ADAPTIVE MUTATION

ANUPAMA CN I M.Sc, BT MCC

INTRODUCTION
EVOLUTION
Merriam-Webster's dictionary gives the following definition of evolution: a theory that the various types of animals and plants have their origin in other pre-existing types and that the distinguishable differences are due to modifications in successive generations. Evolution is the change over time in one or more inherited traits found in populations of individuals. Inherited traits are distinguishing characteristics, for example anatomical, biochemical or behavioural, that are passed on from one generation to the next. Evolution occurs when there is variation of inherited traits within a population over time. The major sources of such inherited variants are mutation, genetic recombination and gene flow. Evolution has led to the diversification of all living organisms from a common ancestor, which are described by Charles Darwin as "endless forms most beautiful and most wonderful". Evolution can simply be defined as the result of environmental stress that selects the fittest. There are four common mechanisms of evolution: Natural Selection: a process in which there is differential survival and reproduction of entities that differ in one or more inherited traits. Selection can act at multiple levels of organization, for example differential survival and/or reproduction of organisms, populations, or gene variants. Genetic Drift: a process in which there are random changes to the proportions of two or more inherited traits within a population. Gene Flow: which is the incorporation of genes from one population into another. Mutation: a process which affects phenotypes expressed across multiple levels of organisation.

MUTATION AND MUTAGENESIS

In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic chemicals, as well as errors that occur during meiosis or DNA replication. They can also be induced by the organism itself, by cellular processes such as hypermutation. Random mutations constantly occur in the genomes of organisms, which produces genetic variation in a population. When mutations occur they can either have no effect alter the product of a gene prevent the gene from functioning. Based on studies in the fly Drosophila melanogaster, it has been suggested that if a mutation changes a protein produced by a gene, this will probably be harmful, with about 70% of these mutations having damaging effects, and the remainder being either neutral or weakly beneficial. We can consider the following broad categories of mutations: SINGLE BASE PAIR CHANGES: Single mutations (or point mutations) involve a change in the identity of a single base or base-pair. Base pair changes are not necessarily deleterious -- a change in genotype need not necessarily cause any change in phenotype. It depends on whether the change occurs within a coding region or within some other important regulatory sequence (e.g. an operator or promoter).There are two types of mutation that change the identity of a base (base-pair) TRANSITIONS:Transitions do not alter the chemical nature of the base. Thus, a purine base is replaced by another purine; a pyrimidine is replaced by a pyrimidine. A -> G or G -> A C -> T or T -> C TRANSVERSIONS: Transversions change the the chemical nature of the base. Thus, a purine base is replaced by a pyrimidine; a pyrimidine is replaced by a purine.

A -> C or A -> T C -> A or C -> G G -> C or G -> T T -> A or T -> G SILENT mutations occur when a base pair change in a coding region does not affect the amino acid that is encoded. For example, the change UGC -> UGU is silent because both are codons for cysteine. Another way in which silent mutations might occur is when the change occurs in a non-coding or non-regulatory region. MISSENSE mutations occur when the base pair change in a coding region results in a change the amino acid that is encoded. How severe the mutation may depends on the nature of the change. For example, the change GAA -> GAU will result in a change from glutamic acid to aspartic acid. One would not expect this change to have a very large effect. However, the change GAA -> AAA would be expected to be significant since it replaces a glutamic acid with a lysine. NONSENSE mutations occur when the base pair change in a coding region results in the creation of a stop codon. Proteins will be truncated as a result of this type of mutation. An example is the change UGC -> UGA.

MUTAGENESIS is a process by which the genetic information of an organism

is changed in a stable manner, either in nature or experimentally by the use of chemicals or radiation. Mutagenesis as a science was developed especially by Charlotte Auerbach in the first half of the 20th century. It was believed that mutagenesis occurs randomly, regardless of the utility of a genetic mutation to the organism. If it is beneficial,or neutral the organism will survive; if it is harmful, the organism will die. Some examples are: TAUTOMERIZATION of either the template base or the base in the nucleotide being added may result in incorrect pairing in the active site of DNA polymerase and, in turn, result in the incorporation of an incorrect base. These types of mistakes are usuallt detected by the proof-reading exonuclease. However, when they are not, they must be repaired by the MISMATCH REPAIR system. DEAMINATION of cytosine can occur spontaneously or as a result of treatment with nitrous acid. This reaction results in the deamination of

