Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from crayfish
Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA
* Author for correspondence (e mail: michele.wheatly{at}wright.edu)
Accepted 6 June 2002
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Summary |
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Key words: heart muscle, cDNA sequence, mRNA expression, tissue specific distribution, moulting cycle, crayfish, Procambarus clarkii
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Introduction |
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Mammalian SERCA is encoded by a family of three homologous and
alternatively spliced genes that encode multiple isoforms with a distinct
pattern of tissue expression (MacLennan et
al., 1985; Brandl et al.,
1986
,
1987
;
Gunteski-Hamblin et al., 1988
;
Burk et al., 1989
;
Wu and Lytton, 1993
;
Wuytack et al., 1994
;
Wu et al., 1995
). SERCA1 is
expressed in fast twitch skeletal muscle. SERCA2 generates two isoforms that
differ in their C-terminal tail. SERCA2a is a muscle-specific isoform with a
short 4-amino-acid (aa) tail; the ubiquitous SERCA2b has an extended tail of
50 residues that provides an additional membrane-spanning stretch placing the
terminus in the ER lumen (Campbell et al.,
1992
). The gene encoding SERCA3 is expressed in
endothelial and epithelial cells of a variety of muscle and non-muscle tissue.
SERCA genes have also been cloned in other vertebrates such as birds, frogs
and fish (Karin et al., 1989
;
Campbell et al., 1992
;
Vilsen and Andersen, 1992
;
Tullis and Block, 1996
).
SERCA has also been characterized in several invertebrate arthropods
including the fruit fly, Drosophila melanogaster (whole body and
presumed primarily muscle; Varadi et al.,
1989; Magyar and Varadi,
1990
; Magyar et al.,
1995
), the brine shrimp, Artemia franciscana (whole body
and presumed muscle; Palmero and Sastre,
1989
; Escalante and Sastre,
1993
,
1994
) and the crayfish,
Procambarus clarkii (axial abdominal muscle;
Zhang et al., 2000
). A single
SERCA gene was identified in Drosophila that had low abundance in all
tissues. In Artemia two SERCA mRNAs (4.5 and 5.2 kb), which are
developmentally regulated, are originated by alternative splicing of a single
gene (homologous to SERCA2a and -b in vertebrates). In
Artemia, the last six amino acids of one isoform are replaced by 30
aa in the other isoform. The 30 aa extension of the Artemia isoform
does not show significant homology with the 49 aa extension of the mammalian
SERCA2b; however, both exhibit hydrophobicity.
In our laboratory we have employed the moulting cycle of a freshwater
crustacean, the crayfish Procambarus clarkii, as a non-mammalian
model to study regulation of expression of Ca2+ pumps (Wheatly,
1996,
1999
). As arthropods,
crustaceans exhibit incremental growth at ecdysis that results in
discontinuous patterns of muscle growth and transepithelial Ca2+
flux. Skeletal muscles can exhibit different growth patterns during moulting
depending upon location (Mellon et al.,
1992
). Abdominal and leg muscles undergo longitudinal growth
during immediate postmoult with cross-sectional growth occurring in late
postmoult/intermoult due to longitudinal myofibrillar splitting in response to
stretching of the new cuticle (El Haj et
al., 1992
). Meanwhile claw muscles undergo a preprogrammed
premoult atrophy to allow removal through the narrow basi-ischial joint
(Mykles and Skinner, 1990
;
Mykles, 1997
).
In an earlier study we characterized the crayfish SERCA from axial
abdominal muscle (Zhang et al.,
2000). Expression was greatest in intermoult and decreased around
ecdysis in response to either hormonal or mechanical stimuli. In order to
determine whether this pattern of expression is replicated in all crustacean
muscle types we elected to characterize a novel SERCA isoform in cardiac
muscle and its expression throughout the moulting cycle. Unlike skeletal
muscle, heart is continuously contractile. Further, since it is not encased in
cuticle, heart muscle may grow continuously rather than incrementally during
the moulting cycle.
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Materials and methods |
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Isolation of total RNA and mRNA
After dissection, tissues were frozen immediately in liquid nitrogen and
stored at -80°C. Total RNA was isolated by acid guanidinium
thiocyanate-phenol-chloroform extraction
(Chomczynski and Sacchi, 1987).
