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Since the 1960’es the conformation and segregation of the chromosome in Escherichia coli has been a subject of interest for many scientists. However, after 40 years of research, we still know incredibly little about how the chromosome is organized inside the cell, how it manages to duplicate this incredibly big molecule and separate the two daughter chromosomes and how it makes sure that the daughter cells receives one copy each. The fully extended chromosome is two orders of magnitude larger than the cell in which it is contained. Hence the chromosome is heavily compacted in the cell, and it is obvious that structured cellular actions are required to unpack it, as required for its replication, and refold the two daughter chromosomes separately without getting them entangled in the process each generation. The intention of the study was initially to find out how the chromosome is organized in the cell by labeling specific parts of it. Later the dynamics of chromosome segregation was included. Investigating chromosome organization by labeling of specific loci was already a widely used technique when I started on this thesis, but the data acquisition and treatment was slow and generally poorly described. There was a great need for an automatic standardized method capable of identifying the number and position of fluorescent foci in cells on photomicrographs fast and precise. A major part of my three-year study was devoted to the development of such a procedure. The result which is described in the accompanying Paper I, is a macro (program) written for the image analysis software Image Pro Plus capable of measuring the physical outline of cells, counting the number of foci within, and measuring their intra-cellular position. 1000 cells are processed in 3 minutes. The development of this fast and reliable method enabled us to start the analysis on the distribution of various chromosomal loci inside slowly growing cells. With the actual counting and measuring no longer being any problem we could easily analyze 14 loci distributed on the E.coli chromosome. More than 15.000 cells were analyzed in total. The results are described in the accompanying Paper II and show clearly that the chromosome is segregated progressively. An unexpected delay between the replication and segregation of markers was also observed and led to a new model on the timing of chromosomal segregation (the Sister Loci Cohesion Model). The results of Paper II also strongly indicated that the chromosome is not replicated in a central factory but by separated and migrating replication forks. A result confirmed by others. Finally we developed a new labeling system compatible with the existing labeling system based on the P1 par system. Using the new system, which is based on the pMT1 par system from Yersenia pestis, we labeled loci on opposite sides of the E.coli chromosome simultaneously and were able to show that the E.coli chromosome is organized with one chromosomal arm in each cell half. This astounding result is described in Paper III. Adding the results of the thesis together with known data results in the following description of the chromosome dynamics of slowly growing E.coli cells: The chromosome of slow growing cells is organized with the origin at the cell center when it is newborn. It has one chromosomal arm on one side of the center and the other chromosomal arm on the other side. The terminus is at the new pole but migrates to the center soon after cell division. Replication is initiated at the origin at the cell center. The duplicated origins stay together for a short while and then migrate to the cell quarters. As the origins migrate away from the center the replication forks split up too and are from this point found on opposite sides of the cell center but randomly distributed. Supposedly the forks track along the two chromosomal arms that are separated to each cell half. As the forks replicate the two arms, the duplicated loci stay together for a while at the non-central position where they were replicated. This delay is the same for all loci. Thus segregation is progressive at a rate comparable to the rate of replication but segregation is delayed with respect to replication. After the delay one of the replicated loci is segregated to the other side of the cell center and the other one stays where it is. This way of segregating the chromosome ultimately leads to the placement of the two arms of the chromosome on each side of the cell quarter. Finally the replication forks meet at the terminus in the cell center and the replication is complete. The terminus does not separate until cell division where after it migrates to the new cell center and the original configuration is re-established.
|Number of pages||90|
|Publication status||Published - Jan 2007|