Delineation of Current Terminology:
Confusion of the Mutation Issue
D. A. Schoch (2011)
WITHIN the last few decades, awareness has developed in the world of genetics having to do with the nature of genetic change. According to classical thought, DNA damaging events and mutations occur randomly throughout the genome of organisms purely by accident. However, a growing body of evidence demonstrates that some genetic change occurs in non-replication, non-random events. The literature gives evidence of two distinct categories of genetic change addressed by the single term “mutation.” These two categories consist of (1) replication-dependent, random chance genetic changes, and (2) non-random chance genetic adaptive change that originate as non-replication dependent changes. Logically, failure to distinguish between these two processes by separate terminology may have caused problems in understanding genetic systems. This paper aims to examine and make delineation between these two phenomena, so further research can proceed with improved knowledge and understanding of genomic processes, which require clear differentiation. Reasonable misunderstanding of many issues concerning heritability, variation, adaptation, and especially mutation, appear as potentially misleading factors without such demarcation. This has the potential of directly affecting cancer research, as well as other pertinent medical fields dealing with genetic diseases.
Keywords: mutation, genetic changes, VGC’s, adaptation, biotic entropy.
WHY do we observe some mutations arriving on the scene exactly when needed for the survival of organisms? If mutations do indeed exist as random, undirected events, why do some demonstrate initiation by genetic mechanisms of the organism? How can mutations exist as both random and non-random genetic events at the same time? Have we discovered a paradox in the nature of genetic change, or are we observing two different phenomena at work? This paper addresses the subject of genetic change. Current belief concerning the nature of mutations has them occurring as random genetic changes with respect to timing and placement within the genome (Elson et al. 2001; Clancy 2008; Hall 1990). In other words, they can hit the genome of an organism whenever and wherever chance might take opportunity. The many causes of mutation, while important depending upon the nature of the topic, are not discussed in this paper. However, it is acknowledged that mutagens can change nucleotides either before, or during, the copying of a gene and if correction enzymes do not catch and rectify these mistakes, the mutation can potentially effect the gene product, which can prospectively effect the health of the organism.
The term “mutation,” as currently used, denotes the accidental, random chance (ARC) copy error events that take place (or solidified by the failure of correction mechanisms) either before or after genetic replication, and has become plastic to the point of including any and all genetic change. Today, most scientists in the disparate fields of biology hold to this definition. However, the latest data demonstrates this view to be in error and in need of revision. For the purpose of clarity, this paper holds the definition of the word “mutation” as strictly ARC replication-dependent errors.
From review of the literature, it is a well attested to fact that some genetic change occurring in organisms are not due to ARC replication-dependent copy errors or DNA damaging events. In the papers on the subject, these genetic changes have been called “adaptive” mutations and “Cairnsian” mutations (in any event, they are still called “mutations”). For the purpose of clarity, it is suggested that these “adaptive” changes be called Variational Genetic Changes (VGC’s), which this paper will adopt throughout the remainder of the discussion. This acronym is suggested because, unlike ARC replication-dependent genetic changes, adaptational and variational genetic changes exist as non-ARC changes that originate from allelic material already defined within the organism’s genome. In short, these variation-dependent genetic changes, rather than replication-dependent genetic changes, are mediated by the organism’s genomic mechanisms.
This paper argues that differential classification and terminology between these two phenomena needs adopting, particularly regarding the observation in the literature that both types of genetic changes have differing mechanisms of origin. This paper demonstrates that two entirely different phenomena exists and argues that the traditionally held view of the definition of mutation subsists in opposition to the evidence in the literature. Because the findings of the data, in which the literature reveals two discrete phenomena rather than only one, go against traditionally held views of the biological community, they have become the seeds of controversy.
