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At the heart of modern biology is a theory most of us take for granted:
neo-Darwinism. Its central idea – that organisms evolve by a combination
of random genetic change and natural selection – has become tantamount to
dogma. So when respectable biologists turn up evidence which appears to
run counter to the creed, people naturally get excited. Splashy articles
appear in newspapers. Commentators gather as though at the scene of a terrible
accident, the running aground of the good ship scientific orthodoxy. Could
He (Darwin) really have been wrong?

So it was in 1988 when John Cairns, a geneticist at Harvard University,
and his colleagues Julie Overbaugh and Stephan Miller published a study
of spontaneous genetic mutations in the bacterium Escherichia coli. Some
of the mutations they reported, instead of being purely chance events, seemed
to be a calculated response to environmental pressure. On the face of it,
this was a blatant heresy – how can an organism sense its needs in a particular
environment and then mutate accordingly? One possibility (unpalatable to
many) was that Lamarck, the 18th century naturalist who suggested that organisms
evolve by acquiring favourable characteristics, was right after all.

Orthodox theory holds that spontaneous genetic change and the environment
behave as two independent forces in evolution. The environment selects mutations
(chance mutations that happen to be beneficial in a particular environment
are more likely to be passed on to future generations than harmful ones)
but it cannot ‘direct’ them. According to Lamarckism, however, the opposite
is true: genetic change occurs in direct response to environmental pressure.
This ‘heresy’ has never mustered much support among modern biologists who
have always been resistant to the idea of the environment restricting genetic
change to beneficial mutations.

Understandably, the Harvard study sparked an extensive and often acerbic
debate in the pages of Nature and other scientific journals. Had Cairns
and his colleagues really found evidence of directed mutagenesis, or was
the whole thing an illusion? Three years on, the debate continues apace.
Other studies, particularly from Barry Hall, a geneticist at the University
of Rochester, New York, have greatly strengthened the Harvard observations
. And as the evidence mounts, geneticists – even staunch neo-Darwinists
– are being forced to re-examine their entrenched ideas about spontaneous
mutations.

Not surprisingly, neo-Darwinism has dominated efforts to trace the origins
of spontaneous genetic mutations. Molecular biologists have revealed that
such mutations arise because the molecular machinery that copies DNA is
error prone. As a cell divides, there is always a small chance of an incorrect
base – one of four types of chemical building blocks in DNA – being incorporated
into the freshly made genome. And although most of these errors are quickly
remedied by enzymes, a few slip through to offspring cells. It is hard to
imagine how this biochemical lottery could be skewed by environmental pressure.

This concern lies at the centre of the current debate. To understand
the issues, one must look back at some earlier experiments. The Harvard
study, despite the controversy it stirred, merely rekindled interest in
the question of spontaneous mutations after a lull spanning more than three
decades. The story really began in the 1940s with Salvador Luria, a biologist
then working at Indiana University in the United States, and a classic experiment
known as the fluctuation test. It was partly for this experiment that Luria
received the Nobel Prize for Medicine in 1969.

The idea of the fluctuation test came to Luria from thinking about gambling
machines. Usually when you pull the handle of a fruit machine the outcome
is no reward. But sometimes you win a little, and even more rarely you hit
the jackpot. Luria reasoned that if conventional evolutionary theory was
correct, then the appearance of specific mutants in a series of similar
cultures of bacteria grown in the laboratory should follow a similar trend:
most cultures should contain no mutants, some a few, and just occasionally
there should be a jackpot – cultures rich in mutants. Jackpots should be
rare, because they depend on a chance mutation happening early in the life
of a colony, but they should at least occur. This would not be true if mutations
arose in direct response to environmental pressure: jackpots would be precluded
by the fact that the number of bacteria ‘challenged’ by the environmental
agent would be roughly the same in each tube.

To put this thinking into practice, Luria grew up a series of cultures
of E. coli. Using a simple assay, he then measured in each tube the number
of bacteria that had mutated so as to be resistant to a bacterial virus
called T1. The results were in line with the fruit machine hypothesis: most
tubes contained few or no resistant bacteria, but a minority contained a
large number. The implication was that genetic mutations do indeed occur
at random during cell growth. However, the conclusion that most biologists
drew from Luria’s experiments was a more extreme one, namely that this is
the only route to spontaneous genetic change.

In retrospect it is easy to see that this generalisation was unjustified.
The limitation of the early experiments of Luria and others was that the
selection methods used to detect mutants – infection by viruses or exposure
to antibiotics – killed sensitive bacteria almost instantaneously. So even
if bacteria were also able to mutate in a more directed fashion, they may
not have had time to do so: Lamarckism had not been given a fair chance.
It was precisely this point which was taken up by Cairns and his colleagues
in 1988. Their experiments exploited a much gentler approach to selection,
one which had been pioneered in the 1950s and early 1960s by Francis Ryan,
a professor of zoology at Columbia University in New York.

