Posts Tagged ‘Adaptive Mutagenesis’

The Amazing History of DR397

Thursday, March 29th, 2012

Drew Reams studies unselected duplication formation, and recently found an eel in the DNA!

Recall that duplication formation is a fundamental small-effect driver of evolutionary adaptation, and TIDs may be the primum mobile of most or all duplications. Drew’s “recalcitrant” duplication strains — those with joints not easily determined by multiplex PCR — are a class which should include TIDs. To analyze them, he sequences their genomes by Illumina technology.

We think TID formation often begins with a snap-back of a 3′ end at a short inverted repeat, forming a stem-loop structure which then initiates DNA synthesis using self as template.

A subsequent template switch restores the fork and finishes the TID. See the excellent article in our Encyclopedia.

The extensive secondary structures at such symmetric TID joints are toxic and rarely observed. Instead, remodelling asymmetric deletions are selected spontaneously, yielding “SJ” (“short junction”) joints. David Leach has shown that sbcCD backgrounds tolerate such structures by not cutting them with the endonuclease product.

Drew wondered what would happen to duplication frequencies in such backgrounds, which may allow the cell to survive the two initial steps of TID formation and increase the yield of duplications. To enhance stem-loop persistance, he also made the cells recQ to prevent stem melting.

He observed a 2-fold increase in duplications in both the chromosome and wild type F’128. The latter contains two IS3 elements and nearly all spontaneous duplications happen by recombination between them. If at least one were removed, the duplication frequency increased by an order of magnitude compared to sbcCD+ recQ+ cells. Something definitely happens in the absence of sbcCD and recQ when IS recombination is blocked

One sequenced duplication was  in F’128 of strain DR397. It had a lacZ read depth 3-fold greater than the chromosome with remaining plasmid DNA about 9-fold greater. In addition, there was a large deletion extending from traI up to lacZ accounting for ~20% of the plasmid.

When we inspected the anomalous read-pair data, we discovered two symmetrical TID joins. We were able to confirm these by showing that reads right at the edge of the deletion window fully contained these joints.

Drew’s suspicions were vindicated. Removing activities which cut stem loop structures or prevent them from folding encourages secondary structure formation and reduces counterselection of the long symmetrical products of snap back and strand switching.

But, what’s the meaning of the enormous deletion? There are a couple of models which come to mind.

Model 1: A snapback at traI followed by another at lac, 5′ resection and ligation of ends will produce a product that matches our observations. It would look something like the following: 

Model 2: A snapback at traI and strand switch at lac will restore the replication fork and lead to a TID. Eventual remodelling by recombination of the flanking repeats will yield our observed product: 

Other related models are possible, with the order of the snapbacks and strand switches reversed, with double strand switches instead of snapbacks, etc. Nevertheless, we favor the second model above, as it only involves two steps to get the essential intermediate, while many generations can pass before selection of the final product.

An interesting aspect of the TID model is the inevitability of the remodeling event. When the origin is itself in the TID, counterselection on a large number of unnecessary genes leads easily to their deletion by recombination. The resulting fitness increase will lead to expansion in the population of the symmetric inversion and eventual extinction of the TID.

-- Eric Kofoid

Kofoid’s Notes for Analytical Genetics, 2009

Wednesday, October 21st, 2009

Here are my notes for a 15′ talk at Analytical Genetics, 2009 (Asilomar). The actual talk had to be pared to 5′ for mysterious reasons!

-- Eric Kofoid

The origin of mutants under selection: Interactions of mutation, growth and selection

Monday, March 9th, 2009

[This is a stub entry I'm making for John under his name. He should re-edit it with his own words. -- Eric]

Here’s the abstract to a new article.

