Intron Positions Research Articles

Complex intron generation in the yeast genus Lipomyces

In primary transcripts of eukaryotic nuclear genes, coding sequences are often interrupted by U2-type introns. Such intervening sequences can constitute complex introns excised by consecutive splicing reactions. The origin of spliceosomal introns is a vexing problem. Sequence variation existent across fungal taxa provides means to study their structure and evolution. In one class of complex introns called [D] stwintrons, an (internal) U2 intron is nested within the 5'-donor element of another (external) U2 intron. In the gene for a reticulon-like protein in species of the ascomycete yeast genus Lipomyces, the most 5' terminal intron position is occupied by one of three complex intervening sequences consistent of differently nested U2 intron units, as demonstrated in L. lipofer, L. suomiensis, and L. starkeyi. In L. starkeyi, the donor elements of the constituent introns are abutting and the complex intervening sequence can be excised alternatively either with one standard splicing reaction or, as a [D] stwintron, by two consecutive reactions. Our work suggests how [D] stwintrons could emerge by the appearance of new functional splice sites within an extant intron. The stepwise stwintronisation mechanism may involve duplication of the functional intron donor element of the ancestor intron.

Comprehensive genomic analyses with 115 plastomes from algae to seed plants: structure, gene contents, GC contents, and introns.

Chloroplasts are a common character in plants. The chloroplasts in each plant lineage have shaped their own genomes, plastomes, by structural changes and transferring many genes to nuclear genomes during plant evolution. Some plastid genes have introns that are mostly group II introns. This study aimed to get genomic and evolutionary insights on the plastomes from green algae to flowering plants. Plastomes of 115 species from green algae, bryophytes, pteridophytes (spore bearing vascular plants), gymnosperms, and angiosperms were mined from NCBI organelle genome database. Plastome structure, gene contents and GC contents were analyzed by the in-house developed Phyton code. Intronic features including presence/absence, length, intron phases were analyzed by manually in the annotated information in NCBI. The canonical quadripartite structures were retained in most plastomes except of a few plastomes that had lost an invert repeat (IR). Expansion or reduction or deletion of IRs resulted in the length variation of the plastomes. The number of protein coding genes ranged from 40 to 92 with an average 79.43 ± 5.84 per plastome and gene losses were apparent in specific lineages. The number of trn genes ranged from 13 to 33 with an average 21.19 ± 2.42 per plastome. Ribosomal RNA genes, rrn, were located in the IRs so that they were present in a duplicate except of the species that had lost one of the IR. GC contents were variable from 24.9 to 51.0% with an average 38.21 ± 3.27%, indicating bias to high AT contents. Plastid introns were present in 18 protein coding genes, six trn genes, and one rrn gene. Intron losses occurred among the orthologous genes in different plant lineages. The plastid introns were long compared with the nuclear introns, which might be related with the spliceosome nuclear introns and self-splicing group II plastid introns. The trnK-UUU intron contained the maturase encoding matK gene except in the chlorophyte algae and monilophyte ferns in which the trnK-UUU was lost, but matK retained. There were many annotation artefacts in the intron positions in the NCBI database. In the analysis of intron phases, phase 0 introns were more frequent than those of phase 2 and 3 introns. Phase polymorphism was observed in the introns of clpP which was derived from nucleotide insertion. Plastid trn introns were long compared to the archaeal or eukaryotic nuclear tRNA introns. Of the six plastid trn introns, one was at the D loop and other five were at the anticodon loop. The insertion sites were conserved among the trn genes in archaea, eukaryotic nuclear and plastid tRNA genes. Current study refurbrished the previous findings of structural variations, gene contents, and GC contents of the chloroplast genomes from green algae to flowering plants. The study also included some noble findings and discussions on the plastome introns including their length variations and phase variation. We also presented and corrected some false annotations on the introns in protein coding and tRNA genes in the genome database, which might be confirmed by the chloroplast transcriptome analysis in the future.

A transcriptome-wide antitermination mechanism sustaining identity of embryonic stem cells

Eukaryotic gene expression relies on extensive crosstalk between transcription and RNA processing. Changes in this composite regulation network may provide an important means for shaping cell type-specific transcriptomes. Here we show that the RNA-associated protein Srrt/Ars2 sustains embryonic stem cell (ESC) identity by preventing premature termination of numerous transcripts at cryptic cleavage/polyadenylation sites in first introns. Srrt interacts with the nuclear cap-binding complex and facilitates recruitment of the spliceosome component U1 snRNP to cognate intronic positions. At least in some cases, U1 recruited in this manner inhibits downstream cleavage/polyadenylation events through a splicing-independent mechanism called telescripting. We further provide evidence that the naturally high expression of Srrt in ESCs offsets deleterious effects of retrotransposable sequences accumulating in its targets. Our work identifies Srrt as a molecular guardian of the pluripotent cell state.

