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Genetic Variation and Molecular Evolution

Werner Arber Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, Switzerland

1 Introduction 3

2 Principles of Molecular Evolution 4 2.1 Evolutionary Roles of Genetic Variation, Natural Selection, and Isolation 4 2.2 Molecular Mechanisms of the Generation of Genetic Variation 6

3 Genetic Variation in Bacteria 7

4 Local Changes in the DNA Sequences 8

5 Intragenomic DNA Rearrangements 9 5.1 Site-specific DNA Inversion at Secondary Crossover Sites 10 5.2 Transposition of Mobile Genetic Elements 11

6 DNA Acquisition 12

7 The Three Natural Strategies Generating Genetic Variations Contribute Differently to the Evolutionary Process 13

8 Evolution Genes and Their Own Second-order Selection 15

9 Arguments for a General Relevance of the Theory of Molecular Evolution for All Living Organisms 16

10 Conceptual Aspects of the Theory of Molecular Evolution 17 10.1 Pertinent Scientific Questions 17 2 Genetic Variation and Molecular Evolution

10.2 Philosophical Values of the Knowledge on Molecular Evolution 18 10.3 Aspects Relating to Practical Applications of Scientific Knowledge on Molecular Evolution 20

Bibliography 21 Books and Reviews 21 Primary Literature 21

Keywords

Biological Evolution A nondirected, dynamic process of diversification resulting from the steady interplay between spontaneous mutagenesis and natural selection.

DNA Rearrangement Results from mostly enzyme-mediated recombination processes, which can be intra- or intermolecular.

Evolution Gene Itsproteinproductactsasageneratorofgenetic variations and/or as a modulator of the frequency of genetic variations.

Gene Acquisition Results from horizontal transfer of genetic information from a donor cell to a receptor cell. With bacteria, this can occur in transformation, conjugation, or phage-mediated transduction.

Natural Selection Results from the capacity of living organisms to cope with the encountered physicochemical and biological environments. Largely depending on its genetic setup and physiological phenotype, each organism may have either a selective advantage or a selective disadvantage as compared to the other organisms present in the same ecosystem.

Spontaneous Mutation Defined here as any alteration of nucleotide sequences occurring to DNA without the intended intervention of an investigator. The term mutation is used here as a synonym of genetic variation.

Transposition DNA rearrangement mediated by a mobile genetic element such as a bacterial insertion sequence (IS) element or a transposon. Genetic Variation and Molecular Evolution 3

Variation Generator An enzyme or enzyme system whose mutagenic activity in the generation of genetic variation has been documented.

The comparison of DNA sequences of genes and entire genomes offers interesting insights into the possible evolutionary relatedness of genetic information of living organisms. Together with a relatively rich database from experimental microbial genetics, conclusions can be drawn on the molecular mechanisms by which genetic variations are spontaneously generated. A number of different specific mechanisms contribute to the overall mutagenesis. These mechanisms are here grouped into three natural strategies of the spontaneous generation of genetic variations: local changes of DNA sequences, intragenomic rearrangement of DNA segments, and acquisition of foreign DNA by horizontal gene transfer. These three strategies have different qualities with regard to their contributions to the evolutionary process. As a general rule, none of the known mechanisms producing genetic variants is clearly directed. Rather, the resulting alterations in the inherited genomes are more random. In addition, usually only a minority of resulting variants provide a selective advantage. Interestingly, in most of the molecular mechanisms involved, the products of so- called evolution genes are involved as generators of genetic variation and/or as modulators of the frequencies of genetic variation. Products of evolution genes work in tight collaboration with nongenetic factors such as structural flexibilities and chemical instabilities of molecules, chemical and physical mutagens, and random encounter. All of these aspects contributing to the spontaneous generation of genetic variations together form the core of the theory of molecular evolution. This theory brings neo-Darwinism to the molecular level. In view of the increasing evidence coming particularly from microbial genetics, knowledge of molecular evolution can be seen as a confirmation of Darwinism at the level of biologically active molecules, in particular, nucleic acids and proteins. Philosophical and practical implications of this knowledge will be briefly discussed.

1 of living organisms. Darwin’s theory of Introduction natural selection brought a new element into the understanding of the long-term Evolutionary biology has traditionally de- development of forms of life. Natural se- voted its major attention to the comparison lection is the result of organisms coping of phenotypical traits of higher organ- with the encountered living conditions that isms, both of those actually living and are dependent on both the environmental of those that have been extinct (paleon- physicochemical conditions and the activ- tological fossils). The resulting theory of ities of all living forms in a particular descent is, together with other criteria, at ecological niche. The Darwinian theory the basis of the systematic classification of evolution also postulated that intrinsic 4 Genetic Variation and Molecular Evolution

