The following is pulse-chase experiment was performed as in Okazaki’s experiment, but in an organism as follows (all other as in regular replication):
• One linear chromosome
• 2 origins of replication spaced 50Kb apart, one firing at t=0 and one in t=10 min.
• Genome replication is completed by t=30minbh
• DNA ligase acts only 5 min after origin has fired
• Molecules of <3Kb can be separated, but >3Kb all pick together and can not be separated by size
The pulse is given for a minute and then the radioactivity is removed.
Drew the expected results in the indicated times: A t=2min, B t=9min, C t=12 min, D t=31
Drew the expected results in the indicated times, after additional pulse is given at t=11: E t=13min, F t=31min
To draw the expected results in the indicated times, we need to understand the process of pulse-chase experiment in this specific organism with the given conditions.
1. At t=0 min: The first origin of replication fires. This initiates DNA replication, and new DNA strands are synthesized. The newly synthesized DNA strands will be labeled with radioactivity during the pulse.
2. At t=1 min: The pulse is given for a minute, labeling the newly synthesized DNA strands. After this minute, the radioactivity is removed.
3. At t=2 min: Only a small part of the genome would have completed replication. The labeled DNA strands will be shorter (less than 3 Kb) and can be separated by size.
4. At t=9 min: More DNA replication will occur, and the labeled DNA strands will increase in length. However, they will still be shorter than 3 Kb and can be separated by size.
5. At t=10 min: The second origin of replication fires. This initiates replication at a different location on the chromosome.
6. At t=12 min: Both origins have fired, resulting in two replicating forks moving in opposite directions. The labeled DNA strands will continue to grow in length, but they will all be less than 3 Kb and can be separated by size.
7. At t=13 min: An additional pulse is given at t=11 min. The newly synthesized DNA strands will be labeled with radioactivity during this pulse.
8. At t=30 min: Genome replication is completed. All the labeled DNA strands will have achieved their full length but will remain less than 3 Kb and can be separated by size.
9. At t=31 min: DNA ligase acts and repairs the nicks in the DNA strands. The labeled DNA strands that were shorter than 3 Kb will now be connected and appear as a single band on the gel.
So, to summarize the expected results:
- A t=2 min: Two separate bands, representing the labeled DNA strands synthesized from the first origin of replication.
- B t=9 min: The two separate bands will grow in size but still be separate from each other.
- C t=12 min: Two separate bands, representing the labeled DNA strands synthesized from both origins.
- D t=31 min: A single band, representing the labeled DNA strands that have undergone DNA ligase repair and reached their full length.
- E t=13 min: After the additional pulse at t=11 min, there will be an additional band representing the newly labeled DNA strands.
- F t=31 min: After DNA ligase acts, all the labeled DNA strands, including those from the additional pulse, will be connected and appear as a single band on the gel.