In silico definition of new ligninolytic peroxidase sub-classes in fungi and putative relation to fungal life style

Ligninolytic peroxidases are microbial enzymes involved in depolymerisation of lignin, a plant cell wall polymer found in land plants. Among fungi, only Dikarya were found to degrade lignin. The increase of available fungal genomes allows performing an expert annotation of lignin-degrading peroxidase encoding sequences with a particular focus on Class II peroxidases (CII Prx). In addition to the previously described LiP, MnP and VP classes, based on sequence similarity, six new sub-classes have been defined: three found in plant pathogen ascomycetes and three in basidiomycetes. The presence of CII Prxs could be related to fungal life style. Typically, necrotrophic or hemibiotrophic fungi, either ascomycetes or basidiomycetes, possess CII Prxs while symbiotic, endophytic or biotrophic fungi do not. CII Prxs from ascomycetes are rarely subjected to duplications unlike those from basidiomycetes, which can form large recent duplicated families. Even if these CII Prxs classes form two well distinct clusters with divergent gene structures (intron numbers and positions), they share the same key catalytic residues suggesting that they evolved independently from similar ancestral sequences with few or no introns. The lack of CII Prxs encoding sequences in early diverging fungi, together with the absence of duplicated class I peroxidase (CcP) in fungi containing CII Prxs, suggests the potential emergence of an ancestral CII Prx sequence from the duplicated CcP after the separation between ascomycetes and basidiomycetes. As some ascomycetes and basidiomycetes did not possess CII Prx, late gene loss could have occurred.

Genome-Wide Identification and Functional Analysis of Carboxylesterase and Methylesterase Gene Families in Peach (Prunus persica L. Batsch).

Carboxylesterases (CXE) and methylesterases (MES) are hydrolytic enzymes that act on carboxylic esters and are involved in plant metabolic processes and defense responses. A few functions of plant CXE and MES genes have been identified but very little information is available about the role of most members. We made a comprehensive study of this gene family in a commercially important species, peach (Prunus persica L. Batsch). A total of 33 peach CXE genes and 18 MES genes were identified and shown to be distributed unevenly between the chromosomes. Based on phylogenetic analysis, CXEs and MESs clustered into two different branches. Comparison of the positions of intron and differences in motifs revealed the evolutionary relationships between CXE and MES genes. RNA-seq revealed differential expression patterns of CXE/MESs in peach flower, leaf, and ripening fruit and in response to methyl jasmonate (MeJA) and ultraviolet B treatment. Transcript levels of candidate genes were verified by real-time quantitative PCR. Heterologous expression in Escherichia coli identified three CXEs that were involved in the hydrolysis of volatile esters in vitro. Furthermore, two recombinant MES proteins were identified that could hydrolyze MeJA and methyl salicylate. Our results provide an important resource for the identification of functional CXE and MES genes involved in the catabolism of volatile esters, responses to biotic and abiotic stresses and activation of signaling molecules such as MeJA and methyl salicylate.

Global Co-transcriptional Splicing in Arabidopsis and the Correlation with Splicing Regulation in Mature RNAs

RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription. However, co-transcriptional splicing has not yet been explored in plants. Here, we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing, which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs. Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency, and introns with alternative 5′ or 3′ splice sites are less efficiently spliced. Furthermore, we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs, and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level. Collectively, our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.

Evolution of Exons and the Exon–Intron Structure of Long Intergenic Noncoding RNA Genes in Placental Mammals

Long intergenic noncoding RNA (lincRNA) genes are abundant in mammals, but their functions remain elusive. The characteristics of lincRNAs can be studied via comparative analysis with those of mRNAs, for which there is a wealth of information. Highly conserved coding sequences (exons) and the exon–intron structure are some typical evolutionary features attributed to eukaryotic mRNAs. The exon structure is conserved considerably less in lincRNA, but to a greater degree than for introns. Comparative analysis of putative intron positions in lincRNA genes in a range of mammalian genomes underscored the high positional conservation for some introns, which appear to be up to 100 million years old. It is therefore possible that primary and/or secondary structure of these molecules confers functional signals.

Exon size and sequence conservation improves identification of splice-altering nucleotides.

