pangloss

This was posted by 'Sumac' before on another board. It is an excellent summary of the 'directed mutation' farce that creationists of all stripes have gobbled up as 'proof' that evolution didn't happen. I did not modify the HTML at all, so it doesn't look as nice as it did originally. I just hope that if anyone replies, we don't get the old 'just look around you' retort regarding 'evidence' for EAM:

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: The phenomenon discussed in the two articles was first described by John Cairns and colleagues (1). I won't present the experiments in detail (unless someone is really interested), but it helps to know that the Cairns experiments were essentially repeats of experiments that had been performed and published over 40 years previous (2). All of the experiments were designed to test whether mutations occurred independent of or in response to selection. Luria and Delbrück's data unquestionably demonstrated that mutations can and do occur independent of selection. Cairns et al.'s data were not as clear cut.

: The key difference between the experiments was that Cairns et al. used a non-lethal selection while Luria and Delbrück used a lethal one (Luria and Delbrück selected for phage resistance; Cairns et al., selected for lactose utilization). Non-lethal selection was important because it allowed the bacteria to remain viable in a so-called stationary state for long periods of time in the selection medium. Although stationary cells are not growing, they are anything but dormant. Stationary cells actively break down and rebuild proteins and other biomolecules and they continuously sample their environment while waiting for any opportunity to begin growing again.

: The vast majority of mutations observed in the Cairns experiments did occur independent of selection, confirming previous conclusions. However, if the bacteria were left in the selection medium for long periods of time (days or weeks), a small number of new colonies would begin to grow suggesting that they contained mutations that occurred after exposure to the selection medium. These low frequency mutations appeared to be specifically responsive to the selection medium which led to the hypothesis that perhaps they were directed to occur by the selection.

: Directed mutation was just one hypothesis of several. Cairns et al. actually favored a model in which a trial-and-error mechanism searched for mutations that would allow the cells to grow. In a commentary that accompanied Cairns's article, Franklin Stahl suggested the possibility that the scavenging state of stationary cells may be conducive to an overall increase in mutation rate (3). Stahl proposed that as soon as a 'good' mutation occurred, normal growth would resume and the mutation rates (and types of mutations) would return to normal.

: Over the next several years following Cairns's discovery, other studies using different types of non-lethal selection were published with similar results. However, none of the studies could demonstrate that the mutations were actually directed to occur. Your first article (the Foster review, ref. 4) compiled data from a number of studies and weighed the data against the hypothesis of directed mutation. Although Foster seemed to like the idea, she was quick to note that directed mutation is "the aspect that is the least supported by the experimental evidence." In the end, Foster favored a model that is a combination of Cairns's and Stahl's hypotheses: the overall mutation rate increases and temporary mutations are tested for benefit against selective pressures.

: From her concluding remarks: <ul><I>The evidence suggests that the role of selection in the mutational process is not to "direct" the process, but to define success. While this may appear to be just a version of "you get what you select for," there is an important distinction. If the idea of transient genetic variants is correct, it is not the organism, but its informational molecules that are under selection. Thus, a nondividing cell is, potentially, multiphenotypic, a characteristic previously thought to be true of the population, not the individual. If a bacterium can, either by design or accident, increase its genetic variability under stress while maintaining its genome more or less intact, this is clearly of evolutionary significance.</I></ul>

: Unfortunately, Foster was sometimes a bit overzealous in pushing her favorite model – even to the point of subtly misrepresenting data (I suspect unintentionally). For instance, when discussing Barry Hall's work on trp reversion (5), she stated, "Hall found no sequence changes in the Trp+ revertants other than the reverted bases themselves, a result which none of the current models for directed mutation can explain." Superficially, this statement is true. Hall didn't find any other sequence changes because he didn't look for other sequence changes. He only sequenced the regions of the genes (TrpA and TrpB) that contained the original trp- mutations. Furthermore, in the TrpA gene, most of the changes were not reversions to the original wild-type sequence, but were other changes that restored enzyme activity. Hall recognized the limitations of his data and acknowledged the possibility that other mutations would be present in the revertants even though he hadn't looked for them. The possibility of second-site mutations was especially likely in a couple of cases where the mutations in TrpA couldn't possibly restore enzyme activity on their own.

