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Biology 1002B Lecture Notes - Experimental Evolution, Gene Duplication, Open Reading Frame

Course Code
BIOL 1002B
Denis Maxwell

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LeCTURE 21: Experimental Evolution
1. Potentiation, Actualization and Refinement
For new ability to appear, the bacterial populations went through three successive evolutionary steps:
Potentiating mutations (of unclear nature) were required for cells to acquire actualizing mutations that
consisted of a specific rearrangement of a few genes and that allowed some growth although poor
on citrate in the presence of oxygen.
Further ‘refining’ mutations, which involved duplications of the rearranged DNA sequence, were needed
for robust growth under such conditions
2. Characteristics of model systems that can be used for experimental evolution
Subject cells and organisms to selective pressure and look at the outcome
Viruses, bacteria, chlamy, drosophila, yeast
Model systems have very fast life cycles
o Short generation time to look at evolution in real time
3. Origins of genetic novelty (variation)
Gene duplication
o One of the two copies usually gets lost through deletion or degeneration
o If it gets retained, only one gets to stay the same
Neo-functionalization: mutate faster than the other gene by changing structure
Sub-functionalization: change promoter/regulation (expressed in different conditions)
Or change both structure and promoter
Genome rearrangement
o Shift promoter closer to another gene
4. Design of Lenski's long term evolutionary experiment (LEE) with E. coli
Can evolution produce adaptation if it depends on random mutations (most of which are harmful)?
Spontaneous mutation usually either bad or neutral
E. Coli has a short generation time and a huge population
o Asexual (no recombination)
12 isogenetic identical populations created from a single cell
o Transfer 0.1 mL (5 million cells) of culture into 9.9 mL media daily
o Freeze every 500 generations/75 days
Compare frozen ancestor with evolved generation
5. Value of cryopreservation to LEE
Low temperatures without causing additional damage caused by the formation of ice during freezing
6. Where citrate enters metabolism
After 30 000 generations Ara 3 changed in turbidity (more cells per mL culture)
o Acquired the ability to use citrate as carbon (Cit +)
Of a genome size of 4.6 million bp, every billion type of mutation was tried many times
o Conclusion: mutation is rare
Citrate in the experiment is used to keep iron in the solution that is needed for ETC
Under oxic conditions, citrate transport gene is not expressed
7. Role of glucose limitation in LEE experiment
Have enough glucose for them to grow for about 8 hours each day
They have 16 hours of opportunity to acquire citrate as a carbon source
8. How to determine if Cit+ phenotype arose from one single mutation or was dependent/contingent on
previous mutations?
Thaw out cells from different generations to compare
Does it grow on citrate agar?
o Sensitive technique to check whether cells use citrate or not
o None slight ability at 32 000 generations yes at 33 000 generations
Prior mutation to get slight ability and improved upon another mutation
If this is a rare mutation, you should get cit+ cells again
o Replay evolution by taking cells from different generations and grow 3770 generations more
All mutations are similar by attacking the same genome
o Only happens after at least 20 000 generations
Not a rare mutation
Need many generations to get a background mutation upon which you build the cit+
9. Genetic changes giving rise to potentiation, actualization and refinement of Cit+ phenotype
Potentiation set genome up for cit+ mutation
Actualization faint positive citrate phenotype
o Promoter that drives rnk is now upstream of citT
o Promoter rnk is constitutive and especially active under oxygen conditions
2 copies of rnk-citT expressed under oxygen conditions to make citT protein
Slight phenotype due to not enough expression of citT
cit+ actualization through genome arrangement
polycistronic mRNA more than one open reading frame to code for more than one
Refinement full function to use citrate
o Increase the number (9 copies) of rnk-citT modules through duplication increases gene
expression of more citrate transporters
10. Why Cit+ lines do not drive Cit- lines to extinction
Cit- is not the dominant form in the new E. Coli
o Not extinct since it is more efficient at glucose utilization
Is it a new species?