Experimental retracement of the origins of a protocell

Journal of Biological Physics 20: 17-36, 1994. © 1995 Kluwer Academic Publishers. Printed in the Netherlands. 17 EXPERIMENTAL RETRACEMENT OF THE ORIGINS OF A PROTOCELL: It Was Also A Protoneuron Sidney W. Fox (+), Peter R. Bahn, Klaus Dose, Kaoru Harada, Laura Hsu, Yoshio Ishima, John Jungck, Jean Kendrick, Gottfried Krampitz, James C. Lacey, Jr., Koichiro Matstmo, Paul Melius, Mavis Middlebrook, Tadayoshi Nakashima, Aristotel Pappelis, Alexander Pol, Duane L. Rohlfing, Allen Vegotsky, Thomas V. Waelmeldt, H. Wax, and Bi Yu Iowa State University, Florida State University, University of Miami, Southern Illinois University, and the University of South Alabama Abstract. Although Oparm used coacervate droplets from two or more types of polymer to model the first cell, he hypothesized homacervation from protein, consistent with Pasteur and Darwin. Herrera made two amino acids and numerous cell-like structures ("sulfobes') in the laboratory, which probably arose from intermediate polymers. Our experiments have conformed with a homoacervation of thermal proteinoid, in which amino acid sequences are determined by the reacting amino acids themselves. All proteinoids that have been tested assemble themselves alone in water to protocells. The protocells have characteristics of life defined by Webster's Dictionary: metabolism, growth, reproduction and response to stimuli in the environment. The protocells are able also to evolve to more modern cells including the initiation of a nucleic acid coding system. Principal spinoffs from the results are revised evolutionary theory, models for protoneurons and networks thereof, and numerous industrial applications of thermal polyamino acids. Life itself has thus been reaffirmed to be rooted in protein, not in DNA nor RNA, which are however crucial to inheritance in modern life as "instruction manuals" (Kornberg). Recognition of the advances have been considerably delayed by the deeply held assumption that life began by chance from random polymerization of amino acids, in contrast to the experimental findings. The concepts of DNA/RNA-first and protein-first are reconciled by a rise-and-fall progression as often seen in biochemical and biological evolution. The fact that amino acids order themselves explains in turn that thermal copolyamino acids are finding numerous applications. The entire sequence of processes (+) To whom correspondence should be addressed: Coastal Research and Development Institute, LSB 124, University of South Alabama, Mobile, AL 36688. 18 SIDNEY W. FOX ET AL, in the proteinoid origins theory is now seen to be highly deterministic, in close accord with Einstein. When one of us (S.W.F.) knew Alexander Ivanovich Oparm in 1956-1980, we had scientific discussions through Nina Oparina, who spoke and taught English and through Raia Fox who can converse in Russian. These conversations contributed to a family type of relationship and elicited the friendly qualities of "Sandro". Scientifically, we agreed upon the proposition that one is not dealing with the origin of life unless his efforts include the cell, which is, most appropriately, the subject of this conference honoring Oparm. The difference in Oparin's approach to the cell and ours is that Oparm's laboratory model was the coacervate droplet that he chose deliberately from components of modem systems, while ours has been the proteinoid microsphere which exists due to fortuitous empirical experiments (Fox et al, 1959), that represent original systems, as explained below. Figure 1. Coacervate droplets of size range 1-100 micron diam. EXPERIMENTALRETRACEMENTOF THE ORIGINSOF A PROTOCELL 19 Figure 2. Uniform proteinoid microspheres of bacterial size range. The theoretical consideration that emphasized the separation of a cell from aqueous solution is explained by the following quotation (p. 320) from Oparm's 1957 book appearing in the year following the first international conference on the origin of life that he organized for Moscow in 1956. "Thus all the evidence now available agrees in indicating that the organic polymers which were originally formed, and in particular the protein-like polypeptides of high molecular weight, must, at some stage in the evolution of carbon compounds, have separated out from a homogeneous solution in the form of multimolecular aggregates similar to the drops of coacervate which are obtained under laboratory conditions." This quotation more aptly describes the proteinoid microsphere than the coacervate droplet, the latter having been adopted by Oparm as his cell model. A detailed description of each kind of experimental model is found in Lehninger's 1975 Biochemistry, pages 1045-1049. There Lehninger described each at length and also stated that the coacervate droplet was an invalid model because it is composed of polymers like gelatin and gum arabic, which are from modern sources that are not conceivable as protobiological. I remember well that Oparm expressed during a visit to Miami his delight that another eminent biochemist, i.e. Albert Lehninger, had published a textbook of biochemistry which was the first to devote an entire chapter to the origin of life. I believe Lehninger's criticism of coacervates did not disturb Oparin; the latter could properly take credit for having shown the way. tn his 1966 book, Origin amt Early Development of Life, Oparin illustrated both