Mutations (replication-dependent, ARC genetic changes), as noted earlier, have a variety of causes, both extra-cellular (such environmental agents as radiation, X-rays, gamma rays, and chemical mutagens) as well as intra-cellular (such as normal metabolic processes, methylation, replication errors, and free radical agents). These DNA damaging events fall into several categories, such as oxidation, thermal disruption, methylation, mismatched bases, deamination, depyrimidination, and depurination. These can all cause various types of accidental random changes, such as substitutions, insertions, deletions, inversions, duplications, translocations, frameshifts, transitions, transversions, CPD’s and PPS’s. All of these can lead to serious defects within the organism incurring them, if they are not arrested by the various repair mechanisms found within cells. Such repair mechanisms as nucleotide excision repair (NER), photoreactivation, base excision repair (BER), nonhomologous end joining (NHEJ), homologous recombination repair (HRR), proofreading enzymes, mismatch repair (MMR), microhomology-mediated end joining (MMEJ), translesion synthesis, DNA damage checkpoints, SOS response, and if necessary, apoptosis.
An organism’s DNA remains in constant exposure to a variety of mutagens that threaten to damage it, therefore, mutations are a part of life. Once the damage occurs, if not repaired, it can lead to mutation. Mutations can lead to hundreds of genetic illnesses and diseases (such as SCA, cancers, and tumors) that seriously hamper the health and well-being of the organism affected, to the point of suffering a painful and early death. Damage to DNA can come from many different sources, both before or during, cellular replication. If correcting mechanisms do not rectify the damage before replication occurs, or immediately afterwards by cellular proofreading enzymes, then from that point forward, the damage will become a replication-dependent mutation. Once the mutation becomes established in the cell (if missed by these correction mechanisms), the cell will no longer recognize it as an error, it will now be considered “fixed” within the DNA.
The term “mutation” has become extremely plastic in definition, and has the potential to produce major concern. The nature of mutations (ARC genetic change) is degenerative and has entropic effects (the inevitable and steady deterioration of physical systems) upon the genome, which I call “biotic entropy” (BE). The concept of entropy (as it has to do with different fields of biology) has been discussed several times in the literature in different contexts and applications (Barton and de Vladar 2009; Iwasa 1988; Xia et al. 2002). BE is accurately compared to the entropy (measurement of “noise” or degradation) in a system of information (Gray 2009), and DNA is indeed a system of information within the organism. Mutation (ARC, replication-dependent genetic change) introduces random “static” or “noise” in the information contained within the genome that, if not arrested by correction mechanisms, can result in catastrophic illnesses and diseases. However, BE is held at bay, normally, by the cellular repair mechanisms working for the survival of the cell, and ultimately, for the survival of the organism.
Since the advent of genetics, we have discovered many things about the genome, even more so now that the Human Genome Project has been initiated and completed. We have learned that DNA is divided up into chromosomes, and further divided into genes. We have learned much concerning the genomic processes that take place through the ancient art of animal husbandry, where skillful hands of animal breeders can bring out and sculpt beautiful variations of animals and birds. We have learned about variations of genes (alleles) and even greater – we are learning about how the genome stores these variant alleles, unexpressed, for future need.
Genes in storage waiting for expression
Research is uncovering the mechanisms of adaptation and gene storage previously hidden. For example, what previously had been thought of as “junk” DNA has been discovered to code for certain RNA sequences, as well as other gene products (Crosio et al. 1996; Huang et al. 2005) in a very highly regulated mechanism of information storage. Most of these sections of DNA that were previously thought of as non-coding regions are introns. Preventing accidental expression of genes when they are not needed is one possible reason genes are broken up by introns, while introns themselves have been discovered to be coding sections in their own right. These are not cases of mutation because they are not DNA injuries or damages, nor are they sought out by repair mechanisms for correction, nor are they replication-dependent changes. On the contrary, they are mediated by genomic mechanisms and carried out with extreme precision and accuracy. It appears that introns themselves are a part of the genomic storage mechanisms that keep unexpressed alleles for future use as the organism may require.