properties of life are not entirely stable chemical stability of biologically ac- and principally identical for all organ- tive molecules. isms of a given species. In the so-called modern synthesis, in which evolutionary 2.1 biology and genetics became integrated, Evolutionary Roles of Genetic Variation, transmissible phenotypic variations repre- Natural Selection, and Isolation senting the substrate of natural selection were explained as due to genetic varia- The long-term maintenance of any form tions (or mutations). Shortly thereafter, of life requires a relatively high stability deoxyribonucleic acid (DNA) was identi- of its genetic information. However, rare fied as the carrier of genetic information. occasional genetic variations occur in all DNA is thus also the target for mutagen- organisms. This gives rise to mixed pop- esis. Within the last few decades a rapid ulations of organisms with the parental development of molecular genetics with genome and organisms having one or novel research strategies leading to ge- more alterations in their genome. These nomics, sequencing of entire genomes, populations are steadily submitted to nat- and functional studies of genes and their ural selection. The experience shows that, products paved the way for hitherto in- in general, favorable genetic variations are accessible knowledge on the basis of life considerably less frequent than unfavor- and its multiple manifestations. This also able ones. The latter provide a selective dis- relates to the process of molecular evolu- advantage. Indeed, genetic variants with tion. A synoptical insight into the various unfavorable genetic alterations will sooner molecular mechanisms contributing to the or later get eliminated from propagating generation of genetic variations represents populations, which will become enriched a molecular synthesis between the neo- for organisms carrying favorable genetic Darwinian theory and molecular genetics. variations. It should be noted that by far This synthesis can confirm the Darwinian not all alterations in the nucleotide se- evolution at the molecular level. quences of a genome will lead to a change in the phenotype of the organism. How- ever, such silent and neutral mutations 2 may later become physiologically relevant Principles of Molecular Evolution in conjunction with still other, upcoming DNA sequence changes. The principles of molecular evolution to Natural selection is by no means a be outlined here are founded on constant element. It varies both in time and in space. This is due to variations 1. the neo-Darwinian theory of evolution in the physicochemical environmental with its three pillars of genetic variation, conditions as well as to variations of the natural selection, and isolation; life activities of all the different organisms 2. the solidly established microbial genet- present in a particular ecological niche ics database; and forming an ecosystem. Since a genetic 3. DNA sequence comparisons with bioin- variation may also affect the influence formatic tools; that the organism exerts on the other 4. physicochemical knowledge on the re- organisms present in the same ecosystem activity, conformational flexibility, and (e.g. think of weeds and pathogenicity Genetic Variation and Molecular Evolution 5 effects, but also of beneficial, synergistic other type of isolation is called reproductive effects), any novel mutation may not isolation. For example, two distantly re- only influence the life of the concerned lated diploid organisms may not be fertile organism itself but also the lives of other in sexual reproduction. But reproductive cohabitants of the same ecological niche. isolation can also be seen in a wider def- The third pillar of biological evolu- inition to seriously limit the possibility of tion – besides genetic variation and natural horizontal transfer of segments of genetic selection – is isolation. Evolutionary biolo- information between two different kinds gists define two different aspects of isola- of organisms. tion. One of these is geographic isolation, As summarized in Fig. 1, genetic vari- which may seriously reduce the number ation drives biological evolution. Com- of potential habitats for an organism. The plete genetic stability would render any

Sources of Modulates Limitation of genetic diversity biological evolution genetic diversity

Geographic Isolation Reproductive

Organisms Ecosystems

Mutation Natural selection Genetic variation

Drives Directs biological evolution biological evolution

Strategies Mechanisms Physico chemical environment Replication Biological environment Local change of infidelities DNA sequence Carrier capacity of biosphere Mutagens - internal DNA - environmental rearrangement Recombinational reshuffling of genomic DNA segments

DNA Horizontal acquisition gene transfer Fig. 1 Synoptical presentation of major elements of the theory of molecular evolution. A number of specific mechanisms, each with its own characteristics, contribute to the four groups of mechanisms of genetic variation listed. Each of the specific mechanisms follows one and sometimes more than one of the three principal, qualitatively different strategies of genetic variation. 6 Genetic Variation and Molecular Evolution

evolutionary process impossible. A very In contrast, it has become a habit in high frequency of genetic variation would molecular genetics to call any alteration rapidly lead to the extinction of the con- in the parental nucleotide sequence of the cerned organisms because of the stated genome a mutation, whether it has pheno- prevalence of unfavorable mutations in the typic consequences or not. There is good spontaneous generation of genetic varia- reason to assume that in most sponta- tions. It is natural selection together with neously occurring mutagenesis events, the the available sets of genetic variants that specific mechanism involved will not pay determines the direction of biological evo- attention to whether the sequence alter- lution, or in other words, the directions ation at the involved target site will cause in which the branches of the tree of evo- a phenotypic alteration or not. Therefore, lution grow. Finally, the geographic and for studies on mechanisms and on the reproductive isolations modulate the evo- statistics of their occurrence, it is indicated lutionary process. to follow the molecular genetics definition of the term mutation, which we use here 2.2 as a synonym of genetic variation. We will Molecular Mechanisms of the Generation use the term spontaneous mutation to label of Genetic Variation any type of DNA sequence alteration unin- tended by the investigator. This definition The concept to be presented here re- says nothing about whether a mutation quires the reader to question some long- relates to a phenotypic change. established textbook knowledge, such as At present, the research on genetic vari- the claim that spontaneous mutations ation mainly follows two strategies. First, would largely result from errors, accidents, the increasing availability of entire and and illegitimate processes. We defend here partial genome sequences offers excellent the alternative view that living nature ac- means to compare regulatory sequence tively cares for biological evolution elements, specific domains of genes, en- tire genes, groups of genes, and entire 1. by making use of intrinsic properties genomes with regard to DNA sequence of matter such as a certain degree of homologies and genome organization. chemical instability and of structural Within a given species, this can reveal flexibility of molecules, and a genetic polymorphism. Between more or 2. by having developed genetically en- less related species, it can give a reliable coded systems, the products of which measure for evolutionary relatedness. For are involved in the generation of ge- example, the molecular clock – an indica- netic variations and in modulating the tor of evolutionary relatedness – is based frequencies of genetic variation. on single nucleotide alterations. Sequence comparisons can often suggest how se- It might be relevant to mention here that quence alterations could have occurred in the term mutation is differently defined in the course of past evolution. Secondly, classical genetics and in molecular genet- a more reliable insight into the genera- ics. In classical genetics, a mutant is any tion of genetic variations can be gained variant of a parental form, showing in by the observation of individual processes its phenotypic properties some alteration of mutagenesis. In view of the large size that becomes transmitted to the progeny. of genomes and of the rare and random Genetic Variation and Molecular Evolution 7 occurrence of spontaneous DNA sequence Selected examples for each of these alterations, this approach is relatively dif- strategies will be discussed in Sects. 4, 5, ficult. However, a rich database is already and 6. available from microbial genetics, particu- larly from bacteria and their viruses and plasmids. Their relatively small genomes 3 are haploid so that phenotypic effects Genetic Variation in Bacteria caused by genetic variation normally be- come rapidly manifested. With appropriate Several seminal discoveries, largely based selection and screening techniques, this on work carried out with microorganisms allows one to identify occasional, function- between 1943 and 1953, were essential ally relevant mutations in populations. On for the later development of molecu- the other hand, investigations on structural lar genetics. alterations in the genomes of individual bacterial colonies, for example, by the 1. It was realized that bacteria and bacte- study of restriction fragment length poly- riophages have genes that can mutate, morphism, can reveal when and where on and that spontaneous mutations in the genome a DNA rearrangement must microorganisms normally arise inde- have occurred. pendently of the presence of selective A quite solid, general result of this type of agents. It was also learned that the experimental investigations reveals that in genetic information of bacteria and of the spontaneous generation of genetic vari- some bacteriophages is carried in DNA ations, it is not just a single mechanism at molecules rather than in other biologi- work. Rather, a number of mechanistically cal macromolecules such as proteins. different processes contribute to the over- 2. The newly discovered phenomena of all mutagenesis. We will discuss selected DNA transformation, bacterial con- examples below. Interestingly, a critical jugation, and bacteriophage-mediated evaluation of the situation shows that the transduction revealed natural means of specific mechanisms of mutagenesis of- horizontal gene transfer between differ- ten depend both on nongenetic elements ent bacterial cells. and on activities of specific enzymes, the 3. It was seen that horizontal gene trans- products of the so-called evolution genes. fer has natural limits, including sys- The multitude of thus identified, distinct tems of host-controlled modification, mechanisms contributing to the forma- which are today known as DNA restric- tion of genetic variations can be grouped tion–modification systems. into three qualitatively different natural 4. Mobile genetic elements were identi- strategies (Fig. 1). These are fied as sources of genetic instability and were seen to represent media- tors of genetic rearrangements. While 1. local changes in the DNA sequences, such rearrangements are often caused 2. rearrangement of DNA segments by transposition, they can also result within the genome, from the integration of a bacteriophage 3. the acquisition by horizontal transfer of genome into the genome of its bac- a DNA segment originating in another terial host strain, which is thereby kind of organism. rendered lysogenic. 8 Genetic Variation and Molecular Evolution

