[Show abstract][Hide abstract] ABSTRACT: The infectious salmon anemia virus (ISAV), which belongs to the new genus Isavirus of the Orthomyxoviridae family, is an important pathogen of the salmon farming industry. Indirect immunofluorescence assays carried out with monoclonal antibodies specific for the nucleoprotein (NP) reveal differential staining of sub-cellular compartments in infected cells. Particularly interesting was the staining of the nucleolus, which showed co-localization with nucleolin in CHSE-214, EPC and SHK-1 cells infected with ISAV. These results were confirmed by co-immunoprecipitation studies showing an interaction between NP and nucleolin. In addition, in situ hybridization carried out with probes specific for each of the 8 RNA segments of ISAV showed that the genomic as well as the anti-genomic strands were also localized in the nucleolus. These results suggest a role of the nucleolus in the replication and/or in the packaging of the ISAV genome.
[Show abstract][Hide abstract] ABSTRACT: Cajal bodies (CBs) are small mobile organelles found throughout the nucleoplasm of animal and plant cells. The dynamics of these organelles involves interactions with the nucleolus. The later has been found to play a substantial role in the compensatory response that evolved in eurythermal fish to adapt to the cyclic seasonal habitat changes, i.e., temperature and photoperiod. Contrary to being constitutive, rRNA synthesis is dramatically regulated between summer and winter, thus affecting ribosomal biogenesis which plays a central role in the acclimatization process. To examine whether CBs, up to now, never described in fish, were also sustaining the phenotypic plasticity observed in nuclei of fish undergoing seasonal acclimatization, we identified these organelles both, by transmission electronic microscopy and immunodetection with the marker protein p80-coilin. We found transcripts in all tissues analyzed. Furthermore we assessed that p80-coilin gene expression was always higher in summer-acclimatized fish when compared to that adapted to the cold season, indicating that p80-coilin expression is modulated upon seasonal acclimatization. Concurrently, CBs were more frequently found in summer-acclimatized carp which suggests that the organization of CBs is involved in adaptive processes and contribute to the phenotypic plasticity of fish cell nuclei observed concomitantly with profound reprogramming of nucleolar components and regulation of ribosomal rRNAs.
Biochemical and Biophysical Research Communications 09/2007; 360(1):40-5. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The complex adaptive mechanisms that eurythermal fish have evolved in response to the seasonal changes of the environment include the transduction of the physical parameter variations into neuroendocrine signals. Studies in carp (Cyprinus carpio) have indicated that prolactin (PRL) and growth hormone (GH) expression is associated with acclimatization, suggesting that the pituitary gland is a relevant physiological node in this adaptive process. Also, the distinctive pattern of expression that carp prolactin receptor (PRLr) protein depicts upon seasonal acclimatization supports the hypothesis that PRL and its receptor clearly are involved in the new homeostatic stage that the eurythermal fish needs to survive during the cyclical changes of its habitat. Here, we characterize the first prolactin receptor gene in a teleost and show that its expression is not associated with alternative promoters, unlike in humans and rodents. Using the regulatory region to direct the transcription of green fluorescent protein (GFP) in zebrafish embryos, we mapped the appearance of this hormone receptor during fish development. This is the first report identifying a fish prolactin receptor gene expressing transcript isoforms encoding for short forms of the protein (45 kDa). These have been found in osmoregulatory tissues of the carp and are regulated in connection with the seasonal acclimatization of the fish.
Journal of Cellular Biochemistry 04/2007; 100(4):970-80. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The eurythermal fish carp (Cyprinus carpio) adjusts to the seasonal changes in the temperature and photoperiod of its habitat through diverse cellular and molecular mechanisms. We have observed that ribosomal biogenesis is modulated during the acclimatization process and correlates with profound phenotypic changes, reflecting a seasonal-dependent ultrastructural appearance of the nucleolar components. Previous studies using classical techniques showed that in winter-adapted carp the nucleolus appears to be segregated. In the present work, we have reassessed the nucleolar ultrastructural organization of the carp in summer- and winter-adapted fish by using more specific cytochemical and immunocytological techniques.