Messenger RNA was separated from total RNA using an oligo-dT cellulose column
(Stratagene; Sambrook et al.,
1989
). RNA or mRNA was quantified spectrophotometrically at
wavelengths of 260 and 280 nm. Only RNAs with an absorbance ratio
A260:A280 of greater than 1.8 were used for further
experiments. The integrity of RNA was confirmed on a 0.72 mol l-1
formaldehyde, 1 % agarose denaturing gel run in Mops buffer (5 mmol
l-1 sodium acetate, 1 mmol l-1 EDTA, 20 mmol
l-1 Mops, pH 6.6).
Amplification of central 460 bp fragment by RT-PCR
First strand cDNA was reverse transcribed from 400 ng of heart muscle mRNA
using the SuperScript II RNase H-reverse transcriptase (Gibco BRL) with
oligo(dT)12-18 as primer. Based on two highly conserved regions of
the published Artemia muscle SERCA sequence (corresponding to
nucleotides 2176-2195 and 2617-2636,
Palmero and Sastre, 1989), two
non-degenerate primers (5'-GAAATTTCCGCTATGACTGG-3' sense and
5'-ACAGTGGCAGCACCAACATA-3' antisense) were designed using Oligo
4.0 software (American Biotechnology Laboratory). These primers targeted a
fragment of approximately 460 base pairs (bp) located between the
5'-(p-fluorosulphonyl)benzoyladenosine (FSBA) binding site and
transmembrane region 7 of a typical SERCA. These primers had been successful
in amplification of a 460 bp fragment of axial abdominal muscle SERCA
(Zhang et al., 2000
) and were
subsequently used to amplify SERCA from heart. Polymerase chain reactions
(PCR; total volume 50 µl) included 2 µl of first strand cDNA reaction,
20 mmol l-1 Tris-HCl, pH 8.4, 50 mmol l-1 KCl, 1.5 mmol
l-1 MgCl2, 0.2 mmol l-1 dNTP mix, 0.1-0.2
µmol l-1 of each primer and 2.5 units of Taq DNA
polymerase (Gibco BRL). PCR was performed by the hot-start method in an MJ
Research thermal cycler. PCR cycles were: 94°C, 3 min followed by 25
cycles of 94°C for 30 s, 53°C for 1 min, 72°C for 1 min, and a
final cycle of 72°C for 5 min. Negative controls in which reactions
contained only one primer or no template cDNA were included. PCR products were
analyzed by electrophoresis on a 0.8-1.0 % agarose gel with 0.5 µg
ml-1 of ethidium bromide in 1x TAE buffer (40 mmol
l-1 Tris, pH 7.2, 40 mmol l-1 sodium acetate and 1 mmol
l-1 EDTA). The DNA bands were visualized with ultraviolet
light.
MarathonTM rapid amplification of cDNA ends (RACE)
Rapid amplification of cDNA ends (RACE) was employed to complete the
3' and 5' regions of the cardiac SERCA. Briefly first strand cDNA
was reverse transcribed by MMOL/LLV reverse transcriptase using a modified
lock-docking oligo(dT) primer. Following creation of blunt ends with
T4 DNA polymerase, the double stranded cDNA was ligated to the
Marathon cDNA adaptor that has the complementary sequence to the Clontech
adaptor primer. PCR amplification of the 5' region was performed on the
resulting template using the Clontech adaptor primer and the gene-specific
primer (5'-TCA GCT TTC TTC AGG GCA GGT GCA TC-3', antisense),
which is designed based on the amplified 460 bp fragment of crayfish heart
muscle SERCA fragment. PCR amplification of the 3' region was carried
out using the gene-specific primer (5'-GAT GCA CCT GCC CTG AAG AAA GCT
GA-3', sense) and the Clontech adaptor primer. PCR conditions were as
follows: one cycle at 94 °C for 3 min; five cycles at 94 °C for 30 s,
72 °C for 4 min; 25 cycles at 94 °C for 30 s, 68 °C, for 4 min;
followed by one cycle at 72 °C for 5 min. PCR products were ligated to PCR
2.1 vector (Invitrogen) for transformation into INVF host cells
(Invitrogen). Each clone was digested with the appropriate restriction enzymes
and subcloned for sequencing. Two or three independent clones containing the
appropriate insert were sequenced from both ends.
