Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14229–14232, December 1996 Colloquium Paper

This paper was presented at a colloquium entitled ‘‘Symmetries Throughout the Sciences,’’ organized by Ernest M. Henley, held May 11–12, 1996, at the National Academy of Sciences in Irvine, CA.

Symmetries throughout organic evolution

ANTONIO GARCI´A-BELLIDO

Centro de Biologı´a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´noma de Madrid, Cantoblanco, 28049-Madrid, Spain

ABSTRACT The biological realm has inherited symme- tions are similarly dextrorotatory. We now know that the tries from the physicochemical realm, but with the increasing prevalence of D-isomers is due to the asymmetry of the active complexity at higher phenomenological levels of life, some catalytic sites of the enzymes that recognize saccharides for inherited symmetries are broken while novel symmetries both their anabolism and catabolism. The reason for the appear. These symmetries are of two types, structural and prevalence of saccharide D-isomers and their derivatives (tar- operational. Biological novelties result from breaking opera- taric acid is one) may be that the corresponding metabolic tional symmetries. They are followed by acquisition of regu- enzymes are encoded by genes all evolutionarily descended larity and stability, in a recurrent process throughout com- from one original prototype. The same may hold for the plexity levels. prevalence of L-phospholipids that are metabolized by many different types of phospholipases. From the predominance of The concept of symmetry implies conservation of shape upon D- over L-isomers of saccharides and of L-phospholipids, we rotation or of isomorphic transformation in interactions, re- learn two important lessons about biological evolution. Ones taining one or several parameters invariant in the transforma- in that molecular recognition provides a profound inertia to tion. These attributes correspond both to topological and evolutionary innovation because it demands conservation of operational properties in the stability and evolution of func- forms of proteins and hence of the encoding genes. The other tional structures. In biology the counterpart of these notions is that the contingent origins of saccharide metabolism am- has synchronic (e.g., anatomic or physiologic) as well as plified asymmetries in the abundance of equally probable, diachronic (e.g., developmental and evolutionary) connota- energetically equivalent, isomers. tions. In dealing with these two connotations, I will try to The same lessons can be draw from the prevalence of L- over distinguish between topological and operational symmetries, D-amino acid isomers in biomolecules. Enzymes involved in being aware that the use of these terms in the context of amino acid metabolism may have derived from a common biology extends beyond the more precise meaning in the prototype that prevailed in the competition between isomeric context of physics. Whereas the notion of topological symme- forms in the primordial organic soup. The consequences were try in biological forms is well documented, that of operational everlasting, since the form of all the proteins whose function symmetry has been, to my knowledge, hardly considered. As in requires amino acid recognition must be complementary to the physics, also in biology, devolving interactions generate breaks form of the L-isomers. Moreover, the helical rotation of long of symmetry. Herein I will discuss briefly a few instances of helical polypeptide chains is derived from the tilt of peptide symmetries and its breaks in biology that have played a central bonds between L-amino acid residues. And that configuration role in organic evolution. is, in turn, basic to many of the elastic properties of proteins Biology deals with forms and their transformations: molec- in structural and functional roles. ular, cellular, histological, organismal, and ecological. Its Similarly, the two polynucleotide chains in the double- structures are grounded in the physical realm and its energetic helical DNA molecule have a clockwise axial rotation based on transactions in the chemical one. In this ascent throughout the tilt imposed by the staggering of successive nucleotides. In complexity levels, we cannot discriminate between historical this case, the degrees of freedom of this rotation are greater contingency and strict causal determination, although the than those available to polypeptides chains because the DNA former seems to play a major role with increasing complexity. molecule can have an opposite rotational torque under phys- Organic evolution devolves from combinatorial propositions iological conditions. The primary helicity of the DNA is a that happened to succeed, i.e., were stable and stayed around consequence of its mode of generation and causes its structural for us to observe and categorize. stability that carries on its higher-order organization allowing for protein recognition