cytosine to uracil which can then base pair with adenine. Since uracil is a "normal" base - although it only normally occurs in RNA - this is potentially a very serious problem. The cell, however, has an enzyme to remove it from DNA: uracil-N-glycosylase. ALKYLATION of DNA can result in mutations in several ways. The addition of bulky alkyl groups by chemicals such as ethyl methane sulfonate (nitrogen mustard gas) or ethylnitrosourea will distort the DNA double helix. The modified nucleotides can be excised but the gaps are often repaired incorrectly. METHYLATION of DNA can be a mixed blessing. It is an important reaction for regulating gene expression in eukaryotes and for marking the host cell DNA in bacteria. However, some methylated bases such as 5-methyl cytosine, are more susceptible to oxidative deamination. For this reason, the methylation system is often accompanied by a special repair system. According to Klug and Cumming, ther are thee types of mutation: Spontaneous Induces Adaptive

In 1988, J. Cairns performed experiments with bacteria that implied that said bacteria could direct their own mutations so that they could cope more speedily with sudden environmental trauma. He proposed that "when populations of single cells are subject to certain forms of strong selection pressure, variants emerge bearing changes in DNA sequence that bring about an appropriate change in phenotype." In Cairns' experiments, bacteria unable to digest lactose were presented with an all-lactose diet. They quickly acquired the mutations needed to digest the only food available. They did not have to wait for random mutations to accidentally hit upon the correct genome changes. This suggests that there exists a particular physiological pathway that responds to a specific selective pressure to produce a mutation conferring the correct phenotype that will alleviate this pressure.

ADAPTIVE MUTATION
It has long been assumed that the force driving evolution is natural selection, not the creation of genetic variants, because the rate of mutation was thought to be constant and unaffected by circumstances (although Darwin himself did not believe this). But, the paper published by John Cairns and his colleagues in 1988 challenged traditional thinking about spontaneous mutation. In that paper, Cairns presented evidence that mutations arise in nondividing, nutritionally deprived cells of Escherichia coli, apparently in response to selective pressure. The Lamarckian idea that these mutations were directed by the selective conditions was based on three observations: mutations arose among nonproliferating cells after selection was applied the presence of the selective agent was required for the mutations mutations that were not selected did not appear during selection. Cairns gave several examples illustrating these points, and other cases quickly appeared in print. Only some of these cases were further investigated, and, in general, one or more of the above criteria has proved not to be true or to be explainable by other causes. The original hypothesis of directed mutation, therefore, has not been supported. Nevertheless much subsequent research has shown that mutation rates can vary, and that they increase during certain stresses such as nutritional deprivation. The phenomenon has come to be called adaptive mutation. Adaptive Mutation is defined as a process that during nonlethal selection produces mutations that relieve the selective pressure, whether or not other, nonselected mutations are also produced. It remains to be seen whether this occurs via an evolved mechanism, or because the cells are simply unable to maintain the integrity of their DNA repair systems. Or it can also be defined as mutations that occur in nondividing or slowly dividing cells during prolonged nonlethal selection, and that appear to be specific to the challenge of the selection in the sense that the only mutations that arise are those that provide a growth advantage to the cell.

EXAMPLES OF ADAPTIVE MUTATION

Examples of adaptive mutation or related phenomena have been reported in the bacteria Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Pseudomonas sp. and Clostridium sp., and, in the eukaryotic microbial species, Saccharomyces cerevisiae and Candida albicans. Whether the phenomenon exists outside of microorganisms is not known, but it seems unlikely to contribute to the evolution of organisms that have their germ lines protected from the environment. Mutations occurring in the nondividing somatic cells of higher organisms, however, could give rise to disorders such as cancer. In the last decade, most research on adaptive mutation has utilized Escherichia coli strain FC40. Consequently, more is known about the mechanisms that produce adaptive mutations in this strain than in any other. Although the results cannot necessarily be extrapolated to other cases, FC40 provides examples of the kinds of mechanisms that can give rise to adaptive mutations.

Escherichia coli strain FC40

Although FC40 is not the strain of E. coli originally described in Cairns, it has proved attractive for studies of adaptive mutation because of its high rate of mutation during selection. The strain is deleted for the lac operon on its chromosome but carries this operon on the F episome, a low-copy conjugal plasmid. The strain cannot utilize lactose (Lac ) because of a + 1 frameshift mutation that inactivates the lacZ gene. During nonselective growth FC40 has a mutation rate to lactose utilization (Lac +) of about 1 per 109 cells per generation; when plated on lactose minimal medium, Lac + revertants arise at a rate of about 1 per 109 cells per hour, and continue to do so at this rate for several days. Yet, during such a selective regime, in a carefully conducted experiment the total number of background (Lac) cells does not change by more than 20%, and there is no evidence of cell death or turnover. In effect, there is a genuine and specific increase in the number of mutational events that produce Lac + revertants. The Lac+ mutations that occur during lactose selection are distinguished from those that occur during nonselected growth by type and by genetic requirements. While insertions and deletions of various types giving a Lac + phenotype occur during nonselective growth, the adaptive Lac+ mutations are almost exclusively

