined by
displacing bound ATP with a large molar excess of
-ATP such that the
back reaction, ATP binding to actin, became negligible
(28). Actin (9
µM) prepared by dialysis against G-buffer (free ATP) was
converted into Mg2+-G-actin and added to
-ATP (0.1
mM), and the fluorescence was measured at respective excitation and
emission wavelengths of 340 and 410 nm at 4 °C in a high pressure cell.
The apparent binding constant and the apparent
V#
in the ATP dissociation reaction were determined as described previously
(29,
30).
Intrinsic Tryptophan FluorescenceThe intrinsic tryptophan fluorescence spectrum of Ca2+-G-actin at 6.25 µM was recorded using a high pressure cell at respective excitation and emission wavelengths of 290 and 320360 nm at 4 °C (31).
DNase I Inhibition AssayThe DNase I inhibition assay was performed as described previously (32). In brief, DNase I, either alone or combined with G-actin at various actin/DNase I ratios, was added to a control or salmon sperm DNA solution (100 mM Tris-HCl, pH 7.6, 4 mM MgSO4, 1.8 mM CaCl2), and the change in absorbance at 260 nm was recorded continuously at 20 °C in a high pressure cell. DNase I activity was calculated from the linear part of the plot of the increase in A260 versus time.
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RESULTS |
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The critical concentrations of actin also increased with high pressure for each species (Fig. 1B). The critical concentrations of the two abyssal actin species were higher than those of other species at 20 MPa and lower pressures, and increased slightly from 0.1 to 60 MPa.
The volume change (V) associated with polymerization at
each pressure was determined from the respective critical concentrations
(Fig. 1C). The two
abyssal actin species had a much smaller
V at each pressure
than did the other species, in agreement with previous reports
(14). The
V of
actins from chicken and non-abyssal species decreased at high pressure,
whereas those from the abyssal species showed little variation.
These observations indicated that, for chicken and non-abyssal species, the
coefficient of compressibility for F-actin was larger than that for G-actin
and that the space produced by actin-actin interactions was reduced by high
pressure. Clearly, there was no such reduction in space in actins from the
abyssal species. Unexpectedly, the V of carp actin, unlike
that of other species, increased with high pressure, which indicated that for
carp the coefficient of compressibility for G-actin was larger than that for
F-actin; in other words, carp G-actin is softer than the G-actin of the other
species. Thus, this would explain why carp actin was able to polymerize at
pressures of only 20 MPa or less.
cDNA Cloning and Deduced Amino Acid SequencesTwo
-skeletal actin isoforms were cloned from each of the two abyssal
species and compared with the two non-abyssal actin species reported
previously (19). Molecular
phylogenic analysis showed that these four
-actin genes from the
abyssal species were all categorized as actin 2. These categorizations were
supported with the comparatively high bootstrap value (72%)
(Fig. 2A). Therefore,
the isoform with an identical amino acid sequence to that of actin 2 of the
non-abyssal species was designated actin 2a, and the other was designated
aemactin 2b and yaqactin 2b for C. armatus and
C. yaquinae, respectively. Consequently, the non-abyssal actin 2 was
re-designated actin 2a. The amino acid sequences of actin 1 and 2a differ by
one amino acid residue at position 155, which is Ala-155 in actin 1 and
Ser-155 in actin 2a (19). The
sequence of actin 2b differs from that of actin 2a by two amino acids (either
V54A or L67P and Q137K) (Table
I). The x-ray crystallography structure of rabbit skeletal muscle
actin shows that residues 54 and 67 are located in a
-sheet of subdomain
2 (residues 3369), whereas residues 137 and 155 are located in the
Ca2+- and ATP-binding sites, respectively
(Fig. 3, A and
B)
(33).