Genes, having formerly been viewed as linear strings of nucleotide bases found in only one place within the genome, have now been demonstrated to sometimes be scattered in pieces (in trans) throughout the chromosomes like data sets on a computer hard drive. The discovery that genes act like computer data sets that can be reunited with each other and activated for expression is giving scientists in genomic studies an entirely different feel for how the genome works from what has been previously believed. Such trans-mediated gene products have been identified in the Drosophila genes mdg4 (Labrador 2001; Dorn et al. 2001) and lola (Horiuchi et al. 2003), and in the C. elegans genes eri-6 and eri-7 (Fischer et al. 2008). It is reported that genes eri-6 and eri-7 produce separate pre-messenger RNA’s that are trans-spliced together to generate a functional mRNA, eri-6/7. One question brought out by this discovery is: will there be proteins found such as eri-6 and eri-7 that are functional in and of themselves, that are also functional when trans-spliced together into a third functional product? It seems this could be a very real possibility. The main concern for this paper, however, is that trans-spliced genes are being labeled as replication-dependent ARC mutations when they are put back together for expression. The question remains, why are they being called mutations when they are obviously directed by the organism’s genomic mechanisms.
What’s more, it has been reported by Chung et al. (2007) that there are reading frames in mammalian genomes that are dual-coding. The authors go on to describe three examples of how human genes (GNAS1, XBP1, and INK4a) are dual-coded, so that there are actually two products coded for within one reading frame, or that reading frames overlap one another, producing two different products. Exactly how much of the human genome has dual-coding within reading frames of genes are unknown at this point in time, however these authors have identified forty so far. How many more mechanisms of allelic storage might be found by future research?
Some of these storage mechanisms have been demonstrated to be mediated by recombination and genomic rearrangements (Foster 2000, 1998; Harris et al. 1996) after being broken apart, presumably to keep them safely inexpressible until needed. Such trans-mediated genes are then spliced together again for expression via recombination or rearrangement mechanisms. Bull et al. (2000) records that recombination-dependent stationary-phase genetic changes take place at multiple sites within the genome. Hall (1998) lists several mechanisms and pathways for these adaptational genetic changes, including base substitutions, frameshifts, excision of mobile elements, and insertion of mobile elements – all mediated by the organism’s genomic mechanisms. Schneider and Lenski (2004) identify insertion sequence (IS) elements mediated by genomic mechanisms that both inactivate genes as well reactivate them when IS elements are excised by those same mechanisms. Schneider et al. (2004) goes on to say that “IS elements are also recognized by the recombination machinery of the cell, leading to complex rearrangements.” IS elements have been demonstrated to be a factor contributing significantly to genetic variability.
McKenzie et al. (2000) have identified such adaptive changes that are controlled by the SOS response system, as well as adaptive changes that require specific recombination proteins. These events are not ARC mutational changes, they are being specifically managed by specific genomic mechanisms under the control of genetic processes. These authors conclude their paper by stating that, “Understanding the regulation of all of the different adaptive or stationary-phase mutation mechanisms will illuminate when, how, and whether cells adjust their mutation rates and mechanisms, thereby inducing heritable changes, and presumably increasing their options for survival.” Since this paper was published, the answer to the mutation rate has been answered, and will be addressed shortly.
The literature is enamored with labeling adaptive genetic changes as “beneficial mutations,” but are they really mutations? VGC’s are beneficial to the organisms in which they occur for survival, but they are not due to replication-dependent ARC mutations. VGC’s have been researched and examined in rich detail (Foster and Cairns 1992; Williams and Foster 1994; Cairns 1998; Sletcha et al. 2002; to name a few).
The data shows three main differences between DNA damage/mutations and VGC’s, first of which is that VGC’s are specific and exact in time and place, while mutations are random chance DNA damaging events. All VGC’s are specific to the exact need of the organism at a specific time. Loewe (2008) reports “The statement that mutations are random is both profoundly true and profoundly untrue at the same time.” This statement was made viewing ARC mutations and VGC’s as one phenomena, yet making the basic random / non-random distinction between the two (as dealing with both time and placement of such genetic changes). Hall (1997) states that “The aspect of adaptive mutagenesis that remains the most contentious is the specificity of adaptive mutations,” while demonstrating that adaptive changes are specific to the “selective challenge[s]” bombarding organisms. In other words, the organism encounters an environmental challenge to survival and its genomic mechanisms meet that challenge by expressing previously stored variational alleles. Hall (1990) demonstrated that such VGC’s only occurred when they were needed by the cell incurring the change. In later experiments, Hall (1998) demonstrated again the difference between replication-dependent changes (mutations) and variation-dependent changes – which is again the specificity of time and place.
