Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 255264
doi:10.1111/j.1745-7270.2007.00277.x
Characterization, evolution and expression of the calmodulin1
genes from the amphioxus Branchiostoma belcheri
tsingtauense
Jing LUAN, Zhenhui LIU*,
Shicui ZHANG, Hongyan LI, Chunxin FAN, and Lei LI
Department
of Marine Biology, Ocean University of China, Qingdao 266003, China
Received: December
26, 2006
Accepted: January
30, 2007
This work was
supported by the Ministry of Science and Technology (MOST) of China and the
National Natural Science Foundation of China (No. 30470203 and No. 30500256)
*corresponding author: Tel, 86-532-82032439;
Fax, 86-532-82032787; E-mail, [email protected]
Abstract Two full-length cDNAs, named CaM1a and CaM1b, encoding
the highly conserved calmodulin1 (CaM1) proteins, were isolated from the cDNA
library of amphioxus Branchiostoma belcheri tsingtauense. There are only
two nucleotide differences between them, producing one amino acid difference
between CaM1a and CaM1b. Comparison of the amino acid sequence of CaM1 reveals
that the B. belcheri tsingtauense CaM1a is identical with CaM1
proteins of B. floridae and B. lanceolatum, Drosophila
melanogaster CaM, ascidian Halocynthia roretzi CaMA and
mollusk Aplysia californica CaM, and CaM1b differs only at one position
(138, Asn to Asp). The phylogenetic analysis indicates that the CaM1 in all
three amphioxus species appears to encode the conventional CaM and CaM2
might be derived from gene duplication of CaM1. Southern blot
suggests that there are two copies of CaM1 in the genome of B.
belcheri tsingtauense. Northern blot and in situ hybridization
analysis shows the presence of two CaM1 mRNA transcripts with
various expression levels in different
adult tissues and embryonic stages in amphioxus B. belcheri tsingtauense.
The evolution and diversity of metazoan CaM mRNA transcripts are also discussed.
Key words amphioxus; Branchiostoma;
calmodulin1; evolution; expression
Calmodulin (CaM) is a calcium-binding
EF-hand protein that mediates the calcium-dependent activity of a variety of
different target enzymes and structural proteins. The primary structure of this
protein has been determined in many organisms from different species and shows
a remarkably high degree of conservation [1]. The protein contains four
conserved canonical calcium-binding domains that might be derived from an
ancestral one-domain precursor through events of gene duplication and
translocation [2,3].
In vertebrates, CaM protein is encoded by
multiple genes. For instance, six genes have been detected in zebrafish [4],
three genes in humans [5–7] and
rats [8,9], at least two genes in frogs Xenopus laevis [10] and
two genes in chickens [11,12]. Interestingly, all of these genes give rise to
identical proteins, and this phenomenon has brought about the hypothesis of
“multigene one-protein” for vertebrate CaM gene families
[13,14]. Although the proteins are present in all cells of all eukaryotes and
they play vital roles in cellular information transduction, the number of CaM
genes in invertebrates is rather small. The exact number of CaM genes
and proteins existing in metazoan is still unknown. It is possible that a
single CaM gene (e.g., Drosophila melanogaster [15], mollusk
Aplysia californica [16], ascidian Ciona intestinalis [1]) or two
genes encode different CaM isoforms (e.g., echinoderm Arbacia punctulata [17],
ascidian Halocynthia roretzi [18], B. lanceolatum and B.
floridae [19]).
Although CaM is ubiquitous, the sizes and
distributions of the transcripts vary in different tissues and embryonic stages
in different species. For example, human CaM1 gene is transcribed into
two mRNAs of 1.7 kb and 4.2 kb. The 1.7 kb mRNA is uniformly present, whereas
the 4.2 kb mRNA is particularly abundant in brain and skeletal muscle [7]. In
chickens, four transcripts of 0.8 kb, 1.4 kb, 1.7 kb and 4.4 kb for CaM1
gene are detected; two major transcripts of 1.4 kb and 1.7 kb are present in
all chicken tissues, whereas the 4.4 kb CaMI transcript is plentiful in
brain [20]. The frog CaM gene is transcribed into five mRNAs of 1.4 kb,
1.6 kb, 2.1 kb, 2.2 kb and 2.7 kb, and a major band of 1.4 kb has been observed
in ovary, testis and brain [10]. In sea urchin, only a single size of 3.2 kb
transcript for the CaM gene is detected in both embryonic and adult
tissues. The mRNA is present in the unfertilized egg at the level of a typical
rare-class mRNA and accumulates approximately 100-fold in pluteus-stage cells
[21]. Fruit fly CaM gene is transcribed into two mRNAs of 1.65 kb and
1.9 kb, and the total amount of mRNA is highest in the larval stage compared to
the embryo stage and the pupal stage [22].