1 base pair deletions in runs of iterated bases. Furthermore, unlike mutation during nonselective growth, adaptive Lac + mutation requires recombination functions, specifically E. colis pathway for double-strand end repair. In addition, the high rate of adaptive Lac+ mutation requires that the lac allele be on the F episome and that conjugal functions be expressed, although actual conjugation is not required. In contrast, when the same lac allele is in its normal position on the chromosome, the adaptive mutation rate to Lac + falls 100-fold, and the mutations are no longer dependent on recombination or conjugal functions.

The accumulation of Lac+ revertants of FC40 during incubation on lactose minimal medium plates. Left axis, circles: the number of Lac+ colonies per 108 cells; right axis, triangles: the number of Lac cells on the plate.

Although adaptive mutation in FC40 might be considered a laboratory artefact, it has several potentially important lessons to teach us. Recombination-dependent mutation might be an important source of spontaneous mutations when cells are not actively replicating their genomes. Error-prone polymerases might increase genetic variation under stress. Conjugal plasmids might be important in the evolution and horizontal transfer of genes.

Role of Recombination in Adaptive Mutation of FC40

In light of the above studies (E.coli FC40), it seems likely that DNA synthesis primed by an invading 3 end during recombination produces the adaptive Lac+ mutations in FC40. The Lac allele is on the conjugal plasmid, F, which carries a special origin for conjugal transfer called oriT. Nicking at oriT by the nickase, TraI, occurs even when the cells are not mating.This nicking can initiate recombination. F also has vegetative origins from which normal replication forks are initiated. Because the Lac allele is slightly leaky, some DNA replication may occur during lactose selection. The best current hypothesis for adaptive mutation in FC40 that accounts for all the genetic requirements is that a replication fork initiated at a vegetative origin encounters the persistent nick at oriT and collapses. The broken arm of the replication fork is repaired by doublestrand end repair, and de novo DNA synthesis initiated by the recombination intermediate produces the Lac+ mutations. The fact that adaptive mutations are eliminated in priA mutants supports this hypothesis.

A model for recombination-dependent mutation. A,B: Collapse of the replication fork at oriT; C,D: RecABCD-catalyzed recombination; E: restoration of the replication fork; F: translocation of the Holliday junction by RuvAB and RecG; resolution of the Holliday junction by RuvC is not shown. Dashed lines are newly synthesized DNA; * at oriT indicates TraI. The polarity of the DNA has been inverted between B and C.

The role of error-prone polymerases in adaptive mutation of FC40

Because the cells are not actively growing, the production of mutations by the recombination-dependent mechanism becomes dominant during lactose selection. But, even so, the mutation rate observed is surprisingly high, equivalent (in mutations per cell per hour) to that observed when the cells are growing exponentially. It seems unlikely that DNA replication and turnover in Lac cells during lactose selection is equal to that occurring during exponential growth. Thus, it appears that the DNA synthesis that gives rise to the mutations is inherently error-prone, refractory to repair, or both. Recently, a new class of error-prone DNA polymerases has been discovered in bacteria and higher organisms, including humans. Because, at least in E. coli, these polymerases are induced by DNA damage and possibly by other stresses, it has been hypothesized that they exist to increase variation in a population during stress. Alternatively, the error-prone polymerases may exist solely to replicate damaged DNA, and their error proneness may be an inevitable consequence of this requirement. It is now known that E. coli has at least five DNA polymerases, three of which are induced as part of the SOS response. Two of the inducible polymerases, Pol IV and V, are error-prone, while one, Pol II, is accurate.In FC40, Pol IV normally accounts for 50% of the adaptive Lac + mutations, but for many more in the absence of Pol II. This means that Pol II and Pol IV are in competition, and Pol II normally limits Pol IV to about 20% of its mutagenic potential. The remaining mutations may be due to Pol III (the replicative polymerase) or to another polymerase. Thus, at least three and possibly more DNA polymerases appear to compete for the 3 ends of recombination intermediates. The adaptive mutation rate is, at least in part, determined by the relative levels of these polymerases. Since induction of these polymerases is under control of the SOS system, which can be induced in nutritionally deprived cells (see below), this is a way that stress can increase mutation rates, as was envisioned many years ago.