Riesenfeld et al. (1997) reports that “adaptive mutations seem to produce only those phenotypes which allow the cells to grow, whereas growth-dependent mutations occur randomly with respect to their effects on fitness” demonstrating that adaptive changes are indeed specific to the needs of the cell. Cairns and Foster (1991) have demonstrated that VGC’s are produced according to the needs of the cell, unlike the nature of the occurrences of ARC mutational changes. Harris (1996) demonstrated that stress responses are initiated to generate the appropriate genetic changes in order to arrest that stress, again showing that VGC’s are directed for the adaptational survival of the cell. Cairns et al. (1988) performing experiments on E. coli, reported the bacteria must have some way of “producing” or “selectively retaining” specific genetic changes for the needs of the cell in order to survive, because the bacteria was producing the exact adaptive changes they needed in order to survive. These adaptive changes are possible because of the genetic variability stored within the chromosomes that can be signaled into expression by environmental stressor cues.
The specificity of time and place of VGC’s are one reason they were first called “directed” mutations, and later as “adaptive” mutations in the papers examining them. When VGC’s were first identified in the literature, it was thought they represented extremely high mutation rates that would have fostered the right mutation for that specific circumstance. However, this hypothesis is no longer used to explain such adaptive changes (Foster 2000). It has been demonstrated experimentally on E. coli that such adaptive changes are not dependent upon increased mutation rates and, in fact, that there were no replication-dependent mutations – a rate of zero, so “no replication means no replication-dependent rate” (Hall 1990). Colby and Williams (1995) also corroborate Hall’s findings.
Second, the fact has been observed that VGC’s are mediated by different genomic mechanisms from those that are replication-dependent ARC mutations. Hall (1998) makes the observation that there are multiple mechanisms and pathways of VGC expression, and none of them are replication-dependent mechanisms which induce ARC genetic changes. Some of these adaptational changes are mediated by specific insertion sequence (IS) elements, via insertion and perfect excision of such mobile elements that turn “on” or “off” specific genes for expression (Schneider and Lenski 2004; Arjan et al. 2004), with over 500 such elements discovered so far. IS elements inactivate genes by means of reading frame disruptions (inserting themselves exactly where they are needed to do so), or reactivate said genes by reversions or the excisions of insertion activities previously mentioned. If it were not for the precision of IS element placements, they would be reeking havoc within the genome by turning on and off genes at random. Hall (1988) identifies IS elements as also providing promoters for specific genes as well as activating “cryptic” genes by inserting themselves into upstream regions of the chromosome.
There is also evidence that some VGC’s are mediated by specific recombination events, making them recombination-dependent genetic changes, and further identifies specific enzymes implicated in initiating such recombination events by nicking (Foster 1998, 2000; Rosenberg et al. 1998). Foster states that recombination events rearrange “existing alleles” and can “create new ones.” Loewe (2008) further elucidates on this phenomena by stating these genetic processes can “lead to the production of new genes by pasting material from different genes together.” I submit that what’s being identified here, is previously inactivated genes through rearrangement processes, stored in-trans, being brought back together again for expression through recombination. Harris et al. (1996) further identify specific enzymes responsible for reading frame disruptions, which initiate recombination-dependent VGC’s. More enzyme-mediated VGC’s have been identified, such as Rpos-dependent events for specific gene expression (Lombardo et al. 2003). McKenzie et al. (2000) ratify that VGC’s are “tightly regulated” responses by genomic mechanisms, not replication-dependent ARC mutational events.
