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Microscope images of plant specimens showing expression of GUS markers, besides being very beautiful, provide useful information regarding various biological processes. However, the information extracted from these images is often purely qualitative, and in many publications is not subjected to quantification. Here, we describe a very simple quantification method for GUS histochemical staining that enables detection of subtle differences in gene expression at cellular, tissue, or organ level. The quantification method described is based on the freely available image analysis software ImageJ that is widely used by the scientific community. We exemplify the method by quantifying small and precise changes (at the cellular level) as well as broad changes (at the organ level) in the expression of two previously published reporter lines, such as the pPILS2::GUS and pPILS5::GUS. The method presented here represents an easy tool for converting visual information from GUS histochemical staining images into quantifiable data and is of general importance for plant biologists performing GUS activity-based evaluation of reporter genes.

Post-translational modification by SUMO is an important mechanism to regulate transcription. Sumoyla-tion has diverse effects on substrate activity, but in most cases reported to date sumoylation of transcription factors correlated with transcriptional repression. Here we describe general strategies to address how post-translational modification by SUMO regulates the activity of a DNA-binding transcription factor.

Reporter gene assays are versatile and sensitive methods of assaying numerous targets in high-throughput drug-screening programs. A variety of reporter genes allow users a choice of signal that can be tailored to the required sensitivity, the available detection apparatus, the cellular system employed, and the required compatibility with multiplexed assays. Promoters used to drive reporter gene expression can be activated either by a broad range of biochemical pathways or by the selective activation of individual targets. In this chapter, we will introduce some of the considerations behind the choice of reporter gene assays and describe the methods that we have used to establish 96-well format luciferase and aequorin assays for the screening of ligands for G protein-coupled receptors.

Herpes simplex virus 1 (HSV-1) is a common and significant neurotropic human pathogen that infects 80% of all persons by adulthood. During acute HSV-1 infection, virus replicates peripherally in epithelia, enters axonal terminals, and is transported retrogradely to sensory nerve ganglia, where HSV-1 may establish latency or progress to life-threatening infection of the central nervous system. Studies of viral and host factors that influence pathogenesis have largely used experimental mouse models that rely on sacrifice of infected mice to determine distribution and titer of virus. Although this experimental paradigm has provided important data, it precludes real-time investigations of the same animal over the entire course of disease progression. This limits potentially significant insights from animal-to-animal variations in host-pathogen relationships. Unexpected sites of infection also may be missed because appropriate tissues are not analyzed for virus. To improve investigations of viral and host factors that determine HSV-1 pathogenesis, we have validated bioluminescence imaging (BLI) as a technique to monitor infection with a recombinant strain KOS HSV-1 virus that expresses firefly luciferase (FL). This imaging technique allows repetitive, noninvasive monitoring of HSV-1 in living mice. In this chapter, we describe the protocols that we use for in vivo BLI of HSV-1 infection.