Science in Society Archive

Life after the Central Dogma

The biotech industry was launched on the scientific myth that organisms are hardwired in their genes, a myth thoroughly exploded by scientific findings accumulating since the mid 1970s and especially so since genome sequences have been accumulating (see Living with the Fluid Genome, by Mae-Wan Ho ).

We bring you the latest surprises that tell you why our health and environmental policies based on genetic engineering and genomics are completely misguided; and more importantly, why the new genetics demands a thoroughly ecological approach.

To Mutate or Not to Mutate

Contrary to views widely held not so long ago, genes do not as a rule mutate at random, and cells may choose what, or at least, when to mutate. Dr. Mae-Wan Ho reports

Non-random 'adaptive' mutations?

The backbone of modern genetics and the neo-Darwinian theory of evolution by natural selection is that gene mutations occur at random, independently of the environment in which the organisms find themselves. Those mutations that happen to be 'adaptive' to the environment are 'selected', while those that are deleterious are weeded out.

The idea that genes do not mutate at random, but 'adaptively', as though 'directed' by the environment in which the organisms find themselves, is so heretical that most biologists simply dismiss it out of hand; or try their utmost to explain away the observations that give life to the idea.

Microbiologist Max Delbrück first used the term 'adaptive mutations' in1946 [1] to refer to mutations formed in response to an environment in which the mutations are selected. The term was adopted more than 40 years later by a research team investigating gene amplification in rat cells [2]. They distinguished between mutations that pre-exist at the time a cell is exposed to a selective environment from those 'adaptive' mutations formed after exposure to the environment.

Other workers have followed the same definition [3, 4]. These 'adaptive' mutations arise in non-growing or slowly growing cells after the cells were exposed to conditions that favour the mutants, preferentially, though not exclusively, in those genes that could allow growth if mutated. Unselected mutations also accumulated in most studies, to varying degrees, so the mutations are not strictly 'directed'. Instead, the cells appear to activate a number of different mechanisms that target mutations to genes, the end result of which is to enable them to grow, which they otherwise would not be able to do.

The archetypal experiment

John Cairns and Patricia Foster created an E. coli strain defective in the lac gene that leaves the cells unable to grow on lactose [3]. They plated out the bacteria on a minimal medium with lactose, and looked for mutants that revert back to normal. As the cells used up the small amount of nutrient they stopped growing. But after some time, mutants began to appear that could grow on lactose. However, the mutations are not strictly directed to the gene in which mutations could be advantageous, as unselected mutations also accumulated. In fact, the mechanisms look like "inducible genetic chaos" according to a reviewer [4].

The defective lac gene in the E. coli strain was in fact a frameshift mutant, in which a small deletion or addition of a nucleotide shifted the whole reading frame of the gene, so it became translated into a totally different enzyme that has little or no ability to break down lactose. This defective lac gene was carried in an F' plasmid involved in bacterial conjugation. Two types of adaptive genetic change are now known to occur in the lac frameshift system: point mutations involving changes in base sequence of the DNA, and gene amplification involving the generation of multiple copies of the defective gene so that large amounts of defective enzyme can still function to metabolise enough lactose to allow the cells to grow.

The point mutation mechanisms are highly diverse, and includes DNA breakage, recombination break repair, genome-wide hypermutation in a subpopulation of cells that give rise to some or all of the adaptive mutants, a special inducible mutation-generating DNA polymerase (polIV or DinB) that has homologues in all three domains of life. There are now many bacterial and yeast assay systems in which adaptive and stationary-phase mutations have been reported, but the mechanisms are largely unknown.

Some of the mechanisms that underlie adaptive genetic change bear similarities to genetic instability in yeast and in some cancers and to somatic hypermutation in the immune system. They might also be important in bacterial evolution to antibiotic resistance, and the evolution of phase-variable pathogens, which evade the host immune system by frequent variation of their surface components.