Third, it has been observed that VGC’s are variation-dependent, non-ARC genetic changes, while mutations are the exact opposite (Riesenfeld et al. 1997). Cairns et al. (1988) also identify VGC’s as being a property of cells in stationary phase, rather than being growth-dependent, which Hall (1997, 1998) confirms. VGC’s have been demonstrated to occur when chromosomes are not being actively replicated in E. coli during nutrition starvation (Williams and Foster 2007). VGC’s are not replication-dependent changes, but have been demonstrated to be stress induced, usually by environmental stressor elements.
Another key difference between these two phenomena is that the cell has a variety of damage repair mechanisms in place to deal with the different kinds of damage that it suffers. In comparison, there are no known attempts by the cell to search out or repair VGC’s – indicating that cells do not recognize them as “damages.” In fact, they are not DNA damage, nor mutations. On the contrary, they are part of normal genomic processes for the continued survival of the organism via adaptation. These discoveries explain the phenomena observed in such cases as E. coli adaptation (Cairns et al. 1988) where the authors make the statement, “bacteria apparently have an extensive armoury of such ‘cryptic’ genes that can be called upon for the metabolism of unusual substrates.” Genes do indeed seem not only to be encrypted, but also broken up and dispersed throughout the genome to be called upon when needed. In every case, the needs of the organism appear to be environmentally induced.
Response to environmental cues
VGC’s in gene expression are induced specifically by the organism’s genome in response to environmental cues for the benefit of the organism. While ARC mutations can also be induced by environmental mutagens, they have the opposite affect. These environmentally cued genetic mechanisms give the organism access to varying phenotypes for survival and adaptability. How genetic mechanisms of adaptation and fitness relate to variation, working in connection with environmental cues, is a long standing question due to the lack of empirical studies documenting the causal relationships between the environment and the molecular underpinnings of fitness related variation (Storz 2007). Storz et al. goes on to identify the “difficulty of integrating molecular data with evidence for causal effects on organismal fitness” and examines specific mechanisms that enable organisms to adapt to their specific environments.
These environmental cueing factors can be altitude (high or low), light cycles or light sensitivity (such as in deep sea dwelling organisms), temperature, diet (such as differing sizes and hardness of seeds impacting the thickness of bird beaks), humidity, hardness of soil, oxygen levels…any number of environmental dynamics (Ralson and Shaw 2008). All of these factors have an effect upon the organism’s phenotype, demonstrating a rich and complex interaction between genes and the environment (Lobo 2008; Lobo and Shaw 2008). These environmentally induced changes in gene expression are called “gene-environment interchangeability” and help the organism to adapt its phenotype to the specific “selective circumstances” they may find themselves in (Leimar 2009). The expression of the needed allele appears to be stress induced, caused by changes in the organism’s environment, which is why such changes are specific to the needs of the organism for its survival.
These traits have been demonstrated by thousands of years of domestic breeding in hundreds of different species, such as bovine, dog, pigeon, etc. The only difference between domestic breeds and wild types is man’s hand culling and bringing out the traits he desires in each specific breed. We create more colorful birds, more milk producing cows, different dog breeds for work or for show, through careful breeding tactics. While, in the wild, these different allelic traits are only expressed in time of stress induced by the animal’s environment – but the genetic mechanisms are the same that operate in both cases. In some cases there are multiple environmental cues that signal genomic changes, such as both photoperiod and ambient temperature affecting the thermogenesis of the Djungarian hamsters (Heldmaier et al. 1982). In their study on domesticated cattle in Europe and Africa, Gautier et al. (2007) concluded from their research and observations that the genetic changes responsible for the variation between each population were not consistent with models considering replication-dependent ARC changes. To use a famous example, in “Darwin’s finches,” what are the environmental cues for the differences in beak size and thickness for each variation of bird on each different island? Presumably, the only cue seems to be the hardness of the seeds on which each island variety has to deal with. If this is true, is the exact environmental cue (for example) how many times a bird has to peck at the outer shell of the seed? Are the vibrations upon the beak from being hit against the hard shell some kind of trigger for beak thickness (in the same way that shivering thermogenesis is activated by the shivering of the body)?
