The structural organization of the ascidian, Halocynthia roretzi, calmodulin genes

Gene 229 (1999) 163–169

The structural organization of the ascidian, Halocynthia roretzi, calmodulin genes The vicissitude of introns during the evolution of calmodulin genes Hajime Julie Yuasa, Hiroaki Yamamoto, Takashi Takagi * Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan Received 6 November 1998; accepted 28 December 1998; Received by T. Gojobori

Abstract Two distinct calmodulin (CaM ) genes are isolated from the ascidian, Halocynthia roretzi, (Hr-CaM A and Hr-CaM B) and those structures are determined. There are three nucleotide substitutions, producing two amino acid differences between Hr-CaM A and Hr-CaM B, and those are corresponding to two of the known eight variable residues among metazoan CaMs. Both HrCaM A and Hr-CaM B are constructed from six exons and five introns, and the positions of introns are identical. The positions of introns of Hr-CaMs are also identical with those of vertebrate CaMs, except third introns. The third introns of Hr-CaMs are inserted at 28bp upstream when compared with vertebrate CaMs. Thus, sliding of the third intron might have occurred in only the ascidian lineage prior to the gene duplication that also occurred only in that lineage. In addition, with the comparison of the intron positions, we attempt to investigate the vicissitude of introns during the evolution of metazoan CaMs. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Exon/intron organization; Intron sliding; Isoforms; Molecular evolution

1. Introduction Calmodulin (CaM ) is a ubiquitous eukaryotic calcium binding protein and one of the most highly conserved molecules. It possesses four calcium binding domains per molecule, called EF-hands, and classified as one of 39 subfamilies of the EF-hand family ( Kawasaki and Kretsinger, 1995). In vertebrate species, CaM is encoded by multiple genes. Three distinct genes (CaM I, II, III ) have been detected for the human (SenGupta et al., 1987; Koller et al., 1990; Rhyner et al., 1994) and the rat (Nojima and Sokabe, 1987; Nojima, 1989), and genes corresponding to two of them (CaM I, II ) have been isolated from the chicken (Simmen et al., 1985; Ye and Berchtold, 1997). The frog, Xenopus laevis, possesses at least two CaM genes (Chien and * Corresponding author. Tel: +81-22-217-6677; fax: +81-22-263-9206; e-mail: [email protected]. Abbreviations: bp, base pair(s); CaM, calmodulin; nt, nucleotide(s); PCR, polymerase chain reaction.

Dawid, 1984), and at least four genes are observed in the teleost fish, Oryzias latipes (Matsuo et al., 1992). These genes obey the multigene one-protein principle, and the amino acid sequences of products from these genes are identical. As an exceptional case, two distinct sequences of CaM were isolated from the electric eel (Electrophorus electricus, Lagece´ et al., 1983; Toda et al., 1985). On the other hand, the number of CaM gene(s) of invertebrates is rather small. In Drosophila (Smith et al., 1987), the mollusc Aplysia (Swanson et al., 1990), and the amphioxus Branchiostoma ( Karabinos and Riemer, 1997), there is only a single copy of the CaM gene and the amino acid sequences of the CaMs from these organisms are identical. Only for the echinoderm, Arbacia, have two distinct CaM cDNAs been reported ( Hardy et al., 1988). The high conservation of CaM sequences make difficult to elucidate the evolutional relationship and diversity of metazoan CaMs. For instance, as Karabinos and Riemer (1997) proposed, two explanations may be possible for the existence of two CaM genes in Arbacia.