sites, complex replication and tran- Structural or Topological Symmetries scription mechanisms, and tertiary folding into chromosomes for cytokinesis. The linear sequence of nucleotide residues in The formation of anisodiametric molecules by the bonding of DNA double strands is on the other hand, constrained by isodiametric atoms is the first symmetry break that gives rise purine–pyrimidine base complementarity required for the to fundamentally asymmetric biological structures. stability of the double helix. But beyond this, the order of The first example of a biologically caused break of molecular nucleotides can in principle be arbitrary. The same freedom of symmetry paradigmatic for many biological structures was nucleotide sequence holds for single-stranded RNA molecules. discovered by Pasteur. Whereas molecules of tartaric acid, can However, in RNA molecules, a new symmetry appears in exist as two optically active, dextro (D) or levo (L) rotatory palindromic nucleotide sequences, at which the single RNA isomers, yeast cells can only metabolize the D-isomers. It so strand folds back on itself by nucleotide base complementarity; happens that all saccharides in biological structures and reac- pins and four-leafed configurations due to palindromic con- figurations are responsible for the stability and molecular The publication costs of this article were defrayed in part by page charge recognition features of RNA molecules. They correspond to payment. This article must therefore be hereby marked ‘‘advertisement’’ in emerging symmetries with profound biological implications. It accordance with 18 U.S.C. §1734 solely to indicate this fact. is precisely these secondary interactions of biological macro-

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molecules (polypeptides and polynucleotides) that have frozen At the next higher or organismic level of biological com- their structural symmetry. plexity, we encounter creatures with bilateral, radial, or helical At the next higher level of biological complexity, we have symmetry. As a rule, bilaterally and helically symmetrical cell organelles and cells lipid–protein membranes, which sep- organisms are motile (swimming or walking) creatures, while arate internal from external aqueous phases and generate in radial symmetry is more often associated with sessile forms. principle isodiametric spheres with minimal surface energies. These symmetries are due to the orientation of the mitotic However, this symmetry is broken by the appearance of planes of the first zygotic divisions, which in turn may be anisotropies. Protista are polarized due to an asymmetric governed by an intrinsic polarity of the egg or by the entry point distribution of cytoskeletal proteins defining an anterior– of the sperm, thus defining an anterior–posterior axis and͞or posterior axis, at the end of which in flagella (in bacteria) or a dorsal–ventral axis of symmetry. Subsequent cell divisions budding zones (in fungi and yeast) distinguish heads from tails. amplify these symmetries by iteration and linear segmentation. This head-to-tail polarity is defined by the intrinsic polarity of Radial cell cleavage gives rise to metamerism along the actin-like helical proteins or by the self-assembly of asymmet- circumference (as in polyps) while cleavage along two orthog- ric proteins complexes. Polarity based on molecular anisotropy onal axes gives rise to bilateral forms. Helical symmetry (as in and molecular recognition has template properties: original molluscs) results from modified radial cell cleavage retaining polarities guide the formation of subsequent ones. At the the helical staggering of successive zygotic divisions along the organelle level head–tail polarity retaining rotational symme- anterior–posterior axis. These symmetries must reflect simple try is a common feature of virus capsids. They result from the structural conditionants because they are easily modified by packing, by molecular matching, of anisotropic proteins, as in mutation. Helical forms of gastropods may have a clockwise or phage particles, with very few steric degrees of freedom. anticlockwise rotation different in closely related species and Alternatively, packing of identical anisotropic proteins gives mutation of particular genes can change the helical polarity of rise to tubes with helical rotational (left or right handed) the offspring. Bilaterally symmetrical organisms may have symmetry, as in virus capsids or flagella. secondary asymmetries in the whole animal, as in flat fishes, The next higher level of complexity is the histological, at or in certain organs, like the heart or the genitalia. In this which asymmetries appear in cells forming part of monolay- example, too, gene mutations are known that reverse the ered tissues. In this example, in addition to head–tail (apical– secondary asymmetry or give rise to a random asymmetry. basal aspects of cells), new left–right (or medial–lateral) and Moreover, radially