The binding of hemimethylated oriC to Escherichia coli membranes has been implicated in the prevention of premature reinitiation at newly replicated chromosomal origins in a reaction that involves the SeqA protein. We describe the resolution of the membrane-associated oriCbinding activity into two fractions, both of which are required for the high-affinity binding of hemimethylated oriC. The active component in one fraction is identified as SeqA. The active component of the second fraction is a previously undescribed protein factor, SeqB. The reconstituted system reproduced the salient characteristics of the membraneassociated binding activity, suggesting that the membraneassociated oriC-binding machinery of E. coli is likely to be a multiprotein system that includes the SeqA and SeqB proteins.In Escherichia coli and most other bacterial species, chromosome replication is initiated at a specific point during the cell cycle (1). It is clear that a mechanism exists to prevent premature reinitiation until the proper time in the cell cycle is reached (2). Several factors have been proposed to play a role in the sequestration of newly replicated origins to prevent premature reinitiation during the eclipse period.The methylation state of the chromosomal origin has been implicated both in the binding of oriC to membranes and in the prevention of premature reinitiation at newly replicated origins. In E. coli, adenine residues within chromosomal GATC sequences are methylated in a reaction catalyzed by Dam methylase. Because the methylation reactions occur after replication, adenine residues within GATC sequences in the newly synthesized strand remain unmethylated for a period of time. The oriC region contains a high density of GATC sites, and methylation of adenine residues at several of these sites is delayed significantly relative to the time of methylation at GATC sites elsewhere in the chromosome (3, 4). As a result, the oriC region of newly replicated chromosomes remains hemimethylated for about 30-40% of the cell cycle.Evidence that hemimethylated oriC does not serve as an effective template for initiation of replication in vivo came from the finding of Russell and Zinder (5) that a fully methylated oriC plasmid was unable to replicate in a Dam

The mtsl gene is specifically expressed in certain metastatic tumors but not in their nonmetastatic counterparts. It is also expressed in several normal ceil and tissue types that exhibit the ability to be motile. The gene was cloned from both mouse and human sources and the 5' flanking regions were sequenced. The sequencing data revealed a 135-base-pair region of high homology between the mouse and human mtl gene. This homology was observed in the vicinity of the TATA box. The 5' region of the mtsl gene was also observed to have a high degree of homology to some known promoter and enhancer sequences. To determine the role this region plays in regulating the transcription of mtsl, promoter analysis was performed. Sixteen constructs were prepared in which the chloramphenicol acetyltransferase gene was fused to different regions of the mouse mtsl promoter. These constructs

SummaryThe lacZ -hobH fusion clone, containing an Escherichia coli DNA segment located at 92 min on the chromosomal map, was screened as a producer of E. coli oriC hemi-methylated binding activity. We have purified the protein encoded by this locus to near homogeneity. The protein corresponds to the monomeric form of a non-specific acid phosphatase (NAP) whose gene has been designated aphA. oriC DNA footprinting experiments showed protection of hemi-methylated probe by partially purified NAP, but not by purified preparations. Yet, gel retardation experiments with an oriC oligonucleotide demonstrated DNA binding activity of purified NAP in the presence of Mg 2þ . This experiment also showed an increased affinity of the protein for the hemi-methylated probe compared with the fully or unmethylated form. Indirect immunofluorescence microscopy revealed the existence of discrete NAP foci at mid-cell in cells with two nucleoids, but at cell poles in those with one nucleoid.