DNA sequencing and sequence analysis
The cDNA clones were sequenced by automated sequencing (Applied Biosystems
Division Model 377, University of Cincinnati, OH). The complete sequence was
analyzed with MacDNASIS software (Hitachi). Sequence homology was revealed
through a GenBank database search using the BLAST algorithm
(Altschul et al., 1990).
Hydropathy analysis was performed with MacDNASIS software
(Kyte and Doolittle,
1982
).
Northern blot analysis
Northern blot analysis was performed to delineate tissue-specific
distribution of the two SERCA isoforms in intermoult crayfish tissues. Total
RNAs (0.2-15 µg) from each tissue (heart muscle, axial abdominal muscle,
eggs) examined was fractionated by electrophoresis through 0.72 mol
l-1 formaldehyde, 1 % agarose denaturing gel run in Mops buffer and
transferred overnight to a Nytran Plus membrane (Schleicher & Schuell) by
capillary elution in 10x SSC (1x SSC is 150 mmol l-1
NaCl, 15 mmol l-1 sodium citrate). RNA was fixed by ultraviolet
crosslinking using a UVC-515 ultraviolet multilinker from Ultra-Lum (120,000
µJ cm-2). RNA molecular mass markers (a 0.24-9.5 kb ladder) were
run along with the samples, then visualized with UV light after staining with
ethidium bromide, and used for the standard curve. The membrane was
prehybridized for 4 h at 68 °C in 6x SSC, 2x Denhardt's
reagent (0.4 g Ficoll type 400, 0.4 g polyvinylpyrrolidone, 0.4 g bovine serum
albumin in 1 l water), 0.1 % SDS and 100 ng ml-1 of denatured
salmon sperm DNA. Hybridization was performed overnight at 68 °C in the
prehybridization solution with 20 ng of SERCA cDNA probe (outlined below) that
was randomly labeled with [-32P]dATP. The membrane was
washed four times for 15 min at 60 °C in 0.1x SSC and 0.1 % SDS.
Membrane was exposed to X-ray film with intensifying screens at -80 °C.
The following fragments from different regions of crayfish SERCA isoforms were
used as probes: (A) a 460 bp fragment corresponding to crayfish nucleotides
2086-2546 that is common to both heart and axial abdominal muscle isoforms;
(B) a 723 bp XbaI to poly(A) tail fragment (nucleotides 3772 to
poly(A) tail) from the 3' untranslated region of crayfish heart muscle
SERCA; and (C) a 515 bp XhoI to poly(A) tail fragment nucleotide 3205
to poly(A) tail from the 3' untranslated region of crayfish axial
abdominal muscle SERCA (Zhang et al.,
2000
).
These fragments were purified from the positive clones using a QIAquick gel
extraction kit (Qiagen) and labeled for 3 h with [-32P]dATP
to a specific activity of 1x109 cts min-1
µg-1 using a random labelling kit (Gibco BRL). The labelled
probe was then separated from unincorporated nucleotides by chromatography on
a Sephadex G-50 Nick column (Pharmacia Biotech). Following high stringency
washes (four times for 15 min at 60 °C in 0.1x SSC and 0.1 % SDS),
membranes were exposed to X-ray film with intensifying screens at -80
°C.
Expression of the heart-specific SERCA was quantified in heart muscle as a function of stage in the moulting cycle using northern blotting. Heart muscle total RNA was isolated from six crayfish in each moulting stage (intermoult, late premoult, 1-2 days postmoult) and hybridized to the heart-specific SERCA probe (B, 723 bp) with exposure to X-ray film for 24 h. To confirm equal loading between samples, 18S RNA was quantified on a corresponding formaldehyde-agarose gel. Total RNA content was determined by OD260 and shown by ethidium bromide staining.
