DNA gyrase stimulates transcription.

The nuclear DNA of HeLa cells can now be isolated unbroken and supercoiled. Using DNA gyrase and the untwisting enzyme, we have prepared an allomorphic series of templates derived from this nuclear DNA, and also from the circular DNA of the bacterial virus, PM2. We have then transcribed these templates using 2 different RNA polymerases--from wheat germ and Escherichia coli. Relaxed DNA is transcribed slowly by both polymerases. Supertwisting the naturally-supercoiled templates with gyrase slightly inhibits transcription by the bacterial polymerase but stimulates dramatically transcription by RNA polymerase II from wheat germ

Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector.

A defective adenovirus (Ad) type 5 E1- vector has been combined with the powerful constitutive cytomegalovirus (CMV) major immediate early (IE) promoter to produce a novel eukaryotic expression system. The Ad vector can replicate to high titres in 293 cells and then be used to infect a wide variety of non-permissive cell types. The Escherichia coli lacZ and CMV IE1 genes have been cloned to generate the Ad recombinants RAd35 and RAd31 respectively. In human fibroblasts infected with RAd35 beta-galactosidase (beta-gal) expression could be detected in virtually 100% of target cells, there was no detectable transcription from the Ad genome and extremely high levels of expression could be achieved with beta-gal representing the predominant cytoplasmic cellular protein. Additionally, a number of agents, including the CMV IE1 gene product (in RAd31) and forskolin, significantly enhanced expression from RAd35-infected human fibroblasts. Lower levels of constitutive beta-gal expression were obtained in RAd35-infected HeLa cells but again expression could be enhanced (up to 60 fold) by chemical inducing agents. Expression from the IE promoter in the Ad vector could be repressed by coinfection with CMV

Electron-microscopy of intact nuclear DNA from human cells.

Structures retaining many of the morphological features of nuclei may be released by lysing human cells in a non-ionic detergent and 2 M NaCl. Such nucleoids contain all the nuclear DNA packaged within a flexible cage of RNA and protein. HeLa nucleoids have been spread at an air-water interface and viewed in the electron microscope. A tangled network of superhelical fibres surrounds the collapsed cage. Irradiation with gamma-rays abolishes supercoiling and treatment with the untwisting enzyme or a low concentration of ethidium reduces it. A high concentration of ethidium induces supertwisting. The nuclear DNA of higher cells can be isolated naked, supercoiled and intact