Doug gets a triple Yally Pally for figuring out the function of the Mysterious Apparatus which has travelled with the lab for years. It is <fanfare of trumpets>… a TLC plate spreader!-- Eric Kofoid
Archive for March, 2009
It just came out in Genetics, click here to get the pdf file.Sophie Maisnier-Patin
[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 a brief review I just wrote with Dan.
Why is natural selection hard to beat and when do you need to beat it?
John R. Roth and Dan I. Andersson
Bacterial genetics defeats natural selection — it uses positive selection to detect large-phenotype mutants without influencing their frequency. Metazoans maintain organism integrity by defeating natural selection on somatic cell growth. Bacterial genetics relies on selection strong enough to prevent growth of both the parent and common slightly-improved mutants. When selective stringency is reduced, frequent small-effect mutations allow growth and initiate a cascade of successive improvements. This rapid response rests on the unexpectedly high formation rate of small-effect mutations (particularly duplications and amplifications). Duplications form at a rate 104 times that of null mutations. The high frequency of small-effect mutations reflects features of replication, repair and coding that minimize the costs of mutation.
The striking effect of small-effect mutations is seen in a system designed by John Cairns to test the effect of growth limitation on mutation rate. A leaky E. coli mutant (lac) is plated on lactose medium. Revertant (Lac+) colonies appear over 6 days above a lawn of (108) non-growing parent cells. These colonies have been attributed to stress-induced mutagenesis of the non-growing parent. This conclusion ignores natural selection, assuming that only large-effect mutants appear– as is true for lab genetic selections. However, selection is not stringent in the Cairns system — small increases in lac enzymes allow growth. Common cells with a lac duplication (and 2x the mutant enzyme level) initiate slow-growing colonies, in which selection drives a multi-step adaptation process – higher amplification, reversion to lac+ and loss of mutant lac alleles. The high yield of revertant colonies under selection does not reflect mutagenesis, but rather the high spontaneous rate of gene duplication (10-5), amplification (10-2/step) and the selective addition of mutation targets (more cells with more mutant lac copies/cell).
Metazoan somatic cells may escape natural selection by the same mechanism. Metazoans reduce the basal level of unexpressed genes 1000-fold (compared to bacteria) by their epi-genetic modification of DNA and histones – making it impossible for small-effect mutations to provide growth.
[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.
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
For the second time, I have been burnt by the presence of a previously inserted cassette in a background I was using for linear transformation. In both cases, it was a pro::spec swap. I noticed an abnormally high number of camR transformants the next day. I printed the colonies to spectinomycin medium , and discovered that most were sensitive! What I had succeeded in swapping was the pre-existing cassette for the new one. Very few of the transformants were those which I had intended.
In parallel experiments, I found that the number of transformants increased over the course of a couple of days. Our standard UNI cassette (with the exception of tetAR) is a drug resistance gene embedded in an otherwise constant context, originally derived from the chloramphicol resistance locus of pACYC184. This means that sequence blocks about 200 bp at each end of the cassettes are always the same. Apparently, these regions are being used as recombination sites during the transformation.
I surmise that lambda red is not causing these insertions, but rather the host recombination system, because of the kinetics of their appearance. We incubate our transformations overnight at 42°C, specifically to eliminate pKD46 — this should confine all transformation to the first few minutes after electroporation. In addition, lambda red contains no mechanism for resecting the 3′ end of the transforming DNA back through the 40 bp intended homology block to make available the core of the cassette for strand invasion. This could only be done by the host after the red system had decayed or been diluted by growth, freeing the cell from red repression of the host system.
If I had used tetAR, I would never have noticed this effect. The only homology blocks available are the UNI ends, too small to be used by the host recombination system, but still requiring host resection to be exposed, which could only happen after elimination of red.-- Eric Kofoid
I have traditionally used 20 mM L-Arabinose to induce lambda red on pKD46. I have also noticed that r-m+ strains grew exceptionally slow during linear transformation. I decided to trace the cause of this defect by comparing their growth along with that of wild type plus or minus ampicillin (used to hold the plasmid) and plus varying concentrations of L-Arabinose. Results: 20 mM L-Arabinose inhibits growth of the r-m+ strain and inhibits yield of both strains. Ampicillin exacerbates the growth inhibition of the r-m+ strain, but not of the wild type. Experiment and data.-- Eric Kofoid
Our UNI CamR, KanR & SpecR cassettes all contain sites for endogenous LT2 restriction endonucleases. I tested the efficiency of transformation of these cassettes into a wild-type background (TT22970) and an r-m+ background (TT22971). My results are consistent with the hypothesis that UNI CamR cassettes are degraded by wild type LT2 when introduced by electroporation. Test description and data.-- Eric Kofoid
[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