PubMed · 1408774
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R N Sandford, G S Elgar. 1992-09-11. A novel method for rapid genomic walking using lambda vectors.. https://doi.org/10.1093/nar%2F20.17.4665
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A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.
We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage lambda DNA. The 3' and 5' extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10-15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100-500 bases are presently detected.
The folding, dimerization and DNA binding equilibria of the bacteriophage lambda Cro repressor have been characterized. Comparison with four engineered variants shows that a folded monomeric species is substantially populated under conditions used for the formation of dimer-DNA complexes. Although Cro dimers are the only DNA-bound species observed in electrophoretic mobility shift assays, cooperativity in Cro-DNA binding isotherms shows that the predominant free protein species is monomeric at nanomolar concentrations. Micromolar dissociation constants for Cro dimers have been measured in the absence of DNA by sedimentation equilibrium and gel filtration chromatography. Denaturation of Cro dimers in the 10 to 100 micromolar concentration range by guanidine hydrochloride (GdnHCl) is well modeled as a two-state process, with folded dimers and unfolded monomers as the only significantly populated species. However, linear extrapolation of this composite unfolding and dimer dissociation free energy predicts a nanomolar dissociation constant in the absence of denaturant. This extrapolation is clearly inconsistent with the DNA binding and hydrodynamic measurements. Our interpretation of these results is that the monomeric species detected in DNA binding and hydrodynamic experiments is predominantly folded. The stability of the folded monomeric species can be calculated as the difference between the dimerization free energy determined from hydrodynamic measurements and the folding free energy extrapolated from GdnHCl denaturation. The calculated stability of the Cro F58W monomer is greater than that of the wild-type Cro monomer. Thus, residue 58, which makes critical intermolecular contacts across the dimer interface, is also involved in intramolecular stabilization of the monomeric intermediate.