the coacervate droplet (Figure 1) and the proteinoid microsphere (Figures 2,3). Here could be seen that coacervate droplets resemble liposomes in their instability and in their 20 SIDNEY W, FOX ET AL. relatively large and heterogeneous size, while the microspheres are stable, uniform and in a range of size that includes bacteria that are regarded as primitive, typically 1-3 microns in diameter. Also shown is the double layer that is characteristic of membranes in the proteinoid microsphere. Figure 3. Proteinoid microspheres as prepared in Oparin's laboratory. Note double layers in optical microscope. Another predecessor of our investigation of cellular origin was the botanist, J.J. Copeland (1936), who in his youth as a ranger-naturalist at the hot springs of Yellowstone Park, suggested: "the probability of the origin of living organisms in the thermal waters .. Copeland was, thus, a pioneer of the concept of a hydrothermal site of life which, with few exceptions (Fox, 1957, 1994a), has been overlooked. One other predecessor was Alfonso Herrera (1942) who reported obtaining amino acids and other products from intermediates such as formaldehyde; this compound was thirty years later identified as a major component of interstellar matter (Fox and Dose, 1977). Moreover, Herrera used only the ambient heat of the Earth, which is ubiquitous, in contrast to the suggestion in an incomplete table of available energies (Miller and Orgel, 1974) that limits terrestrial heat to volcanoes. Rohlfing (1976) showed that temperatures well below 100 ° C. available on the surface of the Earth are sufficient to activate substantial polymerization of amino acids. ORIGINS OF THE MACROMOLECULES OF LIFE: PROTEINS The first macromolecules of life, especially the informational ones, have been widely regarded as nucleic acids in the dominantly considered "chicken-egg problem" (e.g. Eigen, 1971). Our experimental approach has almost uniquely emphasized proteins-first, for the same reason that we believe that life arose before the mechanism to inherit it arose (Fox, 1959); at the molecular level that means proteins before nucleic acids. The EXPERIMENTALRETRACEMENTOF THE ORIGINSOF A PROTOCELL 21 reasoning includes the hypothesis that all proteins, including the first, obtained their information from a molecular evolutionary source, a family of amino acids. Indeed, the beginning of this research was in analytical procedures for identifying the sequences of amino acids to explain the origins of biodiversity (Fox, 1945). The kind of information available from practice of this methodology (Fox, 1945; Edman, 1950) promptly established the nonrandomness of the thermal polyamino acids (Fox and Harada, 1960). In the history of the chicken-egg problem, the possibility of DNA-first ('nakedgene" concept) in life itself was essentially abandoned to an unnamed mineral on an unnamed planet for the polymerization of mononucleotides (Crick, 1981). This occurred about the time that the properties of life (Fox, 1971) had already been observed in a protoprotein protocell by experimental retracement; this protocetl was subsequently shown to be capable of initiating a nucleic acid genetic coding mechanism (Fox, 1981). The inadequacy of RNA parallels that of DNA. In a recent evaluation, Ferris (1993) cites several workers such as Orgel (1986); Joyce et al, 1987) when stating, "There is a growing consensus that the RNA World did not evolve directly from molecules formed by prebiotic processes. --- Indeed, there is evidence to support the hypothesis that it is unlikely that the RNA World evolved from simple prebiotic molecules'. Although not cited by Ferris, some of the earliest evidence of the probable barreness of the RNA route for the first bioinformational macromolecules was determined by simple thermal attempts to polymerize ribomononucleotides (Schwartz and Fox, 1964). Moreover, Jungck and Fox (1973) reported experiments that explained the origins of RNA from prior thermal protein. That life itself is protein in nature as well as an explanation of the confusion that exists on this point has been stated by Kornberg (1989), "What chemical feature most clearly enables the living cell to function, grow and reproduce? Not--- DNA, the genetic material. Despite its glamor, DNA is simply the construction manual that directs the assembly of the cell's proteins. The DNA itself is lifeless---. What gives the cell its life and personality are enzymes.--- Nothing in nature is so tangible and vital to our lives as enzymes , and yet so poorly understood and appreciated by all but a few scientists -°-~. "Enzymes are protein molecules". They are not ribozymes. With the above background we can assert (Fox and Pappelis, 1993) that life and memory are rooted in protein that arose before, not after, the nucleic acid genetic mechanism emerged from its precursors. 