Comparative analysis of DNA loop length in nontransformed and transformed hamster cells

The size of the looped, matrix-attached DNA domains was estimated in both nontransformed and transformed hamster cells. In BHK cells it was observed that in interphase as well as during mitosis, transformed cells, on the average, have shorter loops than nontransformed cells. In CHO cells, however, no alteration of the length of the looped DNA domains was found to accompany transformation. The implications of these observations are discussed in relation to the process of transformation

HL-R5 and HL-D4: Two Differentiation Resistant HL-60 Variants

A wide range of different agents are capable of inducing the onset of HL-60 differentiation along the myeloid lineage. The diversity of these agents has made the analysis of the molecular mechanisms involved in the regulation of the onset and progress of terminal differentiation in these cells difficult. We have adapted the standard soft-agar cloning procedure to enable the single-step selection of clonal populations of spontaneously arising differentiation resistant HL-60 variants. This simple procedure which obviates the need for mutagenesis, long-term exposure to inducing agents or complex manipulations, optimises the chance of obtaining variants with a single lesion blocking the onset of differentiation. The analysis of two variants, HL-R5 and HL-D4, selected by this procedure for resistance to retinoic acid and DMSO, respectively, suggests the existence of different pathways used by the two agents which converge before the onset of terminal differentiation.

Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTAs*). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTAs*. Tetracycline prevents tTAs* from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTAs* binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.