Southern blot analysis
Southern blotting was used to determine whether the heart-and tail-specific
isoforms originated from one or multiple genes. Total genomic DNA was purified
from crayfish muscle following a previously described protocol
(Sambrook et al., 1989). After
electrophoresis, the DNA gel was denatured in denaturing solution (1.5 mol
l-1 NaCl, 0.5 mol l-1 NaOH) for 45 min and neutralized
in 1 mol l-1 Tris-Cl, pH 7.4, 1.5 mol l-1 NaCl for 30
min. The DNA was then transferred to a Nytran Plus membrane (Schleicher &
Schuell) by capillary elution in 10x SSC and hybridized under the same
conditions as described above for the northern blot except that the probe used
was from the 3' terminal of crayfish axial muscle SERCA
(Zhang et al., 2000
),
corresponding to nucleotides 2785-3019. The membrane was washed twice for 20
min in 2x SSC, 0.1 % SDS at 65 °C and once for 20 min in 0.2x
SSC, 0.1 % SDS at 65 °C, then was exposed to X-ray film with intensifying
screen at -80 °C.
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Results |
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Cloning of the complete cDNA sequence by RACE
Following RACE, a 2.3 kb RACE product was obtained from both the 5'
RACE and 3' RACE amplifications. The bands were cloned into pCRII
vector, respectively. Two independent clones containing an insert of
appropriate size were sequenced from both directions following subcloning. The
complete nucleotide sequence and the deduced amino acid sequence is shown in
Fig. 1. The complete crayfish
heart SERCA consists of 4495 bp with a 3060 bp open reading frame, coding for
1020 amino acids. The 5'-terminal 195 bp noncoding region is GC rich. An
in-frame stop codon is situated 25 bases upstream from the start codon. The
initiator Met codon was part of the longer sequence, -CCACCATGG-, which
contains a purine at position -3 and a G at position +4, both of which are
necessary for efficient initiation of translation
(Kozak, 1984). There is an
extremely long 1435-nucleotide 3' terminal noncoding region with a
poly(A) tail. The poly(A) addition signal AATAAA begins 13 bp from the poly(A)
tail.
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Surprisingly, the nucleotide sequence of this clone is almost identical to that of the crayfish axial abdominal muscle SERCA clone up to nucleotide 2980, except for a couple of conservative nucleotide changes and three non-conservative changes (the D in amino acid 57 of axial muscle is changed to Y in cardiac muscle; the T in amino acid 191 of axial muscle is changed to A in cardiac muscle; the H in amino acid 683 of axial muscle is changed to R in cardiac muscle; Fig. 1B). Importantly, from nucleotide 2981 to the end of the clone the cardiac muscle sequence differs completely from that of the axial muscle SERCA. In axial muscle the divergent sequence includes the region coding for the terminal nine amino acids; the heart SERCA sequence codes for 27 additional amino acids in the C-terminal region of the protein that are highly hydrophobic. The relationship between these two crayfish SERCA isoforms is similar to that observed for the two SERCA isoforms in Artemia and mammalian SERCA2 isoforms (Fig. 2). The additional sequences do not show homology to each other among these different animals. However, they share a marked hydrophobic character, which may result in an additional transmembrane domain (Fig. 3).
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A GenBank search using the BLAST algorithm
(Altschul et al., 1990)
revealed that the deduced amino acid sequence of crayfish heart SERCA matched
with more than 30 SERCAs from various invertebrates and vertebrates, of which
Drosophila SERCA showed the highest homologous score of 80%,
Artemia SERCA had 79% identity, frog fast-twitch skeletal SERCA had
73% identity. Mammalian SERCA1 and SERCA2 genes showed approx. 71-72%
identity, and SERCA3 had 68% identity with crayfish heart SERCA.
Northern blot analysis of the tissue distribution of SERCA
isoforms
To distinguish the tissue distribution of cardiac muscle SERCA isoform from
axial muscle SERCA isoform, a northern blot of mRNA from axial muscle, cardiac
muscle and egg was hybridized in individual experiments with cDNA probes
specific to either crayfish heart SERCA (probe B,
Fig. 4B) or crayfish axial
abdominal muscle SERCA (probe C, Fig.
