RESUMO
An increase in the level of the tumor suppressor protein p53 can induce cell cycle arrest or cell death. Although mechanisms for regulating the life span of p53 have been described, there is growing evidence that transcriptional regulation of the p53 gene contributes significantly to controlling p53 protein levels and therefore the fate of a cell. However, the signal transduction pathways that lead to transcriptional activation of the p53 gene are poorly understood. The oncoprotein v-Maf and its cellular counterparts belong to the large combinatorially complex basic leucine zipper family of transcription factors, which include the AP1 family. To date few cellular targets of c-Maf have been identified. It is demonstrated here that v-Maf can bind as a homodimer to a variant Maf recognition element located between -66 and -54 upstream in the mouse p53 promoter. V-Maf and its cellular counterparts are shown to activate p53 expression through this site. The ability of v-Maf to activate p53 expression is modulated by AP1 family members. In addition, overexpression of v-Maf in primary cells leads to a p53-dependent cell death. Thus, Maf and members of the AP1 family are able to regulate p53 expression through this site in the p53 promoter.
Assuntos
Apoptose , Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Galinhas , Sequência Consenso , DNA/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência MolecularRESUMO
Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.
Assuntos
Proteínas E1A de Adenovirus/fisiologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Genes p53 , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação , Fatores de Transcrição E2F , Células HeLa , Humanos , Camundongos , Regiões Promotoras Genéticas , Ratos , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1RESUMO
We have investigated the transcription factor requirements for basal expression of the mouse p53 promoter by using a combination of reporter and electrophoretic mobility shift assays (EMSAs). We have found that only four regions of the promoter bind transcription factors in EMSAs, suggesting that these are the only important factors for basal transcription. These factors are NF1, USF, ETF-like and a novel factor which we have called PF2. Construction of promoter deletion mutants has shown that the absence of the PF2 site completely inactivates the promoter, whereas deletion of other sites, whilst reducing promoter activity, does not. We suggest that this novel transcription factor (PF2) is critical for expression of the mouse p53 promoter.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Regulação da Expressão Gênica , Genes p53 , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , DNA/metabolismo , DNA Recombinante/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Genes Reporter , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFI , Proteína 1 de Ligação ao Retinoblastoma , Deleção de Sequência , Especificidade da Espécie , Fator de Transcrição DP1 , Fatores Estimuladores UpstreamRESUMO
The p53 gene, which is frequently mutated in various tumors, encodes a phosphoprotein thought to have a key role in the regulation of cell proliferation. To explore their biological effects, the HeLa carcinoma line, which does not express p53, was co-transfected with plasmid constructs expressing wild-type or mutant p53 proteins, or unrelated proteins, along with a plasmid conferring resistance to a neomycin-kanamycin antibiotic analog (G418). Both wild-type and mutant forms of p53 stimulated the number of G418-resistant colonies between 5- and 36-fold. Further investigation of colony development revealed that p53 enhanced cell survival, leading to increased colony numbers, but did not stimulate cell growth. Nonetheless, we suggest that an initial slowing of cell growth caused by expression of the unintegrated p53 plasmids renders the transfectants resistant to selection with G418, thus causing a higher frequency of G418-resistant colonies. p53 constructs were found to be expressed transiently in HeLa cells as expected, but the G418-resistant colonies frequently failed to express p53. This loss of p53 expression may be due to negative regulatory effects of p53 on the cytomegalovirus promoter that drives the selection marker.
Assuntos
Genes p53 , Células HeLa/citologia , Proteína Supressora de Tumor p53/genética , Animais , Divisão Celular , Sobrevivência Celular , Expressão Gênica , Humanos , Técnicas In Vitro , Camundongos , RNA Mensageiro/genética , TransfecçãoRESUMO
Adult male zebra finches underwent unilateral denervation of the syrinx or unilateral lesion of the forebrain nucleus HVC known to be important for song control. Disruptive effects on song were greater after right-side than after left-side operations. After denervation of the right half of the syrinx, the fundamental frequencies of all syllables within a song converged on a value near 500 Hz, and nearly all syllables were altered in type. In contrast, the syllables produced after denervation of the left side of the syrinx largely maintained their preoperative frequencies, and fewer syllables changed in type. Unlike nerve sections, HVC lesions did not result in strikingly lateralized effects on syllable phonology; however, HVC lesions did affect the temporal patterning of a bird's song, whereas nerve sections did not, and changes in temporal patterning were more marked after right than after left HVC lesions. Right-side dominance for zebra finch song control is the reverse of that described in other songbird species with lateral asymmetry for vocal communication. We suggest that the need for a dominant side is more important than the side of dominance.
Assuntos
Aves/fisiologia , Encéfalo/fisiologia , Lateralidade Funcional/fisiologia , Vocalização Animal/fisiologia , Anestesia , Animais , Encéfalo/anatomia & histologia , Denervação , Masculino , Traqueia/fisiologiaRESUMO
Process scaleup for stirred-tank animal cell cultures such as suspension and microcarrier cultures often begins at the bench scale in small spinner vessels. In order to initiate process development under the proper conditions, it is essential to know the physical conditions under which the cells are grown. In this article, power inputs and surface oxygen transfer rates to culture medium in 500-mL Corning spinner vessels were determined as a function of the impeller geometry, impeller height, and agitation speed. The results obtained indicate that power dissipation dependency differs from literature correlations and may compromise scale up at constant power input from these vessels. These results are of general utility to researchers using small-scale vessels.