Amphioxus, a cephalochordate, has long been
known as an extant invertebrate that is most closely related to the proximate
ancestor of vertebrates [23,24]. Karabinos and Bhattacharya have suggested the
existence of two CaM genes both in B. lanceolatum and B.
floridae, although it had been previously considered that only a single CaM
gene existed in this taxon [25]. Even though they all belong to the same genus
of Branchiostoma in taxonomic status, the exact number of CaM
genes is sparse in B. belcheri tsingtauense, which is considered
a different species to B. lanceolatum and B. floridae, both at
the molecular level and histological level [26–29]. In addition, the expression pattern of CaM
in amphioxus is still unclear. Our study is driven to explore the answers to
these questions.
In
this study, we isolated two full-length CaM1 cDNAs (CaM1a
and CaM1b) from the cDNA library of amphioxus B. belcheri
tsingtauense, and determined the copy number of the gene and the expression
pattern in different adult tissues and embryonic stages. We also explore the
evolution and diversity of metazoan CaMs.
Material and Methods
cDNA cloning and sequencing
analysis
Gut cDNA library of adult amphioxus B.
belcheri tsingtauense was constructed with the SMART cDNA Library
Construction Kit (Clontech, Palo Alto, USA) using the method described
previously [30]. In a large-scale sequencing of amphioxus gut cDNA library with
an 377XL DNA sequencer (ABI Prism, Foster, USA), more than 5000 clones were
analyzed for coding probability using the DNATools program (http://www.crc.dk/dnatools/downloads/accept.php?accept_url=setup/dt6_setup.exe)
[31].
Initial comparison to the GenBank protein
database was carried out using the BLAST network server at the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/)
[32]. Multiple nucleotide and protein sequences were aligned using the CLUSTAL
method from MegAlign in the DNAStar software package (version 5.0; Dr. Steve
ShearDown, Madison, USA) [33]. A phylogenetic tree was constructed with 1000
bootstrap replicates using the neighbor-joining method (PHYLIP 3.6b software
package, http://evolution.genetics.washington.edu/phylip.html)
[34].
Southern blotting analysis
Genomic DNAs for Southern blotting analysis
were isolated from adult amphioxus and digested with three restriction enzymes
(37 ?C, 20 h): BglII, PstI and HindIII
(1 unit per mg DNA). The digested DNAs were separated on
a 1% agarose gel using 1 TBE (89 mM Tris-borate and 2 mM EDTA) and transferred
onto nylon membranes (Osmonics, Trevose, USA). The membranes were hybridized at
high stringency with the digoxigenin (DIG)-labeled B. belcheri tsingtauense
CaM1a cDNA probe produced with a DIG DNA labeling kit (Roche, Basel,
Switzerland). Hybridized bands were visualized according to the instructions of
the detection kit.
Northern blotting analysis
Northern blotting analysis
Total RNAs were prepared with Trizol
(Gibco, Carlsbad, USA) from various tissues including muscle, notochord,
testis, ovary, gut and gill of adult amphioxus and embryos at four
developmental stages, including blastulae, gastrulae, neurula and 24 h larvae. A
total of 3 mg RNAs each was detected using
electrophoresis and was blotted onto nylon membranes (Osmonics). The blots were
hybridized at high stringency with DIG-labeled B. belcheri tsingtauense CaM1a
riboprobe. The hybridized bands were visualized by BM-Purple (Roche).
In situ hybridization histochemistry
Sexually mature amphioxus were dissected
into three or four pieces and fixed in freshly prepared 4% paraformaldehyde in
100 mM phosphate-buffered saline, pH 7.4, at 4 ?C for 8 h. They were dehydrated, embedded in paraffin,
and sectioned into 6 mm per
slide. The sections were hybridized with the same DIG-labeled B. belcheri
tsingtauense CaM1a riboprobe and the control sections were
hybridized with the sense riboprobe. The hybridized signals were visualized by
BM-Purple (Roche).