Role of Conjugal Plasmids in Adaptive Mutation of FC40

Our original belief that in FC40 only Lac + mutations occurred during lactose selection was based on a failure to find mutations in a control gene (giving rifampicin resistance) among non-reverted Lac cells. The Lac allele is on the conjugal plasmid, F, however, and the target for mutations to rifampicin resistance, rpoB, is on the chromosome. When a suitable target was placed close to the Lac allele on F, non-selected mutations in this gene were readily observed during lactose selection. These mutations occurred at about the same rate and by the same recombination-dependent mechanism as did the Lac + mutations. Mutations at another locus on F also occurs at a high rate. Thus, genes carried on F appear to be highly mutable. F recombines with the chromosome via insertional elements, creating various Hfr strains. When it excises it can carry with it large segments of the chromosome, creating various Fs. The genes on these Fs actively recombine with their homologues on the chromosome. Thus, we might consider F to be an evolution machineit can pick up genes from the chromosome, expose them to a high mutation rate, transfer the genes into new hosts, and recombine the new alleles back onto the chromosome. This is a second way, in addition to induction of error-prone polymerases, that stress could increase mutation rates but, in this case, only of genes carried on the F episome. How common are F plasmids in nature? Although long believed to be rare, recent work revealed that 21% of natural isolates of E. coli carry either F or related conjugal plasmids. Significantly, these plasmids showed evidence of extensive recombination as well as recent horizontal transfer among diverse E. coli strains, and even between E. coli and S. typhimurium. Thus, F may play an important and previously unappreciated role in determining bacterial population structure, in horizontal gene transfer, and in the appearance of new genetic variants.

Venn diagram illustrating the role of transient mutators in adaptive mutation

Adaptive Mutations in the JC Virus Protein Capsid Associated with Progressive Multifocal Leukoencephalopathy (PML)
JC virus is a highly prevalent human polyomavirus. Infection with this virus is generally benign and asymptomatic despite viral persistence in the kidney of many people. However, in immunocompromised individuals, very rarely, the infection can progress to become a potentially deadly brain disease called Progressive Multifocal Leukoencephalopathy (PML). The discrepancy between very high viral prevalence and low incidence of PML suggests that there could be some unique viral characteristics that regulate the progression from the asymptomatic infection to the PML. Identification of such factors will help us to understand the basis of PML development and hopefully will lead to the creation of new diagnostic and treatment tools for managing PML. A part of the viral surface protein that is thought to be responsible for viral interaction with cellular receptors and infection acquires specific mutations that appear to be critical for the development of PML. These mutations are found more frequently than by simple chance and therefore are thought to be positively selected. Based on these results, we hypothesize that the specific mutations in the viral VP1 protein that we have identified are critical for the evolution of JC virus to the version associated with PML. Several amino acids on the surface of the JC virus capsid protein VP1 display accelerated evolution in viral sequences isolated from PML patients but not in sequences isolated from healthy subjects. Strong evidence that at least some of these mutations are involved in binding of sialic acid, a known receptor for the JC virus. Using statistical methods of molecular evolution, a comprehensive analysis of JC virus VP1 sequences isolated from 55 PML patients and 253 sequences isolated from the urine of healthy individuals and found that a subset of amino acids found exclusively among PML VP1 sequences is acquired via adaptive evolution. By modeling of the 3-D structure of the JC virus capsid, its seen that these residues are located within the sialic acid binding site, a JC virus receptor for cell infection. Finally, it can be demonstrate the involvement of some of these sites in receptor binding by demonstrating a profound reduction in hemagglutination properties of viral-like particles made of the VP1 protein

carrying these mutations. Collectively, these results suggest that a more virulent PML causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).

Enhancement of Hepatitis C Virus RNA Replication by Adaptive Mutations

The Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases. Acute infections are usually subclinical or associated with mild symptoms, but the virus persists in more than 70% of infected individuals. The long-term outcomes of these persistent infections are varied, and they can range from an apparently healthy carrier state to chronic active hepatitis, liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication. These viruses have enveloped particles that harbor an RNA genome of positive polarity. The HCV genome has a length of about 9,600

