RESUMO
Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 5 [corrected].
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 4 , DNA Complementar/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Algoritmos , Animais , Northern Blotting , Encéfalo/metabolismo , Clonagem Molecular , Etiquetas de Sequências Expressas , Glutationa Transferase/metabolismo , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Rim/metabolismo , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Miocárdio/metabolismo , Fosforilação , Proteínas Tirosina Quinases , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Distribuição TecidualRESUMO
Using hight-titer recombinant adeno-associated viral vectors (rAAV), we have investigated the feasibility of cancer vaccines from tumor explants. In a first set of experiments, rAAV vectors expressing firefly luciferase reporter genes were used to transduce different human tumor cell lines. At day three post transduction, all of the human tumor cell lines tested showed high levels of luciferase expression. To further evaluate rAAV-mediated gene transfer efficiency into primary tumor cells, we transduced freshly isolated tumor cells from malignant melanoma and ovarian carcinoma patients. As a remarkable result, reporter gene expression in primary tumor cells was significantly higher than in the tested established tumor cell lines. These data could also be reproduced with a rAAV/lacZ vector, since the portion of successfully transduced primary tumor was higher than 90%. Taken together, our data demonstrate that rAAV-mediated gene transfer is a very efficient method for the transduction of freshly isolated human tumor cells and may allow the generation of potent autologous cancer vaccines.