The origin of mutants under selection: Interactions of mutation, growth and selection

Dan I Andersson, Diarmaid Hughes and John R Roth

In microbial genetics, positive selection detects rare cells with an altered growth phenotype (mutants or recombinants).  The frequency of mutants signals the rate of mutant formation – an increased frequency suggests a higher mutation rate.  Increases in mutant frequency are never attributed to growth under selection.  The converse is true in natural populations, where changes in phenotype frequency reflect selection, genetic drift or founder effects, but never changes in mutation rate.   The apparent conflict is resolved because restrictive rules allow laboratory selection to detect mutants without influencing their frequency.  With these rules, mutant frequency can reliably reflect mutation rates. When the rules are not followed, selection rather that mutation rate dictates mutant frequency – as in natural populations.  In several laboratory genetic systems, non-growing stressed populations show an increase in mutant frequency that has been attributed to stress-induced mutagenesis (adaptive mutation).  Since the mutant frequency is used to infer mutation rate (standard lab practice), the rules must be obeyed.  A breakdown of the rules in these systems may have allowed selection to cause frequency increases that were attributed to mutagenesis.  These systems have sparked interest in interactions between mutation and selection. This has led to a better understanding of how mutants arise, and how very frequent, small-effect mutations, such as duplications and amplifications, can contribute to mutant appearance by increasing gene dosage and mutational target size.

-- John Roth

Amplification & Adaptive Mutagenesis

Thursday, March 5th, 2009

[This is just a teaser to get us started -- add to it, change it, throw it away, but please leave something worthwhile behind!]

John Cairn’s once observed that apparently non-growing populations of bacteria spontaneously acquire mutations which enable them to grow on a previously unutilizable carbon substrate.

An early explanation bordered on the metaphysical, invoking an awareness by the non-growing cell of the tantilizing substrate, lactose, and a consequent mutational targeting of a specific gene, lacZ, which, when appropriately modified, would allow growth on this compound.

Many requirements and predictions of this original model were quickly shown to be wrong. DNA replication occured in the “quiescent” cells, which were also growing, albeit at a slow rate. Mutations were not confined to the “targeted” gene alone. Adaptation to growth on lactose would not occur if the gene were on the chromosome; the observed reversion to lactose utilization was only seen when lacZ was on a specialized F plasmid. Additionally, the effect was found only if this plasmid expressed a suite of functions which enabled plasmid replication by rolling-circle synthesis of single-stranded DNA and resulting transfer of this DNA to recipient cells.

Cairn’s descendents have bifurcated two basic models from the original, although a number of others have been left lying in the dust over the years. Both assume that an evolved mechanism senses stress (i.e.,starvation) and directs an increase in mutagenesis. Pat Foster’s model asserts that stress induces rpoS, which in turn makes recombination mutagenic. Susan Rosenberg’s model maintains that a general hyper-mutagenic state is evoked which is independent of rec functions.

We point out that a third model exists which makes no assumption of any evolved stress-sensitive mutagenic mechanism, but instead relies on the bag of genetic tricks described and well verified over the last century. We note that Cairn’s cells are growing slowly, and are replicating their DNA. Duplications in DNA are relatively common and can be amplified during replication. A defective gene which nevertheless sustains slow growth allows an increase in the basal growth rate when duplicated. Selection for faster growth will favor cells containing higher order amplifications of the defective gene. Such cells will sweep the population. The opportunity for true reversion will be roughly the number of such cells times the average amplification factor times the rate of reverting a single gene per generation. Because the amplification factor is under selection and expected to grow with the number of generations,  the probability of true reversion to lac+ will increase substantially over the course of the experiment, accounting for all colonies observed.

We like this hypothesis because it involves no new technology, no magic, and no religion. We have a substantial amount of data supporting it. The requirement that the defective locus be on an F derivative is easily understood by two facts: One, the relatively small region of interest is flanked by exact copies of the insertion element, IS3, which allows easy initial duplication of the region. Two, F is constantly replicating itself by a rolling-circle mechanism generating long single strands. These can induce rearrangements by recombination or annealing. Under selection, this can lead to rapid increase in the degree of amplification, and promote remodeling leading to diminished size of the amplified element, thus minimizing the cost of ancillary gene dosage effects.

-- Eric Kofoid