Merging molecular biology with general bacteriology, basic genetics and sophisticated microscopic and physical techniques discovered the sexuality and circularity of the bacterial chromosome Jacob and Wollman, ; Cairns, ; Hayes, , its replication schedule Helmstetter et al. The numerous scientists who passed through it during their careers Anderson et al. The seminal series of experiments with Salmonella typhimurium published in in two back-to-back articles Kjeldgaard et al.
The stream of articles stemming from the Institute became a flood of crucial information published in the most prestigious periodicals of the time. After physiological manipulations were seemingly exhausted, the use of drugs and mutants became common when the mechanisms of their actions were, or thought to be deciphered. The multi-faceted phenotypes exerted by these lack of specificity and pleiotropism, respectively occasionally remind us to stick to this rule-of-thumb in order to keep interpretations of results as crystal-clear as possible.
This first leg of the journey to understand the logic behind the duplication of a bacterial cell, which took place in the s, is described in this collection by Schaechter , and the other two, partially overlapping legs in the s—by Hanawalt and Helmstetter Phil studied the phenomenon of thymineless-death TLD in thymine-starved populations of thyA mutants Cohen and Barner, employing it to better understand the connection between chromosome replication and cell growth and viability Hanawalt et al.
Thinking rigorously, they derived marker frequency equations Sueoka and Yoshikawa, that survived the test of time. Bidirectionality of the replication has later been demonstrated by various genetic, physiological and microscopic means e. Two essential, unique macromolecules structures exist in a bacterium: DNA nucleoid that stores the genetic information, and the shape-maintaining peptidoglycan sacculus , which also protects the cell from rupture by its osmotic pressure turgor. To survive, the cell must divide after its genome doubles and in a plane between the two emerging sets, hence duplications of the two are coupled, temporally and spatially.
Much effort is expended to discover the mechanism responsible for this coupling, which raises the efficacy of competition among species.
Wild-type E. Slower rates are obtained on poorer sources, whereas adding organic building blocks result in faster rates, the maximum achievable being about 3 h —1 i. A cell divides into two morphologically-identical daughters Trueba and Woldringh, about 20 min designated D after termination of replication hence division follows replication-initiation by about 1 h. It can thus be termed Zaritsky et al. Combining the noted constancy of C and D values Helmstetter et al.
The molecular mechanism regulating initiation of replication, occurring synchronously from all existing oriC copies and once per cell cycle, is under investigation e. This was the current knowledge at the end of , upon the arrival of one of us AZ at Leicester University for graduate studies, supervised by Robert Pritchard, who had established the Genetics Department there merely 4 years earlier 1.
Digressing to some personal involvements, one of us AZ was very lucky to enter the atmosphere inspired by Bob and at the right time to be assigned a project in the just-opened BCD field, about which I had no clue. During 6-years of previous studies — at the Hebrew University of Jerusalem, my M. It may have been important for my active participation in developing CCSim, as described below. Simultaneously, the other CLW extended his biological and microscopic skills at the University of Amsterdam.
There are at least three at that time commonly-accepted ideas that I ruled out during my Ph. My close association with Nanne Nanninga e. In their search to define the structural changes occurring during fixation and dehydration necessary for visualizing the bacterial nucleoid in the electron microscope, the possibilities to study live cells were improved with the reinvention and development of the confocal scanning light microscope CSLM by Brakenhoff see Valkenburg et al.
Back to the main subject, at Leicester, Bob realized existence of literature-recorded contradictory results, the common feature of most is that they were obtained in thymine-requiring strains.
These observations e. This hypothesis could explain all discrepancies and is consistent with lack of active thymine-transport, in E. It was strongly confirmed by four physiological methods, more or less independent of each other Pritchard and Zaritsky, ; Zaritsky, , and later supported by various means in other laboratories reviewed in Zaritsky et al. Thus, the dissociation between syntheses rates of mass and DNA, originally observed by changing the former alone Helmstetter et al.
This method is more amenable to analysis than nutritional shifts because modulating [dTTP] by changing [T] occurs abruptly, without affecting the multitude of metabolic pathways and interactions between them that accompany nutritional shifts Scott and Hwa, In a steady-state exponentially growing culture, concentrations of all cell components increase in parallel to each other and in pace with divisions Campbell, ; Fishov et al.