or helically (strobilar) symmetrical organi- anterior–posterior polarities produce the overall shape of zation in animals and plants can be modified by the secondary epithelia through molecular heterogeneities in the cell mem- appearance of bilaterality. We are beginning to understand the brane. In such epithelial cells, the apical–basal polarity is underlying genetic mechanisms in plants. directly observable under the microscope. Other axial asym- In actual segmented organisms, there are usually local metries are manifest by cell behavior in transplantation or cell heterogeneities in otherwise homologously iterated dissociation-reaggregation experiments. We still do not know metameres. These heterotopies can lead to extreme differ- how these anisotropies are molecularly represented in the cell ences in arthropods. As in the case of cell patterning, these membrane but genetic and biochemical arguments suggest that heterotopies result from local genetic differences in gene they are due to differences in the molecular composition and expression patterns. These patterns correspond to members of molecular recognition of outer membranes of neighboring genes families conserved since the origin of bilateria. cells. Thus, it appears that symmetries and asymmetries in bio- Cell differentiation patterns break the monotonic symme- logical structures up to the organismal level devolve from tries of epithelia. These diversifications, such as the appear- molecular structures that compound their symmetry or assym- ance of sensory elements in epidermis, occur by the differential metry by developmental template mechanisms. The increasing action of genes responding to heterogeneities in cell prolifer- complexity and degrees of freedom of the implementation ation of the epithelium. The observable heterogeneities pos- mechanisms allows for new symmetries from asymmetries or sibly result from local modifications (elimination or duplica- for the generation of novel asymmetries. tion) to regular patterns in evolutionary origin. Appendages such as arthropod limbs result from outgrowths Generative or Operational Symmetries at fixed positions in the two-dimensional monolayers of the integument. Cell proliferation at these positions leads to two Operational asymmetries though less explored, are more in- and a half dimensional monolayers of cells that retain their teresting than structural asymmetries because they have driven anterior–posterior and dorsal–ventral axial polarities in addi- physiological and morphogenetic evolution. tion a novel secondary or proximal–distal polarity. Growth Chemical reactions are reversible and, hence, operationally occurs by cell proliferation throughout the anlage generating symmetrical. Enzyme͞proteins (as well as colloids and other heterogeneities along those two (planar) axes. Similarly local polyionic substrates) can accelerate these reactions, they still evaginations and invaginations give rise to two one͞two- remaining symmetrical. The symmetry is broken, however, in dimensional tissue layers (as in gastrulation), which, in turn, enzymatic tandem reactions that operate far from equilibrium, gives rise to the three-dimensional organization of the embryo. when the reactants include a steady source of free energy or Hence, the morphology of the adult organism is derived mainly the reaction products themselves serve as reactants for further from two and a half dimensional tissue layers. reactions leading to accumulation or use of end products. True three-dimensional structures arise from delamination Directional reactions are at the root of biological syntheses. of cells perpendicularly to a planar cell sheet surface or from They locally concentrate free energy into chemical bonds for apposition of migrating cells of separate origins. For example, further use. the former occurs in the generation of the nervous system (as We do not know how the coupling of DNA, RNA, and in the cerebellum), and the latter occurs in the aggregation and protein first operated in evolution; we can only envisage condensation of mesodermal cells to form muscles or bones. possible scenarios. One such is that it began as a reinforcing That means that the apical–basal axis acquires differential loop in which improved enzymatic efficiency concentrated recognition specificities, manifest in the cell membrane, lead- more energy and, hence, lead to more efficient reactions. ing to cell sorting and apposition. These mechanisms represent Possibly the driving operation improving these energetic trans- paradigmatic cases of how far recognition of molecular an- actions was the possibility of self-replication, a spontaneous isotropy leads to the configuration of two one͞two- and property of RNA molecules. These molecules are capable of three-dimensional organs and body plans. mutating and self-splicing and thus of selecting themselves for Downloaded by guest on October 3, 2021 Colloquium Paper: Garcı´a-Bellido Proc. Natl. Acad. Sci. USA 93 (1996) 14231

