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1

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Eclipse period without sequestration in Escherichia coli (2002)

Olsson, Jan ; Dasgupta, Santanu ; Berg, Otto G. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 44 (2002), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The classical Meselson–Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy (15NH4+ and 13C6-glucose) medium were shifted to light (14NH4+ and 12C6-glucose) medium. The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes. The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02954.x

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2

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Role of the mukB gene in chromosome and plasmid partition in Escherichia coli (2000)

Weitao, Tao ; Dasgupta, Santanu ; Nordström, Kurt

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 38 (2000), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The intracellular locations of oriC and oriR1, the replication origins of the chromosome and plasmid R1, respectively, were visualized by fluorescence in situ hybridization (FISH) in exponentially growing populations of Escherichia coli. The locations of oriC and oriR1 (from a Par+ R1 plasmid) were unique and different in the wild-type host. In a mukB mutant, the positions were perturbed for both origins. The position of oriR1 from a plasmid with active partition (Par+) in the mukB host was as randomized as that of oriR1 from the Par− plasmid in a wild-type host. However, this mukB-induced randomization did not result in unstable inheritance of the Par+ plasmid, as measured by the conventional segregation assay. This might result from the preferential association of the Par+ plasmid with the bigger, decondensed nucleoid-containing daughters during cell division of MukB− cells, whereas the Par− plasmids were distributed at random and were lost by frequently ending up in anucleate cells.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2000.02138.x

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3

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Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity (2001)

Maisnier-Patin, Sophie ; Nordström, Kurt ; Dasgupta, Santanu

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 42 (2001), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non-permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double-strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02718.x

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4

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Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli (1998)

Britton, Robert A. ; Powell, Bradford S. ; Dasgupta, Santanu ; [et al.]

Oxford BSL : Blackwell Science Ltd, UK

Molecular microbiology 27 (1998), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Era is a low-molecular-weight GTPase essential for Escherichia coli viability. The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild-type Era become arrested in the cell cycle at the predivisional two-cell stage. The partially defective Era GTPase mutation (era1) suppresses several temperature-sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division. Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis. Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00719.x

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5

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Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli (1998)

Maisnier-Patin, Sophie ; Dasgupta, Santanu ; Krabbe, Margareta ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 30 (1998), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC. Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1998.01136.x

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6

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Mutual suppression of mukB and seqA phenotypes might arise from their opposing influences on the Escherichia coli nucleoid structure (1999)

Weitao, Tao ; Nordström, Kurt ; Dasgupta, Santanu

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 34 (1999), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: A strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas filamentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing influences on the organization of the bacterial nucleoid.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01589.x

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7

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Direct visualization of plasmid DNA in bacterial cells (1992)

Eliasson, Åsa ; Bernander, Rolf ; Dasgupta, Santanu ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

Molecular microbiology 6 (1992), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase-fluorescence microscopy of DAPI (4′,6-diamidino-2-phenylindole)-stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb01997.x

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8

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Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system (2003)

Olsson, Jan A. ; Berg, Otto G. ; Dasgupta, Santanu ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 50 (2003), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson–Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30°C to 42°C. The mutant plasmid had a similar eclipse at temperatures up to 38°C. At 42°C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350–400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42°C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03683.x

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9

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Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells (1986)

Kuriyama, Ryoko ; Dasgupta, Santanu ; Borisy, Gary G.

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 6 (1986), S. 355-362

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ISSN: 0886-1544

Keywords: centriole ; DNA synthesis ; cell cycle ; Chinese hamster ovary cells ; taxol ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 μg/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.

Additional Material: 6 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970060402

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