Results and Discussion
Identification and evolution
of two amphioxus CaM1 cDNAs
Obtained from the gut cDNA library of the
amphioxus B. belcheri tsingtauense [30], two cDNA clones that encode
CaM1s are named CaM1a (GenBank accession number: AY269783) and CaM1b
(GenBank accession number: EF177448). CaM1a is 1412 bp long and contains
three regions, a short 5‘ untranslated region (UTR) of 64 bp, a longest
open reading frame (ORF) of 450 bp and a 3‘ UTR of 898 bp with a
polyadenylation tail at the extreme 3‘ end. The 3‘ UTR shows the
canonical polyadenylation signal (AATAAA) upstream of the poly(A) tail. CaM1b
is 1459 bp long and contains a short 5‘ UTR of 117 bp, a longest ORF
of 450 bp and a 3‘ UTR of 892 bp with a polyadenylation tail at the
extreme 3‘ end. The 3‘ UTR also shows the canonical
polyadenylation signal (AATAAA) upstream of the poly(A) tail (Fig. 1).
There are only two nucleotide substitutions within their ORFs, producing one amino
acid difference between CaM1a and CaM1b.
Comparison of the B. belcheri
tsingtauense CaM1a and CaM1b with other known CaM1s reveals that the B.
belcheri tsingtauense CaM1a is 100% identical to the CaM1 proteins of B.
floridae (GenBank accession number: Y09863) and B. lanceolatum
(GenBank accession number: Y09880), D. melanogaster CaM (GenBank
accession number: AY118890), ascidian H. roretzi CaMA (GenBank
accession number: AB018796) and mollusk A. californica CaM (GenBank accession
number: AY036120), and the CaM1b differs at only one position, at 138, Asn to
Asp (Fig. 2). The replacement of A with G at the first codon position
generates an Asp instead of an Asn. The CaM1b sequence encoding this
amino acid is further confirmed by genome amplification. Thus, two CaM1
proteins (CaM1a and CaM1b) are obtained from amphioxus B. belcheri
tsingtauense. This finding indicates that the mutation of CaM1b might occur
only in the lineage of amphioxus after the split of the amphioxus from a common
ancestor approximately 550 million years ago. This evidence supports our
previous hypothesis that amphioxus could represent a specialized form radiated
from the chordate ancestor [30,35].
The nucleotide sequence of the coding
regions of CaMs was aligned in DNASTAR (Fig. 3). It has been
found that the B. belcheri tsingtauense CaM1a or CaM1b
have 11 and 21 base substitutions with B. floridae CaM1 and B.
lanceolatum CaM1, respectively. All substitutions appear at the
third codon positions except one position of CaM1b (412, A to G) that
attributes to the single mutation of the CaM1b amino acid sequence. B.
lanceolatum CaM1 and B. floridae CaM1 have 13 base
substitutions that all occur at the third codon positions. It suggested that
the nucleotide sequence mutation between B. belcheri tsingtauense
CaM1s and B. lanceolatum CaM1 is maximal among the three
amphioxus species. This finding reveals the evolutionary relationship of CaMs,
which is still unclear. Table 1 presents the number of substitutions of
amino acids/nucleotides among CaMs in different species. In Table 1, the
CaM1 from each of the three amphioxus species has similar amino acid/nucleotide
substitutions to other CaMs. For example, B. belcheri tsingtauense
CaM1a and CaM1b, B. floridae CaM1 and B. lanceolatum CaM1 show
74, 74, 73 and 70 nucleotide substitutions with D. melanogaster CaM,
respectively. Furthermore, the nucleotide sequence of the respective 5‘
and 3‘ UTRs of CaM1 and CaM2 genes was compared, and they
show 22%–30% identity (not higher than the identity
among their coding regions). In addition, a phylogenetic tree was constructed
using the nucleotide sequence of the coding regions of 28 known CaMs (Fig.
4), and the bacteria Phytophthora infestans CaM was added as
the outgroup on the tree. The results show that B. belcheri
tsingtauense CaM1a and CaM1b cluster together with CaM1s from
the three amphioxus species, and two amphioxus CaM2s are on the separate
branch. Our results also suggest that the amphioxus CaM1s are closer to D.
melanogaster CaM, Caenorhabditis elegans CaM, H. roretzi
CaMA and CaMB than the amphioxus CaM2s. Our findings further show
that the CaM1 sequence in all three amphioxus species appears to be the
conventional CaM, and CaM2 might be the gene duplication product
of CaM1, which is consistent with the previous analyses of Karabinos and
Bhattacharya [19]. We expect to find out the CaM2 in B. belcheri
tsingtauense in our future project. It would be more interesting to further
compare their intron?xon structures of the CaM1 and CaM2
genes in B. belcheri tsingtauense, which is available in B.
lanceolatum [19] and B. floridae (http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Brafl1&id=132038,
and http://genome.jgi-psf.org/cgi-bin/dispGeneModel?db=Brafl1&id=120113).
