Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100°). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (RepIHF) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the RepIHF monomer for iterons was similar to that of wild-type Rep and bent DNA only 14° more strongly than did the wild-type Rep. RepIHF-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How RepIHF can support plasmid replication in the absence of IHF is discussed.
The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic pressure increased the ability of plasmids to transform competent Escherichia coli cells. For pUC18 plasmid, a pressure of 400MPa, and for pBR322, a pressure of 200MPa was found to provide the highest transformation efficiency. The DNA duplexes of the two plasmids were found to be the most stable for melting conditions at these pressures. At pressures higher than these, both the stability of the duplex DNA and the transformation efficiency were affected. The stabilizing effect of high hydrostatic pressure on the hydrogen bond may be responsible for the observed increase in transformation efficiency of the pressure-exposed plasmid DNA. The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques. In agarose gel electrophoresis, pressure-treated plasmids (pUC18 at 400MPa and pBR322 at 200MPa) consistently showed visibly distinct higher mobility compared to untreated plasmids. Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding as is evident from the reduced intensity of fluorescence of the dye bound pressure-treated DNA. Spectroscopic studies using circular dichroism and Fourier transform infrared (FTIR) spectroscopy also showed significant differences in the absorption profiles of pressure-treated plasmids as compared to an untreated control. These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids. On the other hand, the exposure of competent cells of E. coli to a high hydrostatic pressure of 50MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.
The Wis1-Sty1 mitogen-activated protein (MAP) kinase cascade is one of the major signaling systems involved in a wide range of stress responses in Schizosaccharomyces pombe. It is known that Δwis1 and Δsty1 mutants exhibit highly pleiotropic phenotypes, including a phenotype of temperature sensitivity for growth. In this study, we screened multicopy suppressor genes that allow both the Δwis1 and Δsty1 mutants to grow simultaneously at a non-permissive temperature, 37°C. Two such multicopy suppressors were cloned and characterized as sds23+ and hxk2+ genes. The former is known to specify a protein that functions as a multicopy suppressor for mutations of the PP1 protein phosphatase and the 20S cyclosomelanaphase-promoting complex (APC), and the latter encodes hexokinase 2. It was revealed that the multicopy sds23+ gene restored a defect in the mating efficiency caused by the Δwis1 and Δsty1 mutations, whereas the multicopy hxk2+ gene suppressed a phenotype of heat-shock sensitivity for growth of these mutant cells. These findings are discussed with special reference to the Wis1-Sty1 MAP kinase signaling pathway in S. pombe.
The budding yeast Saccharomyces cerevisiae, like many other microorganisms, responds to nutrient starvation by arresting growth and entering into a non-proliferating stationary phase. Studies on the response of S. cerevisiae cells to growth arrest might provide further insight into the non-proliferative states of cells in multi-cellular eukaryotic organisms. Changes might occur at the transcription, translation, and post-translational levels in cells upon entry into the stationary phase. To search for the genes differentially expressed in yeast cells during different growth phases, we have performed systematic Northern hybridization experiments using probes prepared for a large number of genes/ORFs. We have thus isolated and characterized 42 cDNA clones containing genes hyper-expressed in the post-diauxic phase. Some of them have already been characterized, and many others show similarity to known yeast genes or genes of other organisms. However, eleven of them were found to be unrelated to any known genes. We have characterized some of these genes as described below. Also, a possible cis-element for transcriptional regulation was identified.
Carbon material such as graphite and activated charcoal, but not diamond, causes the promotion of growth of certain bacteria under ordinarily non-permissive stress conditions over a distance of several centimeters. Bacillus carboniphilus under the stress of a high KCl concentration and high temperature responded to this remote effect of carbon material with enhanced growth, and thermophile bacterium Bacillus stearothermophilus responded similarly yet moderately under the stress of low temperature. The remote effect of carbon was caused by its activation with external energy, probably of electromagnetic nature, as this effect was markedly decreased by sheltering the experimental system with an iron or aluminum barrier. Carbon material probably transforms the external oscillatory pulses or radiation into a signal exerting, far-reaching, growth-promoting effect upon cells. The most plausible candidate of signals emitted from carbon was considered to be (ultra)sonic.