Ryan’s aim had been to test the assumption that cell growth is essential
for mutagenesis in bacteria. To do so, he studied a mutation that subtly
changed E. coli’s metabolism. The mutation corrected a defective gene, converting
a strain of the bacterium that was unable to synthesise the amino acid histidine
(his-) into one that could (his+). Selecting for this mutation, as opposed
to mutations that confer resistance to antibiotics and bacterial viruses,
offered a crucial advantage: when deprived of histidine, his- bacteria merely
stop growing, they do not die. And that at least gave the bacteria the chance
to adapt.

Ryan’s experiment was relatively simple. He set up several hundred tubes
containing a growth medium supplemented with only a little histidine. He
then inoculated each with a few his- cells and left them to incubate. After
a couple of days most cultures, stunted by insufficient histidine, had barely
grown at all, but about one tube in five was thick with bacteria. These
were the expected hallmarks of orthodox mutagenesis: spontaneous his+ mutations
had occurred at random during the early phase of growth before the histidine
ran out. The resulting mutant bacteria had then been able to grow to saturation
without the need for further histidine.

Up to this point Ryan’s results echoed those of Luria. The new twist
came when he left the tubes to incubate over a period of weeks. More and
more of the tubes became turbid, until after three weeks the number had
doubled. This was odd. It seemed that a special type of ‘late’ mutation
was converting his- bacteria into his+ bacteria, and (more important) doing
so in cells that were not growing. Further experiments established beyond
doubt that the late mutations arose in ‘resting’ cells and were not the
result of very slow cell division.

Ryan’s study provided convincing evidence of the existence of two pathways
to mutagenesis rather than one. The conventional pathway, observed by Luria,
operates during cell growth and so depends on DNA replication; the second
pathway operates in resting cells, in the absence of measurable DNA replication.
By one of those odd quirks of science, however, few of Ryan’s contemporaries
appreciated the importance of this finding. His work sank into obscurity
for 30 years (Ryan died in 1963, aged 47), to be resurrected only last year
– two years after the Harvard team reopened the mutagenesis debate to a
fanfare of publicity.

Cairns and his colleagues covered the same ground as Ryan but added
one crucial element. They asked the question: do late mutations arise only
under selective pressure, to solve the bacterium’s growth difficulties,
or do they happen regardless of such benefits? Ryan had no idea whether
the late mutations were confined to the defective gene blocking the synthesis
of histidine. There was a chance that the mutations were instead scattered
across the entire bacterial genome, and that only a minority of them corrected
the defect, switching the bacterium from his- to his+.

The Harvard team attempted to resolve this uncertainty. Using a genetic
marker similar to Ryan’s, they selected for a mutation which switched a
strain of E. coli known as lac- (which cannot break down lactose) into lac+.
While lac- bacteria need a carbon source other than lactose, lac+ bacteria
are content to grow on lactose. In line with Ryan’s results, Cairns and
his colleagues found that mutations converting lac- bacteria to lac+ bacteria
continued to occur in resting cells whose only carbon source was lactose.
They then went on to test whether these mutations were specific.

Mutating under pressure

First, they took away the lactose to see if it was essential for the
production of late lac+ mutants. It was. Secondly, they examined whether
late mutations occurred in a bacterial gene which had nothing to do with
the metabolism of lactose, and was therefore under no selection pressure.
They did not. On the face of it, the issue seemed clinched: not only could
spontaneous mutations arise in the absence of conventional DNA replication,
as Ryan had shown, but these mutations appeared to be targeted so as to
be advantageous.

The spectre of Lamarckism thus raised, defenders of the faith were quick
to respond. Much of the ensuing debate centred on one question. Did the
late mutations really arise in the absence of conventional DNA replication?
Some suggested that a limited amount of cell growth – and hence turnover
of DNA – may have resulted from bacteria feeding on impurities in the medium,
material from dead cells or even on nutrients produced by a tiny number
of early mutants. This line of criticism, however, turned out to be misplaced.
For unbeknown to most of those involved in the debate, Ryan had already
shown that resting cells mutate, and do so at a rate much higher than one
would expect even allowing for a small amount of residual DNA replication.
A more important question was whether the unexpected mutations were truly
Lamarckian. Here the chief criticism was that the researchers had not looked
at enough genes to be sure the mutations were specific to the gene under
selection pressure.