: Now to your second article (6). Foster and Trimarchi used the same experimental set-up as Cairns, but took it one step further. Bacteria containing a mutant form of the lacZ gene were selected for growth on lactose as before. The lacZ genes from late-appearing revertants were then sequenced and the mutations were analyzed. Out of a total of 54 revertants, six had multi-base insertions and four had multi-base deletions in lacZ. Three of the revertants retained the mutant lacZ gene suggesting that the phenotypic reversion was due to a mutation elsewhere in the genome. The most interesting result was that 41 of the revertants contained single base deletions within lacZ. These deletions were scattered throughout the lacZ gene (41 deletions at 10 different positions) and were found almost exclusively in regions of the gene that contained repeats of a single nucleotide (for instance, CCCC). Coincidentally, another group had been doing pretty much the same thing and published their paper in the same issue of Science (7). Rosenberg et al. identified 86 mutations at 41 different positions, 57 of which were single base deletions spread out over 14 positions. The remainder were larger deletions, insertions, or duplications. The single base deletions discovered by Rosenberg et al. were also predominantly located at regions of repeated nucleotides. Unfortunately, neither Foster and Trimarchi nor Rosenberg et al. looked at mutations outside of the lacZ gene.

: Since that time, more work has been done to understand how stationary bacteria adapt to specific selection media. Huxter presented some of those papers above. The big question was: are the mutations incurred by stationary phase bacteria really directed to occur in specific genes or is the apparent direction an artifact of the experimental set up? In 1997, Torkelson et al. demonstrated that it is the latter (8). Mutations in revertant bacteria are not localized to specific genes, but accumulate throughout the entire genome. The unique nature of these mutations (i.e. the preponderance of single base deletions) is most likely due to increases in specific types of oxidative damage and diminished fidelity of repair and recombination (9, 10). The appearance of specificity comes from the fact that as soon as a positive hit occurs, the bacterium leaves stationary phase and begins to grow on the selective medium. Stahl was right.

: I know I took a long time to get here, but I wanted to (hopefully) make it clear why the examples you provided are not examples of directed mutation. Two facts are worth repeating: 1) the mutations that lead to phenotypic reversion are varied and scattered throughout the relevant genes – they can even be extragenic, 2) unselected mutations accumulate throughout the genomes of revertants. These two facts are clear indications that the mutations leading to reversion are not directed.

: I'll end with two quotes. In a more recent review (11), Foster wrote: <ul><I>Because nonselected mutations arise and persist in the population during selection, a stress-associated general mutational state, strictly speaking, does not meet the original definition of adaptive mutation. However, here I will continue to call the selected mutations "adaptive" to distinguish them from nonselected mutations occurring during selection. This meaning of "adaptive mutation" is the same as that used by evolutionists to distinguish beneficial from neutral or deleterious mutations.</I></ul>

: And finally, from a news article that accompanied your second article (12): <ul><I>What hasn't been narrowed down, however, is a name for this phenomenon. Cairns originally used the term "directed mutation", which to many scientists implied that the bacteria themselves were directing which mutations arose. Others have called it "Cairnsian", "stationary phase" and "starvation-associated" mutation. Foster and Rosenberg both used "adaptive" in print, but on the job in Rosenberg's lab, they call it SPAM–Selection-Promoted Additional Mutations. Foster's idea: Just call it "Fred."</I></ul>