The puzzling phenomenon of division rate-maintenance after a nutritional shift-up Kjeldgaard et al. Specific inhibition of protein or DNA synthesis, however, allows divisions to continue during the D period; these so-called residual divisions cause a decrease in average cell length cf.
Determination of C and D periods for batch cultures of E. Huls et al. From these studies it becomes clear how these cell cycle periods can vary with different strains and growth conditions.
The measurements indicate that the D period is especially variable, making it difficult to generalize the E. When thymine-limited thyA mutants grow at fast growth rates, another puzzling phenomenon appears, namely dissociation between growth and division that is related to replication. Under these conditions, the inter-division time is longer than mass doubling time i.
The 40 years-old observation Zaritsky, a that indicated existence of a minimal possible distance l min between two successive replisomes, promptly explains this phenomenon Zaritsky et al. The question whether the mechanism involved is structural replisome size; Norris et al. Such a breach can be achieved by enhanced initiation frequency Simmons et al.
Release from this situation by restoring the permissive conditions causes a transient increase in the frequency of divisions Zaritsky et al. Our fortuitous encounter at the Luntern Conference in November was very fortunate. In , both of us had already acquired results related to morphometric variations of E.
These and follow-up visits culminated in detailed descriptions of cell dimensional rearrangements during nutritional shift-up experiments Grover et al. One notable outcome of our interactions was implementation of an interactive simulation program Zaritsky et al.
This program implementation was enabled by the recruitment of Norbert Vischer, a computer engineer, by the Amsterdam department chair and faculty dean Nanne Nanninga. Figure 1. All considerations described so far and by the CCSim Figure 2 do not relate to cell dimensions and shape nor to nucleoid segregation.
Future versions of CCSim may be extended to incorporate these aspects. Figure 2. An exponentially growing bacillary cell elongates with unnoticeable change in width, and divides evenly at a perpendicular plane Trueba and Woldringh, The seminal observation Schaechter et al. It was initially interpreted to involve active regulation of length L Grover et al.
This view was later abandoned when peptidoglycan synthesis was demonstrated to be diffuse throughout the cylindrical periphery and only localized during the division process Woldringh et al. With such models in mind, we measured Figure 1 the dimensions of E.
Our nutritional-upshift experiment Woldringh et al. Consequent to this slow adaptation and almost immediate change in the rate of mass synthesis, cell length overshoots, but the mechanism governing this diameter change is still enigmatic.
A diameter increase during the constriction process has also been implied in populations growing in steady state where the cells showed a diameter decrease during elongation see Figure 4 in Trueba and Woldringh, It should be noted that in all these preparations the cells had been fixed with osmium tetroxide and were air-dried, causing their flattening Vardi and Grover, Nevertheless, the measurements compared well with those obtained from hydrated cells with phase-contrast microscopy cf.
Table 3 in Trueba and Woldringh, Figure 3. Electron micrograph of a mixture of two E. The big cells were grown in trypton broth with a doubling time of 22 min; the small cells were grown in synthetic alanine-medium with a doubling time of min. Compare with a similar preparation of mixed populations in Figure 2 of Nanninga and Woldringh Figure 4.
Figure 3 in Nanninga and Woldringh, The nucleoids show up as electron-transparent regions in the air-dried cells, flattened by surface tension cf. Woldringh et al. Red arrows indicate constriction sites, blue arrows, tapered tips. Associated with cell widening, the nucleoids bright areas in Figure 4 start replicating in planes tilted to the long cell axis Figure 4 , rather than parallel to it as during slow growth conditions. The differences in cell dimensions and nucleoids replication-planes are pronounced when thyA cells grow under identical conditions but with limiting [T] that impose slow replication rate compare, e.
These studies clarified that individual cells elongate exponentially i. The results led Koppes et al. Figure 5. Semi-log plot of cell length as a function of cell age cf. Figure 6 in Koppes et al. According to this hypothesis, accumulation of these proteins to a fixed threshold each generation would serve as a trigger for cell division. This proposal, however, does not relate mass growth to the DNA replication cycle, as suggested four decades ago Zaritsky, b.
