RESUMO
HuD belongs to a family of neurospecific RNA binding proteins found in man, frog and fly [49]. To investigate whether this protein is involved in regulation of neuronal differentiation of rodent cells in vivo and in vitro, the cDNA of the rat homolog gene (r-HuD) was cloned, its expression was studied in rat brain and in neurogenic cell lines, and the splicing of its RNA was analyzed. Coding sequences of HuD from man and rat were found to be 99.5 and 95% identical at protein and DNA level, respectively. In rat brain r-HuD transcripts 3.7 and 4.2 kb in length were detected by Northern blot analysis. RT-PCR and in situ hybridization revealed that rodent homologues of HuD transcripts are present in P19 mouse embryo carcinoma and in PC12 rat pheochromocytoma cell lines both able to differentiate into neurons. In contrast, r-HuD transcripts were not detectable in the rat glioma cell line C6. In P19 cells a strong induction of HuD mRNA was observed after triggering neuronal differentiation by retinoic acid, whereas in PC12 cells the mRNA was present before and after nerve growth factor (NGF) induced neuronal differentiation. In both neuronal cell lines and in brain of adult rat and mouse HuD mRNA is alternatively spliced in a region which encodes a proline rich linker domain between the second and third RNA recognition motif. This RNA processing event seems to be differently regulated in PC12 cells on the one hand, and in P19 cells and brain of rat and mouse on the other.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Cerebelo/metabolismo , Primers do DNA , DNA Complementar , Proteínas ELAV , Proteína Semelhante a ELAV 4 , Biblioteca Gênica , Humanos , Hipotálamo/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Neurônios/citologia , Especificidade de Órgãos , Células PC12 , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA/química , Ratos , Ratos Sprague-Dawley , Mapeamento por RestriçãoRESUMO
The physical state of Epstein Barr virus (EBV) DNA was analyzed in the Burkitt's lymphoma (BL) cell line BL60 and in the autologous EBV-immortalized lymphoblastoid cell line (LCL) IARC 277 by Gardella gel analysis, Southern blotting, and nonradioactive in situ hybridization to metaphase chromosomes. Although only episomal viral DNA was detected in the LCL, the 6-12 copies of EBV DNA in the BL cell line are integrated in one site of the host cell genome. The integration site is located near the breakpoint of a translocation 11;19 which is present in this cell line in addition to the BL-specific t(8;22).
