Rb binds c-Jun and activates transcription.

The retinoblastoma protein (Rb) acts as a critical cell-cycle regulator and loss of Rb function is associated with a variety of human cancer types. Here we report that Rb binds to members of the AP-1 family of transcription factors, including c-Jun, and stimulates c-Jun transcriptional activity from an AP-1 consensus sequence. The interaction involves the leucine zipper region of c-Jun and the B pocket of Rb as well as a C-terminal domain. We also present evidence that the complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c-Jun and Rb, inhibits the ability of Rb to activate c-Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c-Jun expression during keratinocyte differentiation.

Poly-L-ornithine-mediated transfection of human keratinocytes.

Human keratinocytes are notoriously difficult to transfect. We have optimized a method for introducing plasmid DNA into keratinocytes that pairs the polycation poly-L-ornithine with a dimethylsulfoxide (DMSO) shock. The optimum poly-L-ornithine conditions for keratinocyte transfection entailed incubating the cells with 12 micrograms/ml poly-L-ornithine and 10 micrograms DNA for 6 h, followed by a 4-min 25% DMSO shock. Based on kinetic studies, 1 h is enough time to produce 10% positive cells in transient transfections, which increases up to an average of 20% after 6 h. Transfected cells survive passaging, and marker plasmids and selection can be used to yield stable transfectants at a rate twofold higher than in cells transfected with polybrene and DMSO. Transient transfection rates were significantly higher using poly-L-ornithine/DMSO than with the polybrene/DMSO or polybrene/glycerol methods previously reported. Overall, transfection mediated by poly-L-ornithine provides an efficient and inexpensive means of transiently or stably introducing DNA into keratinocytes.

Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12.

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21,202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription-translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.