I understand what DAM+ and DAM- are but could someone tell me why in the second
ID: 258992 • Letter: I
Question
I understand what DAM+ and DAM- are but could someone tell me why in the second observation the Dam - transformed cells died?
QUESTION C1 A plasmid deleted of its own origin but bearing oriC behaves exactly like a small E.coli genome (has a copy number of 1; replicates once per cell division -i.e. its timing circuit is identical to E. coli, and it is segregated like the bacterial genome) Here's a set of puzzling observations involving such a plasmid (plasmid X) that carries an AmpR gene. Observation 1: If plasmid X is initially grown and isolated from a dam minus bacteria it can subsequently transform both dam plus (dam+) or dam minus E.coli to give Amp colonies Observation 2: If plasmid X is initially grown and isolated from dam+ bacteria it cannot transform dam minus E.coli to give Ampk colonies. However, it can still transform dam+ bacteria and give AmpR colonies. Why this difference? Using clear labeled diagram(s) to support your answer provide a molecular explanation for this set of observationsExplanation / Answer
DAM in E. coli, represents DNA- adenine Methyl transferase. DAM enzyme or DAM methylases (encoded by DAM gene), can transfer methyl group to adenine residues at N-6 position in sequence GATC, from S-adenosylmethionine (SAM).
The E. coli origin of replication (Ori C), has several GATC sequences, as recognized by DAM-methylases.
The sequence GATC is palindromic:
5’ GATC 3’
3’ GATC 5’
Thus, at the Ori C, the GATC sequence may either remain in different states as:
1. Unmethylated state- none of the GATC sequences are methylated.
2. Fully-methylated state- GATC sequence of both strands are methylated.
3. Hemi-methylated- GATC sequence of one strand is methylated.
As hemi-methylated state Ori C is inactive and cannot carry out replication. While unmethylated, or fully methylated Ori C are active.
Also, the restriction endonuclease sites overlap the Ori C. Under methylated condition, the restriction endonucleases cannot cleave the REN sites, which in turn prevents transformation.
In the give scenario, the origin of replication of the plasmid (plasmid X) is replaced by Ori C.
a. When the plasmid is grown in E. coli cells which are DAM-, there is no methylation by DAM at the Ori C site. Thus, the plasmid can replicate in DAM- cells. Also, REN sites remain free for endonuclease activity during transformation. Thus, transformation occurs and is detected as Ampicillin resistance.
b. When the plasmids are grown and isolated in DAM+ cells, the DAM methylase activity occurs. This restricts transformation to DAM- cells, due to methylation caused modification-dependent restriction system (as DAM+ cells recognizing DAM- as foreign, and REN sites overlapping the Ori C will remain methylated.
Also, due to slower action of DAM, the Ori C remains in hemi-methylated state for a long time, and even if the plasmid enters the DAM- cell, it will not continue replication.
But, transformation will occur incase of DAM+ cells, as unmethylation will occur during transformation and replication (as these cells will not be recognized as foreign).