22 SIDNEYW. FOXET AL. ORIGINS OF METABOLISM Early in this research program it became apparent that reactions of individual biochemical substances tended to follow the same kinds of pathway that are found in living cells (Fox et al, 1957; Fox, 1957). This is not so surprising; it is in a way a molecular reaffirmation of the dictum that ontogeny recapitulates phylogeny. Of the many metabolic reactions that have been found to be catalyzed by thermal proteins (Fox and Dose, 1977) some of the most interesting and unexpected have been those of oxidation-reduction processes. These became much more understandable when the amber color that accompanies all thermal proteins was found to be due to flavins and pteridines (Heinz et al, 1980) synthesized during the heating of the amino acids and incorporated into the polypeptide chains. The color that was at first regarded as a nuisance became a bridge of understanding from premetabolism to metabolism. The evolutionary position of these prosthetic groups is further clarified by the presentation of Kritsky (1994) at this conference. ORIGINS OF THERMAL POLYMERS OF AMINO ACIDS Inasmuch as innumerable investigators since the time of Emil Fischer have shown analytically that protein can be hydrolyzed to amino acids, it seemed logical that the converse synthetic process of combining amino acids to a kind of protein by loss of water was how the first proteins came into existence. We found that such reaction can be retraced in the laboratory if sufficient dicarboxylic amino acid be present in the mixture (Fox and Middlebrook, 1954). We know that the proportions need not be more than a few molar % of each dicarboxylic amino acid (Fox and Waehneldt, 1968), that the copolymerization can include all of the proteinaceous amino acids simultaneously (Fox and Harada, 1958), and that temperature can be well below 100 ° (Rohlfmg, 1976). While amino acids in general had been found to arise (Herrera, 1942; NegronMendoza, this volume) from components prominent in interstellar matter like formaldehyde (Fox and Dose, 1977), adequate proportions of natural aspartic acid and glutamic acid were recovered by hydrolysis from hot aqueous extracts of aseptically collected samples returned from the Moon (Fox et al, 1972). Only protein-type amino acids were found upon hydrolysis of these lunar samples; this was an early example of determinate molecular evolution (Fox, 1994). Of related interest is that substantial proportions of aspartic acid and glutamic acid are recoverable from reactions in simulated atmospheres of Titan (Sagan et al, 1992). FORMATION OF SELF-SELECTED AMINO ACID SEQUENCES Approaches to the origins of cellular life and recognition of the solution obtained to the problem have been inhibited by prevalent mmdsets that focus on the suppositions (a) that life arose from a random (indeterministic) matrix and (b) the related supposition that DNA/RNA preceded protein in evolution. Numerous influential scientists (see Fox and Balm, 1994) have endorsed this latter view. EXPERIMENTALRETRACEMENTOFTHEORIGINSOFA PROTOCELL 23 A first indication that the reactions of various amino acids with each other are determined by stereochemistry, polarity, hydrophobicity, etc. not by playing card probabilities, appeared in reactions of amino acid derivatives under the action of proteases (Fox and Wax, 1950: Fox et al, 1953) in a model of protein synthesis that was originated by Bergmann and Fraenkel-Conrat (1937). Studies that extended the model showed that the amino acids underwent selective reactions themselves, independent of the then popular notion of the specificity of proteolytic enzymes". Using the sequence methods that had been introduced a few years earlier (Fox, 1945) the thermal polymers were found to be highly nonrandom (Fox and Harada, 1960). Indeed, the thermal copolymerization would not have been tested if it were not for the possibility indicated of selective thermal reactions of amino acids among themselves. More recently, Tyagi and Ponnamperuma (1990) found that a simulated prebiotic polymerization of amino acyl adenylates is definitively nonrandom. Furthermore, they showed that the specificity in the sequencing of the resultant polymers of amino acids, while dependent upon reactant amino acid residues for precise structure, is independent of nucleotides included in the reactive amino acyl derivative and also independent of added polynucleotides. The earliest to confirm the self-ordering mechanism were Dose and Rauchfuss (1972). With Zaki (Dose and Zaki, 1971) Dose showed that inclusion of heine in the polymerization yielded oxidation-reduction activities in the polymers. Dose also showed, with Heinz and Ried, that the thermal polymerization of amino acids generates flavins and pterms (Heinz et al, 1980) which in the polypeptide chain function as enzyme cofactors for primitive catalases and peroxidases (Fox and Dose, 1977). In this way can be understood how oxidative as well as hydrolytic and other kinds of metabolism received their start from the first thermal polyamino acids acting as enzymes in cells. Using statistical compilations that had been obtained in abundance from applications of the sequence analysis procedures (Fox, 1945), Ivanov and Ffrtsch (1986) and Ivanov and Ivanov (1991) traced into modem protein synthesis the inheritance of the primordial mechanism of the self-ordering of amino acids. This study protmses to enlarge also the understanding of modern protein synthesis as well as that of serving to integrate the evolutionary developments. A practical consequence of the fact that amino acids order themselves into polymers that have sharply limited heterogeneity is the blossoming of new industrial applications, including many in biomedicine, as manifested in water treatment (Sikes and Wheeler, 1991), in a symposium organized by Sikes (1994) on Polyammo Acids, The Emergence of Life, and Industrial Applications, and by numerous other biomedical applications such as improved drug