Homologous DNA targeting with RecA protein-coated short DNA probes and electron microscope mapping on linear duplex molecules.

We demonstrate that RecA protein-coated, short single-stranded DNA probes paired with a specific homologous DNA sequence in a linear duplex target molecule and accurately targeted the selected DNA sequence. RecA protein-coated complementary ssDNA probes were reacted with linear duplexes, and the homologously paired molecules were observed by electron microscopy. The sites of interaction between the RecA protein-coated DNA probes and the uncoated duplex DNA targets were directly visible on individual target DNA molecules by high-resolution darkfield electron microscopy, without chemical fixation or sample shadowing. The efficiency and specificity of pairing were verified with 446 and 222 base single-stranded DNA probes that shared no homology with one another, and several linear duplex target DNAs with their respective probe homology sites at different locations with respect to the ends of the double-stranded DNA molecules. Measurements of the position of RecA protein-coated probes paired to individual target molecules, observed at high magnification, showed that DNA probes specifically paired at their corresponding homologous target sequences. This RecA protein-mediated DNA mapping method allows homologous sequence positioning and gene mapping on individual double-stranded DNA molecules. Targeting reactions in which two different probe/target sites were 900 bases apart on a single duplex target molecule allowed both sites to be mapped in the same targeting reaction; although targets displaying both probes simultaneously were seen much less frequently than expected. The possible torsional or mechanistic constraints related to these reactions are briefly discussed.

The genome of a baculovirus infecting Tipula paludosa (Meig) (diptera): a high molecular weight closed circular DNA of zero superhelix density.

A characterization of the genome of a baculovirus infecting the diptera, Tipula paludosa Meig, is presented here. It appears to be a double-stranded closed circular DNA. The supercoiled structure has been visualized by electron microscopy. The relaxed molecules have a contour length of 51.2 +/- 1.5 mum which corresponds to a molecular weight of 92 x 10(6) daltons, by taking the value of 6.4 x 10(6) daltons for PM2 DNA used as an internal standard for the spreading. T. paludosa baculovirus DNA has a low G + C content of 27%, as estimated from its buoyant density in CsCl, uv spectrum, and melting temperature. Its sedimentation coefficient in alkaline solution was 294 S, as determined by analytical sedimentation.