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.

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