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Quantification of Actin IsoformsTo identify the expression
ratio of the -actin isoforms, the expression of isoform mRNA was
investigated using quantitative RT-PCR. Quantitative RT-PCR was carried out
with one primer set, which amplified different length products from the two
isoforms, about 480 bp for actin 1 and actin 2b, and about 680 bp for actin
2a, owing to the different lengths of their 3' non-coding regions
(Fig. 2B). Direct
sequencing confirmed that the RT-PCR products were the expected actin
isoforms. The results showed that there was differential expression of the two
isoforms in each species. The expression ratio of actin 2b to actin 2a, or
actin 1 to actin 2a, was 4.3 ± 0.18 for C. yaquinae, 4.1
± 0.083 for C. armatus, and 0.67 ± 0.034 for C.
acrolepis, respectively. We examined further the ratio of protein
isoforms using two-dimensional electrophoresis. The abundance ratio of actin
2b to actin 2a was 4.8 ± 0.087 for C. yaquinae
(Fig. 2C) and 4.5
± 0.18 for C. armatus (data not shown), respectively. The
protein isoforms from C. acrolepis, which have the same isoelectric
point estimated, could not be separated by this electrophoresis method (data
not shown). These results of the electrophoresis were not affected by
dephosphorylation using Escherichia coli alkaline phosphatase (data
not shown). These ratios reflect higher expression of the isoform that is more
essential for the species habitat, as described below.
Quin 2 and Nucleotide Exchange AssaySequence analysis
showed that, although actin 2b from abyssal species has a lysine at position
137, the other isoform and all actins from the other species have a glutamine
at this position, and all actin isoforms from abyssal species have a serine at
residue 155. Actin binds ATP by sandwiching the ATP - and
-phosphates between two structurally equivalent
-hairpins
(residues 1118 and 154161), which belong to homologous
subdomains 1 and 3 (Fig. 3, A and
B) (33,
34). Actin also contains a
tightly bound divalent cation (Ca2+ or
Mg2+) in a deep hydrophilic pocket formed by the
-
and
-phosphates of the bound ATP and actin residues Asp-11, Gln-137,
and Asp-154 (Fig. 3, A and
B) (33,
35). Although actin has a
higher affinity for Ca2+ than for
Mg2+, the much higher cellular concentration of
Mg2+ means that it will be the main occupant of the
divalent cation site in vivo. We also investigated the effect of high
pressures on the divalent cation and nucleotide binding of actin protein at
various pressures (Fig. 4, A and
B). Surprisingly, both dissociation rate constants of
actin from abyssal species were much less affected by high pressures than
those of the other actins, which increased rapidly at pressures greater than
20 MPa. The dissociation rate constant of Ca2+ in
abyssal actins did not differ greatly from that of the other actins at
atmospheric pressure, which means that the actins bind to
Ca2+ with almost the same strength, whereas the
differences in the dissociation rate constant of ATP suggest that Ser-155
actin binds ATP more tightly than Ala-155 actin, as pointed out previously
(19). The apparent
V# value in the Ca2+
dissociation reaction at pressures greater than 20 MPa was estimated as
4.27 ± 1.86 cm3 mol1 for
abyssal species and 154.3 ± 0.583 cm3
mol1 for the other species. The apparent
V# in the ATP dissociation reaction at more than 20
MPa was also determined to be 43.4 ± 0.47 cm3
mol1 or more for abyssal actin, and as
80.4 ± 1.44, 90.0 ± 1.59, and 84.2 ±
0.995 cm3 mol1 for chicken, carp, and
non-abyssal actin, respectively. These results indicated that the smaller
effect in both dissociation rate constants of pressure in abyssal actin
results from Q137K and the differences in both constants among carp, chicken,
and non-abyssal species from A155S.
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Effects of Pressure on Intrinsic Tryptophan Fluorescence
SpectrumActin contains four tryptophan residues (at positions 74,
86, 340, and 356), which are all located in subdomain 1. The emission maximum
of fluorescing tryptophan residues is 350 nm in a neutral water solution but
shifts to shorter wavelengths in a hydrophobic environment, such as the
interior of a folded protein
(36). To investigate effect of
pressures on the actin structure, we measured the intrinsic tryptophan
fluorescence spectrum of Ca2+-G-actin at various
pressures. Although the emission maximum did not shift, the fluorescence
intensity of actins from chicken, carp, and non-abyssal species began to
decrease at only 10 MPa (data not shown), and in particular the decrease for
carp actin was larger than that for chicken and non-abyssal actin; by
contrast, the fluorescence of the abyssal actins did not change even at 60 MPa
(Fig. 5, A and
B). Actin affinity for Ca2+ is
greater than for Mg2+, but there were no differences in
fluorescence intensity between the Ca2+- and
Mg2+-G-actin forms for all actin species (data not
shown). These results indicate that high pressure changes the environment of
the tryptophan residues in actin; in other words, the structure of actin
subdomain 1, which includes one of the -hairpins that sandwiches the ATP
-and
-phosphates and the Ca2+-binding
sites.