higher replication rates. Nucleotide substitutions in RNA already been solved. Since then the expenditure of free energy molecules represent isomeric alternatives, structurally and in driving chemical reactions is minimal because all the energetically equivalent, but from the replication perspective, intermediate steps catalyzed by particular enzymes have al- represent new mutational propositions for further efficiency, ready been tested and selected for maximal efficiency. Organ- leading to further molecular diversity. Since RNA molecules isms can now deal with multiple sources of energy. The act, in addition, as catalysts of extrinsic reactions, i.e., as outcome of this wealth was the explosion in morphological enzymes, the end result of the process is more of the same, the diversity. The main limitations to diversity are internal gen- reaction going in the direction of increasing synthesis. Through erative ones. Multicellular diploblastic metazoans gave rise at primogenial translation, i.e., transferring sequence specificity the beginning of the Cambrian (580 million years ago) to to amino acid coupling in peptide bonds, proteins may have triploblastic metazoa whose three blastoderm layers allow for appeared with the capability of helping RNA synthesis itself, a variety of internal tissues and organs, a supporting skeleton, and vice versa, certain RNA sequences acting as codons sensory and motor nervous systems, specialized sexual organs, (tRNA) for specific amino acid recognition. A new contingent and sexual dimorphisms. In addition, segmentation, i.e., iter- event may have helped to generate molecular diversity–and ation of metameric modules, did allow body-size increase and through it competition and higher efficiency in energy trans- longitudinal specialization. Appendages provided for locomo- action along with further biosynthesis. tory, bucal respiratory, and sensory organs. Epithelial special- Upon the evolutionary invention of reverse transcription of izations gave rise to defense spines and plates and digestive RNA into DNA, the informational stability, through replica- organs. Within 50 million years, the Cambrian explosion led to tion fidelity was ensured. The transformational sequence of the appearance of the major extant phyla: platyhelminths, DNA 3 RNA 3 protein represents a magic loop that pro- arthropods, annelids, molluscs, echinoderms, and chordates. vided for the efficient use and amplification of the reactants, There also arose some groups with different body plans that in the primordial soup. Since the speed of multimolecular have since become extinct. reactions is strongly dependent on the concentration of the Molecular comparisons of genes performing similar func- reactants, their structural association became an obvious step tions in extant organisms (animals or plants) of diverse taxa- in increasing efficiency. Thus, the membranes of archebacteria nomic groups reveals a surprising degree of nucleotide se- may have arisen to enclose loci of association of the reactants quence conservation from the Cambrian ancestors. Thus, we involved in the extraction of free energy from incident photons may ask, which novel genetic operations and which symmetry in photosynthesis or from debris scavenged in the primordial breaks can account for the Cambrian explosion of morpho- soup to self-replicating structures. logical diversity modes? Molecular and developmental genetic In this imaginary process, symmetries become broken. analysis in a few organisms has uncovered a modular nature of Changes in the linear sequences of nucleotides in RNA and the structure of genes, of their functional interactions within DNA, of amino acid residues in proteins, and of complemen- cells, and of the morphologies to which they give rise. This tary base pairing in double stranded DNA generate diversity. modularity is helping us to understand morphogenetic evolu- But mutation, a new source of variation, appeared early in tion. Genes contain in their protein-coding regions amino acid evolution. Mutation is a simple isomeric substitution of nu- sequence motifs that upon translation correspond to protein cleotides, with enormous implications. The genetic repository folds or structural domains. These domains serve particular of different nucleotide sequences related to amino acid se- types of molecular recognition in enzymatic reactions with quences via the genetic code could linearly combine to gen- substrates or between proteins or between proteins and cis- erate an exploding diversity of stereospecific molecular forms. regulatory DNA sequences of genes (consisting of several These associations represent an increase in degrees of com- short polynucleotide motifs that condition its transcription). It plexity at, on, and above the molecular level. In operational so happens that there is only a limited number of protein terms, isomorphism between nucleotide sequences in DNA domains, on the order of 1000–2000 