RESUMO
Transgenic targeting of SV40 large T antigen (Tag) expression to murine cerebellar Purkinje cells induces these normally postmitotic neurons to undergo DNA synthesis and apoptosis. It has been proposed that these effects of Tag are due to the binding of Tag to pRb, which leads to the release and activation of the transcription factor E2F. Here it is reported that E2F and CDC2, the protein product of a gene regulated by E2F, were detectable in the Purkinje cell nuclei of Tag expressing transgenic animals. To directly test whether E2F-1 is part of the mechanism of Tag-induced Purkinje cell degeneration, transgenic mice that overexpress E2F-1 specifically in cerebellar Purkinje cells were generated. Although E2F-1 itself did not affect Purkinje cells, it did accelerate Tag-induced ataxia and Purkinje cell loss, suggesting that E2F-1 can contribute to the mechanism of Tag-induced Purkinje cell degeneration.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Proteínas de Transporte , Moléculas de Adesão Celular Neuronais , Proteínas de Ciclo Celular , Degeneração Neural/metabolismo , Células de Purkinje/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Northern Blotting , Proteína Quinase CDC2/genética , Contactina 2 , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Expressão Gênica/fisiologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Células de Purkinje/química , RNA Mensageiro/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/análiseAssuntos
Processamento Eletrônico de Dados , Processamento de Imagem Assistida por Computador/métodos , Análise de Sequência de DNA/métodos , Biologia Computacional/métodos , Sistemas de Gerenciamento de Base de Dados , Eletroforese em Gel de Poliacrilamida/instrumentação , Análise de Sequência de DNA/instrumentação , SoftwareRESUMO
Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.
