A few good question from 3B and random voices from the grapevines

1) (molecular clock) How can we reconcile the generation time and mutation rate of a particular gene. If a common gene is found in bacteria and an eukaryote, given the relative fast reproduction rate of bacteria, shouldn't the gene accumulate more mutations over the same time span and if so, how does that allow us to decide the point of divergence?


Ans: the generation time and rate of mutation is normalised for a valid comparison to be made btw different organisms!! =)

2) (not impt) why is there two origin of replication in pBluescript?

Ans: Pls ignore f1 origin. pBluescript is what we called a phagemid: a hybrid betw phage and plasmid.
- WIKI: http://en.wikipedia.org/wiki/Phagemid
A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. Phagemids contain an origin of replication (ori) for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles


3) Co-evolution
Each party in a coevolutionary relationship exerts selective pressures on the other, thereby affecting each others' evolution. e.g parasite or mutualism relationship

4) why is the size of the cloning vector important? (sensei)
together with the gene of interest, it is advisable not to have the recombinant plasmid at too biga size because small means higher copy number, greater stability and greater transformation efficiency.

5) why is it that HIV RT is a 3-in-1 model while the RT in cloning we still need to remove RNA (from RNA:DNA hybrid) with RNase H and later use DNA polymerase?

The RT in cloning is a different version and we choose not to use it because if the enzyme has multiple functions, its efficiency (less cDNA) can be compromised when it choose to degrade the RNA of the PolyA tail:OligerT DNA primer instead of elongating the DNA chain.

6) Using terminal transferase: how is the length of CCC or GGGGG ensured (if it is)

ANS: It isn't. They may not be heavenly made matches in terms of length but the gaps at the ends of the 5' strands can be filled in with nucleotides under the catalytic action of DNA polymerase and the nicks in the strands were sealed by DNA ligase.