Evolution as a Learning Process: An Alternative Perspective.

A Short Version of the Thesis Presented at the Poster Session in the Cellular and Molecular Biology Section of the American Association for the Advancement of Science 2003 Annual Meetings Held in Denver CO in February.

A SPECULATION ON EVOLUTION AS A LEARNING PROCESS

Dolores Landy Bentham February 6, 2003

Evolution as a Learning Process D. Landy Bentham

Until RNA showed that its role was more than ancillary, meaning and innovation had been attributed solely to DNA. Changes in DNA were said to be random with respect to the needs of the organism. But since both DNA and RNA are abstraction removes from protein, where the action is, it seems reasonable to propose protein as a non-random source of novelty. However, for novel proteins to be heritable there needs to be feedback to DNA. The oneway only process of protein synthesis, from genetic DNA to RNA to protein (DNA>RNA>protein), proposed by Crick (1970) was replaced by DNA<>RNA>protein when Baltimore (1970) and Temin (1970) discovered reverse transcription of RNA to DNA. In an even more radical departure from Crick's "central dogma", Mekler (1967) hypothesized reverse translation from protein to RNA. (look (1979) and Craig (1981) followed him in this idea. Reverse translation has since been supported by the findings of Nashimoto (2001) and Gold (1998). Sakai (2001) has proposed a calmodulin-mediated reversible non-chromosomal gene expression route in which DNA, RNA or protein can be converted to any one of the other. They have found that a "new gene expression route differing from a conventionally known gene expression route (irreversible chromosomal gene expression route) is present in vivo. . .and that these reactions are all promoted by calmodulin." Sources of novelty in proteins: Resistance (R) to antibiotics may have arisen in proteins rather than DNA. The accepted view is that mutations, which encode resistance, arise spontaneously without reference to the antibiotic. The bacteria in the laboratory setting are said to be naive in this respect. However, bacteria have long lineages and may have met the antibiotic in the wild. Bacteria, fungi, plants

Evolution as a Learning Process D. Landy Bentham

all are equipped with arsenals of antibiotics and R factors to the antibiotics, which they produce for self-defense. The appearance of what seem to be random mutations" may have been the regularly turned on gene for the disarming of the antibiotic. It could also have been the acquisition of a plasmid encoding the R factor (Hardy 1986). But the origin of the R factor remains in question. Some antibiotics may not have correlates in the wild; in this case resistance must arise de novo. Burnet (1964) proposed the neo-Darwinian model for the generation of antibody diversity. In the instructionist model, which it replaced, the antigen is the template on which the antibody forms itself. That is, antibodies arise in proteins rather than randomly in DNA. Current theories, which invoke combinatorial events of germ-line variable genes (Nezlin 2001), don't necessarily rule out that the origin of these genes was in protein templates. The newly formed protein fed back to RNA and DNA would be given access to the genes by a means suggested later in this paper. Long-term memory storage requires protein synthesis (Ungar 1971) (Kandel 2002). Ungar's view that peptides encode long-term memory has been discarded in favor of Kandel's work that shows that it is the strengthening of synapses and the multiplying of connections which requires protein synthesis. Reductionism has served Kandel well (2002). However it has not answered the question of how the memory itself is encoded. Kandel calls the molecular biology of memory storage "a dialogue between genes and synapses", but grants that there is much work to be done to understand the encoding of memory. Moreover, peptides may have been the first molecules of life (Hazen 2001). Since peptides/proteins are the workhorses of life this seems reasonable. DNA had been ruled out, because of its dependence on protein enzymes for replication. Then RNA, with the discovery of

Evolution as a Learning Process D. Landy Bentham

its enzymatic capability, became a leading candidate for first molecule. Lipids have been proposed too (Segre 2001), as well as co-evolution of some of the contenders. However, Orgel (1998) makes a case for peptides as first molecules because they polymerize on minerals especially calcite, which was abundant on the early earth (Hazen, Filley and Goodfriend). With the finding that peptides can self-replicate, their candidacy is bolstered (Lee 1996). Novel proteins, which reverse translate to RNA, and in turn reverse transcribe to DNA complete a circuit of protein synthesis; however, there needs to be access of the DNA to the germ line cells in order for the novelty to be heritable. Lo (2001) discusses circulating DNA. Lavitrano et al. (1997) have shown that pig "sperm cells bind and internalize exogenous DNA". Sin (1998) found in a series of experiments, that foreign DNA, injected into the proximal region of the vas deferens of mouse and rat, was recovered six hours after the injection, in 60% of the sperm. Shamila (1998) found that a foreign gene injected into the testis of fifth instar silkworm larvae, which existed extrachromosomally in the founder larvae," was transmitted to the progeny, indicating that the sperm cells picked up the injected DNA (Sin 1998). Integration of the foreign DNA would be accomplished by "natural genetic engineering" (Shapiro 1998, 2001), just as plasmids become part of the genomes of the prokaryotes, which receive them (Hardy 1986), and endosymbionts become part of the host's genome (Buchner 1953) (Margulis 1989). In summary: As one of the ways of evolution, I propose a learning process in which novelty arises in protein, reverse translates to RNA, reverse transcribes to DNA, and is heritable by gaining access to the germ line cells.

