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- Hjort, Karin, 1965-
(författare)
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The cell cycle of the hyperthermophilic archaeal genus Sulfolobus
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The third domain of life, Archaea is one of the three main evolutionary lineages together with the Bacteria and the Eukarya domains. The archaea are, despite their prokaryotic cell organisation, more closely related to eukaryotes than to bacteria in terms of the informational pathways (DNA replication, transcription and translation). Organisms from the archaeal hyperthermophilic genus Sulfolobus thrives in a hot (80°C), acidic (pH 2-4) and sulphur-rich environment.In my thesis, I have used a variety of different approaches to study the Sulfolobus cell cycle. After dilution of a stationary phase cell culture with fresh medium, synchronous cell cycle progression was obtained. From the synchronised cell culture experiment we could conclude that the major cell cycle events (nucleoid segregation, cell division and chromosome replication) were tightly coupled to each other and to cellular mass increase. Inhibitors of the elongation stage of chromosome replication, and of cell division, as well as drugs arresting the cell cycle in the post-replicative phase, were found in an in vivo screening of a range of antibiotics. The cell cycle was found to be regulated such that the previous cell cycle step had to be successfully accomplished before the next could initiate, except for DNA replication which could occur without an intervening cell division event.The replication pattern of Sulfolobus solfataricus was analysed using a marker frequency assay. From the results, we were able to determine that a single origin is utilized in vivo, that the replication directionality is bidirectional, and also an approximate location of the replication origin within the genome.Intracellular virus production in vivo of SIRV2 (Sulfolobus islandicus rod-shaped virus2) in Sulfolobus islandicus was also analysed. The effects on the host cell were determined, including loss of cell viability, inhibited initiation of replication at virus infection and DNA degradation and loss of cell integrity at the time of virus release. Also, for the first time intracellular virus DNA was visualized with flow cytometry.
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- Olsson, Jan A, et al.
(författare)
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Eclipse-synchrony relationship in Escherichia coli strains with mutations affecting sequestration, initiation of replication and superhelicity of the bacterial chromosome.
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 334:5, s. 919-31
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Tidskriftsartikel (refereegranskat)abstract
- Initiation of replication from oriC on the Escherichia coli chromosomes occurs once and only once per generation at the same cell mass per origin. During rapid growth there are overlapping replication cycles, and initiation occurs synchronously at two or more copies of oriC. Since the bacterial growth can vary over a wide range (from three divisions per hour to 2.5 hours or more per division) the frequency of initiation should change in coordination with bacterial growth. Prevention of reinitiation from a newly replicated origin by temporary sequestration of the hemi-methylated GATC-sites in the origin region provides the molecular/genetic basis for the maintenance of the eclipse period between two successive rounds of replication. Sequestration is also believed to be responsible for initiation synchrony, since inactivation of either the seqA or the dam gene abolishes synchrony while drastically reducing the eclipse. In this work, we attempted to examine the functional relationship(s) between the eclipse period and the synchrony of initiation in E.coli strains by direct measurements of these parameters by density-shift centrifugation and flow-cytometric analyses, respectively. The eclipse period, measured as a fraction of DNA-duplication times, varied continuously from 0.6 for the wild-type E.coli K12 to 0.1 for strains with mutations in seqA, dam, dnaA, topA and gyr genes (all of which have been shown to cause asynchrony) and their various combinations. The asynchrony index, a quantitative indicator for the loss of synchrony of initiation, changed from low (synchronous) to high (asynchronous) values in a step-function-like relationship with the eclipse. An eclipse period of approximately 0.5 generation time appeared to be the critical value for the switch from synchronous to asynchronous initiation.
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