Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays.

Luciferase-based reporter assays are powerful tools for monitoring gene expression in cells because of their ultrasensitive detection capacity and wide dynamic range. Here we describe the characterization and use of a luciferase reporter enzyme derived from the marine copepod Metridia luciferase family, referred to as TurboLuc luciferase (TurboLuc). To develop TurboLuc, the wild-type luciferase was modified to decrease its size, increase brightness, slow luminescent signal decay, and provide for efficient intracellular expression.

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements.

Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication.

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site.

Transfection of keratinocytes abrogates detectable DNA-binding activity of CCAAT displacement protein.

Transfection of keratinocytes with plasmid DNA leads to the loss of detectable DNA-binding activity of CCAAT displacement protein but not of Yin Yang 1, as monitored by electrophoretic mobility shift assay. This phenomenon was found to be attributable to the presence of plasmid DNA in the nuclear extracts prepared from transfected cells. Treatment of these nuclear extracts with DNase I restored the ability to monitor DNA-binding activity of CDP.

Translation and phosphorylation of wheat germ lysate: phosphorylation of wheat germ initiation factor 2 by casein kinase II and in N-ethylmaleimide-treated lysates.

Previously, we observed that N-ethylmaleimide (NEM), a thiol-alkylating agent, was found to stimulate the phosphorylation of several proteins in translating wheat germ (WG) lysates, including the phosphorylation of alpha, the p41-42 doublet subunit, and beta, the p36 subunit, of the WG initiation factor 2 (eIF2). We find now that NEM increases phosphorylation of several proteins significantly in lysates which are moderate or low in their translation compared to optimally active lysates. Heat treatment, which stimulates oxidation of protein sulfhydryls, decreases the translation and phosphorylation ability of WG lysates.