It is at the end of this fruitful period of the occurrence of gene expression at the discoveries, in 1953, that structural anal- relevant time with the needed efficiency. ysis of DNA molecules led to the double- Mutations can affect reading frames as helix model. The filamentous structure of well as control signals, both of which DNA molecules made it clear as to how represent specific DNA sequences. In genetic information could be contained in addition, some specific DNA sequences the linear sequences of nucleotides. The relate to the control of the metabolism of double helical nature of the model also the DNA molecules, in particular, their offered an understanding of semiconser- replication. vative DNA replication at the molecular In the following Sects. 4, 5, and 6, we will level and thus of information transfer describe selected examples of mechanisms into progeny. contributing each in its specific manner to Many classical microbial genetic inves- the formation of genetic variants. We will tigations were carried out with Escherichia group them into the already mentioned coli K-12. Its genome is a single circu- three major natural strategies (Fig. 1), lar DNA molecule (chromosome) of about although, as we will later see, some of the 4.6 × 106 bp. In periods of growth, the rate − specific mechanisms involve more than of spontaneous mutagenesis is about 10 9 one of these strategies. per bp and per generation. This represents one new mutation in every few hundred cells in each generation. E. coli has several 4 well-studied bacteriophages and plasmids. Local Changes in the DNA Sequences This material facilitates investigations on life processes in these bacteria. The process of DNA replication is one Under good growth conditions, the gen- of the important sources of genetic eration time of E. coli, measured between variation, which may depend both on one cell division and the next, is very short, structural features of the substrate DNA on the order of 30 min. Upon exponential and on functional characteristics of the growth, this leads to a multiplication fac- tor of 1000 every 5 h. Thus, a population replication fork. of 109 cells representing 30 generations Some of the ‘‘infidelities’’ of DNA repli- is reached from an inoculum of a single cation are likely to depend on tautomeric cell in only 15 h. This rapid growth rate forms of nucleotides, that is, a structural greatly facilitates population genetic stud- flexibility inherent in these organic com- ies and thus facilitates investigations on pounds. Base pairing depends on specific the evolutionary process. structural forms. Conformational changes On the filiform DNA molecules of E. coli of nucleotides can result in a mispairing and its bacteriophages and plasmids, the if short-living, unstable tautomeric forms genetic information is relatively densely are ‘‘correctly’’ used in the synthesis of stored as linear sequences of nucleotides the new complementary strand upon DNA or base pairs. Genes depend on the replication. For this reason, we do not con- presence of continuous sequences of base sider mutations resulting in this process pairs (reading frames) that encode specific as errors and call them infidelities.DNA gene products, usually proteins, and of replication is indeed one of the sources of expression control signals that ensure nucleotide substitution, and this plays an Genetic Variation and Molecular Evolution 9 important role in the evolutionary devel- later become retrotranscribed into DNA. opment of biological functions. No efficient repair systems are known An inherent low degree of chemical in- toactatthelevelofRNA.Indeed, stability of nucleotides represents another RNA replication shows a higher degree source of mutagenesis. For example, cyto- of infidelity than DNA replication. In sine can undergo oxidative deamination to consequence, genetic information that become uracil. Upon replication, this gives becomes replicated as RNA molecules rise to an altered base pairing and results generally suffers increased mutation rates. also in nucleotide substitution. A relatively large number of internal and Other replication infidelities that may environmental chemicals exert mutagenic relate to slippage in the replication fork effects by means of molecular mechanisms can result in either the deletion or the that in many cases are well understood and insertion of one or a few nucleotides in the often cause local DNA sequence changes. newly synthesized DNA strand. If such Some intermediate products of the nor- mutations occur within reading frames mal metabolic activities of a cell may be for protein synthesis, the phenotypic effect mutagenic and may thus contribute to may be drastic. This is the case when, spontaneous local mutagenesis. The muta- in the protein synthesized from a gene gens of the environment include not only affected by a frameshift mutation, the a multitude of chemical compounds but amino acid sequence downstream of the also ultraviolet radiation and some physic- site of mutation strongly differs from that ochemical constraints such as elevated of the nonmutated product. In addition, temperature, which influence the chem- the size of such a mutated protein is ical stability of nucleotides. Each of these usually altered depending on the chance mutagens and mutagenic conditions con- occurrence of an appropriate stop codon tributes in a specific way to the generation in the new reading frame. of genetic variations. Proofreading devices and other enzy- Again, many of the potential sequence matic repair systems prevent replication alterations brought about by internal and infidelities from producing mutations at environmental mutagens are efficiently re- intolerably high rates. Generally, they act paired by enzymatic systems. However, rapidly after replication by screening for since the efficiency of such repair is imperfect base pairing in the double helix. rarely 100%, evolutionarily relevant mu- Successful repair thereby requires specific tations persist. means to distinguish the newly replicated DNA strand from its template, the comple- mentary parental strand. Because of these 5 correction activities, many primary mis- Intragenomic DNA Rearrangements pairings are removed before they have the opportunity to become fixed as mutations. Various recombination processes are well DNA repair systems modulate the frequen- known to mediate DNA rearrangements, cies of mutagenesis. which often result in new nucleotide Genetic information of some viruses, sequences. and sometimes also segments of genetic While in haploid organisms general information of chromosomal origin, may recombination is not essential for prop- pass through RNA molecules, which may agation, it influences genetic stability at 10 Genetic Variation and Molecular Evolution