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Literature Cited: Baltimore, David. 1970. RNA-dependent DNA polymerases in virions of RNA tumor viruses. Nature 226:1209-2l1. Buchner, Paul. 1953. Endosymbiosis. New York: John Wiley and Sons, Inc. Burnet, F. Macfarlane. 1964. A Darwinian approach to immunity. Nature 203:451. E Cook, Norman D. 1977. The case for reverse translation. J. Theor. Biol. 64: 113-135. Craig, Robin. 1981. The theoretical possibility of reverse translation of proteins into genes. J. Theor. Biol.88:757-760. Crick, F. H. C. 1970. The central dogma. Nature 227: 561-563. Gold, Larry, Craig Tuerk, David Pribnow, Jonathan Drew Smith. 1998. CODEN:USXXAM 5843701 A 1998 1201 Patent written in English. Hardy, K.G. 1986. Bacterial Plasmids 2nd Edition. Wockinham, U.K. Van Nostrand F Reinhold. Hazen, Robert M. 2001. Life's rocky start. Scientific American April:77-85. Hazen, Robert M., Timothy R. Elley, and Glenn A. Goodfriend. 2001. Selective adsorption of L-and Damino acids on calcite: Implications for biochemical homochirality. PNAS 98 10:54875490. Kandel, Eric R. 2002. The molecular biology of memory storage: A dialogue between genes and synapses. Science 294 (5544): 1030. Lavitrano, M., B. Malone, E. Forte, M. Francolini, and C. Spadafora. 1997. Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake. Exp. Cell Res. 233: 56. Lee, David H., Juan R. Granja, Jose A. Martinez, Kay Severin and M. Reza Ghadiri. 1996. A self-replicating peptide. Nature 382:525.

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Evolution as a Learning Process: An Alternative Perspective and Personal Inquiry

"Why would you want to study evo1ution?" Jerome Wolken asked. "Nobody knows how evolution works." Jerome Wolken studied the effect of light on organisms at Carnegie Mellon, Pittsburgh PA, and at the Marine Biological Laboratories (MBL) Woods Hole, MA. In another conversation in the summer of1997, Mary Eubanks said she saw evolution as working in many different ways. She had discovered that corn had originated by hybridization. She was from Duke University in North Carolina and was at the MBL for a workshop on molecular evolution. Despite the views of Wolken and Eubanks, mainstream evolutionary thinking remains decidedly neo-Darwinist; that is, mutations, random with respect to the needs of the organism, are acted on and given orientation by natural selection. By natural selection we mean that those organisms, best adapted to the environment, out-reproduce the competition(Kendrew 1994). Nevertheless, among the ways of evolution cited below, random mutations are not players as sources of novelty. Some of the ways of evolution: Citations refer to those who have either originated or furthered the theory, Hybridization, cross-mating to form new species (Eubanks 1997). Endosymbiosis, merger to form a new species (Buchner 1953) (Margulis 1989). Adaptive mutations (Caims et al. 1988) (Rosenberg et al 1994). The continuum of development and evolution (Gerhart and Kirschner 1997). Morphogenesis (Sheldrake 1995). Regulatory RNA's role in evolution (Eddy 2001) (Mattick 2001). Transposable and retro-transposable elements (McClintock 1957) (Shapiro 1999) Intracellular information processing (Shapiro 2002)
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Inheritance of Acquired Characteristics (Lamarck 1809) (Landman 1993). Reverse translation in evolution (Mekler 1967) (Cook 1977), now known as The RNA/protein symmetry theory (Nashimoto 2001) or SPERT (Systematic Polypeptide Evolution by Reverse Translation) (Gold et al.1998). These ways of evolution are not mutually exclusive and certainly overlap, representing

various aspects of the same phenomenon. The last on the list has engaged my interest for the past 25 years. I was struck by the possibility of reverse translation when first reading about molecular encoding of memory (Milner, Peter M. 1970). If molecules encode memory why would there be no feedback to the genome? Norman D. Cook also takes off from molecular encoding of memory when he makes "The case for reverse translation". Although molecular encoding of memory had been dropped as a line of inquiry in thel970's, it has recently reappeared in the literature. In Science, of 2 August, 2002, Nobelist Eric Kandel and Kausik Si speculate "that a common protein called CREB can self-perpetuate in mammalian brainsalthough cautioning that the evidence is extremely preliminary, they speculate that it might play a role in storing information- in other words, in memory". "Evolution as a learning process" would have been as apt as a title for Cook's article. Peptides change in response to experience to encode the memory of that experience. The peptides are then fed back to the genome by reverse translation to nucleic acids, probably RNA. RNA then reverse transcribes to DNA. In other words, what is learned may become part of DNA. In his article, published in the Journal of Theoretical Biology 1977, Cook cited, among others, the work of Hyden and Egyhazi (1963) for peptide encoding of memory; Mekler (1967) on the hypothesis of reverse translation; and Baltimore (1970) and Temin (1970) on reverse