The speed of which these changes occurred in the finches is simply too fast to be considered strictly replication-dependent ARC mutations, they are more likely VGC changes activated into expression by the individual environmental cues the finches’ genome’s received after being first introduced to the islands. VGC’s are the exact opposite in nature as to their effects when compared with those of ARC mutations. Variational changes are beneficial genetic changes occurring within organisms, the materials of adaptation, while the effects of ARC mutations are nearly always deleterious to the organism. Two different phenomena, with different origins and with different final effects that are opposite one another. These facts seem to make clear that there is a natural distinction between these two genetic phenomena, one that we also need to differentiate between.
It all began with a paper by Cairns et al. (1988) that challenged the currently held view of mutations, and other papers quickly followed presenting concordant observations (Foster 2000). Such papers as these were viewed with much skepticism because they challenged the fundamental premise that all genetic change occurs only randomly as chance events without respect to any advantages they may give an organism (Colby and Williams 1995). Drake stated in her book, The Molecular Basis of Mutation (1970) that “It is clear that the experimental evidence supporting many currently popular hypotheses concerning mutational processes is quite inadequate.” Those words, written almost forty years ago, still appear to hold true today. Nevertheless, clarifying the terminology can advance understanding of biological processes.
Addressing these different classes of genetic change, Hall (1990) stated, “although the two classes of mutations are basically distinct and have different molecular mechanisms…evidence for Cairnsian mutations has now been found in all cases where it has been sought.” According to the evidence in the literature, it appears that we are, indeed, observing two distinct classes of genetic change that must be differentiated between. If not, we could be inundated with perplexity in certain aspects of our research until these distinctions are recognized. For example, Hall himself (1988) illustrates this point in the following quote: “[W]e are ignorant of the fundamental mechanisms and rates of mutations in non-growing but metabolizing cells.” If we are talking about two entirely different phenomena, the mutations Hall addresses here are non-ARC genetic changes (VGC’s), in which there wouldn’t be a mutation rate because these changes occur naturally in populations only when they are cued by the environment. It is important for research to delineate between these two phenomena, for correct “characterization of individual beneficial mutations may lead to the identification of underlying molecular rules and constraints, as well as common adaptive pathways” (Rainey 2000; Travisano 2001; Otto 2002 as reported on by Arjan et al. 2004).
The history of medicine demonstrates a succession of theories of disease and treatment. A strong evidence base is now a requirement for the adoption of new treatments. Similar criteria regarding strength of evidence should be applied to molecular science that now underpins many medical advances. The specificity of non-ARC genetic changes have been demonstrated experimentally (Hall 1997) numerous times by different scientists. Indeed, understanding the differential mechanisms of change, fully and correctly, is critical to the understanding of any genetic phenomena under investigation. Clarity about the origins, mechanisms, and results of both mutations (replication-dependent ARC genetic changes) and VGC’s (non-ARC genetic changes) will improve the quality of biological understanding. Colby and Williams (1995) make the statement, “taking these kinds of mutants into account may therefore be necessary to produce more accurate models of bacterial genome evolution.” Clearly, we are observing two different categories of events. These differences require terminology fit to distinguish the processes taking place. This would also distinguish between the mechanisms and results of each kind of change.
Finally, Hall (1990) states, “Because the randomness of spontaneous mutations forms such a basic part of our view of biological processes, most of us may be more comfortable with an underlying random mechanism than with a directed one. We should be cautious, however, about rejecting the notion of ‘directed’ mutations simply because it makes us more comfortable to do so,” (Hall 1990). This is especially true when considering that these “directed mutations” do not appear to be random mutations at all, having their origins firmly grounded in completely different genetic mechanisms.
I wish to acknowledge and thank Dr. Jerry Bergman, as well as several other anonymous contributors, for their insight, suggestions, and other contributions to this paper. Their assistance has proven invaluable to its production.
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This paper was written on 5/12/2011 and I was never able to get it published through a mainstream science journal, the reason usually cited that it would not contribute anything to mainstream scientific view on the subject matter. I disagree, as well as my peers whom I had review this paper. Therefore, I am “publishing” it here on my blog, that it may make some kind of impact on those who should come across it in their research. Dave Schoch, 10/31/2012.