the population level in various ways as are turned on or off at the same time. a generator of new sequence varieties. Therefore, in an operon fusion, one or For example, it can bring about sequence more genes are put under a different duplications and deletions by acting at transcription control, but the genes per segments of homology that are carried se remain unchanged. In contrast, gene at different locations in a genome. Gen- fusion results in a hybrid gene composed eral recombination is also known to act in of sequence motifs and often of functional the reparation of damage caused to DNA domains originating in different genes. by ionizing radiation. In this case, an in- In site-specific DNA inversion, a DNA tact genome can become assembled from segment bordered by specific DNA se- undamaged segments of sister copies of quences acting as sites of crossing-over the chromosome by homologous recom- becomes periodically inverted by the action bination. The best-known contribution of of the enzyme DNA invertase. Depending general recombination to genetic diversity on the location of the crossover sites, DNA is meiotic recombination bringing about inversion can give rise to gene fusion or random recombinants between the pater- to operon fusion. The underlying flip-flop nal and the maternal chromosomes in system can result in microbial populations diploid organisms. composed of organisms with different phe- Two other widely spread types of recom- notypic appearances: if, for example, the bination systems are dealt with in more DNA inversion affects the specificity of detail in Sects. 5.1 and 5.2: site-specific re- phage tail fibers, as is the case with phages combination and transposition. Both are P1 and µ of E. coli, phage populations with known to contribute to genetic variation. two different host ranges will result. Still other recombination processes, such Occasionally, a DNA sequence that de- as the one mediated by DNA gyrase can, for viates considerably from the efficiently the time being, perhaps best be grouped used crossover site, a so-called secondary as illegitimate recombinations. This group crossover site, can serve in DNA inversion, may contain several different molecular whichthusinvolvesanormalcrossoversite mechanisms that act with very low effi- and a secondary crossover site. This pro- ciency and have remained at least in part cess results in novel DNA arrangements, unexplained. many of which may not be maintained be- cause of lethal consequences or reduced 5.1 fitness; but if a few new sequences are Site-specific DNA Inversion at Secondary beneficial to the life of the organism, these Crossover Sites may be selectively favored. This DNA re- arrangement activity can thus be looked at Genetic fusions represent the results of as evolutionarily important. Since many joining together segments of two genes different DNA sequences can serve in (gene fusions) or of two operons (operon this process as secondary crossover sites, fusions) that are not normally together. An although at quite low frequencies, site- operon is a set of often functionally related specific DNA inversion systems act as genes that are copied into messenger RNA variation generators in large populations (i.e. transcribed) as a single unit. As a of microorganisms. I have thus postulated result of this organization, those genes that this evolutionary role of DNA inver- are coordinately regulated; that is, they sion systems may be more important than Genetic Variation and Molecular Evolution 11 their much more efficient flip-flop mecha- the IS-mediated DNA rearrangements are nism, which can at most help a microbial neither strictly reproducible nor fully population to more readily adapt to two random. Transposition activities thus also different, frequently encountered environ- act as variation generators. In addition mental conditions. As a matter of fact, to DNA rearrangements mediated by the other strategies could be used as well for enzyme transposase, which is usually the latter purpose. encoded by the mobile DNA element Computer-aided comparison of DNA itself, other DNA rearrangements just take sequences quite often reveals that inde- advantage of extended segments of DNA pendent genes may consist of a particular homologies at the sites of residence of domain with high homology and of other identical IS elements at which general DNA sequences showing no significant recombination can act. Altogether, IS signs of relatedness. DNA inversion us- elements represent a major source of ing secondary sites of crossing-over is a genetic plasticity of microorganisms. potential source of such mosaic genes. Transposition occurs not only in grow- DNA inversion can span over relatively ing populations of bacteria but also in large distances in DNA molecules and has prolonged phases of rest. This is readily the advantage of not loosing any DNA se- seen with bacterial cultures stored at room quences located between the two sites of temperature in stabs (little vials containing crossing-over. Deletion formation repre- a small volume of growth medium in agar). sents another source for gene fusion, but Stabs are inoculated with a drop of a bacte- it has the disadvantage that the DNA se- rial culture taken up with a platinum loop, quences between the sites of crossing-over which is inserted (‘‘stabbed’’) from the top are usually eliminated. to the bottom of the agar. After overnight incubation, the stab is tightly sealed and 5.2 stored at room temperature. Most strains Transposition of Mobile Genetic Elements of E. coli areviableinstabsduringsev- Nine different mobile genetic elements eral decades of storage. That IS elements have been found to reside, often in several exert transpositional activities under these copies, in the chromosome of E. coli K-12 storage conditions is easily seen as follows. derivatives. This adds up to occupation A stab can be opened at any time, a small of about 1% of the chromosomal length portion of the bacterial culture removed, by such insertion sequences, also called and the bacteria well suspended in liquid IS elements. At rates on the order of medium. After appropriate dilution, bacte- − 10 6 per individual IS element and ria are spread on solid medium. Individual per cell generation, these mobile genetic coloniesgrownuponovernightincuba- elements undergo transpositional DNA tion are then isolated. DNA from such rearrangements. These include simple subclones is extracted and fragmented transposition of an element and more with a restriction enzyme. The DNA frag- complex DNA rearrangements such as ments are separated by gel electrophoresis. DNA inversion, deletion formation, and Southern hybridization with appropri- the cointegration of two DNA molecules. ate hybridization probes can then show Because of different degrees of specificity whether different subclones reveal re- in the target selection upon transposition, striction fragment length polymorphisms 12 Genetic Variation and Molecular Evolution