The acetylation method provided evidence that the nucleolar organization is different between winter- and summer-adapted carp. In winter-adapted fish the fibrillar component appears as a unique mass surrounded by several granular caps, whereas in summer-adapted carp the fibrillar component forms few cordons surrounded by granular masses. The nucleolar structure and distribution of the condensed chromatin observed varies upon seasonal acclimatization. In winter the nucleolar chromatin is densely packed in masses that surround the nucleolus, whereas during summer it displays a rather looser organization formed by filaments that not only surround the nucleolus, but also go through the nucleolar body. Using the TdT (terminal deoxynucleotidyl transferase)-immunogold labelling technique, we detected condensed and decondensed nucleolar chromatin, and found some labelling of fibrillar components in both seasons. When liver tissue from summer-adapted carp was treated with AMD (actinomycin D), we observed that the rearrangement of the nucleolar components and condensed chromatin were similar to that found in winter-adapted fish, with differences in the distribution of the perinucleolar chromatin.
The acetylation and TdT-immunogold labelling experiments indicated that the rearrangement of the nucleolar components of winter-adapted carp is very similar to the AMD-treated summer-adapted carp nucleolus, with the latter representing the repression of the ribosomal biogenesis that occurs during the cold season. Nevertheless, the distribution of the condensed perinucleolar chromatin in winter-adapted carp compared with AMD-treated cells suggests that the transcription of rRNA genes in winter-adapted fish is less strongly inhibited and does not lead to the classical segregation of the nucleolus of that described after AMD treatment. In addition, we have confirmed that carp hepatocyte nucleoli comprise only two main structural compartments: a fibrillar component and a granular component. Fibrillar centres were not observed.
Biology of the Cell 09/2006; 98(8):457-63. · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protein nucleolin, functionally involved in the main steps of ribosome biogenesis, is codified by a single copy gene in mammals. Here we report that at least three different genes codify for this protein in carp fish (Cyprinus carpio). This is the first description of the genomic organization of nucleolin in a teleost. The carp nucleolin gene includes 8.8 kb and contains 16 exons. Promoter cis regulatory elements are similar to constitutive genes, i.e., a putative TATA box, three G/C boxes, and three pyrimidine-rich boxes. As in other species, carp nucleolin gene introns host three snoRNA codifying sequences: U23 from the H/ACA family and two C/D box snoRNAs, U20 and U82. Both U20 and U82 span a complementary sequence with carp 18S rRNA. Additionally, we identified two cDNAs coding for nucleolin, confirming the existence of several nucleolin genes in carp. Amino acid-derived sequence from carp cDNAs differ from mammal protein because they span additional acidic domains at the amino end, whose functional significance remains unclear. We performed amino acid sequence comparison and phylogenetic analyses showing that the three isoforms of carp nucleolin, which we describe herein, cluster in two groups. cNUC1 probably diverges from cNUC2 and cNUC3 as result of ancestral fish-specific genome duplication, indeed C. carpio is a tetraploid fish.
Biological research 02/2006; 39(2):353-65. · 1.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleolar segregation is the most striking cellular phenotypic feature of cold-acclimatized carp and depicts the cyclical reprogramming that the physiology of the fish undergoes between summer and winter, where a clear differential expression of some nucleolar related genes occurs. We characterized carp nucleolin, a nucleolar protein involved in multiple steps of ribosome biogenesis, and evaluated its expression upon fish acclimatization. We show that the carp cDNA deduced amino acid sequence exhibits the same tripartite structural organization found in other species. Nevertheless, we observed that nucleolin mRNA expression was strongly induced in the cold-adapted carp as was the nuclear protein content, assessed by immunocytochemistry in liver sections. The physiological up-regulation of nucleolin in the cold-acclimatized carp, where rRNA transcription and processing are depressed concomitantly with the nucleolus segregation, is consistent with the notion that nucleolin plays a fundamental role in repressing rRNA synthesis.
Biochemical and Biophysical Research Communications 02/2003; 301(1):152-8. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cascade of mechanisms involving changes in gene expression are substantial to shape the adaptive responses that a eurythermal fish requires upon environmental changes in its habitat. We have previously shown that the cyclical reprogramming of rRNA transcription is a remarkable feature in carp under seasonal acclimatization. Using in situ hybridization and competitive RT-PCR we found significant differences in beta-actin transcripts, generally accepted to be coded by a typical housekeeping gene, in tissues from summer- and winter-acclimatized carp. The physiological differential beta-actin transcription herein reported places us on the alert for the reference genes estimated to be constitutive to quantitatively assess gene transcripts.
