Establishment and characterisation of single cell-derived embryonic stem cell lines from the gilthead seabream, Sparus aurata.

An important bottleneck in fish aquaculture research is the supply and maintenance of embryos, larvae, juvenile and adult specimens. In this context, cell lines represent alternative experimental models for in vitro studies that complement in vivo assays. This allows us to perform easier experimental design and sampling and avoid the sacrifice of animals. Embryonic stem (ES) cell lines have attracted increasing attention because they have the capability to proliferate indefinitely and could be differentiated into any cell type of the organism. To minimise cell heterogeneity and increase uniformity of in vitro studies results, in this manuscript we report the development and characterisation of two single cell-derived ES cell lines (monoclonal) from the morula stage embryos of the gilthead seabream, Sparus aurata, named as SAEC-A3 and SAEC-H7. Both cell lines have been passaged for over 100 times, indicating the establishment of long-term, immortalised ES cell cultures. Sequence analyses confirmed the seabream origin of the cell lines, and growth analyses evidenced their high viability and proliferating activity, particularly in culture medium supplemented with 10-15% fetal bovine serum and 22 °C. Both cell lines showed the ability to generate embryoid bodies and show different sensitivity and response to all-trans retinoic acid. The analysis of epithelial (col1α1) and neuronal (sox3) markers in differentiated cultures revealed that SAEC-A3 tended to differentiate towards epithelial-like cells whereas SAEC-H7 tended to differentiate towards neuronal-like cells. Both cell lines were efficiently transfected with pDsRed2-ER and/or pEGFP-N1 plasmids, indicating that they could represent useful biotechnological tools. Daily expression of pcna showed significant expression rhythms, with maximum levels of cell proliferation during the day-night transition. Currently, these cell lines are being successfully used as experimental models for the study of cellular metabolism, physiology and rhythms as well as for toxicological, pharmacological and gene expression analyses.

Somatotropic regulation of fish growth and adiposity: growth hormone (GH) and somatolactin (SL) relationship.

Growth hormone (GH) and insulin-like growth factors (IGFs) play a major role in fish development and metabolism, and several studies have allowed discernment of a complex and tissue-specific collection of salmonid IGF-I transcripts (Ea-4, Ea-3, Ea-2, Ea-1), which are the result of the alternative splicing of the E-domain region. However, the pattern of IGF-I expression is different in non-salmonid fish, and only one or two transcripts (Ea-4, Ea-2) have been detected in hepatic and extrahepatic tissues of common carp, barramundi, black sea bream and gilthead sea bream. Despite this, when comparisons are made within Mediterranean fish species (European sea bass, common dentex and gilthead sea bream), plasma IGF-I levels are consistent with fish species differences in growth rates. Changes of growth rates, and plasma IGF-I and GH levels are also found in response to changes in diet composition and ration size, which may serve to assess the suitability of feeding regimes in aquaculture practice. Regulation of plasma somatolactin (SL) levels is also examined in gilthead sea bream, and the resulting plasma SL profile differs from that of GH. Thus, in contrast to GH, plasma SL levels augment with the increase of ration size and fish size (advancement of age). A transient increase in plasma SL levels is also found in short-term fasted fish, and this fish peptide may act as an anti-obesity hormone helping to expedite growth-reproductive processes following replenishment of fat stores, and/or mediate the adaptation to fasting until the lipolytic action of GH and/or other endocrine factors is fully accomplished. This agrees with the known increase of plasma SL levels during acute stress and exhaustive exercise. However, a causal link between SL and energy mobilisation (lipid metabolism) remains to be established, and further research is needed to determine the extent to which SL and GH act in a complementary manner to make available metabolic fuels and to regulate body fat mass and feeding behaviour.

Genomic structure and chromosome location of the human gene encoding the zinc finger autoantigen ZNF330.

