REAL-TIME ANALYSIS OF THE TRANSCRIPTIONAL REGULATION OF HIV AND HCMV PROMOTERS IN SINGLE MAMMALIAN-CELLS

The regulation of human cytomegalovirus (hCMV) and human immunodeficie ncy virus (HIV) gene expression has been studied in single intact mamm alian cells. Viral promoters were placed upstream of the firefly lucif erase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon -counting camera system was used to study the level of gene expression in single intact cells, Luciferase expression was studied in the abse nce of activators of viral gene expression, in the presence of the HIV -1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression, In the absence of any activa tor of gene expression, while expression was undetectable in most cell s, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell pop ulation, In the presence of the general activator of viral gene expres sion, sodium butyrate, transcriptional activation from the viral promo ters gave rise to significant and relatively homogeneous levels of luc iferase expression in a majority of cells, The luciferase imaging tech nology was used for the real-time analysis of changes of gene expressi on within a single cell, This non-invasive reporter assay should becom e important for studies of the temporal regulation of gene expression in single cells.