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 9 ) 0 0 02 8 - 1

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The early metazoa had two CaM genes and one of those was lost in most lineages but both were retained in the echinoderms, or the gene duplication occurred in the echinoderms after the echinoderms/chordate divergence. Besides, the cDNA of the ascidian, Ciona intestinalis, CaM was determined recently, and the constructed phylogenetic tree showed a closer relationship of Ciona CaM to vertebrate CaMs than to the other ascidians CaMs (Di Gregorio et al., 1998). There are two theories to explain the evolution of spliceosomal introns: ‘introns-late’ and ‘introns-early’. The former is rather tolerant for the intron insertion and deletion during evolution. The latter basically proposes that the early genes possessed many introns, but most of those were lost during evolution. However, this theory does not deny completely the gain of introns (Long et al., 1995). The investigation of the vicissitude of introns during the evolution may be valuable for the elucidation of the evolution of introns themselves. The insertion and deletion of introns, if they occur correctly, do not affect the gene product, namely they may be ‘neutral’ in evolution, and as they do not occur so frequently, we are hardly confused with the convergent insertion or deletion. Thus, conversely, intron positions may be useful, at least in part, to elucidate the evolution of highly conserved molecules, such as CaMs. In this study, we isolate two CaM genes from the ascidian, Halocynthia roretzi, (Hr-CaM A and Hr-CaM B) and determine those structures. We also attempt to explain the vicissitude of introns during the evolution of metazoan CaMs by comparing the localization of introns.

2. Materials and methods

reverse primer, Hr-CaM A R1; 5∞-CGTTTACCATGTCACATTGC-3∞, complementary to the sequence from nt 1035 to 1054 of the Hr-CaM A cDNA (see Fig. 1). 2.2. Isolation of genomic clones The genomic library was constructed as previously described (Sato et al, 1997), and was screened using the open reading frame of Hr-CaM A cDNA as the probe. The probe was labeled with DIG-DNA Labeling Mixture (Boehringer Mannheim) by PCR using the following primers: a forward primer, Hr-CaM A F1, 5∞-CAGCATCCTAATATTACATA-3∞ (corresponding to the sequence from nt −23 to −4); a reverse primer, Hr-CaM A R2, 5∞-GTTTGTAGATCTATAACCAC-3∞ (complementary to the sequence from nt 448 to 467). Hybridization and washing were carried out according to the manufacturer’s instructions (Boehringer Mannheim) and positive clones were detected with DIG Luminescent Detection Kit (Boehringer Mannheim). 2.3. Detection of Hr-CaM A and Hr-CaM B expression The expression of both Hr-CaM A and Hr-CaM B in body wall muscle was detected by RT-PCR. Hr-CaM A F1 was used as Hr-CaM A/Hr-CaM B common forward primer, and the reverse primers employed were as follows: Hr-CaM A R3, 5∞-CATTCATAAATTTCAGGTTG-3∞ (Hr-CaM A specific primer, complementary to the sequence from nt 870 to 889); Hr-CaM B R1; 5∞-ATGCCACTATTATGCAACGG-3∞ (Hr-CaM B specific primer, complementary to the sequence from nt 863 to 882).

2.1. Cloning of the Hr-CaM A cDNA

2.4. DNA sequencing

The single-stranded cDNA of the body wall muscle was prepared as previously described ( Yuasa et al., 1997). The 3∞-half of Hr-CaM A cDNA was amplified by polymerase chain reaction (PCR) (Saiki et al., 1988) using Ex Taq DNA polymerase ( Takara). The redundant oligomer used for PCR was 5∞-GARTTYYTNACNATGATGGC-3∞, where Y represents C and T, R represents A and G, and N represents A, C, G and T. This was designed based on consensus amino acid sequences among the metazoan CaMs, EFLTMMA (residue 67–73). The oligo-dT adaptor, 5∞-GGGATCCGAATTCT -3∞ was used as another primer. The 17 5∞-upstream of Hr-CaM A cDNA was also amplified by PCR. The Eco RI cassette ( Takara) ligated doublestranded cDNA was prepared as previously described ( Yuasa et al., 1997) and used as template. The primers employed were the cassette-specific primer C1, 5∞-GTACATATTGTCGTTAGAACGCG-3∞ and a

All PCR-amplified products and isolated genomic clones were subcloned to pCR II plasmid vector ( TA-cloning kit, Invitrogen) or pUC18 for sequencing. The nucleotide sequences were determined by the dideoxy chain termination method with Dye Primer Cycle Sequencing Kit (Applied Biosystems) using an automated DNA sequencer (Applied Biosystems 373A).

