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A ten-year search for synchronous cells: obstacles, solutions, and practical applications.

Helmstetter CE - Front Microbiol (2015)

Bottom Line: My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end.In this essay, I recount my personal journey in this obsessive experimental pursuit.That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida Institute of Technology Melbourne, FL, USA.

ABSTRACT
My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end. In this essay, I recount my personal journey in this obsessive experimental pursuit. That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells. Subsequent studies with this methodology led to an understanding of the basic properties of the relationship between chromosome replication and cell division. Accordingly, I end this reminiscence with a simple, fool-proof graphical strategy for deducing the pattern of chromosome replication during the division cycle of cells growing at any rate.

No MeSH data available.


Related in: MedlinePlus

Addition of chromosome configurations to a division cycle construction. Chromosomal DNA is represented by red lines, with small filled circles indicating replication sites. The configurations in rectangles indicate cell divisions. Only one cell is followed after the first division. Number of replication forks per cell (RF/cell) is shown during the division cycle.
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Figure 6: Addition of chromosome configurations to a division cycle construction. Chromosomal DNA is represented by red lines, with small filled circles indicating replication sites. The configurations in rectangles indicate cell divisions. Only one cell is followed after the first division. Number of replication forks per cell (RF/cell) is shown during the division cycle.

Mentions: Thinking is now permitted to complete the exercise because the correctly drawn cycle is in front of us. The first step is to add chromosome configurations during the cycle to the sequences drawn in Figure 5. This process is presented in red in Figure 6, above the I + C + D sequences. Chromosomes are represented by straight lines rather than circles because circles are too hard to draw and unnecessary in this schematic representation. Again start at the beginning of the first sequence at the upper left by drawing a short horizontal line to represent a chromosome with no initiation potential residing in a hypothetical cell. The origin of replication is at the left end of the line, and the terminus at the right. Now, progress to the right, looking vertically as you go, to observe where the cell is located in each of the overlapping I + C + D sequences. At the end of the first I period, the chromosome initiates replication, as indicated by a small filled circle at the left end, and preparation begins for the next initiation event as shown in the second I + C + D sequence. After 20 min of the first C period, the chromosome is half replicated. After 35 min of the first C period, the chromosome is 35/40 replicated and new rounds of replication have initiated due to the second sequence, as indicated by two filled circles at the left end. Thus, the cell has begun to progress through two C periods simultaneously. Replication along the first sequence ends 5 min later so the cell now contains two chromosomes each 5/40 replicated. Twenty minutes later the cell divides due to the first sequence. Each daughter cell contains one chromosome that is 25/40 replicated due to the second sequence, and is also 25/35 of the way along preparation for the next initiation event due to the third sequence. This then is the chromosome configuration in a newborn cell in a culture growing with the given parameters. Chromosome replication during the division cycle of these cells is given by the chromosome configurations between the two vertical division lines. Configurations are shown for three key time points in the cycle, again determined by observing what is happening, vertically, in each I + C + D sequence. Note that cells growing at this rate are progressing along three reproductive paths simultaneously, such that the processes leading to a specific cell division began two division cycles in advance.


A ten-year search for synchronous cells: obstacles, solutions, and practical applications.

Helmstetter CE - Front Microbiol (2015)

Addition of chromosome configurations to a division cycle construction. Chromosomal DNA is represented by red lines, with small filled circles indicating replication sites. The configurations in rectangles indicate cell divisions. Only one cell is followed after the first division. Number of replication forks per cell (RF/cell) is shown during the division cycle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4373376&req=5

Figure 6: Addition of chromosome configurations to a division cycle construction. Chromosomal DNA is represented by red lines, with small filled circles indicating replication sites. The configurations in rectangles indicate cell divisions. Only one cell is followed after the first division. Number of replication forks per cell (RF/cell) is shown during the division cycle.
Mentions: Thinking is now permitted to complete the exercise because the correctly drawn cycle is in front of us. The first step is to add chromosome configurations during the cycle to the sequences drawn in Figure 5. This process is presented in red in Figure 6, above the I + C + D sequences. Chromosomes are represented by straight lines rather than circles because circles are too hard to draw and unnecessary in this schematic representation. Again start at the beginning of the first sequence at the upper left by drawing a short horizontal line to represent a chromosome with no initiation potential residing in a hypothetical cell. The origin of replication is at the left end of the line, and the terminus at the right. Now, progress to the right, looking vertically as you go, to observe where the cell is located in each of the overlapping I + C + D sequences. At the end of the first I period, the chromosome initiates replication, as indicated by a small filled circle at the left end, and preparation begins for the next initiation event as shown in the second I + C + D sequence. After 20 min of the first C period, the chromosome is half replicated. After 35 min of the first C period, the chromosome is 35/40 replicated and new rounds of replication have initiated due to the second sequence, as indicated by two filled circles at the left end. Thus, the cell has begun to progress through two C periods simultaneously. Replication along the first sequence ends 5 min later so the cell now contains two chromosomes each 5/40 replicated. Twenty minutes later the cell divides due to the first sequence. Each daughter cell contains one chromosome that is 25/40 replicated due to the second sequence, and is also 25/35 of the way along preparation for the next initiation event due to the third sequence. This then is the chromosome configuration in a newborn cell in a culture growing with the given parameters. Chromosome replication during the division cycle of these cells is given by the chromosome configurations between the two vertical division lines. Configurations are shown for three key time points in the cycle, again determined by observing what is happening, vertically, in each I + C + D sequence. Note that cells growing at this rate are progressing along three reproductive paths simultaneously, such that the processes leading to a specific cell division began two division cycles in advance.

Bottom Line: My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end.In this essay, I recount my personal journey in this obsessive experimental pursuit.That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Florida Institute of Technology Melbourne, FL, USA.

ABSTRACT
My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end. In this essay, I recount my personal journey in this obsessive experimental pursuit. That narrative is followed by a description of a simplified version of the "baby machine," a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells. Subsequent studies with this methodology led to an understanding of the basic properties of the relationship between chromosome replication and cell division. Accordingly, I end this reminiscence with a simple, fool-proof graphical strategy for deducing the pattern of chromosome replication during the division cycle of cells growing at any rate.

No MeSH data available.


Related in: MedlinePlus