RESUMO
Using transgenic mice, we have defined novel gene regulatory elements, termed "facilitators." These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine deaminase (ADA) gene. Facilitators were essential for gene copy-proportional and integration site-independent reporter expression in transgenic thymocytes, but they had no effect on the enhancer in transfected T cells. Both segments were required. Individual segments had no activity. A lack of facilitator function caused positional susceptibility and prevented DNase I-hypersensitive site formation at the enhancer. The segments were required to be at opposed ends of the enhancer, and they could not be grouped together. Reversing the orientation of a facilitator segment caused a partial loss of function, suggesting involvement of a stereospecific chromatin structure. trans-acting factor access to enhancer elements was modeled by exposing nuclei to a restriction endonuclease. The enhancer domain was accessible to the 4-cutter DpnII in a tissue- and cell-type-specific fashion. However, unlike DNase I hypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an accessible chromatin domain is an intermediate state in the activational pathway. These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a chromatin structure transition that is necessary for the DNase I hypersensitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitators to be present.
Assuntos
Adenosina Desaminase/genética , Elementos Facilitadores Genéticos , Expressão Gênica , Adenosina Desaminase/biossíntese , Animais , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatina/fisiologia , Cruzamentos Genéticos , Desoxirribonuclease I , Desoxirribonucleases de Sítio Específico do Tipo II , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Especificidade de Órgãos , RNA Mensageiro/análise , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Timo/enzimologiaRESUMO
Eleven astigmatic patients seen in a private practice were fit with the Hydron-T custom toric soft lens. The fitting mechanics are described along with the results which illustrate the potential utilization of this lens in the management of patients with high astigmatic errors.