Journal of Cellular Biochemistry 11/2000; 80(2):223-8. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A second Pit-1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon-intron structure was determined, and reverse transcription-polymerase chain reaction (RT-PCR) experiments suggest that only one Pit-1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit-1 gene expression was studied in summer- and winter-acclimatized carp. Quantitative RT-PCR analysis revealed a clear increase of Pit-1 mRNA in the pituitaries from summer-acclimatized carp compared with the winter-adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5'-transactivating region of carp Pit-1 depicted a significantly higher Pit-1 mRNA level in the rostral pars distalis of the summer-acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell- and temporal-specific transcription of Pit-1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat.
Journal of Cellular Biochemistry 01/2000; 75(4):598-609. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We isolated a clone comprising four exons of the carp Pit-1 gene. Using synthetic oligonucleotide probes derived from the carp Pit-1 sequence Pit-1 expression was assessed by in situ hybridization in pituitary sections from summer- and winter-acclimatized carp. Semiquantitative analyses of the hybridization signals revealed a significant higher Pit-1 expression in the proximal pars distalis (PPD) and pars intermedia (PI) of the pituitary glands from summer-acclimatized carp, compared to the winter-acclimatized fish. In both adaptive states, relative to the PPD and PI, only a basal Pit-1 expression was detected in the rostral pars distalis. Thus, during seasonal acclimatization of an eurythermal fish, Pit-1 seems to be involved in the mechanisms that underlie the compensatory response.
Biochemistry and molecular biology international 08/1998; 45(4):813-21.
[Show abstract][Hide abstract] ABSTRACT: In the carp, nucleolar reorganization is one of the most remarkable features of the cellular adjustment that occurs upon seasonal acclimatization. To assess if the level of cellular expression of a specific rRNA is maintained between both seasonal adaptive states, we isolated a carp 5.8S rRNA gene. Sequence analyses showed its high conservation during evolution. The level of expression of 5.8S rRNA was determined by in situ hybridization assays in pituitary sections from summer- and winter-acclimatized carp. The quantification of the positive hybridization signals demonstrated a statistically significant difference between the acclimatization states. Transcription of 5.8S rRNA was considerably higher in summer- than in winter-acclimatized carp. These results indicate that the level of rRNA expression in eurythermal fish is not comparable between seasons and suggest that rRNA genes may be not constitutive. Thus, rRNA transcription should be used with caution as a reference to quantitate transcripts that are modulated as part of the acclimatization process in eurythermal fish.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 01/1997;
[Show abstract][Hide abstract] ABSTRACT: To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.
World Journal of Microbiology and Biotechnology 03/1994; 10(2):149-53. · 1.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: By means of a one-step one-tube extraction from less than 1 mg of tissue it is possible to identify, via the polymerase chain reaction, Renibacterium salmoninarum in salmon with bacterial kidney disease. A 149-bp DNA sequence unique to R. salmoninarum was specifically amplified and its nature confirmed by Southern hybridization using a non-isotopically labelled probe. The sensitivity of the approach allowed the detection of 22 R. salmoninarum cells. The procedure was successfully applied in the identification of the causative agent of bacterial kidney disease in kidney tissue from infected fishes.
[Show abstract][Hide abstract] ABSTRACT: During seasonal acclimatization of eurythermal fish, the nucleolus of the hepatocyte undergoes ultrastructural reprogramming. In winter acclimatized carp, the nucleolar components are segregated, a condition that suggests a decreased transcription of rRNA. The same nucleolar reorganization was observed when pituitary cells from winter- and summer-acclimatized carp were examined. In situ analyses of nucleolar RNA revealed a marked lowering of RNA content in the segregated nucleoli. Accordingly, in vitro synthesis of RNA was shown to be significantly lower in pituitary tissue from cold-acclimatized fish where precursor accumulated. Conversely, in pituitary tissue from summer-adapted fish the rate and extent of synthesis and of rRNA processing was notably higher. The involvement of pre-rRNA processing events during seasonal acclimatization was corroborated by the strong differences of U3 RNA content detected by in situ hybridization in pituitary cells from summer- and winter-fish. When RNA polymerase I activity from both acclimatized states were assayed, no differences were detected. Thus, it appears that in fish RNA polymerase I itself does not play an important role in the control of nucleolar gene expression and the nucleolar gene expression reprogramming that the seasonal rearrangement represents might involve, among the many nucleolar-specific proteins, transcription factors.
Cellular & molecular biology research 02/1993; 39(7):665-74.