Archive for the ‘Adaptive Mutagenesis’ Category

First Solexa Data In!

Wednesday, April 1st, 2009

(For those of you who may have forgotten, Solexa sequencing is a rapid, highly automated method of generating millions of short sequences at random across a DNA sample — often, an entire genome).

I have just received the first set of Solexa data from our collaboration with Fritz Roth and his colleagues, Yong Lu & Joe Mellor. The image below shows “read depth” (the number of runs which cross a given point) in the neighborhood of lacZ for strains TT24815 and TT25790. We expect this measurement to increase in proportion to the degree of amplification. Coverage over non-amplified areas of the plasmid and chromosome exceeded 50-fold for both strains.

Small red arrows show my guess at the amplification endpoints. The TT24815 array stretches from approximately 138256 to 166250 (~28 KB), and TT25790,  from 131600 to 159300 (~28 KB), where the coordinates refer to our standard F’128 sequence counting clockwise from the first nucleotide of IS3A.

Strain TT25790 contains Elisabeth’s known inversion duplication array (EK568), for which we have sequenced a single join point (134075->134087 recombined into 132108<-132098). Small blue arrows show these two tracts. In our simple models of inverted duplication formation, join 1 forms from their recombination, either directly (“Flying Walendas”) or by assymetric deletions of a larger toxic structure (“Slytherin”). Furthermore, all Solexa data in the array should begin at the leftmost blue arrow, gratifyingly close to my guessed endpoint. Join point 2 will be defined by a sequence near the righthand red arrow and its inverted complement at a position yet to be found in the amplified region. I shall go hunting!

Strain TT24815 was chosen for its recalcitrant nature — we were never able to find any join points, but assumed for this reason that it was a likely candidate for an amplified inverted duplication, as crossover sites in these entities are truly difficult to locate and sequence. We were hoping to get two new bits of previously unknown information out of it. Once again, half of each of the join points should be defined by small sequence inversions in the neighborhood of the red arrows, assuming that this is a truly simple array of elements representing one kind of inverted duplication. More hunting!

If you use your browser’s zoom feature, you can inspect the image with better resolution. You can also download a detailed PDF file.


-- 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