Evolution as a Learning Process D. Landy Bentham

transcription. In a paper for an independent I study at Queens College, I cited some of the same sources and came to the same conclusions as Cook. That paper is dated May 1977. I hadn't heard of Mekler, and his speculation on the possibility of reverse translation, but had counted on its possibility. I knew the work of Baltimore and Temin on reverse transcription and of Hyden and Egyhazi and others on peptide encoding of memory. There are gaps in this hypothesis of evolution as a learning process. Neither Cook nor I addressed the question of how the new or altered DNA enters the germ line cells. And if entry were possible, what then? Twenty-five years later there are some answers to these questions and I address them later in this paper. As much as Cook's (and my) hypothesis may have a logical appeal, trends in theoretical biology don't always run a straight course. The following describes the fate of some of the work cited by Cook: Despite the central dogma, which states that protein synthesis proceeds in one direction only, DNA>RNA>Protein (Crick and Orgel), reverse-transcription from RNA to DNA is now part of mainstream biology. This was discovered independently by David Baltimore (1970) and Howard Temin (1972). Reverse transcription was first observed in the RNA Raus sarcoma virus and now infamously in HIV. Reverse transcription is not limited to RNA viruses. The retrotransposon, one of the mobile genetic elements first discovered by McClintock (1948), encodes reverse transcriptase, the enzyme involved in reverse transcription. But the rule against reverse translation from protein to DNA or RNA remained in place until now. In fact it still remains in the minds of most biologists given the results of my searches. Reverse translation of protein to RNA seems to have been lost in the scientific literature. No one I asked knew of it and no searches I tried came up with anything on it When I asked James Shapiro about reverse translation, he suggested that although it was theoretically possible

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to translate backward from protein to RNA- he even spelled out how it could be done- he didn't know of anyone who had tried it (appendix A). He added that I should check with a knowledgeable biochemist before I went too far with it. A biochemist, Lars Backman who responded to my e-mail query, hadn't heard of anyone doing it either and wondered why anyone would want to. He didn't "believe in this idea", but went on to cite the difficulties that could be encountered in trying to reverse translate protein to RNA (appendix B). Of the two other respondents, one found the question "very interesting" with three exclamation points (appendix C) and another referred me back to the central dogma. A search on Sci Finder Scholar at the New York University Library turned up more on "reverse translation". The hypotheses of Mekler, Cook and Craig were raised to the level of theory with the experimental work of Nashimoto (2001) and a method for reverse translating in vitro patented by Gold et al. (1998)Inherent in neo-Darwinist theory, is the genome as a blueprint for life. Sometimes the blueprint is mis-transcribed or damaged, introducing novelty. However, an organism with mistakes in its genome is actually at a disadvantage. Errors mean a loss of information and are therefore deleterious to the organism There is elaborate molecular machinery in place to correct these mistakes (Lowenstein 1999).Some mistakes are neutral, having no effect on the protein product, due to the redundancy of the genetic code- some amino acids are coded for by more than one RNA codon triplet. The rare mistake, which is not neutral, lethally deleterious, nor corrected, is said to be the source of the novelty upon which natural selection works. Should the uncorrected novel gene, when expressed, be advantageous to the organism, in that its bearers out-reproduce the competition, the novelty would be retained and would be spread throughout the population (Kendrew 1994).

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But rather than viewing the genome as a blueprint for life, we see it as a file- the repository of information that a living thing uses to form itself and to function- we may come close to what the evidence reveals (Shapiro 1999, A third way). Although all organisms are made up of cells, cells differ from each other depending on the tissue and organ in which they exist. Each cell accesses the parts of the genome necessary to make it the kind of cell that it needs to be to do the job that it needs to do. The cell must necessarily "know" what parts of the genome to access (Shapiro 2002). Although the cell has some autonomy- it is separated from its environment by a membrane- it is not isolated from that environment. It is inconstant communication with the milieu in which it finds itself. It may receive messages directly from adjacent cells or from a distance by way of the nervous, endocrine, immune, and circulatory systems. The cell therefore looks both outward and inward for the role it plays in the organism and for the means to fulfill that role. The process, which unfolds, is called cellular differentiation. Looking inward the cell calls on the parts of the genome, which spell out how to fulfill its role. Primitive, undifferentiated cells seem not yet to have "decided" on their roles and are hardly distinguishable one from another. But the differentiating cell moves from one stage of its development to the next until its mature appearance is characteristic of its type, and its function appropriate to its kind (Kendrew 1994). The cell must necessarily process thousands of bits of information both from without and within to achieve this end (Shapiro 2002). The genome, far from being the start of a one way street to the production of an organism, is part of a dynamic hierarchical system. Arthur Koestler (1967) invoked the two-headed god, Janus, as a metaphor for this system. The heads of Janus face in opposite directions. One looks down the hierarchy, the other, up. At the cellular level, Janus looks both outward (up) and