Pre-mRNA splicing is regulated through multiple trans-acting splicing factors. These regulators interact with the pre-mRNA at intronic and exonic positions. Given that most exons are protein coding, the evolution of exons must be modulated by a combination of selective coding and splicing pressures. It has previously been demonstrated that selective splicing pressures are more easily deconvoluted when phylogenetic comparisons are made for exons of identical size, suggesting that exon size–filtered sequence alignments may improve identification of nucleotides evolved to mediate efficient exon ligation. To test this hypothesis, an exon size database was created, filtering 76 vertebrate sequence alignments based on exon size conservation. In addition to other genomic parameters, such as splice-site strength, gene position, or flanking intron length, this database permits the identification of exons that are size- and/or sequence-conserved. Highly size-conserved exons are always sequence-conserved. However, sequence conservation does not necessitate exon size conservation. Our analysis identified evolutionarily young exons and demonstrated that length conservation is a strong predictor of alternative splicing. A published data set of approximately 5000 exonic SNPs associated with disease was analyzed to test the hypothesis that exon size–filtered sequence comparisons increase detection of splice-altering nucleotides. Improved splice predictions could be achieved when mutations occur at the third codon position, especially when a mutation decreases exon inclusion efficiency. The results demonstrate that coding pressures dominate nucleotide composition at invariable codon positions and that exon size–filtered sequence alignments permit identification of splice-altering nucleotides at wobble positions.

Cloning and characterisation of the mouse fra-2 gene.

Transcription factor AP-1 is comprised of multiple protein complexes that include members of a family of genes related to the proto-oncogene c-fos. In this report, we have extended the analysis of one member of this family, fos-related antigen-2 (fra-2), by isolating and characterising genomic and cDNA clones encoding the mouse fra-2 homolog. The overall gene structure (number and positions of introns) was similar to that of both the chicken fra-2 gene and other members of the fos family, and the relative positions of putative enhancers in the 5' regulatory region were well conserved between the mouse and chicken fra-2 genes. High levels of fra-2 mRNA were detected in ovary, stomach, small and large intestine, brain, lung and heart. The mouse Fra-2 protein showed 94% and 87.5% conservation with human and chicken Fra-2, respectively, and mouse Fra-2, like the chicken homolog, induced transformation of chicken embryo fibroblasts. The characterisation of the mouse fra-2 gene provides a basis for analysis of Fra-2 function in the whole animal.

In vivo measurements reveal a single 5\u2032-intron is sufficient to increase protein expression level in Caenorhabditis elegans

Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). IME has been measured in cell culture and plant models by quantifying expression of intronless and intron-bearing reporter genes in vitro. We developed hardware and software to implement microfluidic chip-based gene expression quantification in vivo. We altered position, number and sequence of introns in reporter genes controlled by the hsp-90 promoter. Consistent with plant and mammalian studies, we determined a single, natural or synthetic, 5′-intron is sufficient for the full IME effect conferred by three synthetic introns, while a 3′-intron is not. We found coding sequence can affect IME; the same three synthetic introns that increase mcherry protein concentration by approximately 50%, increase mEGFP by 80%. We determined IME effect size is not greatly affected by the stronger vit-2 promoter. Our microfluidic imaging approach should facilitate screens for factors affecting IME and other intron-dependent processes.

High-throughput analysis revealed mutations’ diverging effects on SMN1 exon 7 splicing

ABSTRACT Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene’s exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5′ss) and de novo 5′ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5′ss mutations potentiated the use of three different cryptic 5′ss. Generally, single mutations supporting cryptic 5′ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5′ss. Analyzing double mutants supported the predominating splicing regulatory elements’ effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5′ss can be one of the main factors driving cryptic 5′ss use.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-Like Factors of Eurasian Medicinal Leeches

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

The evolution of the plastid phosphate translocator family.

The plastid phosphate translocators evolved in algae but diversified into several groups, which adopted different physiological functions by extensive gene duplications and losses in Streptophyta. The plastid phosphate translocators (pPT) are a family of transporters involved in the exchange of metabolites and inorganic phosphate between stroma and cytosol. Based on their substrate specificities, they were divided into four subfamilies named TPT, PPT, GPT and XPT. To analyse the occurrence of these transporters in different algae and land plant species, we identified 652 pPT genes in 101 sequenced genomes for phylogenetic analysis. The first three subfamilies are found in all species and evolved before the split of red and green algae while the XPTs were derived from the duplication of a GPT gene at the base of Streptophyta. The analysis of the intron-exon structures of the pPTs corroborated these findings. While the number and positions of introns are conserved within each subfamily, they differ between the subfamilies suggesting an insertion of the introns shortly after the three subfamilies evolved. During angiosperm evolution, the subfamilies further split into different groups (TPT1-2, PPT1-3, GPT1-6). Angiosperm species differ significantly in the total number of pPTs, with many species having only a few, while several plants, especially crops, have a higher number, pointing to the importance of these transporters for improved source-sink strength and yield. The differences in the number of pPTs can be explained by several small-scale gene duplications and losses in plant families or single species, but also by whole genome duplications, for example, in grasses. This work could be the basis for a comprehensive analysis of the molecular and physiological functions of this important family of transporters.