4C) and compared with a probe that was common to both isoforms
(probe A, Fig. 4A),
respectively.
|
Probe A was the 426 bp fragment corresponding to crayfish nucleotides 2086-2546 common to both isoforms. When hybridized with probe A, four bands were determined with molecular masses of 8.8, 7.6, 5.8 and 4.5 kb. In heart muscle this probe recognized a prominent 5.8 kb band, a secondary band at 7.6 kb, and a weak band at 8.8 kb. By comparison, only one prominent band at 4.5 kb was observed in axial abdominal muscle. This probe recognized a prominent band at 7.6 kb in eggs and two fainter bands at 5.8 and 4.5 kb. In summary, the data suggest that there are as many as four different isoforms of SERCA in the three tissues examined. The difference in molecular mass is due mainly to two factors. First, the 5' upstream noncoding region may be much longer than the cloned sequence of 145 bp. Second, the 3' end poly(A) tail often extends to several hundred bases that are not included as part of the cDNA.
Probe B, the 723 bp fragment specific to the 3' noncoding region of crayfish heart SERCA, bound only to the 5.8 kb mRNA in heart muscle. Probe B showed minimal cross-hybridization with the 7.6 kb and a 5.8 kb mRNAs in eggs but no hybridization at all with axial abdominal muscle. Probe C, the 515 bp fragment specific to the 3' noncoding region of the crayfish axial muscle SERCA clone, hybridized strongly with the 4.5 kb mRNA in axial muscle. Probe C also cross-hybridized weakly with a 4.5 kb mRNA in eggs and an 8.8 kb mRNA in heart.
Expression of heart SERCA isoform during moulting stages
Expression of the heart-specific isoform (probe B) was high in intermoult,
decreased in premoult and then was restored in postmoult
(Fig. 5).
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Southern blot analysis of crayfish SERCA gene copy number
The identity of amino acid sequence between crayfish axial and cardiac
muscle SERCA isoforms suggested they may be encoded by one gene. To test this
hypothesis, the genomic DNAs were digested by two different restriction
enzymes, EcoRI and HindIII, and hybridized with a cDNA probe
from a region that is conserved. The result
(Fig. 6) showed only one
hybridization band in each DNA lane, suggesting that these two isoforms (axial
abdominal muscle SERCA and heart muscle SERCA) are encoded by one gene
(Escalante and Sastre,
1994).
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Discussion |
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Little is known about the biological significance of the existence of two
SERCA isoforms. All these genes code for two isoforms that originate by a
similar alternative splicing mechanism, but the C-terminal extensions show
poor conservation. Comparison of the hydropathy plots of the crayfish SERCA
isoforms with that of Artemia and mammalian SERCA2 disclosed that all
the C-terminal extensions have hydrophobic properties
(Fig. 3) that could potentially
form an additional transmembrane domain. Campbell et al.
(1992) reported that the
C-terminal extension of the SERCA2b isoform of birds spans the ER membrane, so
that the SERCA2b isoform has 11 transmembrane domains, whereas SERCA2a has 10
transmembrane domains. Immunocytochemical studies demonstrated that SERCA2a
and SERCA2b have their C-termini on opposite sides of the ER membrane; the C
terminus of SERCA2a is in the cytosol whereas that of SERCA2b is in the ER
lumen. However, functional comparisons have not yet revealed any difference
between the two isoforms. It is possible that this extra transmembrane domain
could change the regulatory properties of the enzyme or its interactions with
other cellular components. From the tissue distribution of these isoforms in
vertebrates and invertebrates, it is hard to connect this extra domain with
any particular tissue. It has been demonstrated that the SERCA2a is expressed
mainly in slow-twitch skeletal, cardiac and smooth muscle; SERCA2b is
ubiquitously expressed and is referred to as a `housekeeping gene'
(Wu et al., 1995
). In this
study, the isoform with the carboxyl extended terminus was expressed
predominantly in heart tissue of crayfish, which seems contrary to the
distribution of mammalian SERCA2. In other invertebrates, Drosophila
and Artemia, tissue distribution data for each isoform is lacking
owing to the difficulty of isolating individual tissues in such small
organisms. Future studies should focus on the tissue distribution of this
SERCA family and localization to specific cell types. This may elucidate
regulatory factors and selective pressures that have contributed to
conservation of SERCA in invertebrates and vertebrates.