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inward (down). Both views inform its mode of existence. At each level of organization the Janus heads look up and down the hierarchy. Stanley Salthe an evolutionary biologist who invokes a similar metaphor wonders where the hierarchy ends. Candace Pert (1997) prefers the metaphor, "network", to "hierarchy, because all systems affect tall others in a more egalitarian way than the word hierarchy implies. Support for the idea of the genome as part of an interactive system rather than a blueprint for life comes from comparisons of DNA from members of different species. Genetic sequences of chimps and humans turn out to be 98.5% identical. Human and mouse sequences are about 60% the same. What has been compared is the protein encoding genes which make up only 3% of the entire human genome. The remaining 97% of the genome is non-protein coding, formerly called junk DNA". These stretches of DNA are described as made up of repetitive sequences (Enard 2002). Obviously we are not so close phenotypically to chimps as we are genotypically (Travis 2000) (Enard 2002). Since these comparisons of genomes were made before the first draft of the human genome project was completed or even well underway, the question arises: Which genes were compared? Were they simply marker genes such as those for hemoglobin of mitochondria? That there is only a small percentage of genes which encode proteins further supports the idea that these genes are more akin to a file, or repository of information, than to a blueprint for life (Shapiro 1999). Therefore, we must look to the whole genome for the answer to the question of the origin of complexity and diversity (Enard 2002). However, non-protein coding DNA has certain characteristics which elevate its status to a role of more importance than that of junk. Some of it instructs DNA transcription to start here and end there or farther along- there, to form two different peptides or proteins (Kendrew 1994).

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Also, when subjected to linguistic analysis, repetitive sequences show characteristics of language (Pennisi 1994).DNA has computational capacity as well and has been used as a test tube computer to solve a complex "shortest distance" problem (Kolata 1994). The relevance of some of these characteristics is unclear. What may be of significance is the finding that it is the nonprotein coding DNA which separates one individual of our species from another. This is the basis for the use of DNA in forensics. Each of us has what we call a "DNA fingerprint" since, like actual fingerprints, no two are exactly alike (Kendrew 1994). Do these differences in our non-protein coding DNA have a meaning larger than that of its usefulness as a forensic tool? Some have thought that it is residual- the DNA chucked out when no longer needed, its origins part of our evolutionary history. Others have thought that the non-protein coding DNA is a repository for new DNA - a warehouse of nucleotide sequences at the ready for use- to signal protein encoding DNA to start or stop at yet other places along its sequence creating novel proteins. Or that it may be used to code for new proteins from scratch. Further, it has been recently discovered that some DNA codes for short non-protein encoding RNA. Like non-protein encoding DNA, the RNA has a regulatory function (Lee and Ambros 2001). These RNAs were the subjects of a review article (Travis 2002) on "biological dark matter" named for the large numbers of these previously undetected genes. These short regulatory RNA's were first discovered in mutants of C. elegans. Mutant worms did not move beyond the first of four larval stages. Instead they repeated the first- getting larger, but not proceeding to the next stage. Victor Ambros (Travis 2002 Darkmatter) of Dartmouth Medical School discovered in the early 90's that a missing bit of DNA, which codes for RNA as its end product was the cause. These bits of regulatory RNA have also been found in bacteria, flies, and

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mammals, including humans. There are also the bits of RNA called introns, which are edited out of RNA before translation. These too may have a regulatory role to play in development. John Mattick (2001) of Queensland University, Brisbane Australia goes further. He believes that these short RNAs may be the answer to the question of the sources of complexity and diversity in evolution. That short RNA s may regulate both development and evolution underlies the notion that development and evolution form a continuum. How does diversity originate? Mattick's answer is that it is certainly not from the increase in the number of genes coding for proteins. He points out that V flies and worms have roughly twice the number of these genes as bacteria. Fish and humans have about the same number, which is twice the number of those of flies and worms. So, they reason, diversity has another source. Ninety eight % of the genome, by their count, is non-protein encoding. (They're factored in the introns.) What this 98% is doing is a matter under study by several labs besides those mentioned above. They include Gary Ruvkun's at Massachusetts General Hospital, Boston; Sean Eddy's at Washington University School of Medicine, St. Louis MO; Gisela Storz and Susan Gottesmarfs at the National Institutes of Health, Bethesda, MD (Travis 2002, Dark matter). More than one evolutionary biologist has noted that a mouse or an elephant can be built from similar proteins (Gerhart and Kirschner 1997). The difference lies inform. Development and evolution, which were historically treated as separate disciplines, have recently merged- each informing the other. The merger has been marked by the publication of a new journal, which includes both "development" and "evolution" in its title and by a nickname, "Evo-Devo". Regulatory genes, those which control development, can have far reaching effects. Note that should the infant chimp retain its head to body ratio and its orthogonal human like face, he would no longer be considered chimp. (Milner, R. 1990).