:
: References:
: 1. Cairns, J., Overbaugh, J., and Miller, S. (1988) The origins of mutants. Nature 335:142-145
: 2. Luria, S.E., and Delbrück, M. (1943) Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28:491-511
: 3. Stahl, F.W. (1988) A unicorn in the garden. Nature 335: 112-113
: 4. Foster, P.L. (1992) Directed mutation: between unicorns and goats. J. Bacteriol. 174:1711-1716
: 5. Hall, B.G. (1991) Adaptive evolution that requires multiple spontaneous mutations: mutations involving base substitutions. Proc. Natl. Acad. Sci., USA 88: 5882-5886
: 6. Foster, P.L. and Trimarchi, J.M. (1994) Adaptive reversion of the frameshift mutation in Escherichia coli by simple base deletions in homopolymeric runs. Science 265: 407-409
: 7. Rosenberg, S.M., Longerich, S., Gee, P., and Harris, R.S. (1994) Adaptive mutation by deletions in small mononucleotide repeats. Science 265: 405-407
: 8. Torkelson, J., Harris, R.S., Lombardo, M.-J., Nagebdran, J., Thulin, C., and Rosenberg, S.M. (1997) Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation. EMBO J. 16: 3303-3311
: 9. Bridges, B.A., Foster P.L., and Timms A.R. (2001) Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40. Mutat Res 473: 109-119
: 10. Harris, R.S., Feng, G., Ross, K.J., Sidhu, R., Thulin, C., Longerich, S., Szigety, S.K., Hastings, P.J., Winkler, M.E., and Rosenberg, S.M. (1999) Mismatch repair is diminished during stationary-phase mutation. Mutat. Res. 437: 51-60
: 11. Foster, P.L. (1998) Adaptive mutation: has the unicorn landed? Genetics 148: 1453-1459
: 12. Culotta, E. (1994) A boost for "adaptive" mutation. Science 265: 318-319

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: <font size=small>Posted on: 2:49 pm on November 1, 2001</font>
: <ul>John Paul quoting who?:<I><b>(1988) "Adaptive evolution that requires multiple spontaneous mutations. I. Mutations involving an insertion sequence," Genetics, vol. 120, pp. 887-897. In the last, Hall investigated the occurance of the precise deletion of IS103 in the presence of saline. 2 mutations had to occur in order for Hall's strain to metabolize salacin. The sequence IS103 had to be precisely deleted, then the right nucleotide had to be changed or else a sequence called IS1, or another one called IS5, had to be inserted into the cryptic regulatory gene. Hall tried to measure the spontaneous rate of the precise deletion of IS103 but found it to low to measure (he gave it a probability of less than 2 in a trillion). In the absence of saline these two mutations occur in the same cell with a chance less than 10 to the -19th. If the two are indeed independent of each other, then the chance of the rifgt double mutation in at least one cell of the population in two weeks (many generations of bacteria) is about one in 30 million. But wait! In two weeks Hall found that 60% of his colonies underwent both mutations and could metabolize salacin. Imagine that. Just looking at the IS103 deletion, which was to low to measure before, 89% of the colony underwent that deletion in 8-12 days. </b></I></ul>

: It is true that Hall's initial data concerning the bgl operon could be interpreted as directed mutation. However, as we saw with the Cairns model, critical evaluation of the data and additional experimentation have shown that Hall's results were artifacts of the experiment and are not really evidence of directed mutation.

: As with the Cairns experiments, Hall was studying the reversion of a mutant phenotype back to the wild-type phenotype. The E. coli strain used for these studies(chi342LD) is a derivative of the K12 strain and was selected for its inability to metabolize salicin (a beta-glucoside sugar). The bgl operon (the unit of genes that metabolize beta-glucosides) of chi342LD contains two mutations: a point mutation in bglR and an insertion in bglF (Hall refers to this insertion as IS103 in early papers and IS150 in later papers...does anyone know why the nomenclature changed?). In order for chi342LD to metabolize salicin <I>efficiently</I>, these two mutations must be compensated for somehow.