Intron-mediated regulation of β-tubulin genes expression affects the sensitivity to carbendazim in Fusarium graminearum.

The plant pathogenic fungus, Fusarium graminearum, is known to have two β-tubulin genes (named Fg-β1tub and Fg-β2tub). Mutations in Fg-β2tub rather than in Fg-β1tub have been shown to confer resistance to carbendazim (MBC), even though Fg-β1tub has higher homology than Fg-β2tub to the β-tubulin isotypes related to benzimidazole resistance in other fungi. However, sequence alignment of β-tubulin isotypes related to benzimidazole resistance showed that the number and position of introns in Fg-β2tub are more consistent than Fg-β1tub to those in other β-tubulin genes. In detail, Fg-β1tub lacks three introns, i.e., intron i3, i4, and i6 corresponding to positions in Fg-β2tub of F. graminearum. To investigate the effects of the divergence introns on the function of β-tubulins in F. graminearum, a strategy of intron deletion and insertion was used. Our results showed that deletion of the second intron from Fg-β1tub gene increased Fg-β1tub expression levels leading to increased sensitivity to MBC. Besides, inserting the divergence introns into Fg-β1tub can increase Fg-β1tub expression leading to increased sensitivity to MBC. In addition, intron-mediated Fg-β1tub gene expression requires a splicing-competent intron within the body of the host gene. Furthermore, the insertion and deletion of introns in Fg-β1tub gene have no significant effect on hyphal growth, conidiation and virulence in F. graminearum. Thus, we proposed that introns may be among the factors contributing to the evolution and functional divergence of two β-tubulin genes and also significantly regulate the expression of β-tubulin genes, which, in turn, affects sensitivity to MBC fungicides in F. graminearum.

Genome streamlining via complete loss of introns has occurred multiple times in lichenized fungal mitochondria.

Reductions in genome size and complexity are a hallmark of obligate symbioses. The mitochondrial genome displays clear examples of these reductions, with the ancestral alpha‐proteobacterial genome size and gene number having been reduced by orders of magnitude in most descendent modern mitochondrial genomes. Here, we examine patterns of mitochondrial evolution specifically looking at intron size, number, and position across 58 species from 21 genera of lichenized Ascomycete fungi, representing a broad range of fungal diversity and niches. Our results show that the cox1gene always contained the highest number of introns out of all the mitochondrial protein‐coding genes, that high intron sequence similarity (>90%) can be maintained between different genera, and that lichens have undergone at least two instances of complete, genome‐wide intron loss consistent with evidence for genome streamlining via loss of parasitic, noncoding DNA, in Phlyctis boliviensisand Graphis lineola. Notably, however, lichenized fungi have not only undergone intron loss but in some instances have expanded considerably in size due to intron proliferation (e.g., Alectoria fallacina and Parmotrema neotropicum), even between closely related sister species (e.g., Cladonia). These results shed light on the highly dynamic mitochondrial evolution that is occurring in lichens and suggest that these obligate symbiotic organisms are in some cases undergoing recent, broad‐scale genome streamlining via loss of protein‐coding genes as well as noncoding, parasitic DNA elements.

Cerebral hypomyelination associated with biallelic variants of FIG4.

The lipid phosphatase gene FIG4 is responsible for Yunis-Varón syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G > A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read-through from exon 20 into intron 20 and truncation of the final 115 C-terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.

Effect of the thymine-DNA glycosylase rs4135050 variant on Saudi smoker population.

