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Lecture 6

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University of Toronto St. George
Larry Moran

Bch 447 lecture 6 Richard lenski in 1988 he decided to do an evolutionary experiment. So, he started growing a culture of bacteria for many generations. He got 12 flasks and placed e-coli b in there. He seeded 12 flasks at 100ml each. The flasks had very low glucose... Therefore limitting the growth. He also placed citrate in the medium to chelate heavy metals as bacteria are sensitive to heavy metals. He let flasks sit on a bench, and every day he would dilute the cultures 1:100. Every day there were 6.64 generations of e-coli. The experiments is still going 60000 generations later.. It's 2014 now. He would take alliquotes of each of the flasks and freeze them once every 8000 generations. The bacteria were under a lot of selective pressure... It makes sense that the bacteria using the glucose more efficiently will grow more quickly and dominate. After a year or two... All twelve of the cultures would become cloudier much faster than at the beggining, probably using glucose more efficiently. Most of the cultures had similar mutations. Rbs mutation : ribose utilization operon... Important for ribose formation SpoT mutation... It's a guanine nucleotide with two phosphate groups at the 5' and 3' end and it is an activator of operon transcription... It activates genes for carbon metabolism. MalT... Maltose... Important for the uptake of 6 carbon sugars. Mutations here retrieve sugars other than glucose.... The bacteria fed on their dead friends. Also modifications of nad formation and pyruvate shuttling... Cell wall biosynthesis genes. Plasmid regulation.. Topoisomerase helps unwind. Basically all of the cultures had common mutations. But each flask had a few mixtures of surviving bacteria with different genes. One of the cultures after 30000 generations the ara3 type bacteria became very dense. These guys had aqcuired a mutation allowing them to grow to extensive densities. The culture was citrate plus.... The bacteria mutated to use citrate as a carbon source along with glucose. Note that the other cultures did not aqcuire this mutation. Firstly, looking at the bacterial genomes they asked... What is the random rate of mutation in these cultures... By comparing the genomes from previous generations. We know that mutations will occur.... And that some will be fixed. Further more is there a background neutral mutation rate that is constant... Remember this is different because there is hardly any junk DNA in e coli. It may be that every mutation that occurs may modify a gene. So, they decided to look at the synonymous mutations that occured in every generation... Mutations that didn't change the amino acid sequence. So, what's the rate of fixation of these mutations. So, they took all 941000 synonymous sites in e coli and compared them. About 1/5 of the genome. They looked at 8 of the cultures. And they asked how many synonymous alleles were there fixed accross all of them... They didn't use the other 4, because four of the flasks had differences in their mutation rate. So, they compared accross 300000 generations. The equation is on the sheet... They found that the mutation rate was consistent with the sequencing. As per the equation if we are looking at neutral mutations the fixation rate should be equal to the mutation rate. Which it is. It appears that synonymous mutations here are essentially neutral.... Note though that sometimes synonymous substitutions can be selected for. Four of the cultures had mutations that changed the mutation rate 100 fold. The mutation rate became 10^-8. It's likely because the bacteria wanted to mutate faster Under the low glucose selective pressure. These e coli have way more mutations per generation. The downside here is genetic load. The prediction is that once the beneficial mutations required for survival in low glucose arise... The population would revert the mutation rate to normal once more. Wilga's said that we should see evidence in our 8 cultures that are now under normal mutation rate.... We may be able to see evidence of mutate DNA machinery... To show that perhaps those 8 flasks underwent a higher rate of mutation and reached their improved phenotype... At which point they reverted back to a normal mutation rate. Perhaps we can see a transient period of hypermutation in the genome. We don't select for mutations in DNA machinery.... But they accidentally occur.... And we get hyper mutation.... If this hypermutation is beneficial... Then that species survives. Linkage disequilibrium... The bad mutation is carried along with the good mutation.... That is the mutant DNA machinery inducing hypermutation get
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