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DNase I Inhibition AssayActin 2b of the two abyssal species contains either a V54A substitution or an L67P substitution (Table I). The x-ray crystallography structure shows that these residues are located in subdomain 2 (residues 3369) (Fig. 3A). In actin-DNase I interactions, DNase I primarily contacts the DNase I-binding loop (residues 4048) in subdomain 2 and interacts slightly with Thr-203 and Glu-207 in subdomain 4 (residues 181269) (33, 37). To investigate whether these substitutions, V54A or L67P, affect the actin-DNase I interaction, we performed a DNase I Inhibition assay at various pressures (Fig. 6). Notably, we found that the abyssal actin inhibited DNase I even at 60 MPa, whereas the other actin species showed little activity at 60 MPa. In addition, carp actin was almost inactive at 30 MPa. The decrease in DNase I inhibition activity of actin proteins from 0.1 to 60 MPa was greater in C. armatus actin (23.9%) than in C. yaquinae actin (17.2%), in agreement with the fact that C. yaquinae lives at greater depths (38). These decreases should depend on the denatured state of actin 2a by high pressures, which exist slightly in muscles of both C. yaquinae and C. armatus as shown by two-dimensional electrophoresis (Fig. 2C). Our finding clearly indicates that the actins from abyssal species were bound to DNase I even at 60 MPa, which suggests that the substitutions in subdomain 2 of actins from abyssal species are most likely to reduce the increased volumes that occur with the interaction of actin with DNase I. However, the mechanism underlying the reduction in volume remains unclear.
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DISCUSSION |
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Subdomain 2 (residues 3369) is probably the most flexible of the
actin subdomains (39), and its
conformational change after ATP hydrolysis and Pi release from
ADP-Pi during actin polymerization plays a critical role in
filament dynamics (40,
41). Residues 4145 in
subdomain 2 of one actin molecule interact with two other actin molecules, one
at residues 166169 and the other around residue 375
(42). Our results show that
the DNase I-binding loop (residues 4048) in subdomain 2 of the abyssal
actin can interact with DNase I at high pressure levels; therefore, we propose
that abyssal actin also undergoes actin-actin interactions at high pressure
without a large increase in volume. Clearly, such a possibility is provided by
the V54A or L67P substitutions found in the -sheet of subdomain 2 of
actin from the abyssal species. We speculate that subdomain 2 of abyssal actin
is more compact and has reduced space in the
-sheet, because these
substituted residues do not normally form
-sheets. However, the exact
mechanistic details remain to be determined.
Our findings indicate that the dissociation of ATP and
Ca2+ in particular in actins from abyssal species is
less affected by pressure than in actins from other species. This tolerance to
high pressure is due especially to the A155S and Q137K substitutions in actins
of abyssal species. These substituted amino acids, which increase the negative
value of the V# of both the
Ca2+ and the ATP dissociation reactions, prevent these
reactions from being strongly accelerated by high pressures. The
Ca2+ lies below the bound ATP on the pseudo-2-fold axis
and is coordinated by four water molecules, which are held in place through
interactions with the side chains of Asp-11, Gln-137, and Asp-154, and two O
atoms from the
- and
-phosphates of the bound ATP
(33,
34). Actin, Hsp70 molecular
chaperones, hexokinase, and sugar kinases share structural homology and can,
therefore, be considered members of a superfamily
(43,
44). In this superfamily, the
amino acid in the equivalent position to Gln-137 in most actin is usually Asp
or Glu, i.e. a negatively charged residue. Surprisingly, actins from
abyssal species have the positively charged residue Lys at position 137. The
Q137K substitution thus changes the coordination of Ca2+
with the protein. The repulsion between Ca2+ and Lys-137
would prevent the Ca2+ from being pushed into the bottom
of the interdomain cleft by high pressures. On the other hand, another study
showed that the rate of ligand entry from the solvent to the protein interior
is increased if the space available to the ligand in the protein pocket is
increased by substituting an amino acid residue with a smaller side chain
(45). The substitution of
amino acids with larger side chains
(46), Q137K and A155S, might
prevent both the Ca2+ and the ATP dissociation reactions
from being accelerated by high pressures. Previous studies have reported that
high pressure can slightly affect the apparent Km
of substrate values of some dehydrogenases from deep-sea fishes
(59).