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However the body plan plays out in development, there have to be raw materials available from which the body makes itself. Important among these materials are amino acids. At some point there had to have been the invention of proteins.RNA has been suggested as the first molecule of life because it can act as an enzyme that edits parts of itself out and splices disparate parts together (Cech 1986). With the help of enzymes it can also replicate itself and code for proteins. Until this discovery of the ribozyme, peptides and proteins were considered to be the sole molecules capable of enzymatic activity. But as discussed above, by current neoDarwinist theory, DNA, not RNA, is the sole molecule of protein invention, and novelty is random with respect to needs. According to this theory there is no feed back to DNA. However, with the discovery of reverse transcription from RNA to DNA there are at least some cases where what is encoded in RNA is fed back to DNA. This discovery places RNA in a pivotal position in the process of protein synthesis. It can not only translate to protein; it can transcribe back to DNA. For this reason it has been put forward as the first molecule of life (Orgel 1998). Even though RNA as enzyme supports the case for RNA as the first molecule of life, there have been other arguments against it because of the lack of cytosine in the early environment (Shapiro, R.1999). As important as RNA is as an enzyme, most of the enzymatic jobs of the cell fall to peptides or proteins. Since this is the case, it seems reasonable to suggest that the origins of novelty lie not only in DNA and RNA, but in protein itself. Segre et al. (2001) suggest that even lipids in the early earth had some enzymatic capability. DNA has a few non-random tricks up its sleeve, as well, under the rubric "directed mutations". These mutations are, in fact, in response to need. And far from being the inevitable end product of a synthesis which is, for the most part, one-way, protein too edits itself, paralleling, as it does, RNA editing. Inteins are edited out and exions remain to fold into useful proteins, sometimes with the help of

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chaperones" or chaperonins (Kendrew 1994). And even end products are subject to change. Proteins may substitute one amino acid for another. (Sahl and Bierbaum.1998) In short, proteins are not just products; they are part of the process of creating themselves. Relevant to the idea of proteins' creating themselves is the "Instructionist" model for the generation of antibody diversity. In this model it is the antigen which instructs antibody formation. Although this model was discarded in favor of the "clonal selection model (Bumet 1964), in its time it was championed by among others, Linus Pauling. Lending new support to the old instructionist model is the work of Mosbach (1998) and others (Ansell 1996). Mosbach has actually synthesized antibodies from templates. Although they do not themselves extrapolate their in-vitro findings to explain the generation of antibody diversity, these findings suggest that a new look at the instructionist model may be in order. The contrasting clonal selection model is a neo-Darwinian application to the study of the immune system. Antibodies are generated in random diversity and selected for by their specific antigens. Clones, of the immuno-competent cell producing the relevant antibodies, are produced. This theory gained wide acceptance, but the recent "imprinting" of antibody mimics from templates suggests that the instructionist model may have something to say for itself after all. Steele (1998) for one, has invoked the workings of the immune system to support his theory of somatic mutations fed back to the genome. He invokes RNA with its capacity to reverse transcribe to DNA as the principle player in this scenario. His theory is much like neoDarwinism with one exception: Random mutations, which occur in somatic cells, among them immuno-competent cells, are acted on by natural selection. Because these acquired characteristics (the random mutations in the somatic cell genome) are inherited, his theory has been characterized as Lamarckian. However, because it is nevertheless based on random

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variation acted on by selection, it has more in common with neo-Darwinism than with Lamarckism. I am invoking the generation of antibody diversity to a non-Darwinian end. If the immune system works according to the instructionist model, there needs to be feed- back, not only from RNA to DNA(reverse transcription), but also from the template formed protein to RNA or to DNA (reverse translation). This latter was not supposed to happen, but hypothesis has gained support with the work of Larry Gold, Craig Tuerk et al. (1998) among others. Their acronym for the process- SPERT (Systematic Polypeptide Evolution by Reverse Translation) is given along with patent application information. This I work was carried out at Nexstar Pharmaceuticals, Inc, USA. Bacterial resistance to antibiotics is another example of seemingly non-Darwinian forces at work. No sooner is a new antibiotic devised then bacteria find a way to disarm it. According to neo-Darwinism, bacteria, before dividing, occasionally make a mistake in transcription, substituting one nucleotide for another. Over time an accumulation of mistakes, add up to something useful to the bacterium. A sugar that was previously indigestible becomes useful or anantibiotic is disarmed by the creation of a new enzyme. These changes are said to be random with respect to the need to digest the sugar or to disarm the antibiotic. They occur in the absence of the sugar or the antibiotic. Sometimes these changes take place at regular intervals (Lederberg 1952). However, bacteria have long lives. Their life histories stretch back eons. Bacteria split in two to reproduce. The mother cell duplicates the genome before dividing so that each daughter cell receives a full complement of genes. Even though the mutation occurs in the absence of the sugar or the antibiotic in the experimental set-up, bacteria as well as other organisms in the wild produce antibiotics. There is no guarantee that the bacterium in question has not encountered