The tissue distributions of the crayfish SERCA isoforms seem more
complicated than that of Artemia. Northern blot analysis revealed a
broad expression of this gene in egg, heart and axial muscle. Tissue-specific
expression of the isoforms is apparent with a pattern resembling that in
vertebrates (Zhang et al.,
2000). The isoform-specific probes (B and C) confirmed that the
original 3865 bp clone from the axial muscle corresponds to the 4.5 kb
transcript and the new 4495 bp clone isolated from cardiac muscle corresponds
to the 5.8 kb transcript, respectively. In eggs, the 4.5 kb and 5.8 kb RNAs
hybridized to probes B and C, respectively, indicating that the SERCA axial
muscle type transcript and cardiac muscle type transcript are formed during
the early stage of crayfish development. A strong band shown in eggs (7.6 kb)
may be a precursor of these two transcripts suggesting possible developmental
regulation of the gene.
Expression of the heart-muscle-specific SERCA isoform varied as a function
of the moulting cycle (Fig. 5).
The expression pattern was similar to that reported for the axial abdominal
muscle isoform in the transition from intermoult to premoult
(Zhang et al., 2000), namely
that expression was high in intermoult and decreased significantly in
premoult. However, in the postmoult period these two isoforms exhibited
different expression patterns. While axial abdominal muscle SERCA remained
downregulated in the first 1-2 days postmoult and required 2 weeks for
recovery to intermoult levels, the cardiac SERCA isoform expression rapidly
returned to (and even exceeded) intermoult expression within 2 days.
While the ultrastructure of the intermoult crayfish heart is well described
(Komura, 1969; Howse et al.,
1971a
,b
;
Anderson and Smith, 1971
), and
it has been established that the crustacean heart grows indefinitely in
proportion to the body mass in species exhibiting indeterminate growth
(Wilkens and McMahon, 1994
),
it is unknown whether the crustacean heart grows incrementally (like somatic
muscles) or continuously. Since growth of the heart is not restricted by
encasement in cuticle, continuous growth is possible.
Interpretation of the SERCA expression data is probably best explained by
considering the relative contractility of the different muscle types during
the moulting cycle. A study of cardiac function during moulting in blue crabs
(de Fur et al., 1984) showed
that heart rate decreased significantly in premoult; however, within 24 h of
ecdysis it had recovered dramatically, associated with the need to deliver
oxygen to metabolizing tissues. In immediate postmoult a large increase in
hydrostatic pressure was associated with increased stroke volume. The heart
muscle is attached via alary ligaments to skeletal elements that
would impose additional stretching. So, by all indicators, cardiac function
may be temporarily reduced in immediate premoult, but recovers rapidly after
ecdysis for reasons of physiological necessity. By comparison, contractility
of skeletal muscles is visibly reduced for several days surrounding ecdysis as
crustaceans appear relatively inactive or quiescent for 2-3 days following
ecdysis. Somatic muscles are typically stretched in postmoult following
skeletal expansion. Muscle flexion of lobster carpopodite extensor muscle has
been shown to upregulate actin mRNA expression and myofibrillar growth within
1-2 weeks (Harrison and El Haj,
1994
) coincidentally the time frame for recovery of axial
abdominal SERCA expression. Therefore the patterns of SERCA expression in at
least these two different muscle types could be directly attributable to
muscular contractility. Examination of claw muscle might further our
understanding of SERCA expression; claw muscle atrophies in premoult to enable
extrication through a narrow opening.
The Southern blot suggested that crayfish axial muscle SERCA and cardiac
muscle SERCA are encoded from one gene. This gene shows higher homology with
Drosophila (80 %) and Artemia (79 %) than with the
vertebrate genes (72 %); it shows similar homology with the three vertebrate
genes. In a separate study of the evolutionary relationships among all SERCA
sequences (Wheatly et al.,
2001) we have determined that the gene duplications of SERCA into
the three homologues (SERCA1, SERCA2 and SERCA3) occurred
within vertebrates. A single SERCA gene is found in invertebrates that is
equally related to vertebrate SERCA-1, -2 and -3. These data
confirm an earlier hypothesis (Escalante
and Sastre, 1993
) that there is a unique ancestral SERCA gene that
gave rise to the three genes in vertebrates and to a single invertebrate gene.
The same alternative splicing was preserved in the invertebrate gene and
vertebrate SERCA2 gene, while it was lost during evolution of
vertebrate SERCA1 and SERCA3.
The SERCA sequence from crayfish Procambarus clarkii cardiac muscle has been accepted by GenBank (Accession number AF025848).
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Acknowledgments |
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References |
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