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R G Wake

Publications and source records attributed to R G Wake.

At least 73 records · Page 4Linked to original sources
PubMed1984

Impediment to replication fork movement in the terminus region of the Bacillus subtilis chromosome.

The terminus regions of the chromosomes of three strains of Bacillus subtilis 168 were radioactively labelled by supplying [3H]thymine towards the end of a round of replication. These strains lacked or contained the prophage SP beta c2. Following restriction endonuclease digestion of the purified DNA and fluorography, an SP beta c2-related perturbation of the terminus-labelling profile was observed, which was completely consistent with the previously suggested existence of an impediment to replication fork movement (terC) within a BamHI 24.8 X 10(3) base fragment (Weiss & Wake, 1983). The present data suggest that terC is located within the 11.4 X 10(3) base BamHI + SalI double-digest portion of this BamHI fragment.

Bacillus subtilis↗
PubMed1984

A unique DNA intermediate associated with termination of chromosome replication in Bacillus subtilis.

A unique DNA structural intermediate associated with termination of replication in Bacillus subtilis has been identified. This was achieved by probing for the Bam HI DNA fragment within which the terminus of the chromosome (terC) resides. The intermediate migrates more slowly than the bulk of digested chromosomal DNA upon electrophoresis. It is more readily detected in DNA from a merodiploid strain in which the clockwise fork should be immobilized at terC for longer than usual, and it is destroyed by S1 nuclease. The intermediate may be a forked or related structure. These findings provide evidence for the existence of a specific, sequence-based termination site that blocks or severely impedes the movement of at least the clockwise fork, which is the first to encounter it.

Bacillus subtilis↗
PubMed1984

Physical map of the Bacillus subtilis replication terminus region: its confirmation, extension and genetic orientation.

The library of Bacillus subtilis DNA previously cloned in the cosmid vector pHC79 has been screened for the replication terminus region using a higher level of terminus probe. 24 of 48 recombinant cosmids which gave a positive response had restriction fragment compositions consistent with their inserts originating from or extending into the terminus region for which a 150-kb restriction map has already been constructed (Weiss and Wake, 1983). DNA spanning terC, the site of termination, appears to be missing from the library, although DNA to either side of terC has been cloned. A detailed analysis of four of the newly identified recombinant cosmids has confirmed most of the previous 150-kb map and allowed it to be extended to 180 kb. Physical linkage of the two cosmid inserts that most closely approach terC on each side has been demonstrated. The location of the genetic marker gltA and the orientation of the restriction map relative to the genetic map of the B. subtilis chromosome have also been established.

Bacillus subtilis↗
PubMed1983

Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome.

The Bacillus subtilis 168 dna-1 chromosome was labelled during sporulation with [3H]thymine for five minutes immediately before termination of replication. The isolated radioactive DNA was cleaved with BamHI (or SalI) and the resulting restriction fragments separated by agarose gel electrophoresis. The individual fragments, fractionated into a series of slices cut from the gel, were then cleaved with SalI (or BamHI) and the double-digest fragments identified by electrophoresis and fluorography. All major fragments and most minor ones present in a whole double-digest were assigned to BamHI and SalI parents. Such information enabled the construction of an unambiguous restriction map of 150 X 10(3) bases of the approximately 250 X 10(3) bases of DNA labelled in the five minutes. In conjunction with published data on the order of replication of restriction fragments as termination is approached, it was clear that most (105 X 10(3) bases) of the mapped DNA was replicated by a major fork moving in one direction towards a BamHI 24.8 X 10(3) base fragment. The 45 X 10(3) bases extending to the other side of this region were labelled only slightly, and presumably was replicated by a fork that approached the other in an opposite direction until its progress was blocked or severely impeded within this region at a site, referred to as terC, sometime (less than 5 min) earlier. The regions of the map replicated in the final 2.5 and 1.0 minute by the major fork were also identified.

Bacillus subtilis↗
PubMed1983

Cloning DNA from the replication terminus region of the Bacillus subtilis chromosome.

DNA from the Bacillus subtilis 168 prototroph, SB19, was partially cleaved with MboI and cloned into the BamHI site of the Escherichia coli cosmid vector, pHC79. [3H]thymine-labelled DNA from the replication terminus region of the B. subtilis dna-1 chromosome was used to identify, by hybridization, clones harboring recombinant cosmids carrying inserts from the terminus region. Restriction maps have been constructed for two such cosmids carrying overlapping DNA inserts that cover or extend into four of the previously identified five SalI fragments which are replicated last. The composite map of the cloned region, together with the available data on the replication order of fragments within it, is consistent with its replication being achieved by the unidirectional movement of a fork through it and towards the late replicating 16.2-kb SalI fragment. Most, if not all, of the terminus sequences in at least one of the recombinant cosmids are missing from a viable strain of B. subtilis that carries a deletion in the SP beta-gltA region of the chromosome.