In the experiment, Lac+ mutants that existed before exposure to the lactose plates form visible colonies by about two days. The colonies that emerged after 2 days fall into two classes. Most of the Lac+ colonies (~160 /108 cells at 10 days) are adaptive point mutants, which occur by a recombination dependent mechanism and produce compensatory frameshift mutations. On later days (from ~4), an increasing fraction (up to ~35 out of a total of ~160 on day 10) of the colonies are not point mutants but amplifications (20-50 direct repeats) of a 7-40kb region of DNA that contains the lac frameshift gene, which provides sufficient gene activity to allow growth on lactose medium. The number of E. coli cells does not increase during the first five days.

A profusion of mechanisms

There are many ways to generate adaptive mutations.

Interestingly, adaptive point mutations in the lac system requires homologous recombination proteins of the E. coli RecBCD double-strand break-repair system which is widely involved in gene conversion and recombination (see "How to keep in concert", this series). Double-strand ends could be generated during DNA replication by a number of different mechanisms.

The adaptive Lac+ point mutations that revert a framewhift allele are nearly all -1 deletions (deletion of a single nucleotide) in small mononucleotide repeats, whereas the pre-existing (non-adaptive) Lac+ reversions are heterogeneous. Mononcleotide repeat instability is thought to reflect DNA polymerase errors, which is consistent with the requirement of a special error-prone DNA polymerase (polIV) for adaptive mutations.

The 'SOS response' is the bacteria's response to DNA damage or the inhibition of DNA replication. It involves de-repression of at least 42 genes that carry out DNA repair, recombination, mutation, translesion DNA synthesis (synthesis across non-repaired or damaged DNA) and prevent cell division.

Global hypermutation is thought to occur in a subpopulation of the cells. This is because the frequencies of unselected mutations are about two orders of magnitude higher among Lac+ mutants than in the main population of Lac- starved cells. These results mean that stationary-phase mutations in this system are not directed exclusively to the lac gene, and both adaptive and neutral mutations are formed. Some or all of the adaptive mutants arise in a subpopulation that is hypermutable relative to the main population.

The subpopulation of cells that are transiently mutable is estimated to be between 10-3 and 10-4 of all cells. Despite that, the frequency per unit length of DNA in the genome is markedly uneven, with definite hotspots and coldspots, perhaps depending on the proximity to double strand breaks (DSBs) in DNA that are generated.

Gene amplification is 'adaptive' in the sense that it only occurs in response to the selective environment. Cells carrying the amplification are not hypermutated in unselected genes, and neither the SOS response nor polIV is required. Dependence on homologous recombination is implied in that adaptive Lac+ colonies do not appear in the absence of RecA and RecBCD enzyme, and RuvAB and C recombination proteins.

Similar findings in bacteria isolated from the wild

Until 2003, the phenomenon of adaptive mutations has been observed only in laboratory strains. But researchers from the University of Paris, France, and the National University of Mexico (UNAM) reported similar stress-inducible mutagenesis in stationary-phase bacterial colonies grown from strains culled from the wild [5]. This provides evidence that most natural isolates of E. coli from diverse habitats worldwide increase their mutation rates in response to the stress of starvation.

A total of 787 E. coli isolates were collected from habits including air, water and sediments, and the guts of a variety of host organisms. Colonies formed during the exponential growth phase were subjected to starvation during a prolonged stationary phase, and the production of mutants was monitored in the starved aging colonies. The vast majority of colonies showed an increased number of mutants. In a sample of colonies, the authors were able to link the increased mutagensis to starvation and oxidative stress by showing that either additional sugar or anaerobic incubation could block the increased mutagenesis.

The bacteria were highly variable in their inducible mutator activity. The frequency of mutations conferring resistance to rifampicin (RifR) in day 1 (D1) and day 7 (D7) was measured. For all strains, the median values of RifR mutations were 5.8 x 10-9 on day 1, and 4.03 x 10-8 on day 7, an increase of 7 fold, while the median number of colony-forming units increased 1.2-fold. In comparison, the E. coli K12 MG1655 lab strain showed a 5.5-fold increase in frequency of RifR and a 1.7 fold increase in colony forming units. Constitutive mutator strains having a D1 mutation frequencies >10-fold or >100-fold higher than the median D1 frequency of all the strains represented 3.3% and 1.4% of isolates respectively. The D7/D1 mutation frequency ratio showed that 45% of strains had more than a 10-fold, and 13% more than a 100-fold increase in mutagenesis over 7 days. Interestingly, constitutive mutagenesis and MAC (mutagenesis in aging cells) showed a negative correlation.