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that antibiotic or that sugar in its long past. Given that only a small fraction of the bacteria of the world have been described and an even smaller fraction have been cultured in the laboratory, it seems that the assumption that there has been no previous contact is insupportable. Rather than a mutation, random with respect to a need, a gene may be turned on or off at regular intervals or, in the case of the conversion to lactase producing E coli, by a shift in frame (Cairns, Overbaugh and Miller 1988) (Foster and Thaler 1994). The frame-shift itself could be thought of as a turning on of a gene, which in fact already exists. Bacteria also exchange genes promiscuously, even crossing over species lines. Plasmids, small rings of DNA, are passed from bacterium to bacterium by a process known as "conjuga1ion". The plasmid is a duplicate, so that the donor retains a copy of the gene. The recipient may incorporate the DNA of the plasmid into its own genome by natural genetic engineering (Shapiro 1999 Transposable elements) and may now become a donor. Even this exchange of genes is far from random. A bacterium lacking a bit of DNA sends a signal, which another responds to by supplying the appropriate plasmid. Even though the occurrence of new genes in bacteria can be explained by turning on an existing gene, or shifting frame or exchanging plasmids, the question that still remains is how the enzyme was invented in the first place. Of course the immune system and bacterial resistance make up only part of the picture. The resolution of the problem of the encoding of instinct could be instructive to evolutionary theory. No one can deny that there is such a thing as instinct; that is, inherited knowledge. Birds build nests according to an inborn plan; birds sing song, which has an inherited, as well as a learned part. Border collies herd other dogs as well as children when there are no sheep to herd. German Shepherds, on the loose, herd cars.

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How are these characteristics, this knowledge, these ways of being encoded? They must necessarily be encoded in the genome of the germ line cells. That is what is passed on from one generation to the next. Because neo-Darwinism holds that information encoded in the genome is generated by random events, the question of how learned information gets into DNA of the germ line cells is rendered moot. Germ line cells are theoretically inviolate, uninfluenced by their environments according to August Weismann (1891-1892). If this were indeed the case, we should not expect to find molecular receptors on the membranes of germ line cells. But in fact they are there by the thousands. Receptors are invitations to molecules to enter. If Weismann were right and they have no purpose, why are they there? Frank Y. T, Sin (1998) reviews "Interactions between exogenous DNA and sperm of vertebrates and invertebrates", but plays down their relevance to evolution even though in the cases of injection of foreign DNA into the proximal region of the vas deferens of mouse and rat "over 60% of the spermatozoa were found to have the exogenous DNA". Also testicular injection of fifth instar silkworm larvae with a foreign gene resulted in transmission of it to their progeny. If genes can gain access to germ line cells, one wonders how the genes get there. An article, which appeared in Science News October 7, 1989, was entitled "DNA's Extended Domain, Sightings of cell-surface DNA turn scientific orthodoxy inside out". The reporter, Ingrid Wickelgren , states that "Although most scientists still think of this vital nucleic acid as residing only within cellular confines, accumulating evidence- some nearly 20 years oldindicates some DNA exists outside that domain, securely anchored to cell membranes". Then the question arises- how do the nucleic acids get to the cell membrane? Recent published Annals of the New York Academy of Sciences, has as its subject circulating nucleic acids. Putting it all

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together: Nucleic acids circulate, attach to cell membranes, and gain access to cells, even germ line cells, by molecular receptors. Going back to proteins and their capacity for self creation, Leslie E. Orgel (1998), in an about face on his early championing of an RNA world, has lately declared that in the twenty years or so since its conception there has not been a shred of evidence to support it. Meanwhile his interests have centered on the self-assembly of peptides on minerals common in the early earth. There has been experimental support for this theory (Hazen (2001). These findings bolster the notion that proteins rather than RNA were the first molecules of life. We have already touched on the formation of proteins on templates (Mosbach). Let's consider that these new proteins may reverse translate to RNA and then reverse transcribe to DNA, which have entry to germ line cells. If this were the case, the back-flow of information from protein to germ line cell would be complete. This, followed by the insertion by recombination of the new DNA to the genome, is all that is required for the inheritance of a novel protein. Recombination of DNA occurs naturally (Shapiro, 2002). The finding that genes of plasmids are inserted into the genomes of bacteria is now part of mainstream biology (Travis 2000), as is the insertion of genes of endosymbionts into the genomes of organisms. In turn, excess or redundant DNA of the endodsymbiontis discarded (Margulis 1989). Although the neo-Darwinian theory of evolution is still considered incontestable by many mainstream biologists there are still unknowns which may relate to evolution. In Nature Reviews Genetics of December 2001, Sean Eddy of the Howard Hughes Medical Institute and Department of Genetics, Washington University School of Medicine defines Cajal bodies (coiled bodies) as "Nuclear organelles of unknown function". Also unknown is the function of proteins which surround the RNA mass of the large sub-unit of the ribosome, the organelle where RNA is

