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
ISIS Report 15/09/04
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
version of this paper is posted on ISIS members website.
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
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
Microbiologist Max Delbrück first used the term adaptive
mutations in1946 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. 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. 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. 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.
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
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
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 bacterias 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
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. 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 hosts
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.
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
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, or at any rate, provoked, and highly
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. 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