nucleotides, and it carries a single long open reading frame (ORF) that is flanked at both termini by nontranslated regions (NTRs). Viral proteins are generated as a polyprotein precursor that is translated via the internal ribosome entry site (IRES) located in the 5 NTR.It permits the direct binding of the 40S ribosome subunit in the absence of additional translation factors . The polyprotein precursor is cleaved by viral and host cell enzymes into at least 10 different products. The structural proteins that are located in the amino-terminal region of the polyprotein are the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are separated from the structural proteins by the short hydrophobic polypeptide p7, which has an unknown function . The development of selectable subgenomic HCV replicons for the first time enabled the study of viral RNA replication in cell culture .These replicons are composed of the 5 NTR, which directs translation of the gene encoding the neomycin phosphotransferase; the IRES of the Encephalomyocarditis virus (EMCV), which directs translation of the HCV NS3NS5B region; and the 3 NTR. Upon transfection of cells of the human hepatoma cell line Huh-7 and selection with G418, cell lines that carried high levels of selfreplicating HCV RNAs could be established. Subsequent analyses of the sequences of HCV RNAs isolated from selected cell lines revealed that these replicons harbored cell culture-adaptive mutations. For instance, a single amino acid substitution in the NS5B RdRp increased the efficiency of colony formation (ECF) 500-fold compared with that of the unaltered replicon RNA. However, the mechanism by which this cell culture adaptation was achieved remained unknown. In this study we show that cell culture-adaptive mutations enhance RNA replication. Using a transient-transfection assay without subsequent selection of cells, we demonstrate a clear correlation between the level of cell culture adaptation and RNA replication. However, within selected cell lines that were obtained after transfection of replicons with different levels of adaptation, the differences in the replicon copy numbers per cell varied only slightly. These results suggest that the host cell plays an additional important role in determining the replication level.

Adaptive Mutations in Sindbis Virus E2 and Ross River Virus E1 That Allow Efficient Budding of Chimeric Viruses
Alphavirus glycoproteins E2 and E1 form a heterodimer that is required for virus assembly. It has been studied that adaptive mutations in E2 of Sindbis virus (SIN) and E1 of Ross River virus (RR) that allow these two glycoproteins to interact more efficiently in a chimeric virus that has SIN E2 but RR E1. These mutations include K129E, K131E, and V237F in SIN E2 and S310F and C433R in RR E1. Although RR E1 and SIN E2 will form a chimeric heterodimer, the chimeric virus is almost nonviable, producing about 107 as much virus as SIN at 24 h and 105 as much after 48 h. Chimeras containing one adaptive change produced 3 to 20 times more virus than did the parental chimera, whereas chimeras with two changes produced 10 to 100 times more virus and chimeras containing three mutations produced yields that were 180 to 250 times better. None of the mutations had significant effects upon the parental wild-type viruses, however. Passage of the triple variants eight or nine times resulted in variants that produced virus rapidly and were capable of producing >108 PFU/ml of culture fluid within 24 h. These further-adapted variants possessed one or two additional mutations, including E2-V116K, E2-S110N, or E1-T65S. The RR E1-C433R mutation was studied in more detail. This Cys is located in the putative transmembrane domain of E1 and was shown to be palmitoylated. Mutation to Arg-433 resulted in loss of palmitoylation of E1. The positively charged arginine residue within the putative transmembrane domain of E1 would be expected to alter the conformation of this domain. These results suggest that interactions within the transmembrane region are important for the assembly of the E1/E2 heterodimer, as are regions of the ectodomains possibly identified by the locations of adaptive mutations in these regions. Further, the finding that four or five changes in the chimera allow virus production that approaches the levels seen with the parental SIN and exceeds that of the parental RR illustrates that the structure and function of SIN and RR E1s have been conserved during the 50% divergence in sequence that has occurred.

CONCLUSION
Comparative biochemistry demonstrates that the metabolites, complex biochemical networks, enzymes and regulatory mechanisms essential to all living cells are conserved in amazing detail throughout evolution. Thus, in order to evolve, an organism must overcome new adverse conditions without creating different but equally dangerous alterations in its ongoing successful metabolic relationship with its environment. Evidence suggests that stable long-term acquisitive evolution results from minor increases in mutation rates of genes related to a particular stress, with minimal disturbance to the balanced and resilient metabolism critical for responding to an unpredictable environment. Microorganisms have evolved specific biochemical feedback mechanisms that direct mutations to genes derepressed by starvation or other stressors in their environment. Transcription of the activated genes creates localized supercoiling and DNA secondary structures with unpaired bases vulnerable to mutation. The resulting mutants provide appropriate variants for selection by the stress involved, thus accelerating evolution with minimal random damage to the genome. This model has successfully predicted mutation frequencies in genes of E. coli and humans. Stressed cells observed in the laboratory over hundreds of generations accumulate mutations that also arise by this mechanism. When this occurs in repair-deficient mutator strains with high rates of random mutation, the specific stress-directed mutations are also enhanced. This stress-directed mutations are ADAPTIVE MUTATION.

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