(RFLPs), which are indicative of the occur- as a plasmid. In these experiments, IS2 rence of mutations during storage. was the most active element and it mainly If this method is applied to subclones inserted into a few preferred regions of the isolated from old stab cultures, and P1 genome, but each time at another site. if DNA sequences from residential IS The used insertion targets did not show elements serve as hybridization probes, any detectable homology or similarity an extensive polymorphism is revealed. with each other. This is another good None or only little polymorphism is seen example for an enzymatically mediated with hybridization probes from unique variation generator. The experiment as chromosomal genes. Good evidence is such identifies IS transposition as a major available that transposition represents a source of lethal mutagenesis. major source of this genomic plasticity There is no evidence available that bacte- observed in stabs, which at most allow rial mobile genetic elements would play an for a very residual growth at the expense essential role in the bacterial life span ex- of dead cells. One can conclude that tending from one cell division to the next. the enzymes promoting transposition are However, these elements are major players steadily present in the stored stabs. Indeed, in the evolution of bacterial populations. the IS-related polymorphism increases As we have seen here, they contribute to linearly with time of storage for periods as intragenomic DNA rearrangements. De- long as 30 years. In a culture of E. coli strain pending on the target sequences involved, W3110 analyzed after 30 years of storage, the resulting mutations may often be lethal each surviving subclone had suffered on by interrupting essential reading frames or the average about a dozen RFLP changes as expression control regions. Favorable mu- identified with hybridization probes from tations may be relatively rare, but these can eight different residential IS elements, of contribute to evolutionarily advantageous which IS5 was the most active. Lethal developments of the genome. In Sect. 6, mutations could of course not be identified we will see that mobile genetic elements in this study. also play important roles in the natural Lethal mutations that affect essential strategy of DNA acquisition. genes for bacteriophage reproduction can be accumulated in the prophage state of the phage genome in its lysogenic 6 host. Such mutants can be screened DNA Acquisition for their inability to produce infective phage particles upon induction of phage While the mutagenesis mechanisms be- reproduction. Experiments were carried longing to the strategies of local changes out with E. coli lysogenic for a phage in the DNA sequences (Sect. 4) and of in- P1 derivative grown in batch cultures at tragenomic DNA rearrangements (Sect. 5) ◦ 30 C for about 100 generations allowing are exerted within the genome and can for alternative periods of growth and rest. affect any part of the genome, an addi- Most of the independent lethal mutants tional strategy of spontaneous sequence could thereby be identified to be caused alterations depends on an external source by the transposition of an IS element of genetic information. In DNA or gene from the host chromosome into the P1 acquisition, genetic information indeed prophage that is maintained in its host originates from an organism other than Genetic Variation and Molecular Evolution 13 the one undergoing mutagenesis. In bac- keep the rate of DNA acquisition low, and teria, DNA acquisition can occur by means at the same time they stimulate the fixation of transformation, conjugation, or virus- of relatively small segments of acquired mediated transduction. In the latter two DNA to the receptor genome. This strat- strategies of horizontal gene transfer, a egy of acquisition in small steps can best plasmid or a virus, respectively, acts as offer microbial populations the chance to natural gene vector. occasionally extend their biological capaci- The association and dissociation of chro- ties without extensive risk of disturbing the mosomal genes with natural gene vectors functional harmony of the receptor cell by often arises from transpositional activities acquiring too many different functions at and from general recombination acting at once. These considerations have their rel- IS elements that are at different chromo- evance at the level of selection exerted on somal locations. These mechanisms have the hybrids resulting from horizontal DNA been well studied with conjugative plas- transfer. This selection represents one of mids and with bacteriophage genomes the last steps in the acquisition process. serving in specialized transduction. For DNA acquisition by horizontal gene example, composite transposons, which transfer is a particularly interesting source are defined as two identical IS elements of new genetic information for the receptor flanking a segment of genomic DNA (of- bacterium because the chance that the ten with more than one gene unrelated acquired DNA exerts useful biological to the transposition process), are known functionsisquitehigh–mostlikely,ithas to occasionally transpose into a natural already assumed the same functions in the gene vector and, after their transfer into a donor bacterium. receptor cell, to transpose again into the re- An interesting hypothesis links the ceptor chromosome. Hence, together with universality of the genetic code with the other mechanisms, such as site-specific important role played by horizontal gene and general recombination, transposition transfer in the evolutionary development represents an important promoter of hori- of the living world. According to this zontal gene transfer. view, those organisms using the most Several natural factors seriously limit common genetic language would, in the gene acquisition. Transformation, con- long term, be able to profit best from jugation, and transduction depend on the increasing worldwide pool of genetic surface compatibilities of the bacteria in- functions under the pressure of adapting volved. Furthermore, upon penetration of to changing living conditions. donor DNA into receptor cells, the DNA is very often confronted with restriction endonucleases. These enzymes cause a 7 fragmentation of the invading foreign The Three Natural Strategies Generating DNA, which is subsequently completely Genetic Variations Contribute Differently to degraded. Before fragments become de- the Evolutionary Process graded, however, they are recombinogenic and may find a chance to incorporate all or Biological evolution is a systemic process. part of their genetic information into the As outlined above, many different specific host genome. Therefore, we interpret the mechanisms contribute to generate ge- role of restriction systems as follows: they netic diversity that represents at any time 14 Genetic Variation and Molecular Evolution

