(1) We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC.
(2) The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction.
(3) Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located.
(4) To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine.
(5) By screening these libraries, a series of partially overlapping clones which covered the terC region was isolated.
(6) This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC.
(7) The effect of deletion or modification of DNA sequences on either side of terC (defined by the sequence location of the arrested clockwise fork junction) has been investigated.
(8) Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42 degrees C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks.
(9) The 250-bp segment immediately ahead of terC encompassed the previously identified inverted repeat region as well as potential promoters for the transcription of an adjoining open reading frame (ORF).
(10) The terC-containing portion of this plasmid has been subcloned within each of two overlapping fragments of DNA, 1.75 and 1.95 kb, again in E. coli plasmids.
(11) Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.
(12) Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress.
(13) Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.
(14) The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision.
(15) 60%) of that predicted for a complete block to movement of the clockwise fork at the replication terminus, terC.
(16) A procedure is described for relocating a functional terC-region to various sites on the Bacillus subtilis chromosome, and in alternative orientations.
(17) The terC-deletion strain of Bacillus subtilis 168, SU153 [Iismaa and Wake, J. Mol.
(18) With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site.
(19) 195 (1987) 299-310] was used for the re-insertion of a 1.75-kb segment of DNA containing terC at a site approx.
(20) On the basis of hybridization to synthetic oligonucleotides corresponding to known positions in the terminus region sequence it has been shown that neither the leading nor lagging strands, as they approach terC, traverse the distal inverted repeat, IRI.