Studies carried out since then, by Hall and Cairns himself, have largely
laid this concern to rest. Biologists are slowly coming round to the idea
that in certain circumstances bacteria may mutate in an apparently directed
fashion. So, if one provisionally accepts the evidence, how can we explain
it using our present knowledge of molecular biology? It is easy to picture
mutations creeping into a genome during replication, but much harder to
explain their appearance in resting cells. Most of the theories advanced
so far have attempted to defuse the controversy with explanations based
on purely random cellular processes.

One such theory, proposed independently by Frank Stahl, a geneticist
at the University of Oregon, Oregon, and Lars Boe, of the Technical University
of Denmark in Copenhagen, is shown in the figure. Stahl and Boe argue that
the genomes of resting cells, instead of having a fixed structure, in fact
have a highly dynamic one. The resting cell is constantly replacing short
stretches of the DNA in its genome with freshly synthesised strands of DNA.
Moreover, this process is extremely error prone, so incorrect bases sneak
into the strands as they are synthesised. As a consequence, pairs of ‘mismatched’
bases appear at various points along the genome.

It is from these mismatched base pairs that the ‘directed’ mutations
originate, say Stahl and Boe. Most of the mismatches will be quickly corrected
by the cell’s repair enzymes. The only way a mismatch can be converted into
a permanent mutation is if a round of conventional DNA replication occurs
before the repair enzymes have a chance to act. But what could prompt a
resting cell to replicate its DNA? One answer is a mismatch which happens
to solve a cell’s growth problems. The likelihood of such a mismatch becoming
‘fixed’ by DNA replication would probably increase as a resting cell ages
and its repair mechanisms slow down.

As an illustration, consider Ryan’s experiments. The his- strain of
E. coli carries a defect in a gene needed for histidine synthesis, so the
cell cannot grow without histidine. At some point during incubation, however,
a mismatched base becomes inserted into one strand of the gene’s DNA which
just happens to correct the defect. The result? The cell synthesises a small
amount of histidine, there is a round of cell division and the mismatched
base becomes frozen into the bacterium’s genome. The key point is that only
mismatches which solve the bacterium’s growth problem can be fixed in this
way, so mutations are confined to genes which are under selection pressure.

Another explanation, and one initially favoured by Hall and myself,
is based on a hypothetical ‘hypermutable’ state. The gist of it is this.
As a colony of bacteria ages, a fraction of its cells might enter a precarious
state in which the synthesis of single strands of DNA is far more error
prone than usual. If the error rate were high enough, the DNA repair mechanisms
in such cells would be overwhelmed, and a range of random mutations would
suddenly appear. Many of these would prove lethal: the only way out of the
hypermutable state would be if a mutation happened to solve the bacterium’s
growth problem. The thing that distinguishes this explanation from that
of Stahl and Boe is that it predicts the appearance of late mutations at
sites all over the genome. The evidence so far suggests that late mutations
occur predominantly in the gene (or genes) under selection pressure.

Both explanations are easy to accommodate within orthodox theory. Indeed
there is not so much as a hint of the environment directing mutations. Random
errors enter the genomes of resting cells, and stable mutants are selected
from these in the traditional way by the environment. The process appears
to be Lamarckian but only because the cells are starved of nutrients and
need a mutation in order to grow.

There is some evidence that a cell’s propensity for directed mutagenesis
depends on its ability to produce certain proteins involved in mismatch
repair. As yet, though, firm experimental support for any of the random
mechanisms suggested is lacking, so it is impossible to rule out more heterodox
ideas. The most intriguing suggestion in this vein – but one which is no
longer taken very seriously – is that the directed mutations might originate
in RNA, rather than DNA. The synthesis of messenger RNA – the halfway stage
between a gene and the protein it produces – is even more error prone than
the synthesis of DNA. An error that happened to remedy a defective messenger
RNA might just be sufficient to kick start a few rounds of cell division.
Any cells temporarily invigorated in this way might then copy the information
encoded in the RNA – including the error – back into DNA, which would fix
the mutation. The heterodox element is that in copying the information,
the cells ignore RNA messages that do not carry beneficial errors. In some
way they are able to ‘sense’ what genetic information they need in order
to grow.

In making sense of directed mutations, one thing should be borne in
mind: geneticists may be experts in manipulating growing bacteria but they
are still novices in dealing with bacteria at rest – their natural state
in soil, water and the gut. Many surprising findings are likely as they
acquire this expertise; but in the meantime we should treat all results
with caution. At the same time, biologists need no longer be alarmed at
the mere notion of directed mutagenesis, for cells are almost certain to
possess enzymes capable of altering genetic structure in the absence of
cell division – the key to finding an orthodox explanation of the phenomenon.
Once heralded a blasphemy, Lamarckian adaptation may eventually be seen
as an inevitable consequence of molecular genetics.

Neville Symonds is Emeritus Professor of Microbial Genetics at the University
of Sussex