domains, and hence of and RNA and amino acid sequences in polypeptide chains is molecular recognition features. These domains remain con- a symmetrical phenomenon. The transformation is based on a served in evolution possibly because mismatches between universal genetic code. As such it is stable, although we do not protein partners in their transaction leads to failures in func- know whether that stability results from an inertia based on tion. This necessity for conservation represents a large inertial once contingently acquired stereospecific matching main- component of evolution. tained because the consequences of changing it would be One way to generate diversity in the face of this inertia is to largely deleterious. duplicate the gene modules, followed by slight modification of The most important symmetry break in biology is the their individual structure and secondary modulation of their contingent consequence of mutation because it led to diversity function. Another way is to reshuffle these modules (exons) and higher complexity with more efficient ways of replicating and generate chimeric transcriptional units giving rise to faithfully and capturing more surrounding available energy. complex proteins. In addition, reshuffling of cis-acting pro- Thus, photosynthesis in biosynthesis and the oxidation– moter regions of a gene can bring it under the control of novel reduction processes in metabolism were efficient conse- trans-acting regulatory protein. While gene duplication fol- quences in prokaryotic cells. Eukaryotic cells appeared as lowed by mutation can give rise to diversity in the number of compounds of three prokaryotic symbionts, some reduced to related functions of the original protein, differential splicing of cell nuclei, others reduced to plastids for photosynthesis or to exons encoded in the same gene can give rise to several mitochondria for oxidative-reductive chemical reactions. This different proteins. Thus, a large fraction of the vertebrate prokaryotic symbiosis represents another instance of symme- genome encode for numerous (up to a thousand) members of try breaking, which leads to a further increase in the phenom- the same protein family. Yet the total genome complexity has enological levels of complexity. The origin of multicellular increased very little in number of genes, from about 4000–5000 eukaryota lies in yet another symmetry break that leads to a in bacteria, to 6000 in yeast, to 15,000–18,000 in nematodes higher level of biological complexity. By comparison the and insects, and about 60,000 in primates. The apparent subsequent appearance of a large diversity of multiple metazoa increase in morphological complexity from monocellular eu- and metaphyta is a trivial step representing combinatorial karyotes to metazoa was attended by a less than 10-fold variations of conserved generative mechanisms. increase of magnitude in the number of genes. When 700 million years ago evolution put multicellular High mutation rates, attributable to both nucleotide substi- organisms on the scene, the fundamental problems of energy tutions and DNA segment rearrangement, such as transloca- fixation and transaction, metabolism, and biosynthesis had tions and a gene conversion, provide a continuous source of Downloaded by guest on October 3, 2021 14232 Colloquium Paper: Garcı´a-Bellido Proc. Natl. Acad. Sci. USA 93 (1996)

novel genetic propositions for evolution. The viability of these protostomia). These ‘‘selector’’ genes implement topographi- propositions is limited only by their immediate phenotypic cal differentiation by locally controlling syntagmata of histod- effects. A dysfunctional type of mutation in the protein-coding ifferentiation genes. Cell groups become isolated in modular regions may set off a rapid selection for a compensatory entities (developmental compartments) associated with the mutation in that protein’s interacting partner (so called co- specific expression of those territorial (‘‘selectors’’) genes in all evolution). A mutation in the regulatory region of a gene, in their cells. These cell groups are polyclonal in origin and contrast, resolves in the disappearance of one phenotypic become established making use of specific cell recognition character in the cell lineage in which the gene is normally labels, adhesion modules of complementarily matching spe- acting; the appearance by mutation of a new short sequence in cific ligands and receptors (selector dependent) of neighboring the regulatory region of a gene may result in the appearance cells. of a new phenotypic character in a new cell lineage or at later Cell proliferation within modules is indeterminate but the branchings of it. The later types of genetic changes leave cells cannot cross module borders, which later correspond to overall development unperturbed but provide an opportunity segmental or subsegmental entities in the adult patterns. Cell for the appearance of large morphogenetic novelties. The