Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

SUMMARY: Three myosin heavy chain isoforms with different actin-activated Mg2+-ATPase activities were found in the fast skeletal muscle from carp (Cyprinus carpio) acclimated to 10 and 30°C. The composition of three types of myosin heavy chain was dependent on acclimation temperature,demonstrating the presence of temperature-specific myosin isoforms in carp. Subsequently, the temperature-dependence of the sliding velocity of fluorescent F-actin in myosins isolated from 10°C- and 30°C-acclimated carp was measured. At 8°C, the filament velocity was three times higher for myosin from 10°C- than from 30°C-acclimated fish. Activation energies (Ea) for the sliding velocity of F-actin were 63 and 111 kJ mol-1 for myosins from 10°C- and 30°C-acclimated fish, respectively. Activation energy for actin-activated Mg2+-ATPase activity was 0.46 kJ mol-1 in myosin from 10°C-acclimated fish and 0.54 kJ mol-1 in myosin from 30°C-acclimated fish. The inactivation rate constant(KD) of Ca2+-ATPase was 7.5×10-4s-1 at 30°C for myosin from 10°C-acclimated fish, which was approximately twice that for myosin from 30°C-acclimated fish. It is suggested that these differences in thermostability reflect a more flexible structure of the myosin molecule in cold-acclimated carp, which results in a reduced activation enthalpy for contraction and, hence, a higher sliding velocity at low temperatures. Structural analysis of cDNAs encoding the carp myosin heavy chain demonstrated striking differences in two surface loops of myosin subfragment-1 (S1), loops 1 and 2, between the 10°C and 30°C types, which were predominantly expressed in carp acclimated to 10°C and 30°C, respectively. Chimeric myosins composed of Dictyostelium discoideum myosin backbones with loop sequences of carp S1 heavy chain isoforms demonstrated that the diversity of the loop 2 sequence of carp S1 affected the Vmax of actin-activated Mg2+-ATPase activity.

SUMMARY: Molluscan smooth muscle can maintain tension over extended periods with little energy expenditure, a process termed catch. Catch is thought to be regulated by phosphorylation of a thick filament protein, twitchin, and involves two phosphorylation sites, D1 and D2, close to the N and C termini,respectively. This study was initiated to investigate the role of the D2 site and its phosphorylation in the catch mechanism. A peptide was constructed containing the D2 site and flanking immunoglobulin (Ig) motifs. It was shown that the dephosphorylated peptide, but not the phosphorylated form, bound to both actin and myosin. The binding site on actin was within the sequence L10 to P29. This region also binds to loop 2 of the myosin head. The dephosphorylated peptide linked myosin and F-actin and formed a trimeric complex. Electron microscopy revealed that twitchin is distributed on the surface of the thick filament with an axial periodicity of 36.25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch.

MicroRNAs (miRNAs) in fish have not been as extensively studied as those in mammals. The fish species Takifugu rubripes is an intensively studied model organism whose genome has been sequenced. The T. rubripes genome is approximately eight times smaller than the human genome, but has a similar repertoire of protein-coding genes. Therefore, it is useful for identifying non-coding genes, including miRNA genes. To identify miRNA expression patterns in different organs of T. rubripes and give fundamental information to aid understanding of miRNA populations in this species, we extracted small RNAs from tissues and performed deep sequencing analysis to profile T. rubripes miRNAs. These data will be of assistance in functional studies of miRNAs in T. rubripes. After analyzing a total of 139 million reads, we found miRNA species in nine tissues (fast and slow muscles, heart, eye, brain, intestine, liver, ovaries, and testes). We identified 1420 known miRNAs, many of which were strongly expressed in certain tissues with expression patterns similar to those described for other animals in previous reports. Most miRNAs were expressed in tissues other than the ovaries or testes. However, some miRNA families were highly abundant in the gonads, but expressed only at low levels in somatic tissue, suggesting specific function in germ cells. The most abundant isomiRs (miRNA variants) of many miRNAs had identical sequences in the 5′ region. However, isomiRs of some miRNAs, including fru-miR-462-5p, varied in the 5′ region in some tissues, suggesting that they may target different mRNA transcripts. Longer small RNAs (26–31 nt), which were abundant in the gonads, may be putative piRNAs because of their length and their origin from repetitive elements. Additionally, our data include possible novel classes of small RNAs. We elucidated miRNA expression patterns in various organs of T. rubripes. Most miRNA sequences are conserved in vertebrates, indicating that the basic functions of vertebrate miRNAs share a common evolution. Some miRNA species exhibit different distributions of isomiRs between tissues, suggesting that they have a broad range of functions.