deliver),, antiaging, nerve repair (Fox and Bahn, 1994), and many patents. ORIGIN OF THE CELL The two representations of the first cell that have dominated the textbooks and other expositions for more than thirty-five ),ears are the coacervate droplet and the proteinoid microsphere. Again, as Lehninger (1975) pointed out, the coacervate droplet is helpful 24 SIDNEYW. FOXET AL. as a model of a modem cell but, being made of polymers obtained from modem cells rather than from those of polymers that evidently retrace the protobiological type, they are not germane to the question posed by this symposium: the origin of thefirst cell. In using modem systems as a guide to the first system, Oparin was acting analytically in the manner that is characteristic of biologists who back-extrapolate from current biological research. Our approach differed in at least two important aspects: (a) we tested various possibilities for the first bioinformational beginnings (Fox, 1953) and found one (thermal protein) of which the forward evolution could be retraced, and (b) we found fortuitously in subsequently repeated experiments how the functionally pregnant precursors of the first cell could have given birth to the first cells in a maximally simple way appropriate for the primitive Earth (Fox, 1960). By now, numerous variations in morphology of the first cell (Figures 4-6), as exemplified by the proteinoid microsphere, are visible in numerous textbooks (Fox and Dose, 1977) and in the paper of Pappelis (1994) at this meeting, and by other authors. Additional variations are seen in Figures 7-10. Rohlfing (1975) has made microspheres from lysine rich proteinoid; these resemble coacervate droplets. Figure 4. Scanning electron nucrograph of centrifuged proteinoid microspheres. These are slightly under 2 microns in diam. Figure 5. Transmission electron micrograph of proteinoid microspheres and section of electron micrograph of Bacillus cereus. Right hand micrograph shows an assembly of microspheres after interior material has been allowed to diffuse out. The double layers are visible in the boundaries which are estx~ially lasting. Lower left-hand micrograph shows a section of proteinoid microsphere at the first stage of diffusion outward. Upper EXPERIMENTALRETRACEMENTOFTHEORIGINSOF A PROTOCELL 25 left-hand micrograph is a section of Bacillus cereus, from a journal. Figure 6. Proteinoid microspheres showing internal layering and various phases of binary fission. During the course of making thermal proteins and washing out the test tubes with water, Jean Kendrick and Fox (1988) observed that the tubes had a milky layer. Although not bacteriologists, we were in our laboratory doing amino acid assays by growth of Lactobacillus arabinosus (De Fontame and Fox, 1954). The milky layer suggested a suspension of bacteria. When examined under the microscope (Fox, 1988), components of the colloidal suspension indeed looked like bacteria, albeit a bit too perfect. Subsequently, we learned that the boundary is a double layer, that the laboratory protocells join and adhere in various ways, what functional properties the units have, and that variations m morphology resulted when the amino acid mixture subjected to heating is varied. We also identified some amino acid compositions that yield axon-like outgrowths from the systems, and that all of the cell-like structures tested generate electrical signals. The double layer (Figures 3,4,and 6) that appears so spontaneously in the microspheres is functionally membranous. VvNle the many catalytic activities of the proteinoids are much weaker than those in modern cells, the membrane selectivities and activities, such as cellular proliferation and electrical signalling of microspheres from proteinoid, appear to be more intense. Selective diffusion and related phenomena have been published (Fox et at, 1969). The probability that the first cell not only was, but looked much like, a proteinoid microsphere is heightened by the fact that artificial fossilization of the laboratory products (Francis et al, 1978) closely resemble, in limited variations, ancient microfossits from ancient strata (Berra, 1990; Fignre 7). 26 SIDNEYW. FOX ET AL. Figure 7. On the left: nested, vacuolated, and clustered microfossils published by micropaleontologists. On the right: photomicrographs of nested, vacuolated, and clustered proteinoid microspheres found to resemble microfossils after the latter were published. MEETING A DEFINITION OF LIFE If we are to understand how the first cell emerged from its evolutionary matrix, and if we are also to recognize the solution to this problem when it appears, it is helpful or obligatory to have a definition of life that can elicit general agreement. In the purely analytical phase of the history of biology, that has been difficult; some biologists have considered the construction of a definition to be futile. In recent years the sense of futility has been replaced by a wide, yet narrowing, range of definitions. Most notable is the fact that the definition m Webster's International Dictionary (Gove, 1966) is receiving serious attention. The Webster definition comprises functional aspects that are common to virtually all definitions. The definition in this source lists the characteristics of life as metabolism, growth, reproduction, and responsiveness to stimuli. If this definition is to be criticized, a mare objection is that Webster's definition