Our results suggest a mechanism for stabilizing enzyme-substrate interactions
under elevated pressure.
The Q137K and A155S substitutions indicate that other actin species than
abyssal actin have larger cavities in the protein pocket, owing to the
presence of amino acid residues with smaller side chains. The results from the
Quin 2 and nucleotide exchange assays show, however, that these cavities
remain intact at high pressures. On the other hand, our observations here
(Fig. 4, A and
B) and previous studies show that abyssal actins form
tighter associations with ATP and Ca2+ than do other
actin species (19,
47). The structure of actin is
maintained not only by its internal weak bonds but mainly by bound ATP and
Ca2+
(28). Thus, the tightly bound
ATP and Ca2+ in abyssal actins presumably maintain the
actin structure from inside, rather like a pillar. In support of this, carp
G-actin is much softer (Fig. 5, A
and B), and the V of carp actin increased
with high pressure (Fig.
1C), which indicated that for carp the coefficient of
compressibility for G-actin was larger than that for F-actin. Thus, Ala-155
actin would explain why carp actin was able to polymerize at pressures of only
20 MPa or less.
The Ala-155 actin variant is not disadvantageous for freshwater fishes such
as carp, because they do not experience high pressures; however, it would be
disadvantageous for many marine fishes. The molecular phylogenetic tree for
actin genes clearly shows that actin 2a diverged from actin 1
(Fig. 2A). Because all
-actins cloned from the freshwater fishes so far are the Ala-155
variant, this suggests that Ala-155 actin is necessary for living in
freshwater conditions and Ala-155 actin found in marine fishes may be a
remnant from an ancestral fish. Deep-sea fishes, which live in an environment
where actin 1 does not function, probably have the gene for actin 1 but do not
express it. In the superfamily, including actin, Hsp70 molecular chaperones,
hexokinase, and sugar kinases, the amino acid in the equivalent position 155
in actin is usually an uncharged polar or nonpolar amino acid
(43,
44). Therefore, the Ala-155
actin found in many fishes would be accepted without the problem in the
polarity. We do not, however, understand the full significance of the alanine
residue at position 155 in actin in freshwater fishes.
A large portion of our data in this study was collected from a mixture of
actin isoforms isolated from muscle, which we were unable to separate by
biochemical techniques. Although the expression of recombinant proteins in
microorganisms is a powerful tool, the system for actin, and in particular
-skeletal actin, has been not perfected as yet because of various
problems, such as the formation of inclusion bodies and the low yield of
expressed protein (48). We are
therefore optimizing this system for future studies.
There are a multitude of actin-binding proteins
(49), and, as a result, actin
is one of the most conserved proteins with only limited variable positions.
Our results have shown that a novel function that enables species to adapt to
a new environment can evolve in a protein by a very few amino acid
substitutions in key functional positions. Moreover, the molecular
phylogenetic tree based on the -actin coding region shows that actin 2a
diverged from actin 1 and that actin 2b diverged from actin 2a, again by a
very few amino acid substitutions (Table
I). The previous study indicated that freshwater fishes had only
Ala-155 actin, i.e. actin 1
(19). Thus, when the teleosts
advanced from freshwater to sea and continuously from surface to abyssal zone,
they should duplicate the
-actin gene each time. These observations are
consistent with Perutz's theory of protein speciation
(50), the theory of gene
duplication (51), and the
prediction from the molecular phylogenetic tree based on mitochondrial DNA
analysis (3).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
To whom correspondence should be addressed. Tel./Fax: 81-45-788-7654; E-mail:
takam{at}affrc.go.jp.
1 The abbreviations used are: RT, reverse transcription; V,
volume change; Quin 2,
8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N, N,
N', N'-tetraacetic acid; V#,
activation volume;
-ATP, 1,N6-etheno-ATP.
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ACKNOWLEDGMENTS |
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REFERENCES |
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