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translated to protein. This was reported in the August 11, 2000issue of Science. The appearance of the RNA was as had been expected, but the proteins were a surprise: "In many places, the proteins give the impression of a man-of-war Jelly fish," says Peter B. Moore, Yale University. "Instead of being the compact globular structures that most proteins are, these things will often have a globular part and then there will be a little strand that extends deep into the structure of the ribosome."These novel protein structures appear to stabilize the ribosome, while the RNA assembles amino acids into peptide chains. (Science News, Vol. 158.) The phrase "they appear to stabilize the ribosome" suggests that the function of the protein structure is not in fact known and is assumed to be a structural component rather than functional. What may be more important than what is not known, is what IS known. James A. Shapiro in a debate in the Boston Review: Is Darwin in the details? (1999) points to four categories of molecular discoveries which were unknown when the neo-Darwinian theory of evolution was formulated: The Organization of the Genome. Repair capabilities of the cell. Mobile genetic elements and natural genetic engineering. Cellular information processing. Given both what is not yet known in biology and what has become known since the formulation of neo-Darwinism it seems ostrich-like to continue "believing in" an explanation which falls short of even being a theory, according to Poppers definition(Soka1 1999). A theory must be falsifiable; that is, it must be testable in such a way that either a positive or a NEGATIVE result is possible. Neo-Darwinism is not testable by this criterion. It is time to look to other models or theories to begin to explain the complexity and diversity of living things. Frequently we hear of brains being compared to computers in which synapses are switches and that memory is only a matter of strengthening those, which are used, or losing those, which are not used-, a matter of selection. Again this follows the model of neo-Darwinism. Matt Ridley has this to say:
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It is misleading to think of a brain as a computer for many reasons, but one of the obvious is that an electrical switch in a computer is just an electrical switch. A synapse in a brain is an electrical switch embedded in a chemical reactor of great sensitivity. (Genome p. 162) James Shapiro, however, uses the computer analogy to advantage in discussing information processing in the cell. Given that there is such a thing as artificial intelligence, he considers how reasonable it is to see that the cell in all of its complexity has at least this capacity for intelligence. If intelligence consists of taking in information, remembering it, making connections among the things learned, making decisions based on what is learned, and giving out information as needed, the cell does indeed have this capacity for intelligence (Shapiro 2002). In more complex organisms such as mammals, the brain is the organ specializing in all of the above, but intelligence resides elsewhere in the body as well. In humans, the enteric nervous system has as much nervous tissue as the spinal cord (Gershon 1998). The heart has something akin to intelligence (Pearsall 1998) as do all systems, organs, tissues, and cells. Analogously all parts of the cell participate in its intelligence not just the cell brain" or nucleus. It is the interaction of all the molecules of the cell which make up this intelligence. Very important among these molecules are the peptides and proteins. Even DNA cannot replicate without protein enzymes. Enzymes are the organic catalysts which speed up reactions between and among molecules to make life possible. RNA can also act as a catalyst as described above. But the primary catalysts are proteins and peptides. Proteins are also the molecular motors within cells, which move other proteins along actin, fibrils also made up of proteins. The protein molecules decorated with sugar recognition sites let in information. Protein molecules may be the information itself. Proteins, sugars, lipids, nucleic acids are all essential to the life and function of the cell. But none is independent of

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proteins which as catalysts are involved in the construction of all the others including protein molecules themselves. To sum up: Maybe Jerome Wolken was right in saying that no one knows how evolution works and maybe Mary Eubanks was right too in saying that it works in many ways. As a start to understanding one of the ways in which evolution may work I have discussed some of the non-random events which can be sources of innovation, with emphasis on evolution as a learning process. This is in contrast to current neo-Darwinist theory, which states that novelty arises randomly with respect to the needs of the organism and that orientation of evolution is the result of natural selection (differential reproduction rates). I offer an alternative hypothesis: The centerpiece of evolution as a learning process is a homeodynamic genome, within a dynamic cell, in which all factors in the process of protein synthesis and heredity influence each other. Dolores Bentham, August 31, 2002

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Pert, Candace B. l997. Molecules of emotion. New York: Scribner. Popper, Karl R. 1959. The logic of scientific discovery. London: Hutchinson. Ridley, Matt 2000. Genome: the autobiography of a species in 23 chapters. NewYork: Harper Collins. Rosenberg, Susan M., Simonne Longerich, Pauline Gee, Reuben S. Harris. 1994. Adaptive mutation by deletions in small mononucleotide repeats. Science. 265:405-407. Sahl, Hans-Georg and Gabriele Bierbaum. 1998. Lantibiotics: Biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria. Annu. Rev. Microbiol. 52:41-79. Segre, Daniel, Dafna Ben-Eli, David W. Deamer and Doron Lancet. 2001. Origins of life and evolution of the biosphere 31: 119-145. Netherlands Kluwer Academic Publishers. Shapiro, James A. 1999. More articles on evolution. A third way. Boston Review University of Illinois Press. September 7. Travis, John. 2000. Human, mouse, rat...What's next? Scientists lobby for a chimpanzee genome project. Science News 158:236-238. _____Biological dark matter 2002. Newfound RNA suggests a hidden complexity inside cells. Science News 16:24-25. Varmus, Harold. 1987. Reverse transcription: Years ago a group of viruses was found to convert RNA into DNA, reversing the usual sequence of molecular events. Now it appears this process is native to other viruses and many higher organisms. Scientific American September:56-65. Weismann, August. 1891-1892. Essays upon heredity and kindred biological subjects 2 vols. Ed. E. B. Poulton et al. Oxford: Oxford University Press.