does not include structural aspects of life such as cellularity and membranicity. These latter, however, can be regarded as implicit because cells and membranes are essential to the functional characteristics. On a personal level, perusal of the list of editors and consultants for Webster reveals a very. large number of eminent biologists and also a large number of eminent chemists but no comparable individuals who are expert in both functional biology and structural chemistry. The characteristics of life mentioned by Kornberg in his book on For The Love of En~mes (1989) comprise biofunctionality, growth, and reproduction. These are the same as three of the four attributes set forth in the definition of Webster. All four of these have been shown to be met by the proteinoid microsphere (Fox and Dose, 1977; Fox, 1988). At this meeting, Eirich (1994) has listed Webster's four characteristics and then has added as a fifth the quality of rhythmicity. We agree with this expansion of the definition, and would point out that rhythmicity m electrical activity has often been seen in microspheres (Fox, 1988, p. 177). Thus, even as the definition of a living microsystem displays variation from one author to another we see a common core of characteristics for all definitions and that the microsphere meets all of them. Moreover, it has become apparent that the most lasting EXPERIMENTALRETRACEMENTOF THE ORIGINSOF A PROTOCELL 27 definition will be that derived from the synthetic rather than the analytical approach, as in organic chemistry. The evidence points to the possible inference that the first cell had discernibly a greater list of attributes than later evolved cells which tended to be more specialized. It can be said that the unit that meets the definitions of life provides evidence of other attributes that can now be tested one at a time. On this basis, we can understand that textbooks such as that of Berra (1990) refer to this work as explaining by experimental retracement of evolution how the first cells came into existence; personal credit for the scientific observance has been designated to one author in the 1992 Who's Who (Canning and Marks, 1992) and later editions although this credit belongs to all of the names cited in this paper. THE PROTOCELL WAS ALSO A PROTONEURON Of all the defined characteristics of life in Webster and other dictionaries, the most significant may well be the responsiveness to stimuli. The salutary response is that of electrical signalling. The search for such activity m protemoid microspheres was suggested by the late H. Burr Steinbach of the University of Chicago, who also saw the possibility of relationships of chemical compositions of polymers in excitable cell membranes to their activity (Stembach, 1965). This type of study was begun in 1971 with Ishima (Ishima et al, 1981) and continued in collaboration with a series of electrophysiotogical experts. The protemoid cell membranes were seen to be excitable from the outset and, as discussed above, the molecular antenna in the polypeptides has been established as that of flavins and pterins. Worth emphasizing is that the same amino acids that enable the thermal copolymerization, aspartic acid and glutamic acid. are the ones that are known to be the center of excitability (Vaughan et al, 1987). The excitability in a proteinoid microsphere compared to that of the crayfish stretch receptor neuron is displayed in action potentials in numerous textbooks; the comparison was composed by Pol (Figure 8). The rhythmicity that Eirich (t994) spoke about here is shown in Figure 9. This is from a microsphere composed only of aspartic acid, glutamic acid, and arginine but, of the many thermal polymers tested, all have been found to contain the pigments and all to have electrical activity m the presence of small amounts of light (Fox, 1988; 1992). The nature of the reaction yielding ravin deserves further attention. 28 SIDNEY W. FOX ET AL. CRAYFISH lores i20mV 2:2:1 Figure 8. Action potentials from crayfish stretch receptor neuron (above) and from proteinoid microsphere (below). By Alexander Poi. i.v ~_ c ~qr,,-- "-" : ,Ta,,'~ '- Figure 9. Rhythmicity in electric discharge of microsphere of thermal poly (asp, glu, arg). In addition to the activity in all of the protoneuron retracements, the units have a considerable tendency to associate. The inherent tendency of the microsphere to form what looks like a primitive type of gap junction (Figure 10) has been often seen (Fox and Dose, 1977) in the lab and in textbooks. The more recently studied formation of dendritic associations (Figure 11) is related to the tyrosme content (Pappelis, this meeting). EXPERIMENTALRETRACEMENTOF THEORIGINSOF A PROTOCELL 29 Figure 10. Association of proteinoid microspheres. Stained with Crystal Violet. Suggests primitive gap junctions. A common phenomenon among microspheres. Figure 11. Dendritic associations between microspheres composed of thermal poly (asp,glu,leu,tyr). This effect is not as pervasive as that of Figure 10, and deserves more research. ORIGINS OF THE GENETIC CODING MECHANISM (MOLECULAR HEREDITY) The genesis of informational molecules prior to the cell is in its broad defmition genetic. Some definitions of life include power of heredity in the first cell. This inclusion in the definition may be arbitrary, i.e. the first cell could have had the functions of life without being able to pass those abilities