RESUMO
We describe in detail an automated and highly sensitive functional assay for calcium-coupled receptors (those receptors whose activation results in an increase in intracellular calcium levels) utilizing coelenterazine-charged aequorin as a probe for intracellular calcium levels ([Ca(2+)](i)). The assay was originally established to investigate Galpha(q)-coupled prostanoid receptors, which are members of the G-protein-coupled receptor (GPCR) superfamily, signaling through elevation of [Ca(2+)](i), initially focusing on the human EP(1) prostanoid receptor (hEP(1)). The parental human embryonic kidney cell line 293-AEQ17, developed by Button and Brownstein (Cell Calcium 14, 663-671, 1993), constitutively expresses apoaequorin and was used to develop a clonal cell line which stably coexpresses hEP(1). This cell line was used to optimize assay parameters in order to maximize accuracy and throughput in an automated 96-well format with the result that each 96-well plate can be completed in 70 min. Use of this flexible system will greatly simplify the functional analysis of GPCRs and other receptors which when activated result in increases in [Ca(2+)](i).
Assuntos
Equorina , Cálcio/análise , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/metabolismo , Linhagem Celular , Humanos , Medições Luminescentes , Sondas Moleculares , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP1 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidade e EspecificidadeRESUMO
Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by fluorescent in situ hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7, FRA7H. A region of 161 kb spanning FRA7H was defined and sequenced. Several regions with a potential unusual DNA structure, including high-flexibility, low-stability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published FRA3B sequence and the putative partial FRA7G, which also revealed an impressive cluster of regions with high flexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility.