the substrate for natural selection. The also be expected to bring about a novel building up of functional complexity is a biological function. However, this kind of stepwise process, in which many random long-term process can gain efficiency only attempts of genetic alterations become once natural selection starts to be exerted rapidly rejected, while relatively few novel on such upcoming function. sequences are approved as favorable by In contrast, the reshuffling of DNA natural selection and are maintained and segments within the genome can be amplified. The genome can thus be seen as considered as a tinkering with existing a cabinet in which key information from fa- elements, whereby favorable gene fusions vorable historical developments is stored. and operon fusions may occasionally Stepwise, additional favorable information result. DNA rearrangement can also be is added. In the context of changing selec- thesourceofgeneduplicationandhigher tive conditions, stored information, having amplification, which are widely recognized lost its functionally beneficial relevance, contributions to the evolutionary progress. may be deleted or favorably altered. As we In Sect. 6, we have already pointed to the have seen, a multitude of different mech- evolutionarily high efficiency of horizontal anisms are behind this dynamic process. gene transfer. As a matter of fact, DNA For a better understanding of the events, acquisition allows the recipient organism we have grouped the identified mecha- to share in the success of evolutionary de- nisms into three major natural strategies velopments made by others. In drawing of genetic variation: local changes in the the evolutionary tree of bacteria, DNA ac- DNA sequences, intragenomic DNA rear- quisition should be accounted for by more rangements, and DNA acquisition. These or less randomly adding temporal horizon- three strategies have different qualities tal shunts between individual branches. It with regard to their contributions to bi- must be kept in mind that usually only ological evolution. small DNA segments flow through such ThelocalDNAsequencechangeis shunts in horizontal gene transfer. probably the most frequently involved Several of the specific mechanisms of strategy of genetic variation. Indeed, its genetic variation employ, strictly speak- frequency, which depends primarily on ing, more than one of the three strategies intrinsic properties of matter, chemical shown in Fig. 1. In transposition of IS instability and conformational flexibility, elements, for example, a chromosomal would be intolerably high if it would not be DNA segment consisting of the mobile modulated by efficient enzymatic systems genetic element can undergo a translo- of DNA repair. Local sequence changes cation and thereby become inserted at bring about nucleotide substitution, the a new target site. As a rule, the target deletion and the insertion of one or a few sequence thereby gets duplicated, which base pairs, or a local scrambling of a few usually involves a few nucleotides. Thus, base pairs. These sequence changes can this transposition event will consist of both contribute to a stepwise improvement of a DNA rearrangement and a local sequence a biological function. It must be kept in change. mind that the functional test for such As far as we know, most of the well- improvement is carried out by natural studied microbial strains use in parallel selection. In principle, a long series of each of the three natural strategies for stepwise local sequence changes could the generation of genetic variations. In Genetic Variation and Molecular Evolution 15 addition, bacteria very often use not only factors, specific products of genes are very one, but several different specific mech- often at work. These gene products can anisms for mutagenesis by each of the belong to systems for repair of DNA dam- strategies. Dissimilar specific mechanisms age and will, in this case, modulate the often work with different efficiencies as frequency of mutagenesis. Similarly, re- reflected by their contribution to the over- striction enzymes seriously reduce both all mutation rate. For any given strategy, the chance of DNA acquisition and the it might be less relevant which specific size of a DNA segment that may even- mechanism is at work than the fact that tually be acquired by the recipient cell. the particular strategy finds its application Other gene products such as transposases with an evolutionarily useful efficiency. and other mediators of DNA recombina- In other words, specific mechanisms may tion act as generators of genetic variations. substitute for each other within a strategy, Since variation generators and modula- at least to some degree. This rule does tors of the frequencies of genetic variation not apply between the strategies because are evolutionary functions, we call the of the difference in the qualities of their underlying genetic information evolution evolutionary contributions. genes. In the microbial world, these evolu- The efficiency displayed by a given tion genes generally play no essential role specific mechanism of spontaneous mu- in the physiology of individual lives go- tagenesis may depend on both internal ing by cell division from one generation (e.g. availability of enzymes that medi- to the next. Under laboratory conditions, ate mutagenesis) and external factors (e.g. neither restriction–modification systems environmental stress). It is also to be nor enzymes for DNA rearrangements are noted that some mechanisms may act needed for the propagation of bacteria. The more or less randomly along a DNA role of such enzyme systems is primarily molecule, while other mechanisms may evolutionary and becomes manifest at the show regional or site preferences for their level of populations. activities. In view of these considerations, We assume that evolutionary genes are we tend to assume that an evolutionar- themselves submitted to selective pres- ily fit (or well prepared) organism should sure. However, such selection cannot best be able to use a few specific muta- follow the rules of direct selection for genesis mechanisms for each of the three improvements of essential functions such strategies to generate genetic variations. In as those of housekeeping genes. Rather, Sect. 8, we will explain what we mean by the selection for the presence and im- evolutionary fitness. provement of a variation generator will be exerted at the level of populations. Clearly, it will also be an individual that may one 8 day undergo a mutation, which improves Evolution Genes and Their Own an evolutionary function. This function Second-order Selection will also be exerted in its progeny in which appropriate genetic variants of genes for di- The attentive reader will have seen in the rectly selected products will be either more description of some specific mechanisms or less abundant. Any genetic alteration contributing to the spontaneous mutagen- that affects an evolution gene and proves in esis that besides a number of nongenetic thelongtermtobeofhigherevolutionary 16 Genetic Variation and Molecular Evolution