on. We recognize however, that the proteinoid microsphere, or perhaps a somewhat specialized form of it, can extend those attributes into later generations. While the inherent presence of properties of life in a proteinoid microsphere were shown by 1971 (Fox) the ability to bequeath them to later generations was shown by 1981 (Fox). In 1973, Jungck and Fox demonstrated that lysine-rich proteinoids can catalyze the synthesis of oligonucleotides from ATP, whereas the direct production of peptides from oligonucteotides was not known. Nakashima and Fox (1980) reported that peptides can be made under the catalytic influence of the same lysine-rich proteinoids in a protobiotic mode. Syntheses of components of nucleotides from amino acids and of polynucleotides in the biotic era in cells has already been well charted (Zubay,1993). 30 SIDNEYW. FOXET AL. RANDOMNESS OR REALITY A widespread belief in a random matrix has been overturned by the experimental production of a cellular system that meets definitions of life; the reactions identified in the retracement of the origins of the first cell, as indicated above, have been definitely shown to be nonrandom. Numerous influential scientists have subscribed to the concept of random beginnings (see Tyagi and Ponnamperuma, 1990; Fox and Balm, 1994). Examples are those of Miller and Orgel (1974) who title chapters in part "Random Polymers" and refer to thermal proteins as "random polypeptides" (p.144), of Crick, 1981 (p. 83) who writes of the "accidental polymerization" that might have produced "proteinoid molecules', of Eigen (1986, p.25) who states that his theories have (belatedly) shown that the "landscape is not random', and De Duve (1991, p. 116) who refers to the ~random assembly process" of protein synthesis with "coding still to come'. THE MOLECULAR BASIS FOR BIODIVERSITY The impetus for the beginning (Fox, 1945) of the research reported here was a desire for the development and application of chemical methods for protein primary sequence determination that might explain the diversity and specificity of various biological forms and their biochemical components. In the course of the research indications have appeared of molecular bases for such diversity (Fig. 11). They are the result of varied compositions of amino acid in polymers that assemble in the various manners shown and in others. Conditions of formation of the microspheres play a secondary, role in the morphology. Evidently, aspartic acid and glutamic acid, which are two amino acids essential for the copolymerization and are also amino acids significant in the excitability of the proteinoid microspheres, play a role in the conjugation of microspheres. The formation of connections is aided by hydrophobic amino acids such as tryptophan. In Fig. 11 is seen the effect of a substantial proportion of tyrosine in the polymer. This type contributes to the formation of networks that can be found in the preparations, whereas dendricity tends to be absent when tyrosine is not included. It had long been known that a change in one amino acid in a chain of 576 residues in hemoglobin was sufficient to specify sickle-cell anemia in otherwise healthy hemoglobin (Ingram, 1957). It is our later studies that indicate that a single type of amino acid specifies protocellular morphology. BASIC STEREOCHEMISTRY DOES NOT NEED CHIRALITY Two mare kinds of sterex~hemistry relate to amino acids. The one most often considered is that of chiratity which distinguishes the enantiomorph of amino acids and other biochemical substances. One form is dominant in modern organisms. The other kind of stereochemistry is that which distinguishes one amino acid from the other by virtue of the sidechains. In examining the functions of chiral amino acids m thermal polymers in EXPERIMENTALRETRACEMENTOF THE ORIGINSOF A PROTOCELL 31 protobiogenesis, we do not see total prohibition of functions in polymers from racemic, or partly racemic, amino acids. The absence of single chiral types can be responsible for much lower activity in enzymic and hormonal polyamino acids but not for an absence of such function. The cytological activities such as reproduction and adhesion seem not to be impaired qualitatively by racemic components, however. The question of the origin of chirality need not be placed before that of the origin of life. Langenbeck (1955) is responsible for the concept that chirality developed during biotic evolution (Fox et al, 1956), an explanation extended by Bonner (1991). The other kind of stereochemistry, that of the sidechains was, however, extremely fundamental. It, plus deployment of charges in the molecules were the determinants of self-selection (self-ordering) in the selective thermal polymerizations of amino acids. A RECONCILIATORY RESOLUTION OF THE CHICKEN-EGG PROBLEM On looking back or, better yet, looking back to a starting point and then looking forward by retracement of evolution, we can see that the existence of the chicken-egg problem of which came first: protein or nucleic acid, can be explained. The two views can be reconciled. The problem is understood by a principle that is common to evolutionary biology in its widest span, that of rise-and-fall. When we survey the evolutionary life span of developments on the Earth we see rise-and-fall. We are acquainted with it in the rise-and-fall of the Roman empire, the Mayan empire, the Third Reich, etc. We even see rise-and-fall