Background: We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discretē uorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions.

SummarySeqA protein, which binds to hemi-methylated GATC sequences of DNA, is localized to discrete fluorescent foci in wild-type Escherichia coli cells. In this work, we observed cellular localization of the SeqA-Gfp fusion in living cells. SeqA-Gfp was localized to a discrete focus or foci in wild-type and seqA null mutant cells, but the fusion was dispersed in the whole cell in dam null mutant cells lacking Dam methyltransferase. These results were consistent with the previous description of the localization of SeqA by immunofluorescence microscopy. Time-lapse experiments revealed that duplicated SeqA-Gfp foci migrated rapidly in opposite directions. Flow cytometry demonstrated that the fusion restored synchronous replication of chromosomal DNA from multiple origins in seqA null mutant cells, indicating that SeqA-Gfp is biologically active. Immunoprecipitation of the fusion from cell extracts using anti-Gfp antibody indicated that the fusion was assembled with the wild-type SeqA protein.

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SummaryWe analysed Escherichia coli cells synchronized for initiation of chromosomal DNA replication by fluorescence in situ hybridization (FISH) using fluorescent DNA probes corresponding to various chromosomal regions. Sister copies of regions in an approximately oriC-proximal half of the chromosome are cohesive with each other after replication until the late period of chromosome replication. Sister copies of regions relatively close to the terminus are also separated from each other in the same late period of replication. It is important that sister copies in all the tested regions are thus separated from each other nearly all at once in the late period of chromosome replication. These results are consistent with results obtained by FISH in randomly growing cultures. Cohesion of sister copies in an oriC-close region is observed in a dam null mutant lacking DNA adenine methyltransferase the same as in the parental isogenic dam 1 strain, indicating that the cohesion is independent of DNA adenine methyltransferase. This further implies that hemimethylated DNA-binding proteins, such as SeqA, are not involved in the cohesion. On the other hand, the cohesion of sister copies of the oriC-close region was not observed in mukB null mutant cells, suggesting that MukB might be involved in the chromosome cohesion.

The sopAB operon and the sopC sequence, which acts as a centromere, are essential for stable maintenance of the mini-F plasmid. Immunoprecipitation experiments with purified SopA and SopB proteins have demonstrated that these proteins interact in vitro. Expression studies using the lacZ gene as a reporter revealed that the sopAB operon is repressed by the cooperative action of SopA and SopB. Using immunofluorescence microscopy, we found discrete fluorescent foci of SopA and SopB in cells that produce both SopA and SopB in the presence of the sopC DNA segment, but not in the absence of sopC, suggesting the SopA-SopB complex binds to sopC segments. SopA was exclusively found to colocalize with nucleoids in cells that produced only SopA, while, in the absence of SopA, SopB was distributed in the cytosolic spaces.

We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible conserved regions was validated. We present several promising antiviral siRNA candidates that effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic HIV-1 group M strains.

To clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sister oriC copies of the Escherichia coli chromosome.We have recently demonstrated that sister copies of the oriC-proximal half of the Escherichia coli chromosome are cohesive with each other and that the cohesion is released almost all at once in the late period of chromosome replication (5, 15). The MukFEB complex (17,18) participates in the sister chromosome cohesion (15). Sister chromosomes form two separated nucleoids after release from the cohesion. Sister copies of the oriC region are localized in the pole-proximal regions of each nucleoid, and sister copies of the replication-terminal region are localized near the constriction site before cell division (2,11,12,13).F plasmid DNA molecules are partitioned with fidelity to both daughter cells during cell division cycles owing to F plasmid's own partition system. The partition system, constituted of the plasmid genes sopA and sopB and the cis-acting plasmid region sopC, is essential for the stable maintenance of the plasmid during proliferation (9, 14; for a review, see reference 4). The sister copies of mini-F plasmid are replicated in the mid-cell position and migrate in opposite directions up to the 1/4 and 3/4 cellular positions without coupling but with cell elongation and the formation of tethered copies at the cellular positions, where they remain until the cell divides (11). Movement to these positions is not coupled with cell division. Single fluorescent foci representing mini-F plasmid DNA were seen to be localized in mid-cell, whereas pairs of fluorescent foci were seen to be localized at the 1/4 and 3/4 cellular positions within the nucleoid. In contrast, DNA molecules of a mini-F plasmid lacking the partition genes are randomly localized in cytosolic spaces of the cell poles. The partition system of mini-F is thus essential for the proper localization of plasmid molecules (4, 11).Mini-F plasmid can be replicated at all stages of the division cycle, and the plasmid is segregated in a nonrandom fashion similar to that of the E. coli chromosome and minichromosomes (3,8). Three models can be imagined concerning the separation and migration of replicated sister copies of mini-F plasmid. First, sister copies of mini-F are separated from each other and migrate from mid-cell to the 1/4 and 3/4 cellular positions immediately or shortly after replication, being independent of the mechanism of host sister chromosome cohesion. Second, sister copies of mini-F are cohesive with each other, and the cohesion is released at the same time that host sister chromosome cohesion is released by an unknown cellular signal. In the second model, a common cohesion mechanism might act for both mini-F and the E. coli chromosome. Third, sister copies of mini-F are cohesive for a ...