Bacillus subtilis↗
PubMed1983

Timing and other features of the action of the ts1 division initiation gene product of Bacillus subtilis.

The ts1 division initiation mutation of Bacillus subtilis 160 was transferred into a thymine-requiring strain of B. subtilis 168. Aspects of the role and timing of the action of the ts1 gene product in relation to septum formation were studied by comparing the behavior of this new strain with that of the isogenic wild type after outgrowth of germinated spores. The ts1 gene product was shown to be required for the asymmetric division which occurs in the absence of chromosome replication, in addition to normal division septation. The time interval between completion of the action of the ts1 gene product and initiation of the first central division septum was estimated to be less than 4 min at 34 degrees C, and it is possible that an active ts1 gene product is required until the commencement of septal growth. Recovery of septa after transfer of outgrown spores (filaments) from the nonpermissive to the permissive temperature was also examined. During recovery, septa formed at sites which were discrete fractional lengths of the filaments, with the first septum located at the most polar of these sites. The data have been interpreted in terms of the formation of potential division sites at the nonpermissive temperature and the preferred utilization, upon recovery, of the most recently formed site. Recovery of septa at the permissive temperature occurred in the absence of DNA synthesis but was blocked completely by inhibitors of RNA and protein synthesis. It is possible that the only protein synthesis required for recovery of septa is that of the ts1 gene product itself.

Bacillus subtilis↗
PubMed1982

Conformation and segregation of nucleoids accompanying cell length extension after completion of a single round of DNA replication in germinated and outgrowing Bacillus subtilis spores.

When germinating spores of the temperature-sensitive DNA initiation mutant of Bacillus subtilis TsB134 are shifted to the restrictive temperature at a time such that just one or two rounds of replication are accomplished, the completed, nonreplicating nucleoids that form eventually adopt a doublet conformation. This conformation has now been observed after fixation by glutaraldehyde or osmium tetroxide, as well as by Formalin as found previously. The doublet was observed in media of different degrees of richness and under both light and electron microscopes. Electron micrographs of serial sections through the doublet were consistent with its formation by the gradual pulling apart of a single mass of DNA into two lobes. A systematic study was made of the effect of the time of shifting from the permissive to the restrictive temperature and of the restrictive temperature used on the number of nucleoids segregating within the outgrowing rod. It was established that the doublet nucleoid behaved as a single unit in replication control and segregation in both rich and poor media. Measurement of the relative position of the two segregating nucleoids within the outgrowing rod after completion of just one round of replication yielded quantitative information on the segregation and cell length extension processes. Segregation was accompanied by cell length extension at approximately equal rates on both sides of each nucleoid. Furthermore, the data were consistent with an exponential increase in such an extension with time over the early and major portion of the period studied, but it was not possible to rule out other models of length extension.

Bacillus subtilis↗
PubMed1981

Characterization and mapping of temperature-sensitive division initiation mutations of Bacillus subtilis.

Two temperature-sensitive, filamenting mutants of Bacillus subtilis (ts1 and ts12) have been shown to be defective in the initiation of septation. Recombination index mapping showed that these mutations mapped in two different but closely linked genes. A third proposed initiation mutation, tms-12, probably maps in the same gene as ts12. Another proposed initiation mutation was not linked with these genes by transformation, indicating that there was a minimum of three genes involved in the initiation of division. PBS1 transduction mapping located these three genes close to the pyr cluster.

Bacillus subtilis↗
PubMed1979

Completed Bacillus subtilis nucleoid as a doublet structure.

When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.

Bacillus subtilis↗
PubMed1977

Initiation of deoxyribonucleic acid replication in germinating spores of Bacillus subtilis 168 carrying the dnaB (Ts)134 mutation.

The nature of the deoxyribonucleic acid synthesis reported by others to occur at 45 degrees C in germinating spores of the temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 45 degrees C. At a slightly higher temperature, 49 degrees C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts(+) strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 45 degrees C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions.

Bacillus subtilis↗
PubMed1976

Segregation of Bacillus subtilis chromosomes radioactively labeled during the first round of replication after germination of spores.

Spores of Bacillus subtilis W23 thy his were allowed to incorporate [3H]thymine for short periods of time either continuously from, or soon after, the start of the first round of replication after germination. They were then transferred to nonradioactive medium to allow growth into microcolonies (up to 12 cells), which were autoradiographed. The relative numbers of various types (major versus minor) of grain clusters associated with individual microcolonies throughout the populations were scored. Analysis of the results showed clearly that, in the majority of spores at least, only one chromosome was undergoing replication soon after the start of deoxyribonucleic acid synthesis. Furthermore, under the conditions used, no evidence for initiation of replication of a second chromosome within 25 min after the first could be obtained. Accepting that B. subtilis spores are essentially homogenous in deoxyribonucleic acid content, the results support the conclusion that the spore contains only one copy of the chromosome, not two.

Bacillus subtilis↗