Dynamic expression pattern of the growth hormone receptor during early development of the Chilean flounder.

The entire cDNA sequence of the growth hormone receptor (GHR) of the Chilean flounder (Paralichthys. adspersus) was cloned by RT-PCR and RNA ligase rapid amplification of 5' and 3'ends. The deduced amino acid sequence contains 641 residues and codes for the GHR1 form. The receptor includes all the structural domains and motifs responsible for its interaction with the growth hormone and growth signal transduction. Sequence comparison revealed 95 and 88% identity with other flat fish such as the Japanese flounder and Atlantic halibut respectively, but decreased to 41% with the GHR of other teleosts such as salmon. In addition we performed a phylogenetic analysis of this receptor in teleosts. RT-PCR experiments were performed to study the expression of GHR1 mRNA in different tissues of juvenile fish, detecting the transcripts in all tissues investigated with higher expressions in the liver, brain and gonads. Additionally, using whole-mount in situ hybridization in larvae stages, we observed an on and off GHR1 mRNA expression pattern. This novel finding evidences that during early development of a teleost, GHR1 is transiently expressed in somites, a source of muscle, bone and spinal chord precursors cells, suggesting a relevant role of GH in fish development. GHR1 was also temporally detected in the notochord, intestines, brain and retinal layers, before its ubiquitous establishment.

Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus).

The full length cDNA sequence of the myostatin gene was cloned from a teleostean fish, the Chilean flounder (Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5'- and 3'-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including the structural domains responsible for its biological activity. Amino acid sequence comparison revealed high sequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Gene expression analysis showed that cfMSTN mRNA is present in a wide variety of tissues in juvenile fish. In addition, we assessed the spatial expression pattern of the MSTN mRNA during embryos and larval stages through whole mount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days post fertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye, were positive for MSTN mRNA. Our results contribute to the knowledge of the MSTN system in larval and juvenile stages; in particular the strong expression observed in the notochord suggests that MSTN, in synchronization with positive growth signals, may play an important role in the control of the development of larvae somites.

Seasonal environmental changes regulate the expression of the histone variant macroH2A in an eurythermal fish.

Adaptation to cold and warm conditions requires dramatic change in gene expression. The acclimatization process of the common carp Cyprinus carpio L. in its natural habitat has been used to study how organisms respond to natural environmental changes. At the cellular level, adaptation to cold condition is accompanied by a dramatic alteration in nucleolar structure and a down regulation of the expression of ribosomal genes. We show that the enrichment of condensed chromatin in winter adapted cells is not correlated with an increase of the heterochromatin marker trimethyl and monomethyl K20H4. However, the expression of the tri methyl K4 H3 and of the variant histone macroH2A is significantly increased during the winter season together with a hypermethylation of CpG residues. Taking into account the properties of macroH2A toward chromatin structure and dynamics and its role in gene repression our data suggest that the increased expression of macroH2A and the hypermethylation of DNA which occurs upon winter-acclimatization plays a major role for the reorganization of chromatin structure and the regulation of gene expression during the physiological adaptation to a colder environment.

Differential profile of ultrasound findings associated with malignancy in mixed and solid thyroid nodules in an elderly female population.

Objective. Ultrasonographic characteristics are associated with thyroid malignancy. Our aim was to compare the diagnostic value of ultrasound features in the detection of thyroid malignancy in both solid and mixed nodules. Methods. We prospectively studied female patients (≥50 years) referred to ultrasound-guided fine needle aspiration biopsy. Ultrasound features considered suspicious were hypoechogenicity, microcalcifications, irregular margins, high anteroposterior (AP)/axial-ratio, and absent halo. Associations were separately assessed in mixed and solid nodules. Results. In a group of 504 elderly female patients (age = 69 ± 8 years), the frequency of malignant cytology was 6%. Thirty-one percent of nodules were mixed and 60% were solid. The rate of malignant cytology was similar for mixed and solid nodules (7.4 versus 5.8%, P: 0.56). While in mixed nodules none of the ultrasound characteristics were associated with malignant cytology, in solid nodules irregular margins and microcalcifications were significant (all P < 0.05). The combination of irregular margins and/or microcalcifications significantly increased the association with malignant cytology only in solid nodules (OR: 2.76 (95% CI: 1.25-6.10), P: 0.012). Conclusions. Ultrasound features were of poor diagnostic value in mixed nodules, which harbored malignant lesions as often as solid nodules. Our findings challenge the recommended minimal size for ultrasound-guided fine needle aspiration biopsy in mixed nodules.

Gene structure of the carp fish ribosomal protein L41: seasonally regulated expression.

The seasonal acclimatization of the carp fish demands physiological compensatory responses. The process involves profound nucleolar adjustments and remarkable changes in rRNA synthesis, which affect ribosomal biogenesis. We have documented that protein kinase CK2, whose activity is related to ribosomal protein L41 and the regulation of rRNA synthesis, was expressed in notably higher amounts in summer-acclimatized carp compared to the cold-season adapted fish. Thus, we approached the study of the functional genomics of carp L41 protein. We report the first cloning of a fish L41 gene encoding the highly conserved 25 amino acids, including approximately 1700 bp regulatory upstream region and the 3(') polyadenylation signal, plus the isolation and characterization of two different L41 cDNAs. We found a clear differential expression of L41, which follows the same pattern as protein kinase CK2beta that transcribes at higher levels in the summer-acclimatized carp than it does in the winter-adapted fish.

