Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy.

We studied theta-mode DNA replication in p15A-based Escherichia coli plasmids by analyzing their replication intermediates using a combination of neutral agarose 2D gel electrophoresis and electron microscopy. Our analysis: (1) confirms the original assignment of various features of the 2D gel pattern; (2) shows that while one replication fork progresses around the plasmid DNA, the other is immobile, as if the replication were unidirectional; and (3) reveals that termination often occurs at a location away from the replication origin, suggesting that the replication of our plasmids is, in fact, bidirectional, the two forks being active at different times.

Structural analysis of membrane-bound retrovirus capsid proteins.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles.

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid.

Masking generates contiguous segments of metal-coated and bare DNA for scanning tunneling microscope imaging.

To date, no microscopic methods are available to confirm scanning tunneling microscope (STM) images of DNA. The difficulties encountered in repeating these images may be attributed to inadequate distribution of molecules on the substrate, poor adhesion to the substrate, or the low conductivity of the molecules. However, these factors are difficult to assess in an STM experiment where they may act simultaneously.

Deposition and imaging of metal-coated biomolecules with the STM.

We have applied a simple and reliable procedure for imaging biomolecules with the scanning tunneling microscope (STM). The biomolecules are adsorbed on glow-discharged mica, then coated with a thin film of platinum-carbon. We have tested this method with linear and circular (plasmid) DNA molecules.