Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup).

HSP90 proteins are chaperones that play a pivotal role in controlling multiple regulatory pathways such as stress defense, hormone signalling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, two cDNAs encoding for cytosolic HSP90, referred to as HSP90AA and HSP90AB, have been sequenced. Main features and sequence identities with other fish and mammals are described. Phylogenetic analysis grouped both genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, HSP90AB was constitutively expressed with lower transcript levels in skeletal muscle. In contrast, HSP90AA was mainly expressed in heart, skeletal muscle and skin. During metamorphosis, HSP90AB mRNA levels did not change whereas HSP90AA transcripts decreased significantly at the beginning of metamorphosis with the lowest mRNA levels at the metamorphosis climax. Due to the role of thyroid hormones (THs) on sole metamorphosis, the transcriptional regulation of HSP90 genes by THs was evaluated. Larvae exposed to the goitrogen thiourea (TU) exhibited higher HSP90AA mRNA levels than untreated control. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels with respect to the untreated control. Unlike HSP90AA, the transcript levels of HSP90AB did not vary under any treatments. The response of both HSP90 genes to thermal stress in post-metamorphic individuals was also studied. A heat shock treatment (+7.9 degrees C for 1 h) rapidly activated HSP90AA (but not HSP90AB) transcription, reaching a peak after 30 min and declining expression levels progressively in the following 24 h. No significant changes in HSP90AA or HSP90AB transcript levels after a cold shock (-10 degrees C for 1 h) were observed. Overall, these results demonstrate that HSP90AA transcription is down-regulated by THs and up-regulated after a heat shock in Senegalese sole.

Isolation and characterization of polymorphic microsatellites for the bastard sole (Microchirus azevia).

The bastard sole (Microchirus azevia) is a species of commercial interest in Spain. Nevertheless, little information is currently available about the genetic characteristics of wild populations. In this survey, we have developed eight new microsatellites using an enriched genome library protocol. Primers were screened on a total of 54 individuals from two wild populations (Mediterranean and Atlantic) from the south coast of Spain, revealing six to 18 alleles per locus with expected heterozygosities ranging from 0.51 to 0.94. These markers can potentially be useful tools for use in population genetic studies.

Molecular characterization and gene expression of six trypsinogens in the flatfish Senegalese sole (Solea senegalensis Kaup) during larval development and in tissues.

The application of large-scale genomics to Senegalese sole (Solea senegalensis) has allowed for the identification of six different trypsinogen genes. The catalytic triad (His-57, Asp-102, and Ser-195) and other residues required for trypsin functionality were conserved across all trypsinogens. Sequence identities, charges and phylogenetic analysis allowed them to be classified into three groups: group I or anionic trypsinogens (ssetryp1a, ssetryp1b and ssetryp1c), group II or cationic trypsinogen (ssetryp2) and group III or psychrophilic trypsinogens (ssetryp3 and ssetrypY). The expression profiles of these genes were studied in juvenile tissues and during larval development using a real-time PCR approach. In juvenile fish, trypsinogens were expressed mainly in the intestine. Transcripts of ssetryp1c were the highest in all tissues except in brain where those of ssetryp2 were the most abundant. During larval development, ssetryp1 variants and ssetryp2 transcript levels increased from 2 to 6 days after hatching, and decreased thereafter. In contrast, transcripts of group III trypsinogens increased slightly or not significantly in premetamorphosis and decreased at metamorphosis. The expression levels ssetryp3 and ssetrypY were the lowest in larvae (from 172- to 1391-fold lower than ssetryp1 and ssetryp2). In contrast, they were expressed at a similar level as ssetryp2, although lower than ssetryp1, in juvenile tissues.

Phylogenetic differentiation between Atlantic Scomber colias and Pacific Scomber japonicus based on nuclear DNA sequences.

In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus, and S. japonicus. Yet, some fish taxonomists have recently recognized Scomber colias, inhabiting the Atlantic Ocean, as a separate species from S. japonicus, distributed in the Pacific Ocean. Such proposal was based on significant mitochondrial DNA divergence as well as great phenotypic variation among individuals from these two ocean basins. However, in the absence of nuclear DNA data this issue remains still controversial. In this study, a phylogenetic analysis of nuclear 5S rDNA sequences was performed. A total of 30 individuals of S. colias collected in the Atlantic and 34 specimens of S. japonicus from the Pacific were characterized. Moreover, nine individuals of Pacific S. australasicus and eight of Atlantic S. scombrus were included. Maximum likelihood, maximum parsimony, and neighbor-joining analyses revealed the presence of two well-supported distinct clades corresponding to S. colias and S. japonicus, respectively. Altogether, morphologic and genetic data are in agreement with the recognition of two different species, S. colias in the Atlantic, and S. japonicus in the Pacific.