lineage borders become major organizative entities. Exchange latter type of genetic changes surely constitutes the most of signals at either side of these borders drive differential gene important driving operation in organic evolution. activity and the release of growth factors (acting as diffussible Morphological diversity devolves from combinatorial asso- morphogens or as ligands) operating on the receptors of next ciations of a few genes acting in discrete groups in cells, along cells in transmission cascades. Thus, the territorial specifica- cell lineages in development. Combinations of modular ele- tion of cells by ‘‘selector’’ genes is implemented in cell behavior ments is the basis for the formation of protein complexes acting and hence in developmental operations. Following further cell as stable cell structures (such as the cytoskeleton) or as proliferation, a new subdivision of territories appear, each ‘‘agglomerates’’ (such as ribosomes and transcription and specified by new selector genes. In this way, genetic specifi- replication complexes). Because of the constraints imposed by cation of modules results from combinations of syntagmata molecular recognition, these complexes remained largely in- operating in the same cells. Segmental or periodic diversity, variant in the course of evolution. Transient associations already recognizable in Cambrian taxa, must have used these between genes and proteins are combinatorial devices, assign- genetic operations because the selector genes involved are ing several formerly independent acting genes to one working present in the derived forms of common ancestors. Local team (‘‘syntagma’’) by subjecting them to the control of the modifications of these genetic–developmental operations, fun- same regulatory genes operating on their common DNA damentally by combinatorial displacements, have since di- promoter regions. Individual syntagmata are conserved func- rected the currently observable morphological diversity. Their tional entities, modules, performing the same genetic opera- tion throughout evolution. The DNA motifs of these common association and dissociation of syntagmata in cells of different promoter regions can be several in number, thus exposing a cell lineages determined the fantastic morphological diversity single gene to several controlling genes. Cell proliferation of the Cambrian explosion. Hence, the extant morphological during development allows novel uses of these syntagmata in world is then the result of combinatorial diversification of a different cell lineages (positions) and at different developmen- few, functionally successful, molecular interactions. tal times, while always retaining the same basic molecular The evolution across several levels of complexity from atoms recognition specificity. A given cell of a particular lineage may to molecules, to cells, to organs, and to organisms is an iterated thus be under the control of several active syntagmata. Cell– symmetry break. And possibly new breaks of symmetry un- cell interactions may induce those syntagmata and hence derlie the emergence of new organic properties like the differential cell behavior as defined by combinatorials of organization of neurons in brains, behavior of animals con- syntagmata. forming societies, or groups of species interacting in ecosys- The genetic operations defined by active syntagmata corre- tems. It is a mystery how the increase in complexity levels spond to functional modules in developmental operations. occurred, what the driving force of organic evolution was, and Thus, cell division, cell differentiation, cell adhesion, cell in particular how amplification in morphological diversity recognition, signal transduction, nerve growth guidance, and happened to arise. The Cambrian explosion took place within even sexual dimorphism are the work of syntagmata. In a only 20–50 million years and in a rather homogeneous marine growing number of cases, we find that members of syntagmata world. Selection from or adaptation to new niches seems to have remained the same since Cambrian times. have been of little importance. Rather it seems that the Cambrian organisms devised supracellular modules, seg- generation of new morphologies through new genetic associ- ments, and developmental compartments. The locally differ- ations, i.e., the propositional aspect of selection, may have entiated properties of body parts along the anterior–posterior played the dominant role. Since molecular recognition en- and the dorsal–ventral axes as well as features of organs (such coded by genes appears to override physical or external as heart and eye) are now known to be controlled by members specifications. Organisms seem to be more dependent on their of gene families that have been conserved from the ancestors generative mechanisms than on the external morphologies to of the modern triploblast grade (including both deutero- and which they give rise. Downloaded by guest on October 3, 2021