If P-sector proteins are at a fixed number per cell, then they would become diluted during the interdivision time molecules fixed, but cell volume increases. Therefore, it is not clear how it could result in their accumulation to trigger division. Other aspects of this idea have recently been rebutted in more details Zaritsky, Coupling between DNA replication and cell elongation could be obtained by the nucleoid occlusion mechanism that is being relieved when daughter nucleoids are segregating apart Mulder and Woldringh, ; Nanninga et al.
In other words, a length increment of the nucleoid would be sensed rather than a length increment of the cell. Youngren et al. However, while during slow growth all newborn cells can be assumed to contain nucleoids with the same amount of DNA, this will not hold for fast growth showing multifork replication. Here, stochastic premature or postponed division of mother cells will produce small and large daughter cells, respectively, with different amounts of DNA per nucleoid and thus different stages of segregation.
Here, sensing of a constant length increment is starting at the last initiation of DNA replication. How a size increment rather than a critical size is monitored and whether nucleoid segregation is involved in such a model remains to be seen. Thus I follow my lectures with videos and classroom discussions, and their homework consists of finding cool biology videos or articles and posting the links on the classroom blog for all to see. A couple of times I used malaria as a thread that connected all the topics - from cell biology to ecology to physiology to evolution.
I think that worked well but it is hard to do. They also write a final paper on some aspect of physiology. Another new development is that the administration has realized that most of the faculty have been with the school for many years.
We are experienced, and apparently we know what we are doing. Thus they recently gave us much more freedom to design our own syllabus instead of following a pre-defined one, as long as the ultimate goals of the class remain the same. I am also worried that, since I am not actively doing research in the lab and thus not following the literature as closely, that some of the things I teach are now out-dated. Not that anyone can possibly keep up with all the advances in all the areas of Biology which is so huge, but at least big updates that affect teaching of introductory courses are stuff I need to know.
I need to catch up and upgrade my lecture notes. And what better way than crowdsource! So, over the new few weeks, I will re-post my old lecture notes note that they are just intros - discussions and videos etc. If I got something wrong or something is out of date, let me know but don't push just your own preferred hypothesis if a question is not yet settled - give me the entire controversy explanation instead.
If something is glaringly missing, let me know. If something can be said in a nicer language - edit my sentences. If you are aware of cool images, articles, blog-posts, videos, podcasts, visualizations, animations, games, etc. And at the end, once we do this with all the lectures, let's discuss the overall syllabus - is there a better way to organize all this material for such a fast-paced class.
Today, we continue with the cell biology portion of the course - covering the way cells communicate with each other, something that will come up over and over again for the rest of the course.
See the previous lectures:. Biology and the Scientific Method. BIO — Cell Structure. What is the mechanism of a red flag, or danger signal that activates a checkpoint? How does it alert the cell? This starts a that temporarily halts S phase progression. Therefore, ATR is also known as the S phase "checkpoint kinase. ATR kinase acts in several ways to keep the replication process intact. One hypothesis is that phosphorylation of one or several of the Mcm subunits prevents the CMG complex from unwinding more and more DNA.
This action effectively stops the process so that it can be repaired before proceeding. Currently, many researchers are trying to better understand the mechanisms of crosstalk between ATR and the replication machinery Forsburg ; Bailis et al. Nature Reviews Molecular Cell Biology 9 , doi So why would normal cells need ATR?
There are other circumstances that cause replication to go awry. One is that the DNA template somehow becomes defective during replication, and causes the polymerase to pause Figures 3 and 4a. For example, a DNA base can be chemically modified or spontaneously altered. Scientists use the term "stalled forks" for areas of replication forks where DNA polymerization is halted. Little is known about the phosphorylation targets that lie further downstream of Chk1, but when scientists observe Chk1 phosphorylation in cells, they conclude that cells are actively trying to protect replication forks with DNA lesions.
What happens when ATR function goes awry? A DSB is a catastrophic event because it ruins the replication fork. Under these circumstances, cells activate the ATM kinase Figure 4, on the right.