in the galaxies. We see rise-and-fall in the life of a single individual. Typically, a human grows into his twenties, after which he begins a long decline of stature; the generalization is confused by the fact that each of a number of minor riseand-fall progressions begins at a different stage. This can be explained in the context of biochemical and therefore biological evolution as the rise-and-fall of glucose. When this planet supported plants only, photosynthesis resulted in the formation of glucose and energy-rich derivatives for which the energy was obtained from the Sun. Later, when animals appeared in evolution, they broke down the compounds that had been synthesized by plants and released carbon dioxide and water, which had been the original reactants for the formation of energy-rich substances (Zubay, 1993). We may therefore infer that overall bioevolution itself is a progression of riseand-fall. When we adopt this precept we can see that the rise was in part the nonrandom formation of protein that resulted from amino acid self-ordering. In evolution, those proteins went on quickly to become ceils with, in some qualitative degree, all of the properties of proteinaceous cells as we know them. The formation of proteins led in the cell to another class of substance, DNA/RNA, as the progression headed into the falling phase. The retracement experiments reported hereto infer that the first phase of rise proceeded on the basis of the nonrandom synthesis of protein informed by its evolutionary precursors, a family of amino acids, and that in the falling phase these processes were reversed as in the overall evolution of sugar synthesis. The Central 32 SIDNEY W. FOX ET AL. Dogma mechanism as we know it came into existence. We need to look for further evidence; we can see already that the chicken-egg problem may be reconciled by an evolutionary rule of nature. EPILOG The process of experimental retracement of evolution to and from the first cell has generated salient overviews, o f which some are presented above. Very striking is one of the last papers in this program that features the views of Dyson as stated in the latter's book on The Origin of Life (1985). The views expressed in our paper are quite critical but central to why the scientific answer to how life began was so long in coming and was then slowly recognized. Like some other physicists and chemists who ignore or underrate biology, Dyson treats the problem with little attention to biological relevance. He does not explain how the first cell, the first unit of life, came into existence; in other words, his emphases are not cell-centered as are those of this conference. Secondly, he has virtually nothing to say about the biologists' understanding of functions of the cell. He is not alone. Eigen (1985), who would defer the cell to late in evolution, which is logical for a theorist who recognizes the complexity of the cell, has come finally to the view that precellular nature is nonrandom. While Dyson does correctly emphasize the somewhat heretical view of proteinfirst that has recently gained popularity, he allows young students of science to believe that that understanding arose from theoretical consideration rather than from empirical experimentation (Fox and Harada, 1960), such as has been throughout the history of science the hallmark of innovative awarenesses. In his preface, Dyson states, "In my survey of experiments and ideas I make no attempt to be complete or even to be fair. I apologize in advance to all the people, living and dead, whose contributions to knowledge I shall ignore, especially to J.B.S. Haldane, Desmond Bernat, Sidney Fox, Hyman Hartman, Philip Anderson and Stuart Kauffman." Because of the effect this can have on young students and others in promoting an underevaluation of empiricism, I for one reject Dyson's apology. One of the main lessons from the empirical experimentation is that the transition from precursor to first cell was almost instantaneous. As long as the transition was not identified, it was logical to assume that the production of the precursors and that of the cell consumed the millions of years that are sometimes assigned to it theoretically. Another new view is that complexity was maximal at the outset. Without some awareness of this through experiment it was logical to assume that complexity was built up through an association of simplicities. This now appears to have indeed been true for the chemical reactions in the rising phase until the first cell resulted; about that time EXPERIMENTALRETRACEMENTOFTHEORIGINSOFA PROTOCELL 33 complexity gave way to an assembly of specializations in what in part was a falling phase. But we must look to the future to understand this overview in full. Finally, the fact that this research has been largely under the auspices of an agency that wants to know about life in the Universe merits comment. An original objective in the synthetic phase of the research was to develop useful analogs of protein. Soon thereafter, the next objective was to understand how life began anywhere. The subject of life in the Universe has in general been fiddled with speculations and suppositions. This research program has told us some of what we know about potential evolution to life on our Moon (Yuasa and Oro, 1981). For the Solar System and especially beyond it, the research is bristling with hints about how to look for evolving life in its