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We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.Although considerable indirect evidence suggests that initiation of eucaryotic DNA replication occurs at specific nucleotide sequences, or origins (2), only recently have direct tests for initiation sites become available. Both Huberman et al. (7) and Brewer and Fangman (1) recently reported the use of two-dimensional (2D) gel electrophoretic methods which allowed localization of the replication origins and termini (sites where replication forks stop) used in vivo by Saccharomyces cerevisiae plasmids. Both methods show promise for application to eucaryotic chromosomal DNA. This report provides additional details of the method of Huberman et al. (7) and a current assessment of its applicability to higher eucaryotic organisms.A hypothetical stretch of chromosomal DNA containing a replication origin and two sites for a restriction endonuclease (R) is shown in Fig. 1. The short segments labeled 1, 2, and 3 are assumed to be available for use as hybridization probes, and the entire restriction fragment is assumed to be replicated by forks traveling from left to right. DNA is isolated from cells (which must be growing but need not be synchronized), cut with restriction enzyme R, and enriched for fork-containing molecules by chromatography through benzoylated, naphthoylated DEAE-cellulose (BND-cellulose). The planar, aromatic benzoyl and naphthoyl groups can interact (in a manner similar to base stacking in native DNA) with the unpaired bases present in single-stranded DNA. Replicating molecules contain small single-stranded regions at their forks. These and other molecules with single-stranded regions are eluted from BND-cellulose with caffeine, which competes with the unpaired bases for the aromatic groups on BND-cellulose.The replication-fork-enriched DNA molecules are then electrophoresed through a neutral agarose gel (Fig. 1D). Nonreplicating molecules form a sharp band (closed vertical bar), whereas the replicating molecules form a trailing smear. The position of a replicating molecule in the smear is determined by its extent of replication: molecules beginning replication migrate just behind the nonreplicating molecules, whereas almost fully replicated molecules are ...

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A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r 2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.

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