Chapter 5, Problem 11P

A.

Summary Introduction

To explain: The reason why in the two experiments, some regions of the tracks are dense with silver grains (dark), whereas others are less dense (light).

Concept introduction:  Radioactive substances have decay emissions in the form of β particles and γ-rays. This property is exploited to study DNA replication where one of the molecules of the DNA is replaced with a radioactive element and the decay emissions are captured in a photographic plate to study the rate of replication.

Explanation of Solution

Given information: The DNA synthesis in tissue-culture cells is investigates with $\text{H}$ -thymidine was used to radioactively label the replication forks. Autoradigraphy was performed. The emulsion sensitive to radioactive emissions therefore, $\text{H}$ -thymidine labeled DNA is observed as tracks of silver grains. Due to stretching the replication bubbles are stretched and the daughter duplexes lie side by side undistinguishable from each other.

The cells are pretreated in order to synchronize all cells to the beginning of S phase. In the first set of experiment, the synchronizing block is released and $\text{H}$ -thymidine is added immediately. After 30 minutes, the medium is washed to reduce the total concentration of thymidine. Post 15 minutes, the DNA is prepared for autoradiography. In the second set of experiment, the synchronizing block is released and $\text{H}$ -thymidine is added after 30 minutes. After 30 minutes, the medium is washed to reduce the total concentration of thymidine. Post 30 minutes, the DNA is prepared for autoradiography.

The silver grains (darks) tracks in the autoradiograph that are dense refers to the segments of DNA that had undergone replication when the $\text{H}$ -thymidine concentration was high. The less dense (light) tracks in the autoradiograph refers to segements of DNA that has undergone replication when the $\text{H}$ -thymidine concentration was low.

B.

Summary Introduction

To explain: The difference between the first experiment that had tracks with central dark section and light sections at both ends and the second experiment that had dark sections with light section at only one end in the autoradiograph.

Concept introduction:  Radioactive substances have decay emissions in the form of β particles and γ-rays. This property is exploited to study DNA replication where one of the molecules of the DNA is replaced with a radioactive element and the decay emission are measured is captured in a photographic plate to study the rate of replication.

Explanation of Solution

Given information: The DNA synthesis in tissue-culture cells is investigates with $\text{H}$ -thymidine was used to radioactively label the replication forks. Autoradigraphy was performed. The emulsion sensitive to radioactive emissions therefore, $\text{H}$ -thymidine labeled DNA is observed as tracks of silver grains. Due to stretching the replication bubbles are stretched and the daughter duplexes lie side by side undistinguishable from each other.

The cells are pretreated in order to synchronize all cells to the beginning of S phase. In the first set of experiment, the synchronizing block is released and $\text{H}$ -thymidine is added immediately. After 30 minutes, the medium is washed to reduce the total concentration of thymidine. Post 15 minutes, the DNA is prepared for autoradiography. In the second set of experiment, the synchronizing block is released and $\text{H}$ -thymidine is added after 30 minutes. After 30 minutes, the medium is washed to reduce the total concentration of thymidine. Post 30 minutes, the DNA is prepared for autoradiography.

 Due to the $\text{H}$ -thymidine labeling scheme preformed during the two sets of experiments, there is difference in the tracks with respect to the arrangement of light and dark sections

First experiment:

• $\text{H}$ -thymidine was added immediately after the synchronizing block was released. Thus, the replication forks were formed in the presence of $\text{H}$ -thymidine and the DNA polymerase added these $\text{H}$ -thymidine. Therefore, a central dark region in the track is seen.
• The medium was washed and was incubated for 15 minutes post which they were prepared for autoradiography. During this incubation time, the replication continued with less $\text{H}$ -thymidine. Hence, the light color track as the replication continues away from the initial origin of replication.

Second experiment:

• $\text{H}$ -thymidine was added post 30 minutes after the synchronizing block was released. Thus, the replication forks were formed in the absence of $\text{H}$ -thymidine initially and the DNA polymerase added normal thymidine. tThe replication forks were formed in the presence of $\text{H}$ -thymidine and the DNA polymerase added $\text{H}$ -thymidine post 30 minutes when the radiolabeled thymidine was added. The medium was washed and was incubated for 30 minutes post which they were prepared for autoradiography. During this incubation time, the replication continued with less $\text{H}$ -thymidine. Hence, there is an dark section with light section at one end.

C.

Summary Introduction

To estimate: The rate of fork movement (µm/min).

Concept introduction:  Radioactive substances have decay emissions in the form of β particles and γ-rays. This property is exploited to study DNA replication where one of the molecules of the DNA is replaced with a radioactive element and the decay emission are measured is captured in a photographic plate to study the rate of replication.

Explanation of Solution

 The rate of fork movement can be found out approximately with the given information about labeling time and the length of the labeled sections.

First experiment:

$\begin{array}{l}\text{Length}\text{of}\text{the}\text{segment}\text{=100μm}\\ \text{Labeling}\text{period}\text{=}\text{45min}\\ \end{array}$

There are two replication forks for each replication bubble; therefore each replication fork will synthesize half of the total length of the segment that is 50 µm

$\begin{array}{c}\text{Rate}\text{of}\text{replication}\text{fork movement}\text{=}\frac{\text{Length}\text{of}\text{the}\text{segment}\text{synthesized}\text{by}\text{one}\text{replication}\text{fork}}{\text{Labeling}\text{period}}\\ \text{=}\frac{\text{50}\text{μm}}{\text{45}\text{min}}\\ \text{=1}\text{.1}\text{μm/min}\end{array}$

Second experiment:

Here, only one replication fork synthesizes the full length of the DNA segment.

$\begin{array}{l}\text{Length}\text{of}\text{the}\text{segment}\text{synthesized by}\text{one replication fork}\text{=50μm}\\ \text{Labeling}\text{period}\text{=}\text{45min}\\ \end{array}$ $\begin{array}{c}\text{Rate}\text{of}\text{replication}\text{fork movement}\text{=}\frac{\text{Length}\text{of}\text{the}\text{segment}\text{synthesized}\text{by}\text{one}\text{replication}\text{fork}}{\text{Labeling}\text{period}}\\ \text{=}\frac{\text{50}\text{μm}}{\text{45}\text{min}}\\ \text{=1}\text{.1}\text{μm/min}\end{array}$

Conclusion

The rate of fork movement for both experiments is 1.1µm/min

Summary Introduction

To explain: The estimates from the two experiments agree and if the information can be used to gauge the time it would take to replicate the entire genome.

Explanation of Solution

The given estimates do agree but this information is insufficient to estimate the time period required to replicate the entire genome. The additional information required are total number of active origin of replication and the distribution of active origin of replication along the entire genome.