We have recently described a novel zinc finger cDNA, ZNF330, which was immunologically characterized as a new human autoantigen, highly conserved during evolution from nematodes to humans. The protein was found at the nucleolus and the cytoplasm in interphase and transiently associates with centromeres in mitosis as determined by immunofluorescence analysis. We now describe that the association of ZNF330 with the nucleolus but not with the cytoplasm is RNA-dependent as shown by RNAse treatment of fixed culture cells, since ZNF330 localization was unaffected by DNAse treatment. We also report the cloning, structural organization and chromosome location of the human ZNF330 gene. The gene is comprised of 10 exons and spans approximately 16 kb of genomic DNA. The conserved residues forming nine CXXC motifs are contained in exons 3 to 9. Several major transcription initiation sites were located 126, 124 and 121 bp upstream of the translation initiation codon ATG, as determined by primer extension analysis. The human ZNF330 gene was mapped by FISH to chromosome 4q31.1-->q31.2, the site of the FRA4C locus previously described as a common fragile site for acquired chromosome instability in humans.

The accuracy of segregation of human mini-chromosomes varies in different vertebrate cell lines, correlates with the extent of centromere formation and provides evidence for a trans-acting centromere maintenance activity.

We show that the accuracy of mitotic segregation of three engineered, mapped human mini-chromosomes differs between human, mouse and chicken cell lines. We have studied the cause of these differences by analysing the extent of centromere formation on one mini-chromosome immunocytochemically. In human and chicken cell lines the mini-chromosomes segregate accurately and form centromeres but in one mouse cell line centromere formation is undetectable and mitotic segregation is inaccurate. These results indicate that the centromere is maintained by an activity that functions in trans and varies either in amount or specificity between different cells. Structurally defined mini-chromosomes may allow this activity to be studied.

Gene structure, chromosomal localization and immunolocalization of chicken centromere proteins CENP-C and ZW10.

We determined the genomic structures and complete sequences of the coding regions of the chicken CENP-C and ZW10 genes. These two genes encode proteins that are thought to be involved in maintaining the fidelity of chromosome segregation. The chicken CENP-C gene is 30 kb in length and contains 19 exons. The chicken ZW10 gene spans 10 kb and contains 15 exons. The 5'-untranslated regions of these genes contain several binding sites for transcription factors such as Sp-1, E2F, p300, and members of the GATA family. By fluorescence in situ hybridization (FISH) analysis, the CENP-C was mapped to chromosome 4 and the ZW10 gene was mapped to a microchromosome. Antibodies against the chicken ZW10 protein revealed a cell cycle-dependent staining pattern in DT40 cells. ZW10 protein was distributed throughout the cytoplasm of DT40 cells during interphase. In most metaphase cells, ZW10 proteins appeared equally divided between the centromere and the spindle apparatus. During anaphase, chicken ZW10 proteins were no longer localized near chromosomes or the mitotic apparatus but were present diffusely in the cytoplasm.

Molecular analysis of the 5' region of human ribosomal transcription factor UBF.

Upstream binding factor, UBF, is a nucleolar autoantigen involved in the transcription of ribosomal DNA genes. Previously, human genomic clones served to demonstrate that an alternative pre-mRNA splicing of a single gene is used to form UBF1 and UBF2. Here, to complete characterizing the 5'end genomic organization of this nucleolar transcription factor, lambda clones containing the human UBF gene were isolated from a human placenta genomic library using a hamster UBF cDNA as a probe. An additional PCR product was isolated from HeLa genomic DNA to cover the first translated 60 nt of the gene containing the ATG initiation codon. We have also determined the transcription start site of the gene by primer extension analysis at nt 188 upstream from the start ATG codon. It served first, to identify an untranslated initial exon on the UBF gene covering the first 121 nt of human UBF cDNA, and also to establish the sequence of the proximal promoter. The human UBF promoter lacks a TATA and CAAT boxes but contains multiple binding sites for SP1, AP1, AP2, TFIID, NF-1 and a single site for NFAT-1. Consequently we have defined the first five exons of the human UBF gene covering 7.5kb. The complete gene now consists of 20 exons with intervening sequences and spans approximately 15kb of DNA.