3. Results and discussion 3.1. Hr-CaM A cDNA and genomic structure With the redundant primer-used PCR amplification, we could detect only one sort of CaM cDNA, named Hr-CaM A. The Hr-CaM A cDNA is composed of 1100 nucleotides (Fig. 1), and the deduced amino acid sequence is identical with Drosophila, Aplysia and

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Fig. 1. Comparison of cDNA and deduced amino acid sequences of H. roretzi CaMs. Upper lanes: Hr-CaM A cDNA and deduced amino acid sequence; lower lanes: Hr-CaM B cDNA and deduced amino acid sequence. Identical nucleotides and amino acids to those in CaM A are indicated by dots (.), and gaps are inserted for maximal similarity and shown by bars (–). In the downstream region from the position indicated by an upward arrow (( ), no significant similarity is observed between Hr-CaM A and Hr-CaM B. The typical polyadenylation signals (AATAAA) are underlined. The initiator methionine (residue −1, parenthesized ) must have been removed after translation. The downward arrows (3) indicate the positions of introns in both Hr-CaM A and Hr-CaM B genes.

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Fig. 2. Comparison of genomic structures of Hr-CaM A and Hr-CaM B. Restriction and exon/intron maps of the Hr-CaM A (upper) and HrCaM B ( lower) genes. Only Eco RI ( E ) and Hinc II (Hc) restriction sites are indicated. Exons are shown by boxes. The 5∞- and 3∞-flanking regions and introns (In 1–In 5) are shown by bars, and the length of each intron is indicated.

Branchiostoma CaMs. The identity of the nucleotide sequences among open reading frames of these four species CaM cDNAs is listed in Table 1. In spite of their phylogenetic relationship or distance, the values are almost equal (about 80% identity). This might arise from high conservation of CaM amino acid sequences, namely only the synonymous substitution might be permitted, and the reverse mutation occurred repeatedly. Using Hr-CaM A cDNA as a probe, we isolated seven clones from the genomic library of H. roretzi. According to the length of partially PCR amplifiedfragments of those clones using several primers sets, they were divided into two groups (data not shown). The one group, two out of seven clones, contained a full length of Hr-CaM A gene. The nucleotide sequence of 5317 bp was determined, and the genomic structure showed that it is divided into six exons by five introns (Fig. 2). All introns start with gt and end with ag, and according to the nomenclature of Kretsinger and Nakayama (1993), the introns positions are −10/0,

Table 1 The identity (%) among open reading frames of invertebrates CaM cDNAs

Hr-CaM A Amphioxus Drosophila

Amphioxus

Drosophila

Aplysia

80

81 84

80 78 79

1.01/1, 2.04/0, 3.12/0 and 4.21/1.1 There is no sequence discrepancy between the nucleotide sequences of exons and that of cDNA. 3.2. Hr-CaM B genomic structure and expression The other group of genomic clones, five out of seven, contained partial or full length of CaM gene, but distinct from Hr-CaM A, so named Hr-CaM B. The nucleotide sequence of 5739 bp was determined, and the genomic structure of Hr-CaM B showed that it is also divided into six exons by five introns (Fig. 2). The intron positions are identical with those of Hr-CaM A, but different in length except for the second intron. As their second introns are rather short (78 bp, see Fig. 2), from the functional standpoint, any deletion may not be permitted for the correct splicing event (in vertebrate, the minimum length for splicing of introns is approximately 70 bp, Kennedy and Berget, 1997). Further, the random insertion of an intron would be less probable in this short region than into other longer introns. 1 The positions of introns are indicated according to the nomenclature of Kretsinger and Nakayama (1993). The first number indicates the number of the EF-hand domain sequentially numbered from N to C. The second number (following the period) shows the number of the residue within the domain, which is generally constructed from 29 residues. The last number (following the slash) is the phase: 0 means the intron lies between triplet codons, 1 means between first and second nucleotides of the codon, and 2 means between second and third. For example, 4.21/1 means domain IV, 21st residue and phase 1. −10/0 means phase 0, 10 residues before the beginning of domain I.