The results show that the gene organization of CaM2 differs from that of
CaM1: CaM2 in B. lanceolatum and B. floridae has
three introns, whereas CaM1 has four introns.
Copy number of the amphioxus CaM1
gene
To analyze the copy number of B. belcheri
tsingtauense CaM1 gene, we used the DIG-labeled cDNA probe of B.
belcheri tsingtauense CaM1a to hybridize digests made from amphioxus
genomic DNA by the restriction enzymes BglII, PstI and HindIII.
Two hybridization bands were observed on a Southern blot of the BglII
and PstI restriction digest, and three bands were seen in the HindIII
digest alone (Fig. 5). The presence of the three bands generated with
the enzyme HindIII is possibly due to HindIII’s restriction site
on the B. belcheri tsingtauense CaM1 intron, as the same
restriction site of HindIII has also been seen in the B. lanceolatum
CaM1 intron [25]. These findings suggest the presence of two copies of
the CaM1 gene in the genome of amphioxus B. belcheri tsingtauense.
A high level of nucleotide sequence homology (98.3% identity), which has been
noticed between CaM1a and CaM1b cDNAs, raises the possibility
that the two genes might be the products of a gene duplication event that
occurred only in the lineage of amphioxus.
Expression analysis of the
amphioxus CaM1a gene
To detect the distribution of CaM1a mRNA
in tissue, we applied in situ hybridization on tissues of adult
amphioxus B. belcheri tsingtauense, using a specific probe, the B.
belcheri tsingtauense CaM1a cDNA (Fig.6). The results show a
strong expression of CaM1a in ovary, hepatic caecum, hind-gut, testis,
gill, endostyle and theca of notochord. A weak expression is found in neural
tube, muscle and notochord. It would also be interesting to test for coexistent
expression between CaM1a and CaM1b in amphioxus B. belcheri
tsingtauense in further studies.
In addition, the expression patterns of CaM1a
in different adult tissues and embryonic stages in amphioxus B. belcheri
tsingtauense were analyzed using Northern blot. As shown in Fig.7,
two different sizes of mRNA are seen at 1.4 kb and at 3.2 kb. The 1.4 kb mRNA
shows strong signals in the tissues of testis and gill, although weak signals
are seen in other tissues including gut, muscle, notochord and ovary. During
embryonic development, the 1.4 kb mRNA starts from extremely low density in
blastulae and gastrulae, gradually increases in neurula, and reaches its
maximum at 24 h larvae.
In contrast, the 3.2 kb mRNA is transcribed
significantly in ovary, immaterially low in gut and gill, and rarely in testis,
muscle and notochord. Its expression pattern is notably different to that of
the 1.4 kb mRNA in adult amphioxus tissues. However, the expression pattern of
the CaM1a mRNA (the 3.2 kb and the 1.4 kb mRNA together) agrees
consistently with the result detected by in situ hybridization. The
expression pattern of the 3.2 kb mRNA is similar to that of the 1.4 kb mRNA in
each stage of the embryo, showing the weak appearance from blastulae to
gastrulae, then a significant increase in neurula, and finally reaching the
maximum elevation in 24 h larvae. The progression of the positive signal of
both mRNA, starting from the neurula stage, probably reflects the relationship
between the differentiation of the neural system and the onset of gene
transcription. These results concur with the expression pattern of CaM transcript
in ascidians that are developmentally regulated and specifically restricted to
the larval neural system [1].
We propose that the two different sizes of
mRNA for CaM1a in amphioxus B. belcheri tsingtauense might have
arisen from a common nuclear precursor of the gene through differential
polyadenylation. It has been reported that three different CaM mRNAs in
eel electroplax tissue are derived from a single nuclear transcript of
approximately 5500 nucleotides, which represents a primary transcript of the
gene [36]. However, further experiments are needed to determine whether the two
different sizes of mRNA for CaM1a in amphioxus B. belcheri
tsingtauense originated from one gene.
Acknowledgements
We thank Dr. Xiangning Li, who works in the Center for Scientific Review of National
Institutes of Health (Bethesda, USA), for his critical reading of the
manuscript.
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