RESUMEN
The development of more efficient, ethical, and effective means of assessing the effects of chemicals on human health and the environment was a lifetime goal of Gilman Veith. His work has provided the foundation for the use of chemical structure for informing toxicological assessment by regulatory agencies the world over. Veith's scientific work influenced the early development of the SAR models in use today at the US Environmental Protection Agency. He was the driving force behind the Organisation for Economic Co-operation and Development QSAR Toolbox. Veith was one of a few early pioneers whose vision led to the linkage of chemical structure and biological activity as a means of predicting adverse apical outcomes (known as a mode of action, or an adverse outcome pathway approach), and he understood at an early stage the power that could be harnessed when combining computational and mechanistic biological approaches as a means of avoiding animal testing. Through the International QSAR Foundation he organized like-minded experts to develop non-animal methods and frameworks for the assessment of chemical hazard and risk for the benefit of public and environmental health. Avoiding animal testing was Gil's passion, and his work helped to initiate the paradigm shift in toxicology that is now rendering this feasible.
Asunto(s)
Alternativas a las Pruebas en Animales , Relación Estructura-Actividad , Pruebas de Toxicidad/métodos , Simulación por Computador , Modelos Químicos , Relación Estructura-Actividad Cuantitativa , Medición de RiesgoRESUMEN
We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86.
Asunto(s)
Citomegalovirus/genética , Proteínas Inmediatas-Precoces/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Antígenos Transformadores de Poliomavirus/metabolismo , Secuencia de Bases , Citomegalovirus/metabolismo , ADN Viral , Humanos , Datos de Secuencia Molecular , Factores de Transcripción/metabolismoRESUMEN
The early proteins of simian virus 40 (SV40) large T and small t antigen (T/t antigen) can each cause the transcriptional activation of a variety of cellular and viral promoters. We showed previously that simian cellular DNA-binding factors (the Band A factors) bind to sequences within the SV40 late promoter which are important for transcriptional activation in the presence of the SV40 early proteins. Band A factors isolated from simian cells which produce T/t antigen (COS cells or SV40-infected CV-1 cells) have altered binding properties in comparison with the factors from normal simian cells (CV-1). This suggests that the transcriptional activation mediated by T/t antigen may be due to either modification of existing factors or induction of new members of a family of factors. We have purified the Band A factors from both COS and CV-1 cells and have determined the binding site by methylation interference and DNase protection footprinting. The COS cell factors have altered chromatographic properties on ion-exchange columns and have higher-molecular-weight forms than the CV-1 cell factors. Major forms of the CV-1 factors migrate between 20 and 24 kilodaltons, while the COS factors migrate between 20 and 28 kilodaltons. The binding sites for the factors from CV-1 and COS cells are similar, covering a rather broad region within the 72-base-pair repeat comprising the AP-1 site and the two-octamer binding protein (OBP100/Oct 1) sites, OBP I and OBP II. Specific binding competition analyses indicate that the two general regions within the binding site (the AP-1-OBP II site and the OBP I site) each retain partial binding ability; however, the factors bind best when the two regions are adjacent in a relatively specific spatial arrangement. The binding site for the Band A factors corresponds very well to sequences necessary for the activation of the late promoter as defined by deletion and base substitution mutagenesis studies (J. M. Keller and J. C. Alwine, Mol. Cell. Biol. 5:1859-1869, 1985; E. May, F. Omilli, M. Emoult-Lange, M. Zenke, and P. Chambon, Nucleic Acids Res. 15:2445-2461, 1987). These data, in combination with the data showing that the Band A factors are modified or induced in the presence of T/t antigen, strongly suggest that T/t antigen mediates its transcriptional activation function, at least in part, through the Band A factors.