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H.J. Yuasa et al. / Gene 229 (1999) 163–169 Table 2 Variable residues among metazoan CaMs Species position

60

74

78

97

99

130

143

147

Ref.

Vertebrate Eel (pCM116) Ascidian (Hr-CaM A)a Ascidian (Hr-CaM B) Ascidian (C. intestinalis) Sea urchin alpha Sea urchin beta Scallop Sea anemone

N N N N N N N D B

R K R R R R R R R

D D D E D D E D Z

N N N N N N N D B

R R F F F R F F F

I I I I V I I I I

Q Q T T N ? A T K

A A S C N ? S S S

Lagece´ et al. (1983) This work This work Di Gregorio et al. (1998) Hardy et al. (1988) Hardy et al. (1988) Toda et al. (1981) Takagi et al. (1980)

a The amino acid sequence of Hr-CaM A is identical with those of Branchiostoma (amphioxus), Drosophila and Aplysia.

The genomic DNA-derived cDNA sequence of HrCaM B is aligned with Hr-CaM A cDNA ( Fig. 1). The polyadenylation site is not identified, but the typical polyadenylation signal (AATAAA) is observed only once within about 2 kbp of the downstream region from the termination codon ( Fig. 1, underlined ). Between open reading frames of Hr-CaM A and Hr-CaM B, there are three nucleotide substitutions, producing two amino acid differences (positions 78 and 147). Among metazoan CaM amino acid sequences, variants have been observed at only eight positions out of 148 residues ( listed in Table 2), and substitutions of Hr-CaM B correspond to two of these positions. So, it is supposed that Hr-CaM B is functional molecule, although the functional difference between Hr-CaM A and Hr-CaM B is unknown. Indeed, the expression of Hr-CaM B is observed at least in body wall muscle, though the amount of expression is slightly smaller than Hr-CaM A (Fig. 3). Thus, the ascidian, H. roretzi possesses at least two distinct CaM genes, and both genes may be

functional. It may be noted incidentally that CaM is a single copy gene in the other species of the ascidian, Ciona intestinalis (Di Gregorio et al., 1998). Multiple CaM genes are commonly observed in vertebrate species, but these genes encode identical proteins in general. The only other occurrences of two distinct sequences of CaM are in the electric eel (E. electricus) and in the sea urchin (A. punctulata), so H. roretzi is the third case. 3.3. Comparison of intron positions among metazoan CaM genes The intron positions of various species CaM genes whose structures have been determined are listed in Table 3. From the comparison of intron positions, the following hypotheses are supposed for the vicissitude of introns during evolution of CaM genes. The positions of the first and the last introns (−10/0 and 4.21/1) are identical in all CaM genes. As for the second intron (1.01/1), only Drosophila lacks the second intron (1.01/1). This is parsimoniously explained by a single loss in the Drosophila (perhaps insecta or higher taxonomic group) lineage. Similarly, the absence of the third intron (2.13/1) in the rat CaM II gene can be parsimoniously interpreted as a result of a single loss. All the other CaM genes except H. roretzi possess the third intron positioned at 2.13/1, and besides, this intron is also observed among troponin C ( TnC ) genes, an Table 3 The intron positions of metazoan CaM genes

Fig. 3. PCR amplification of Hr-CaM A and Hr-CaM B cDNAs. The cDNA prepared from the body wall muscle was used as template. Lane M, molecular markers. Lane A, Hr-CaM A specific reverse primer, Hr-CaM A R3 was used. Lane B, Hr-CaM B specific reverse primer, Hr-CaM B R1 was used. As the forward primer, Hr-CaM A F1;HrCaM A/Hr-CaM B common forward primer was employed in each reaction.