value will be maintained and will provide a nucleotide). In many cases, nongenetic an evolutionary benefit to the carrier of factors and products of evolution genes co- the involved gene. In the long run, this operate in the formation of genetic variants will lead to fine-tuning of the evolutionary at physiologically tolerable and evolution- functions of both variation generators and arily beneficial levels. This is, for example, of modulators of the frequency of genetic thecaseinspontaneousmutagenesisbyan variation. The underlying indirect selec- environmental mutagen when some of the tion based on the cells ability to provide primary damage on the DNA gets success- genetic variants at a well-balanced level is fully repaired while some other damage called second-order selection. leads to a fixed mutation. We must be aware that some gene The theory of evolution postulates that products may exert their essential func- life on Earth started almost four billion tions for the benefit of both the life years ago with primitive, unicellular mi- of the individual and the evolutionary croorganisms. It is in the first two billion progress of the population. In these cases, years that microbes must have developed we assume that evolutionary selection the basis for the actual setup of evolution- is exerted for both kinds of functions ary strategies and the underlying evolution and will eventually bring the gene to a genes. One can postulate that the acquired fine-tuned state to optionally carry out evolutionary capacities could have allowed its functions for each of the different some microbial populations to undergo a purposes. However, as we have already division of labor in more and more sta- mentioned, a number of gene products in- ble associations of cells. This development volved in genetic variation are inessential might later have led to multicellular or- for the lives of individual bacterial cells. ganisms. In this kind of development, Similarly, the products of many house- the evolutionary fitness of the involved keeping genes are inessential for biological organisms might have been an impor- evolution. tant precondition. At still later stages of further evolutionary development, the three natural strategies for generating ge- 9 netic variations (Sects. 4 to 7) must have Arguments for a General Relevance of the continued exerting their evolutionary in- Theory of Molecular Evolution for All Living fluence together with some other factors Organisms such as the formation of symbiotic asso- ciations. As a matter of fact, we attach Largely on the basis of evidence from mi- considerable evolutionary relevance to en- crobial genetics, we have so far postulated dosymbiosis of higher organisms with that the products of a number of evolu- bacteria. Such situations of cohabitation tion genes contribute, each in its specific may form an ideal condition for occasional way, to the generation of genetic variants at horizontal gene transfer between the close evolutionarily useful frequencies. Thereby, associates. the sources of mutagenesis may relate ei- A scientifically justified quest for fur- ther to the activity of the evolution gene ther experimental proof of the postulates product itself (e.g. a transposase) or to a of the theory of molecular evolution re- nongenetic factor (e.g. a chemical muta- mains quite difficult to be answered. gen or an intrinsic structural flexibility of Clearly, there is a need for research on Genetic Variation and Molecular Evolution 17 the spontaneous generation of genetic 10 variation in higher organisms, ideally at Conceptual Aspects of the Theory of the level of the genomes. While this Molecular Evolution is already quite difficult in microorgan- isms, it is of increased perplexity with With reference to Sect. 2.2, it is fair to again the much larger genomes of higher or- explicitly state that for the time being, ganisms. However, sequence comparisons evolution genes and evolution functions now offer fruitful ways to search for se- are a concept rather than a fully proven fact. quence homologies, sequence similarities, This concept is based on a particular way to single nucleotide polymorphisms, as well interpret numerous available experimental as for traces of intragenomic DNA rear- data. We will briefly analyze the difficulties rangements and of horizontal transfer of to clarify the situation in a scientific genetic information. Data so far available debate. This will be followed by pointing to areinsupportoftheprinciplesofmolec- philosophical and more practical values of ular evolution outlined in Sect. 2, which a deeper understanding of the molecular are likely to be valid for any kind of living processes that drive biological evolution. organism. Some of the general evolutionary strate- 10.1 gies developed in microorganisms must Pertinent Scientific Questions have also turned out to be useful for the de- velopmental and physiological processes In the history of scientific investigations, at somatic levels of higher organisms. biologists have often searched for evidence The generation of antibody diversity in that living organisms would be able to the immune systems of vertebrates by specifically modify, or adapt, their genetic genetic rearrangements and so-called so- information in order to better cope with matic mutagenesis is a good example. upcoming changes in the living condi- Another example is the enzymatic re- tions. Most of these attempts have failed to pair of DNA damage caused in somatic give the expected response. In other cases, cells by external mutagens such as UV where a certain degree of specific adap- irradiation. tation could be observed, specific causal These considerations illustrate that explanations have sometimes been found whatever gene function may prove to upon deeper investigation. However, there be useful for whatever particular pur- is at present no good scientific evidence pose, it may be evolutionarily retained for a general rule that genetic alterations and in the course of time further fine- would always be directed toward a specific tuned. We have already encountered this goal. This situation favors the view that principle in the microbial world, where spontaneous mutations affect DNA more we have postulated multifunctional en- or less randomly, which is in line with the zymes (such as working both for the general observation that only a minority of physiology of the cells and for an evo- spontaneous mutants prove to be favorable lutionary task) to become evolutionarily under the encountered living conditions. improved both by direct and by second- The postulate of evolution genes that act order selection for the various functions. as generators of genetic variations may be This may also be the case in higher a surprise in this context. This has to do organisms. with a widely followed concept of genetic 18 Genetic Variation and Molecular Evolution

