Guest guest Posted September 16, 2004 Report Share Posted September 16, 2004 > 15 Sep 2004 18:06:06 -0000 > To Mutate or Not to Mutate > press-release > > > The Institute of Science in Society Science Society > Sustainability http://www.i-sis.org.uk > > General Enquiries sam Website/Mailing > List > press-release ISIS Director > m.w.ho > ======================================================== > > > > 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 http://www.i-sis.org.uk/fluidGenome.php). > 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. > > Death of the Central Dogma > http://www.i-sis.org.uk/DCD.php > Caring Mothers Reduce Response to Stress for Life > http://www.i-sis.org.uk/MCDIRTS.php > Subverting the Genetic Text > http://www.i-sis.org.uk/RNASTGT.php > To Mutate or Not to Mutate > http://www.i-sis.org.uk/TMONTM.php > > > > ISIS Press Release 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 > > A fully referenced version of this paper is posted > on ISIS > members' website > http://www.i-sis.org.uk/full/TMONTMFull.php. > Details here http://www.i-sis.org.uk/membership.php. > > 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 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 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. 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. > > 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, 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. 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. > > > ======================================================== > > This article can be found on the I-SIS website at > http://www.i-sis.org.uk/TMONTM.php > > If you like this original article from the Institute > of > Science in Society, and would like to continue > receiving > articles of this calibre, please consider making a > donation > or purchase on our website > > http://www.i-sis.org.uk/donations. > > ISIS is an independent, not-for-profit organisation > dedicated to providing critical public information > on > cutting edge science, and to promoting social > accountability > and ecological sustainability in science. > > If you would prefer to receive future mailings as > HTML > please let us know. If you would like to be removed > from our > mailing list at > > http://www.i-sis.org.uk/mailinglist/.php > ======================================================== > > CONTACT DETAILS > > The Institute of Science in Society, PO Box 32097, > London > NW1 OXR > > telephone: [44 20 8643 0681] [44 20 7383 3376] > [44 20 > 7272 5636] > > General Enquiries sam Website/Mailing > List > press-release ISIS Director > m.w.ho > > MATERIAL IN THIS EMAIL MAY BE REPRODUCED IN ANY FORM > WITHOUT > PERMISSION, ON CONDITION THAT IT IS ACCREDITED > ACCORDINGLY > AND CONTAINS A LINK TO http://www.i-sis.org.uk/ > > Quote Link to comment Share on other sites More sharing options...
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