The ␤-subunit of DNA polymerase III is located as one or two condensed clusters within the nucleoidoccupied space in exponentially growing cells of Escherichia coli. When chromosome replication is terminated after incubation at nonpermissive temperature in a temperature-sensitive dnaC mutant, the ␤-subunit is located in the cytosolic spaces of the cell poles.The dnaN gene in Escherichia coli encodes the ␤-subunit of DNA polymerase III. A dimer of the ␤-subunit forms the DNA sliding clamp. The dimer is loaded to DNA in an ATP-dependent manner catalyzed by the ␥ complex (a subassembly of DNA polymerase III) to form the preinitiation complex. In the next step, the DNA polymerase III core that catalyzes DNA synthesis associates with the preinitiation complex to form the initiation complex. The ␤-subunit exists as 300 to 5,000 dimers per cell (for reviews, see references 1, 6, 7, and 9). In this work, we have analyzed the subcellular localization of the ␤-subunit in exponentially growing wild-type cells and temperature-sensitive dnaC mutant cells synchronized for initiation of chromosome replication.Subcellular localization of the ␤-subunit of DNA polymerase III and SeqA in exponentially growing cells. We analyzed the subcellular localization of the ␤-subunit of DNA polymerase III by indirect immunofluorescence microscopy (4) using rabbit anti-␤-subunit polyclonal antibody. In exponentially growing cells of strain YK1100 (a tryptophan-deficient mutant derived from W3110 [11]) at 37°C in M9 glucose medium supplemented with L-tryptophan (50 g/ml), the ␤-subunit formed one or two distinct condensed clusters within the nucleoid. Besides clear clusters, a portion of ␤-subunit molecules was distributed in polar cytosolic spaces in some cells (Fig. 1A and Table 1). The average length of cells with two ␤-subunit clusters was longer than that of cells with one ␤-subunit cluster (Table 1 and (Table 1 and Fig. 2A). This result eliminates the possibility that this type of cells without clusters was in a specific stage of the cell cycle, for example, the D period (the nonreplication period under the slow-growth conditions at a 55-to 60-min doubling time). Presumably, it is difficult to detect faint clusters, because of a high background of ␤-subunit dispersed throughout the whole cell.In a rich medium (L medium), cells had one, two, three, and four clusters of the ␤-subunit (Table 1) (Table 1). The average length (3.06 Ϯ 0.86 m) of cells without clear clusters was similar to that (3.21 Ϯ 0.85 m) of total cells with one, two, three, and four clusters (Table 1 and Fig. 2B). It is unlikely that this type of cells without clusters was in a specific stage of the cell cycle.We previously reported that SeqA is localized as discrete foci in growing YK1100 cells. SeqA forms clusters with hemimethylated nascent DNA segments behind replication forks (4, 10; for a review, see reference 3). We therefore analyzed the subcellular localization of the SeqA protein as a landmark of hemimethylated nascent DNA segments in the cell cycle. As shown in...