General method for plasmid construction using homologous recombination.

We describe a general method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with > 80% efficiency. Quantitative data on the influence of DNA concentration and overlap length on the efficiency of recombination are presented. Using a simple procedure, plasmids were shuttled from yeast into E. coli for subsequent screening and large-scale plasmid preps. This simple method for plasmid construction has several advantages. (i) It bypasses the need for extensive PCR amplification and for purification, modification and/or ligation techniques routinely used for plasmid constructions. (ii) The method does not rely on available restriction sites, thus fragment and vector DNA can be joined within any DNA sequence. This enables the use of multifunctional cloning vectors for protein expression in mammalian cells, other yeast species, E. coli and other expression systems as discussed. (iii) Finally, the technology exploits yeast strains, plasmids and microbial techniques that are inexpensive and readily available.

hns, rpoS and lrp mutations affect stationary-phase survival at high osmolarity.

The survival of enteric bacteria during stationary phase requires the expressions of different genes than those required for growth during log phase. Genes coding for functions protecting cells from environmental stress are expressed during the onset of stationary phase. Many promoters of these genes require sigma s, the product of the rpoS gene, for transcription. During stationary phase, we found that strains lacking the neutral, histone-like, DNA-binding protein H-NS (hns- rpoS- double mutants) are more viable at high osmolarity at 37 degrees C than hns+ rpoS- cells. We did not observe differential viability at high osmolarity at 30 degrees C or at normal osmolarity at 37 degrees C. We showed that this effect is due to the absence of H-NS. The leucine-responsive regulatory protein acts without H-NS to protect a strain containing an rpoS mutation from death in stationary phase at high osmolarity. We present evidence that hns- rpoS double mutants can synthesize the log phase osmotic shock protective system.