: The experiment in the original paper (Hall, Genetics 120: 887-897, 1988) was fairly simple. Essentially, Hall plated chi342LD on medium that contained salicin as the sole carbon source and looked for bacteria that could grow. Hall found that, during a period of two to three weeks on the selection medium, approximately one out of every 100,000,000 cells plated would acquire mutations that could overcome the two defects in the bgl operon. Many, but not all, of the revertants contained deletions that excised all or a part of IS103 from bglF. Many, but not all, of the revertants contained point mutations in bglR.

: The quote (from who?) above is deceptive for a number of reasons. First, the observed deletions of IS103 were not "precise". Deletions of variable size were detected by Hall and others. The only common feature of these deletions were that they retained the reading frame of bglF and removed the STOP codons in IS103. In addition, there was no specificity to the revertant mutations in bglR. A number of different mutations all gave rise to the Sal<sup>+</sup> phenotype.

: Another deceptive statement in the quote concerns the 60% number. While it is true that 60% of the colonies plated on the selective medium acquired mutations, only one or a few cells in each colony were actually revertants. Hall described these events as "papillae" which means that a single cell began to grow while the remainder of the colony remained in stationary phase. The rate of reversion was <I>not</I> 60%; it was 0.000001% (10<sup>-8</sup> x 100). This is still several orders of magnitude faster than the reversion rate without selection, but I will get to that in a moment.

: The last sentence in the quote is not only deceptive, it is outright wrong. Hall found that between 1% and 10% of the cells in each colony acquired a deletion that removed all or a part of IS103 from bglF, not 89% (where did that number come from?!?). Of these, only a very small number acquired the second mutation necessary for complete phenotypic reversion. The rate of acquisition of the second mutation was similar to the normal rate of reversion in bglR.

: So what's going on? As we discussed before, it has been shown that cells in stationary phase have a much higher rate of mutation than growing cells and that the high mutation rate is especially apparent when one looks at deletions. Deletion of all or a part of IS103 from bglF occurs more often in cells plated on the selection medium because deletion rates in general are higher. Mittler and Lenski (Nature 356: 446-448, 1992) showed that chi342LD cells that had excised IS103 from bglF could grow slowly on salicin even if there was no reversion in bglR. Just a little growth on salicin would be enough to provide an advantage to IS103 deletions versus other types of deletions and, thus, would allow for an enrichment of these mutations in the population. The second mutation - reversion of bglR on selection medium - occurred at rates similar to reversion without selection.

: Therefore, it looks like what is really happening is that an increased rate of non-specific deletions, together with the normal rate of non-specific point mutations, combine to give the appearance of an induced phenotype. But it is really just an artifact of the selection. The selection does not induce a specific type of mutation, it only allows the "correct" ones to grow once they occur.

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: <font size=small>Posted on: 8:31 am on June 14, 2001</font>
: I think it is worth noting that the debate among scientists over whether or not the phenomenon discovered by Cairns is the result of directed mutation is being hashed out in journals such as Nature, Science, PNAS, Genetics, Journal of Bacteriology, Mutation Research, EMBO Journal, etc. There are a few scientists such as Hall who favor the directed mutation interpretation (despite the fact that the evidence seems to be against them), yet they do not have any trouble publishing their data and opinions about the data in these mainstream journals. Directed mutation runs against the current paradigm, yet the editors of the journals feel that the data are compelling enough to be worthy of debate. So, you see, there is no conspiracy by evolutionists to keep conflicting data out of mainstream journals. The conspiracy is to hold conflicting data to the same standards of quality as supporting data. If directed mutation turns out to be a real phenomenon (and I'm not yet convinced that it is), then it must be incorporated into the paradigm. Even the most diehard evolutionists understand this.

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pangloss

Looks like mturner is playing his ignore card here, too.... <img src="graemlins/banghead.gif" border="0" alt="[Bang Head]" /> <img src="graemlins/banghead.gif" border="0" alt="[Bang Head]" />