Species (type)

Positions of introns

Human (I, III ) Rat (I, III ) Rat (II ) Chicken (I, II ) Amphioxus Hr-CaMs Aplysia Drosophila

−10/0 −10/0 −10/0 −10/0 −10/0 −10/0 −10/0 −10/0

(–) shows the absence of intron.

1.01/1 1.01/1 1.01/1 1.01/1 1.01/1 1.01/1 1.01/1 –

2.13/1 2.13/1 – 2.13/1 2.13/1 2.04/0 2.13/1 2.13/1

3.12/0 3.12/0 3.12/0 3.12/0 – 3.12/0 – –

4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1

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Fig. 4. Schematic presentation of hypothetical vicissitudes of introns during evolution of CaM genes. Points of each hypothetical intron insertion/deletion/sliding are marked on the molecular phylogenetic tree. The tree is constructed based on the conventional phylogenetic tree among Phyla, and the length of branches does not reflect phylogenetic relationship or distance among them.

evolutionarily closely related molecule to CaM ( Yuasa et al., 1998). In contrast, the third introns of both HrCaM genes are inserted at a unique position: 2.04/0. This may be explained by the ‘intron sliding’ that occurred in only the ascidian lineage, and the following gene duplication created two CaM genes. Thus, the existence of two CaM genes in H. roretzi may have arisen from a gene duplication that occurred only in the ascidian lineage. According to case studies of discordant introns (Stoltzfus et al., 1997), most of reported cases of ‘intron sliding’ are artifacts, namely they arise from errors of sequencing or alignment. In this case, the distance between two introns (2.04/0 and 2.13/1) is so long (28 bp) and, CaM is a highly conserved molecule, that such errors of sequencing or alignment have never occurred. However, the evidence for putative intron sliding is weak, and the mechanism of it is unclear, so it may be appropriate to change the term of ‘intron sliding’ to ‘separate loss and gain’ (Stoltzfus et al., 1997). The absence of the fourth intron (3.12/0) has been a common feature among invertebrate CaMs (Aplysia, Drosophila and Branchiostoma); however, it is broken by the presence of it in the ascidian CaMs. With regard to the absence of the fourth intron of the amphioxus CaM gene, it may be proper to consider this as arising from a single loss that occurred in the amphioxus lineage. This is because the fourth intron is commonly observed among chordate CaM genes except amphioxus, and, in addition, the CaM-like genomic fragment isolated from the amphioxus possesses the

fourth intron at 3.12/0 ( Karabinos and Riemer, 1997). Namely, the structure of the CaM gene of the chordate ancestor is supposed to have been six exons and five introns. On the other hand, there may be two phylogenetic schemes to account for the absence of the fourth intron in invertebrate (Drosophila and Aplysia) CaM genes. In the first scheme, the structure of the CaM gene of the metazoan ancestor was six exons and five introns, and a single loss of the fourth intron occurred in the invertebrate (or protostome) lineage after invertebrate/chordate (or protostome/deuterostome) divergence. Alternatively, the early metazoan CaM gene was divided into five exons by four introns, and a single gain of the fourth intron occurred within the chordate (or deuterostome) lineage. The above hypothetical vicissitudes of introns are summarized in Fig. 4. To elucidate the whole aspect of metazoan CaM evolution, it is necessary to determine structures of CaM genes from other species, both deuterostome and protostome. Hence, the intron positions may be good landmarks to investigate the evolutional relationship of metazoan CaM genes.

Acknowledgement This work was supported in part by a grant from the Japan Society for the Promoting of Science.

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