Using a modified version of the Meselson and Stahl protocol, W. Fangman and coll

Question

Using a modified version of the Meselson and Stahl protocol, W. Fangman and colleagues were able to show that different yeast ARS sequences (replication origins) fire at specific times during S-phase (e.g., early, middle or late). In class we learned that ~400 ARSs, spread over 16 chromos, have been identified and their sequence is known. Devise a protocol that will allow you to assess when in S-phase particular ARSs fire. Hints: a) Specific DNA sequences can be detected in complex DNA mixtures by a number of techniques (e.g., Southern blot or PCR; coming later in course). Just assume you can do this. b) Yeast cells in culture can be cell cycle synchronized. e.g., The mating pheromone -factor will block a cells in G1, right before they are to initiate S-phase (and the block is reversible).

Explanation / Answer

The cells can be synchronized using -factor will block a cells in G1. By blocking the cells at different time of S- phase and extracting the DNA from these stages and non denaturing electrophoresis can be done.

The ARS regions of the yeast DNA can be utilized on southern blot against oligonucleotide probe of ARSs we can prove whether the ARSs fires at different times.

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