Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 100

It is important to know when replication occurs during the cell cycle. Experiments were designed to determine the relationship between the time of chromosome replication and the cell cycle in E. coli (see Helmstetter and Cooper, Suggested Reading). The conclusions are still generally accepted, so it is worth going over them in some detail. The investigators recognized that if the DNA content of cells at different stages in the cell cycle could be measured, it would be possible to determine how far chromosome replication had proceeded at that time in the cell cycle. Since bacterial cells are too small to allow measurements of DNA content in a single cell by the methods they had, it was necessary to measure the DNA content in a large number of pooled cells. However, cells growing in culture are all at different stages in their cell cycles. Therefore, to know how far replication had proceeded at a certain stage in the cell cycle, it was necessary to synchronize the cells in the population so that all were the same age or at the same point in their life cycles at the same time.

Helmstetter and Cooper accomplished this by using what they called a bacterial “baby machine.” Their idea was to first label the DNA of a growing culture of bacterial cells by adding radioactively labeled nucleosides and then fix the bacterial cells on a membrane. When the cells on the filter divided, one of the two daughter cells would no longer be attached and would be released into the medium. All of the daughter cells released at a given time would be newborns and so would be the same age. This means that cells that divided to release the daughter cells at a given time would also be the same age and would have DNA in the same replication state. The amount of radioactivity in the released cells was then a measure of how much of the chromosome had replicated in cells of that age. This experiment was done under different growth conditions to show how the timing of replication and the timing of cell division are coordinated under different growth conditions.

Figure 1.23 shows the results of these experiments. For convenience, the following letters were assigned to each of the intervals during the cell cycle. The letter I denotes the time from when the last round of chromosome replication initiated until a new round begins. C is the time it takes to replicate the entire chromosome, and D is the time from when a round of chromosome replication is completed until cell division occurs. The top of the figure shows the relationship of I, C, and D when cells are growing slowly, with a generation time of 70 min. Under these conditions, I is 70 min, C is 40 min, and D is 20 min. However, when the cells are growing in a richer medium and are dividing more rapidly, with a generation time of only 30 min, the pattern changes. The C and D intervals remain about the same, but the I interval is much shorter, only about 30 min.

Figure 1.23 Timing of DNA replication during the cell cycle, with two different generation times (A and B). The time between initiations (I) is the only time that changes. See the text for definitions of I, C, and D.

Some conclusions may be drawn from these data. One conclusion is that the C and D intervals remain about the same independent of the growth rate. At 37°C, the time it takes the chromosome to replicate is always about 40 min, and it takes about 20 min from the time a round of replication terminates until the cell divides. However, the I interval gets shorter when the cells are growing faster and have shorter generation times. In fact, the I interval is approximately equal to the generation time—the time it takes a newborn cell to grow and divide. This makes sense because, as discussed below, initiation of chromosome replication appears to occur every time the cells reach a certain size. They reach this size once every generation time, independent of how fast they are growing.

Another point apparent from the data is that in cells growing rapidly with a short generation time, the I interval can be shorter than the C interval. If I is shorter than C, a new round of chromosomal DNA replication will begin before the old one is completed. This explains the higher DNA content of fast-growing cells as compared to slowgrowing cells. It also explains the observation that genes closer to the origin of replication are present in more copies than are genes closer to the replication terminus.

Despite providing these important results, this elegant analysis does not allow us to tell whether division is coupled to initiation or termination of chromosomal DNA replication. The fact that the I interval always equals the generation time suggests that the events leading up to division are set in motion at the time a round of chromosome replication is initiated and are completed 60 min later independent of how fast the cells are growing. However, it is also possible that the act of termination of a round of chromosome replication sets in motion a cell division 20 min later. Multiple laboratories are working to resolve these issues.