information as a strict program for the ful- endogenous viruses such as retroviruses fillment of a specific task. This definition that reside in many higher organisms. does apply to many housekeeping genes, Again, one may wonder if these viruses the products of which efficiently catalyze primarily fulfill evolutionary functions for a reaction that reliably always yields the the evolutionary development of their same product. This is not how a variation hosts or whether they should rather be generator that does not work efficiently and looked at as parasites that may carry out that yields a different product from case some evolutionary function by accident. to case functions. A good example is the While prokaryotic organisms have transposition of mobile genetic elements. genomes that are relatively densely However, not all scientists see the pri- packed with functional genes, many mary function of a mobile genetic element higher organisms have extended segments in genetic variation. Rather, some col- of intergenic DNA sequences, some leagues interpret IS translocation, which of which are highly repetitive. Some often goes along with the replication of the of these noncoding sequences are element, as a selfish activity. This interpre- highly homogeneous with regard to tation considers mobile genetic elements their nucleotide composition. While the as parasites with the argument that their biological roles played by noncoding activity would often harm their host cell. regions are still not well understood, it This discussion shows that the concept has been postulated that compositional of evolution genes cannot easily be de- constraints may influence natural fended by referring to scientific evidence. selection. These aspects have not been Rather, the concept reflects an attitude of covered in this article and they may the observer of natural events. According to the view defended in this article, nature more specifically relate to the molecular actively cares for biological evolution. The evolution of higher organisms, in addition products of evolution genes are actively to the principles outlined here largely involved in generating different kinds of on the basis of evidence from microbial genetic variants at frequencies insuring genetics. both a certain genetic stability required for maintaining the concerned form of life and 10.2 a low frequency of genetic variations as the Philosophical Values of the Knowledge on driving force of evolution. This interpreta- Molecular Evolution tion recognizes biological evolution as an essential principle of self-organization of One of the central questions of human life on Earth. curiosity is to know where life – and more Another pertinent question that cannot specifically human life – comes from. The find an easy scientific answer relates to Darwinian theory opposed to the idea of a the evolutionary function of viruses. At specific act of creation for each particular least some viruses are clearly identified form of life, the alternative explanation of a to sometimes act as gene vectors in steady evolutionary development implying horizontal gene transfer. Some of them the descent of the actual species from com- also temporarily integrate their genome mon ancestors. The directions of evolution into the host genome. This relates to the are thereby given by natural selection act- lysogenic state of bacteria as well as to ing steadily on all available forms of life Genetic Variation and Molecular Evolution 19 including all present variants. Until re- genes or more generally of evolutionary cently, the sciences could not specifically functions of many different gene products. explain how genetic variants are generated. A recently published book devoted to these With recently developed research strate- exciting insights is entitled Darwin in gies, molecular genetics can now fill this the Genome. gap. The branch of science called molecular The high philosophical value of this ex- evolution explains that there is not just a tension of our worldview is obvious and single source for genetic variants. Rather, merits to be widely discussed and eval- many different specific mechanisms con- uated in its various cultural dimensions. tribute to the generation of genetic variants One interesting aspect is the implied du- at low frequencies. These mechanisms fol- ality of the genome. Indeed, evolution low one and sometimes more than one genes are located together with all the basic strategies of evolutionary develop- other genes on the genome and on ac- ment. These are local changes in the DNA cessory DNA molecules such as plasmids sequences, rearrangements of DNA seg- and viral genomes. While probably a major ments within the genome, and acquisition part of gene products carries out functions of segments of foreign DNA by hori- to benefit the cell and the individual, of- zontal gene transfer. As a general rule, ten a multicellular organism, probably a spontaneous mutagenesis is not specifi- minority of gene products works for the cally directed; it is at least to some extent biological evolution of the concerned pop- random, so that only a minor fraction of ulation. Note, however, that the generation spontaneous genetic variants turn out to of a novel genetic variant obviously also oc- be favorable for the concerned organism curs in an individual cell. But this act of and thus provide it with a selective ad- creation has only a small chance to bring to vantage. Nevertheless, new knowledge on the concerned cell and to its descendants the precise molecular mechanisms of the a selective advantage. More often, the mu- generation of genetic variations provides tation is unfavorable and renders the life strong evidence that, in many cases, spe- of the concerned organism more trouble- cific enzymes are involved – the products some. As long as the spontaneous rate of of evolution genes. These products work in mutagenesis remains low (mostly thanks tight collaboration with nongenetic factors to the intervention of evolution genes), that can be intrinsic properties of matter or unfavorable mutations are tolerable at the environmental conditions. This view of the level of propagating populations. evolutionary process represents the core of Incidentally, the situation described here a theory of molecular evolution and it can offers a possible explanation to the quite be seen as an extension of neo-Darwinism difficult theodicean question: why does at the molecular level. God, despite His love for the human It should be clearly stated that the creature, admit that physically evil events theory of molecular evolution does not such as a mutation causing an inherita- explain the origin of life. It can, however, ble disease can occur to individuals? As a explain that biological evolution exerted matter of fact, Genesis describes creation in all living beings is a steady, dynamic as a stepwise process, which implies the process that is actively promoted not only permanent evolutionary expansion of the by intrinsic properties of matter but also by diversity of life forms. Genesis also states the intervention of products of evolution that God evaluated this system as good. 20 Genetic Variation and Molecular Evolution

Hence, biological evolution occurs accord- there, one can conclude that from an ing to God’s intention to amplify diversity inoculum with one single bacterial cell, of life on our planet. Occasional unfavor- one can theoretically expect to obtain 1030 able mutations affecting rare individuals cells within only 50 h. In reality, growth in populations is a sacrifice brought to the will be stopped much earlier by lack of creative force residing in the system of nutrition, but this reflection illustrates molecular evolution. well the enormous internal forces for In brief, the genetic information con- expansionofagivenformoflife. tained in each genome – of bacteria as well Similarly, a high potentiality for evolu- as of all higher organisms – represents an tionary expansion toward more diversity internal duality. It serves individuals for resides in the mechanisms of molecular the fulfillment of their individual lives evolution that are described in this article. and it serves populations for a slow but These mechanisms can serve us as a basis steady expansion of life forms and thus of to better understand both the origin and biodiversity. the steady replenishment of biodiversity as well as the internal limits set to evo- 10.3 lutionary expansion. This knowledge can Aspects Relating to Practical Applications andshouldincreasinglybeusedasaback- of Scientific Knowledge on Molecular ground for any measures taken toward the Evolution protection of biodiversity and of habitats for diverse forms of life. Last but not the Living organisms today occupy an amaz- least, a better understanding of the evo- ing variety of ecological niches on our lutionary process can be of help to render planet Earth. These niches include ex- the development of agricultural and related treme physicochemical conditions such as practices more sustainable. elevated temperatures, high pressure, and Genetic engineering offers ample new quite unusual compositions of chemical possibilities for the sustainable produc- elements. However, despite the intrinsic tion of medical drugs, to obtain food of potential of the living world to evolutionar- higher quality, and to reduce the nocent ily expand, one can estimate the carrying impact of the human civilization on the en- capacity of the planet for life to be in the vironment. The serious reservations made order of 1030 living cells. Although this is a by large parts of the human population very large number, it seriously limits free impede many of the proposed biotech- expansion of life in its various forms. nological applications. A part of these The following reflections should help concerns refer to unpredictable long-term illustrate this statement. An adult human effects of genetically modified organisms being carries in the order of 1013 cells. The (GMO) that are released into the environ- human population today, thus occupies a ment, such as in agricultural applications. share of about 1023 cells of the available Scientific assessments of long-term and, 1030.Incidentally,thishappenstobe in particular, evolutionary effects of such close to the average available for each applications are thus required. Knowledge of the estimated 107 different species of the natural strategies of molecular evo- of organisms on the planet. Bacteria lution can provide a good basis for such propagate by cell division as outlined in studies. As a matter of fact, in genetic en- Sect. 3. 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