Human mini-chromosomes with minimal centromeres.

We have introduced a 6.5 Mb human mini-chromosome with a complex centromere structure into DT40 cells and have used sequence targeting and telomere-directed chromosome breakage to dissect the sequence requirements for centromere function. These experiments proved that a vertebrate centromere with two blocks of functional alphoid DNA separated by 2.5 Mb can exist as a stable structure in some but not all vertebrate cells. Further experiments indicated that recovery of chromosomes with less than approximately 100 kb of alphoid DNA is very inefficient, suggesting that a functional centromere requires a minimum of approximately 100 kb of alphoid DNA. Mini-chromosomes with minimal centromeres segregate accurately in some but not all vertebrate cells and should be useful for the detection of sequence-specific factors required for vertebrate centromere maintenance.

CENP-C is necessary but not sufficient to induce formation of a functional centromere.

CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase-anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase-anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle.

Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay.

A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respectively. Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 micrograms/ml), similar to that reported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.

Cloning and expression of somatolactin, a pituitary hormone related to growth hormone and prolactin from gilthead seabream, Sparus aurata.

A pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of the teleost pituitary. The function of this protein is uncertain. Clones coding for SL were isolated and sequenced from a gilthead seabream pituitary cDNA expression library. The nucleotide sequence of the larger cDNA isolated was 1.5 kb containing a 0.8-kb 3'-untranslated region and two potential polyadenylation signals (AATAAA). The mature polypeptide is composed of 207 amino acids, and a signal peptide of 24 residues was also found in the SL precursor. A potential N-glycosylation site Asn-Lys-Thr was identified in gilthead seabream SL. A comparison of the SL amino acid sequences of several fishes indicated that seven cysteine residues are characteristically present in all the SLs so far isolated. Six of those residues are present in homologous positions in SL and GH Sparus aurata proteins. SL and GH from S. aurata showed a 43% homology at the nucleotide level and 22% identity at the amino acid level. Expression of recombinant SL (rSL) in Escherichia coli and isolation from inclusion bodies led to a monomeric form of SL identical in electrophoretic mobility to one of the two forms of the native SL secreted from gilthead seabream pituitaries cultured in vitro. Further, a native glycosylated modified SL secreted in vitro as shown by N-glycosidase treatment was identified. Specific anti-SL antibodies that discriminate well against gilthead sea-bream GH and PRL in immunoblotting were also raised against rSL.

Growth hormone as a function of age and dietary protein: energy ratio in a marine teleost, the gilthead sea bream (Sparus aurata).

We examined in a factorial design the effect of dietary protein (45%, 52% and 60%) and lipids (8%, 12%, 17%) on growth performance and circulating growth hormone (GH) levels of fingerling sea bream (5-month-old) fed to satiation with self-feeders. Daily weight gain (2.6-2.9%) and feed gain ratio (1.1-1.3) of fish fed high protein-low lipid diets were comparable to those found in fast growing strains of rainbow trout. However, increasing hyperphagia in association with the decrease of daily weight gain and feed conversion efficiency were found with the decrease of dietary protein:energy ratio. This growth impairment was linked to increased concentrations of circulating GH, which would exacerbate glucose and lipid intolerance. We consider the elevated concentration of circulating GH to be a risk factor leading to some state of metabolic starvation, in which feeding behavior and feed conversion efficiency are largely altered. From our results, it can be also concluded that circulating and pituitary GH availability decreases progressively from 1- to 3-year-old fish. This blunted GH synthesis and release is discussed in relation to age decrease in the optimum dietary protein:energy ratio.

Cloning and sequencing of the genes encoding the hamster ribosomal transcription factors UBF1 and UBF2.