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Bacterial Resistance

There are few that can resist giving bacteria credit for ingenuity, guile, even brilliance, when describing how these otherwise lowly creatures can get around antibiotics. No sooner is an antibiotic extracted, synthesized or invented, when up pop bacteria that knowhow to disarm it. If there is an enzyme, which destroys bacteria's ability to produce cell wall, there are bacteria which "know how" to render that bit of biochemistry useless. The swiftness with which that news travels from cell to cell, bridging not only individuals, but even species is remarkable. It used to be said- and still is- that what happens is this: Bacteria, before dividing occasionally make a mistake in transcription, substituting one nucleotide for another in a gene. Over time these changes build up and eventually an accumulation of mistakes add up to something useful to the bacterium. That could mean that it is now able to use a sugar that it couldn't use before by synthesizing an enzyme previously absent or it could mean, more significantly for us, that the bacterium has created and enzyme capable of dismantling an antibiotic. These changes are random with respect to the need to digest a sugar or to disarm an antibiotic. They occur in the absence of the sugar or the antibiotic. Sometimes these changes take place at predictable time intervals. There was an ingenious test of this devised by the Nobelist Joshua Lederberg, (Replica Plating and Indirect Selection of Bacterial Mutants, March 1952) in which he showed that in the laboratory, bacteria, which have not previously been exposed to an antibiotic, convert to forms resistant to that antibiotic. These tests were done to show that mutations take place without reference to need. The problem with that line of thought is that bacteria have long lives. Their life histories stretch back eons. A bacterium simply splits in two to reproduce, so that each daughter cell is

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half the mother. The mother has duplicated the genetic material before dividing, so that each daughter receives a full complement of her genes. Even though the mother has given up her own existence, she has become her daughters, and so it goes through the millennia. For just as many eons other organisms have been around. Bacteria as well as other organisms produce what we now call antibiotics. In the life histories of these organisms there has been a myriad of opportunities for contact. Some have been for the good- mergers with blue-green algae which became plastids that yielded the plants as we know them, and mergers with other bacteria which became mitochondria, the energy producing machines of the cell. But some of the contact resulted in all-out war; antibiotics are the artillery in that war. So, the ingeniously devised test to ensure that bacteria have never been in contact with a particular antibiotic is flawed. It works only for the present laboratory situation. It doesn't take into account all the other opportunities for contact over the organism's long history. What seems a regular appearance of mutants, with or without contact with whatever may have influenced the change, could be seen as something else. Instead of a random mutation, the change in an organism's adapting to a situation to a sugar, an antibiotic- could simply be the turning on of a gene at regular intervals. This may be what happens. Genes are not after all static entities which change willy nilly, but dynamic systems parts of which may be turned on or off. They can be rearranged and parts of them can be passed around. A small piece of genetic material in a ring may be passed from bacterium to bacterium in "conjugation". There is exchange of genetic material in bacterial after all. In a phenomenon, which resembles mating, rings of genetic material, called plasmids, are injected by one bacterium into another. These plasmids may contain the genes for disarming antibiotics, for digesting a sugar or even for adding a bit of toxic punch to the bacterium's existing arsenal. The

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donor has copied these plasmids first, so there is one to give away and one to keep to be copied again as needed. Some bacteria have thus acquired the means to render all known antibiotics ineffectual. There is no need to invoke chance mutations to explain an organism's seemingly sudden genetic change. Whatever was needed may have been simply provided by turning on a gene or acquired from another in conjugation. These too could happen at regular intervals as the "mutations" were. All of the proceeding deals with the question of what laboratory results may or may not mean, but there still remains the question of how the bacteria developed the enzymes to digest a sugar or to breakdown an antibiotic in the first place. Somewhere in its long history it has encountered the substance for the first time, This must still go on. Some antibiotics that have been engineered by people are novel. No organism, so far as we know, has developed them until now. What now? Are we still to believe that enzymes to disarm them are randomly produced by point mutations in the genome? Mutations are nearly always, if expressed, lethal to the organism, which carries it. Rare are those which are beneficial. Some work, now discounted for reasons discussed elsewhere in this paper, but which may still have merit; showed that RNA itself could be changed by learning. Whether or not it is the RNA that changed itself or was influenced to change by other factors, that it can change and be reverse transcribed to DNA, provides an alternate way to introduce novelty into a genome. There are even hints that it may be possible to translate protein back to RNA. These are only hints, but in any case they are paths to explore. The possibility of reverse translation of protein to RNA is discussed elsewhere in this paper.

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