PubMed · 1436055
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X H Guo, E J Huff, D C Schwartz. 1992-10-29. Sizing single DNA molecules.. https://doi.org/10.1038/359783a0
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It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity. The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.
An alternative chromatographic procedure for the separation of large double-stranded DNA molecules was discovered recently and called "slalom chromatography". This fractionation is based on a new hydrodynamic process that is determined by the progression of the mobile-phase flow through the interstitial spaces created between the highly packed particles inside the column. Here, the separation is treated as the result of a slowing down of the large double-stranded DNA fragments in relation to their size with the flow direction changing around the particles. A model, based on the concept derived from the reorientation time of macromolecules, was adequate to describe the hydrodynamic phenomenon. This model constitutes an attractive tool to enhance the expansion of this chromatographic procedure and provide valuable information on the dynamic behavior of biological polymers.
An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.