various stages and how to interpret the results. That body of potentials has become large enough that it deserves another review, if not a book, BRIEF SUMMARY Since the only life of which the origin merits realistic consideration is that of cellular life, this paper focusses on the two most widely examined models for the first celt. These are the coacervate droplet of Oparin and the proteinoid microsphere from our laboratory. Reasons for regarding the coacervate droplet as irrelevant to the .first cell are again expressed. The origins of proteinoid microspheres on the primitive Earth, the finding of characteristics of living systems therein, and the numerous spinoffs are discussed. ACKNOWLEDGEMENTS. The principal coworkers in the thirty-five (or fifty) year research of this review are listed as co-authors. The dominant proportion of these associates and generous supplies of equipment etc. have been financed by grants from NASA during the period 1960-1992, by NFCR. IBM, the Longevity Foundation, and a number of other sources cited in original papers. Inasmuch as studies of the origins of life have required administrative protection, special thanks are expressed to President Emeritus Henry. King Stanford of the University of Miami (1964-1981) and to Dean John Yopp of Southern Illinois University (1989-1993) for both protection and support. For his help in confirming and extending original observations for NASA, fundamental credit belongs to Dr. Richard S. Young. Helpful suggestions are acknowledged from many, such as Dr.H.B.Steinbach, and a PhD chairman T. H. Morgan. Critics have also been helpful. in addition to those included in the authorship line and regarded arbitrarily as major contributors to experiments and interpretations, other significant contributors to at least one paper each who should also be mentioned are: T. Adachi, D.E. Atkinson, G. Baumann, S. Brooke, F. Denes, R. Fiorovanti, T. Fukushima, J.R. Grote, P.E.Hare, F.Hefti, M-W. Ho, P. Hoagland, M. Ingrain, A. Khoury, E. Lederer, R.J. McCauley, P.O. Montgomery, G. Mueller, C. W. Pettinga, B.J. Price, J. W.Ryan, P. Saunders, A. W. Schwartz, W.D. Snyder, W. Stillwetl, W.P. Stratten, F. Suzuki, R. H. Syren, G. Vaughan, C.T. Wang, C. Warner, H. Wax, A.L. Weber, C.R. Windsor, M. Winitz, A. Wood, A. Yuki, S. Yuyama, and V. Zworykin. 34 SIDNEY W. FOX ET AL. It was o f course infeasible to check with all those listed for a p p r o v a l o f this paper, so a reader may not a s s u m e any o f them to be responsible for the integrated interpretations herein. Special thanks are h o w e v e r rendered to D r s . D . L. R o h l f m g and T . O. F o x for their critical r e v i e w o f the penultimate manuscript. T h a n k s are e x p r e s s e d to J e r r y D i x o n for careful f o r m a t t i n g and typing o f the manuscript. References Bergmann, M. and Fraenkel-Contat, H. 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W (1953) A correlation of observations suggesting a familial mode of molecular evolution as a concomitant of biologicalevolution.Amer. Naturalist 87, 253-256. Fox, S.W (1957) The chemical problem of spontaneous generation.J. Chem. Education 34, 472-479. Fox, S.W. (1959) Biologicalovertones of the thermal theory,of biochemical origins.Bull. Amer. Inst. Biot.Sc. 9, 20-23. Fox, S.W. (1960) How did life begin? Science 132, 200-208. Fox, S.W. (1971) Chemical origins of cells-2. Chem. Eng. News 49, 46-53. Fox, S.W. (1974) Aleksandr Ivanovich Oparin, Ency.ciopedia Britannica, 15th edition, pp. 577-578. Fox, $.W. (1981) A model for protocellular coordination of nucleic acid and protein syntheses. In M. Kageyama, K. Nakamura, T. Oshima, and T. Uchida (eda.) Science and Scientists. Japan So. Soc. Press, Tokyo. pp. 39-45. Fox, S.W. (1988) The Emergence of Life/Darwinian Evolution From the Inside. Basic Books. Fox, S.W. (1992) Thermal proteins in the first life and in the "mind-body" problem. 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(1994) Contribution of coenzym¢ molecules to the evolution of photoreceptors. This conference. Langenbeck, W. (1935)D/e Organischen Katatyzatoren. Springer, Berlin, p. 97. Lehninger, A.L. (1975) Biochemistry, Worth and Co,. New York. Miller. S. and Orgel, L. ~1974) The Origins of Life on the Earth, Premice-Hall. Nakashima, T. and Fox. S.W. (I980) Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid. J.Molec. Evolution 15, 161-168. Negron-Mendoza, A. (1994) Alfonso L. Herrera, a Mexican pioneer in the studies of the origin of life. This conference. 36 SIDNEY W. FOX ET AL. Oparin. A.I. (1957) The Origin of Life on the Earth. Academic Press. Oparin, A.I. (1966)/'he Origin and Early Development of Life (transl), Meditsina, Moscow. Orget, L. E. (1986) RNA catalysis and the origin of life. J. Theoret. Biol. 123, 127-149. Pappeiis, A. (1994) Domain prmolife: protocetls and metaprotocells within thermal protein matrices. This conference, p ~ m . p. 5. Rohtfing,D.L. 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Molec. Evolution 30, 391-399. Vaughan, G., Przybylski, A.T., and Fox, S.W. (1987) Thermal proteinoids as excitability-inducing materials. Bio~ystems 20, 219-223. Yuass, S. and Oro, J. (1981) HCN as a possible precursor of the amino acids in lunar samples. In M. Kageyama, K. Nakamura, T. Oshima, and T. Uchida (eds.) Science and Scientists Iapan So. Soc. Press, Tokyo, pp. 31-37. Zubay, G. (1993) Biochemistry, third edition, Win. C.Brown, Publ.