Genomic organization of the rDNA cistron of the teleost fish Cyprinus carpio.

The seasonal adaptation of the teleost Cyprinus carpio to the cyclical changes of its habitat demands physiological compensatory responses. The process involves profound nucleolar adjustments and remarkable changes in rRNA synthesis, which affects ribosomal biosynthesis. In this context, we have demonstrated that the synthesis of several proteins involved in ribosomal biogenesis as protein kinase CK2, ribosomal protein L41 and nucleolin, as well as U3 snoRNP, are differentially regulated in summer-acclimatized carp compared to the cold-season adapted fish. To understand the mechanisms involved in the seasonal regulation of rRNA gene transcription, we have been studying the carp rDNA cistron structure. Because the cis-elements that regulate the expression of the tandem organized ribosomal genes are located in the non-transcribed intergenic spacer (IGS), we analyzed the primary structure of the carp rDNA gene IGS. The gene organization is similar to that described from other vertebrate species, including numerous repetitive sequences, the transcription start site, and some potential cis-elements such as ribosomal enhancers, proximal terminator and transcriptional terminators. Ribosomal DNA is a remarkable case of gene duplication and has been used as a model to test the concerted evolution theory. We performed sequence comparison analyses of 18S rRNA coding sequences from carp with different species, data with which an unrooted phylogram was constructed.

In situ hybridization of somatolactin transcripts in the pituitary glands from acclimatized carp (Cyprinus carpio)

We isolated and cloned a carp somatolactin SL DNA fragment, of which 78 per cent of the nucleotides were identical to the corresponding salmon SL sequence. The results obtained upon Northern blot hybridization of carp pituitary RNA allowed the identification of two transcripts as described for other fish. When the content of SL transcripts in pituitary sections from summer- and winter-acclimatized carp was quantified by in situ hybridization assays, we found no significant differences between the two seasons. In salmonids, plasma SL reaches higher levels in summer than in winter in synchrony with the water temperature cycle; in the eurythermal carp, however, the complex adaptive responses imposed by seasonal environmental changes do not seem to include the regulation of the somatolactin detected with the probe used at the transcriptional level in pituitary glands.

Genomic organization of the rDNA cistron of the teleost fish Cyprinus carpio

The seasonal adaptation of the teleost Cyprinus carpio to the cyclical changes of its habitat demands physiological compensatory responses. The process involves profound nucleolar adjustments and remarkable changes in rRNA synthesis, which affects ribosomal biosynthesis. In this context, we have demonstrated that the synthesis of several proteins involved in ribosomal biogenesis as protein kinase CK2, ribosomal protein L41 and nucleolin, as well as U3 snoRNP, are differentially regulated in summer-acclimatized carp compared to the cold-season adapted fish. To understand the mechanisms involved in the seasonal regulation of rRNA gene transcription, we have been studying the carp rDNA cistron structure. Because the cis-elements that regulate the expression of the tandem organized ribosomal genes are located in the non-transcribed intergenic spacer (IGS), we analyzed the primary structure of the carp rDNA gene IGS. The gene organization is similar to that described from other vertebrate species, including numerous repetitive sequences, the transcription start site, and some potential cis-elements such as ribosomal enhancers, proximal terminator and transcriptional terminators. Ribosomal DNA is a remarkable case of gene duplication and has been used as a model to test the concerted evolution theory. We performed sequence comparison analyses of 18S rRNA coding sequences from carp with different species, data with which an unrooted phylogram was constructed

Uso de sondas no radiactivas para la determinación del número de copias de genes y para el diagnóstico de enfermedades moleculares / Use of non radioactive probes to assess gene copy numbers and for the diagnosis of molecular diseases

Se sintetizó un análogo fotoactivable de biotina, el que se utilizó para marcar sondas de ácidos nucleicos. La marca se reveló con dos sistemas de detección avidina-peroxidasa y estreptavidina-fosfatasa alcalina, siendo ésta última la que demostró una mayor sensibilidad. Los plasmidos pSS1.8 y pSP64/U1 fueron fotobiotinilados y utilizados en ensayos de hibridación en gota con DNA extraido de leucocitos humanos. Despues de la incubacion con estreptavidina y fosfatasa alcalina biotinilada, la actividad de la enzima se reveló con un sustrato soluble. Los resultados obtenidos demuestran diferencias cuantitativas consistentes con el número de copias para globina y U1snRNA humano. El plasmido pSS1.8 fotobiotinilado se utilizó para identificar fragmentos de restricción de DNA genómico alterados en un paciente afectado de anemia de células falciformes. El gen de la globina mutado se detectó por digestión del DNA del paciente con la endonucleasa de restricción Dde I, seguido de una hibridación "Southern" con la sonda marcada