The MAC was genome wide in a large fraction of natural isolates. There was no significant correlation between MAC and phylogeny. The host's nutrition might explain some of the variation of MAC. For example, bacteria from the guts of omnivorous species like human beings have weaker stress-inducible mutator activities than those from carnivores.

The mechanisms for generating mutations looked even more diverse than in the laboratory strains [6].

Wider significance of adaptive mutations

Amplification is an important manifestation of chromosomal instability prevalent in many human cancers, and DSBs in DNA are also involved. Induction of mammalian amplification by selective agents is correlated with the ability of those agents to produce chromosomal breaks.

The adaptive point mutation mechanism at lac might be relevant to microbial evolution, particularly of pathogenic bacteria. Many phase variable pathogens have simple repeated sequences that flank genes that they regulate by frameshift mutation.

These 'contingency genes' used under stress provide phase variations that allow evasions of the immune system. Two of them, Neisseria meningitides and N. gonorrhoeae, have one or more genes homologous to dinB. For many pathogenic bacteria, antibiotic resistance is also achieved by point mutation mechanisms and could be induced adaptively. Even antibiotics that cause lethality can be merely bacteriostatic at lower concentrations, such that stress-promoted mutation mechanisms might be significant in the development of resistance in clinical environments.

In multicellular eukarytoes, parallels between adaptive mutation and cancer have been noted, the key being that acquisition of mutations in growth-limited state (stress) allows cells to proliferate.

Humans have three E.coli polIV homologues of unknown function, in the DinB/UmuDC/Rad30/Rev1 superfamily of DNA polymerises, as well as a homologue known to carry out translesion synthesis (the tumour suppressor protein XP-V). DinB1 or polk, a true DinB orthologue, is found in germline and lymphoid cells. More and more geneticists now think that mutation is regulated [7], or at any rate, provoked, and highly non-random.

Indeed, in one study on 12 long-term E coli lines, 36 genes were chosen at random, and 500 bp regions sequenced in four clones from each line and their ancestors [8]. Several mutations were found in a few lines that evolved mutator phenotypes, but no mutations were found in any of the 8 lines that retained functional DNA repair throughout the 20 000 generations experiment. This confirms the low level of 'spontaneous' or unprovoked mutation.

Article first published 15/09/04

  1. Delbrück M. Cold Spring Harbor Symp. Quant. Biol. 1946, 11, 154 (cited by Rosenberg SM, 2001).
  2. Tlsty T D, Margolin B H & Lum K. Differences in the rates of gene amplification in nontumorigenic and tumorigenic cell lines as measured by Luria-Delbrück fluctuation analysis. Proc. Natl Acad. Sci. USA 1989, 86, 9441-5.
  3. Cairns J. & Foster P L. Adaptive reversion of a frameshift mutation in Escherichia coli. Genetics 1991, 128, 695-701.
  4. Rosenberg SM. Evolving responsively: adaptive mutation. Nature Reviews Genetics 2001, 2, 504-15.
  5. Bjedov I, Tenaillon O, Gerard B, Souza V, Denamur E, Radman M, Taddei F and Matic I. Stress-induced Mutagenesis in Bacteria. Science 2003, 300, 1404-7.
  6. Rosenberg SM and Hastings PJ. Modulating mutations rates in the wild. Science 2003, 300, 1382-3.
  7. Drake JW. Spontaneous mutation. Ann. Rev. Genet. 1991, 26, 126-46.
  8. Elena SF and Lenski RE. Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation. Nature Reviews Genetics 2003, 4, 457-68.

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