The RNA polymerase I transcription factor, UBF, belongs to a family of high-mobility-group DNA-binding proteins. Here, a human autoantibody reactive with the nucleolus organizer regions (NOR) was used to select cDNA clones encoding the hamster transcription factors, UBF1 and UBF2. Comparison at the nucleotide level showed a high degree of homology with other mammalian upstream binding factors (UBF) already identified. The deduced amino acid sequences are identical for both UBF1 and UBF2, except for a 37 amino acid insertion found in UBF1. This insertion is completely conserved among mammalian UBF1 which indicates a putative role of this region on the function of this transcript.

Bacterial production and purification of the fish pituitary hormone somatolactin.

Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. A Solea senegalensis somatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL in E. coli BL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization-renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secreted in vitro by cultured pituitaries from sole. Another native SL expressed in S. senegalensis represented a glycosylated modified hormone as shown by N-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions.

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.

Cloning, expression, and characterization of a recombinant gilthead seabream growth hormone.

cDNA clones coding for the gilthead seabream (Sparus aurata) growth hormone (sbGH) were isolated from a pituitary expression library using a flounder cDNA probe. The nucleotide sequence of a GH cDNA clone containing an insert of 896 nucleotides was determined. The cDNA encoded a polypeptide of 204 amino acids including a signal peptide of 17 amino acids and contained a 5' and a 3' untranslated region of 48 and 233 nucleotides, respectively. The mRNA determined by Northern blot was approximately 1 kb. Amino acid sequence homologies of 97.1% with red seabream GH, 88.9% with the tuna GH, and 67% with the coho salmon GH was found. Transient expression of a sbGH cDNA was done in HeLa cells by induction with a vaccinia virus system, and the expressed GH was detected by immunofluorescence and immunoprecipitation with a specific antibody to the native sbGH. The sbGH cDNA was expressed in Escherichia coli by using the pGEX-3X and the pET-3a expression systems. The recombinant sbGH expressed in the pET-3a system was similar, if not identical, to the native hormone when analyzed by homologous radioimmunoassay and receptor binding assay.

Cloning of a somatolactin-encoding cDNA from sole (Solea senegalensis).

From a Solea senegalensis cDNA expression library, clones encoding somatolactin (SL), a new pituitary hormone belonging to the growth hormone/prolactin family, were isolated and analyzed. Northern blot analysis showed a unique 1.0-kb mRNA species. The sole SL 778-bp cDNA encoded full-size S. senegalensis SL (ssSL) (230 amino acids), including seven Cys and two potential glycosylation sites. A consensus polyadenylation signal, AATAAA, was found. Protein homology and DNA sequence alignment of SL cDNAs from other evolutionarily distant marine fishes suggest that the SL sequence is highly conserved.

Cloning of the sole (Solea senegalensis) growth hormone-encoding cDNA.

We report here the complete nucleotide (nt) sequence of a cDNA clone encoding Solea senegalensis growth hormone (sGH) isolated from an expression library prepared from sole pituitary gland poly(A)+RNA. The library was screened using a flounder GH cDNA. The cDNA sequence containing an insert of 769 nt was found to encode a polypeptide of 203 amino acids (aa), including a signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 17 and 119-nt long, respectively. Northern blot hybridization detected a 0.9-kb RNA species. The sGH cDNA sequence shows homologies of 80.9, 76.9, 73.8 and 64.2% with the GH of tuna, gilthead seabream, flounder and rainbow trout.

Immunochemical studies of histone H5 from Halobatrachus didactylus.

1. Histone H5 from Halobatrachus didactylus was isolated by using perchloric acid (PCA) extraction of fish liver nuclei and trichloroacetic acid (TCA) precipitation. 2. A polyclonal antiserum was generated by immunizing rabbits with the antigen purified from SDS-PAGE. 3. By immunofluorescence the serum stains erythrocyte nuclei from H. didactylus but it does not react with mammalian cells. 4. By Western blotting, the anti-H5 antibody reacts with the isolated antigen at high titers. 5. Digestion of histone H5 with pepsin and cyanogen bromide suggests that the epitopes are located in the globular and C-terminal region of the H5 molecule excluding the N-terminal.