BackgroundThymine‐DNA glycosylase (TDG) is an essential DNA‐repair enzyme which works in both epigenetic regulation and genome maintenance. It is also responsible for efficient correction of multiple endogenous DNA lesions which occur commonly in mammalian genomes. Research of genetic variants such as SNPs, resulting in disease, is predicted to yield clinical advancements through the identification of sensitive genetic markers and the development of disease prevention and therapy. To that end, the main objective of the present study is to identify the possible interactions between cigarette smoking and the rs4135050 variant of the TDG gene, situated in the intron position, among Saudi individuals.MethodsTDG rs4135050 (A/T) was investigated by genotyping 239, and 235 blood specimens were obtained from nonsmokers and smokers of cigarette respectively.ResultsT allele frequency was found which showed a significant protective effect on Saudi male smokers (OR = 0.64, p = 0.0187) compared to nonsmoking subjects, but not in female smokers. Furthermore, smokers aged less than 29 years, the AT and AT+TT genotypes decreased more than four times the risk of initiation of smoking related‐diseases compare to the ancestral AA homozygous genotype. Paradoxically, the AT (OR = 3.88, p = 0.0169) and AT+TT (OR = 2.86, p = 0.0420) genotypes were present at a higher frequency in smoking patients aged more than 29 years as compared to nonsmokers at the same ages.ConclusionDepending on the gender and age of patients, TDG rs4135050 may provide a novel biomarker for the early diagnosis and prevention of several diseases caused by cigarette smoking.

GeMoMa: Homology-Based Gene Prediction Utilizing Intron Position Conservation and RNA-seq Data.

GeMoMa is a homology-based gene prediction program that predicts gene models in target species based on gene models in evolutionary related reference species. GeMoMa utilizes amino acid sequence conservation, intron position conservation, and RNA-seq data to accurately predict protein-coding transcripts. Furthermore, GeMoMa supports the combination of predictions based on several reference species allowing to transfer high-quality annotation of different reference species to a target species. Here, we present a detailed description of GeMoMa modules and the GeMoMa pipeline and how they can be used on the command line to address particular biological problems.

DUCHENNE MUSCULAR DYSTROPHY - GENETICS: P.222Influence of the intronic breakpoint of the DYS 45-55 exon deletion on the clinical phenotype

Multiexon skipping is a novel approach in the treatment of DMD that can be achieved with the new Crispr-Cas9 technology. In particular, 63% of DMD mutations are located between exons 45 to 55 and spontaneous deletion spanning that region occurs in asymptomatic subjects or with variable Becker phenotype. We evaluate a cohort of subjects with a 45-55 deletion sustaining the hypothesis that intronic breakpoint positions might influence the phenotype. We analyzed the exact position of the del 45-55 intronic breakpoints in 10 index patients, first performing arrays and multiple PCRs to restrict the breakpoint areas, followed by amplification and sequencing of the junction deletions and the stretch spanning each breakpoint. Finally we reviewed the clinical and cardiac features. Seven patients shared the same breakpoints in both introns but presented variable clinical manifestation: 2 asymptomatic, 3 paucysymptomatics and two manifesting mild BMD phenotypes with associated cardiomyopathy in one of them. Two patients harbored a different deletion point that affected the regulatory regions of the dystrophin isoform Dp140. One of them manifested mild muscle symptoms (myalgia) with evident cognitive impairment. The other was asymptomatic but presenting an incipient dilated cardiomyopathy. The last patient showed a proximal insertion of a stretch from the deleted region in the 44-45 intronic breakpoint and manifested a BMD phenotype with an early onset cardiomyopathy. We cannot certainly affirm that the position of the intronic breakpoints in patients with deletion 45-55 is an absolute determinant of the phenotype, nevertheless we found several associations suggestive of some kind of influence.

Multiple calmodulin genes of the Pacific abalone, Haliotis discus hannai (Mollusca: Vetigastropoda: Haliotidae)

ABSTRACTIn this study, we identified four canonical calmodulin genes in the Pacific abalone, Haliotis discus hannai. Their full-length cDNAs were variable in the 5′ and 3′ untranslated regions, but highly similar (91–97%) in the coding region. Each of the genes encoded 149 amino acids, with 93–97% similarity among themselves and 94–98% similarity with human CAM I. There were 54 substitutions distributed unevenly throughout the coding regions, found mostly in the third codon position. Gene structure analysis revealed that each of the calmodulin genes comprised five exons and four introns. The intron positions and phases were identical and there were no introns in the fourth exon. The corresponding introns differed in their sequences and sizes. Expression profiles of nine tissues from abalone revealed that the calmodulin genes were transcribed in common in gill and mantle tissue, but differentially in the other tissues. A phylogenetic analysis based on the amino acid sequences revealed that calmodulin C was the most common isoform in Gastropoda and calmodulin was the most diverged isoform. An in silico analysis of the calmodulin genes identified paralogous genes in other Haliotis species, indicating that gene duplication might have occurred in the last common ancestor of Haliotis.Abbreviations: ORF: open reading frame; RACE: random amplification of cDNA end; TSA: transcriptome shotgun assembly; UTR: untranslated region