It does so by phosphorylating checkpoint kinase 2 Chk2 , a protein that triggers a cascade of phosphorylation events that ultimately result in the repair of the DSB. Interestingly, when Chk2 triggers events that ultimately repair a DSB, another event also takes place. This event is the phosphorylation of the well-known p53 Caspari This observation is a clue that repairing DSBs may have something to do with preventing the formation of tumors. Together with a variety of other molecules, ATR and ATM kinases are key factors for the surveillance of DNA replication, and prevent chromosome breakage in dividing cells.
However, during repair processes, chromosome fragments can be improperly joined together. Indeed, some scientists consider that such mistakes enable some degree of genetic evolution by creating new and different genetic sequences.
Nevertheless, if even a single cell in our body makes a mistake and fuses DNA fragments to each other that are not supposed to be joined, the rearrangement can be sufficient to deregulate normal cell division. If multiple changes of this type accumulate, then this single cell can eventually turn into a tumor. In these affected individuals, the cellular surveillance system described above is defective and no longer provides full protection from random events that affect DNA replication.
For example, the name of the ATM protein derives from the affliction that results from a mutated ATM protein: ataxia telangiectasia. In this disease , patients suffer from motor and neurological problems, and they also have what is known as a genome instability syndrome that genetically predisposes them to developing cancer Shiloh With these observations, it may be possible to create new ideas for novel diagnostics and therapies for cancer that specifically track these potent molecules.
The process of DNA replication is highly conserved throughout evolution. Investigating the replication machinery in simple organisms has helped tremendously to understand how the process works in human cells. Major replication features in simpler organisms extend uniformly to eukaryotic organisms, and replication follows fundamental rules. During replication, complex interactions between signaling and repair proteins act to keep the process from going awry, despite random events that can cause interruption and failures.
Discovering the exact repair mechanisms that help keep DNA intact during replication may help us understand the mechanisms of tumor growth, as well as develop strategies to detect or treat cancer.
Alberts, B. DNA replication and recombination. Nature — Anderson, S. Metabolism of Okazaki fragments during simian virus 40 DNA replication. The Journal of Biological Chemistry — Bailis, J. Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote S-phase genome stability.
Molecular and Cellular Biology 28 — doi Bochman, M. The Mcm complex has in vitro helicase activity. Molecular Cell 31 — The Mcm complex: unwinding the mechanism of a replicative helicase. Microbiol Mol Biol Rev 73 — Caspari, T. How to activate p Current Biology 10 R— doi Chattopadhyay, S. Molecular Biology of the Cell 18 — doi: Cimprich, K. This process involves replication of the cell's chromosomes, segregation of the copied DNA, and splitting of the parent cell's cytoplasm.
The outcome of binary fission is two new cells that are identical to the original cell. In contrast to prokaryotic cells, eukaryotic cells may divide via either mitosis or meiosis.
Of these two processes, mitosis is more common. In fact, whereas only sexually reproducing eukaryotes can engage in meiosis, all eukaryotes — regardless of size or number of cells — can engage in mitosis.
But how does this process proceed, and what sorts of cells does it produce? During mitosis, a eukaryotic cell undergoes a carefully coordinated nuclear division that results in the formation of two genetically identical daughter cells.
Mitosis itself consists of five active steps, or phases: prophase, prometaphase, metaphase, anaphase, and telophase. Before a cell can enter the active phases of mitosis, however, it must go through a period known as interphase , during which it grows and produces the various proteins necessary for division.
Then, at a critical point during interphase called the S phase , the cell duplicates its chromosomes and ensures its systems are ready for cell division. If all conditions are ideal, the cell is now ready to move into the first phase of mitosis. This page appears in the following eBook.
Aa Aa Aa. Walther Flemming's drawing of chromosomes. What happens during mitosis? Figure 1: During prophase, the chromosomes in a cell's nucleus condense to the point that they can be viewed using a light microscope.
Prophase is the first phase of mitosis. During this phase, the chromosomes inside the cell's nucleus condense and form tight structures. In fact, the chromosomes become so dense that they appear as curvy, dark lines when viewed under a microscope Figure 1.
Because each chromosome was duplicated during S phase, it now consists of two identical copies called sister chromatids that are attached at a common center point called the centromere.
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