Question 7: Biosynthesis of Phosphatidic Acid in Liposome Compartments – Toward the Self-Reproduction of Minimal Cells

Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive ‘synthetic cells (minimal cells)’ approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes – like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Question 7: Construction of a Semi-Synthetic Minimal Cell: A Model for Early Living Cells

Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named “Puresystem” (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30–20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

An Interpretive Review of the Origin of Life Research

Life appears to be a natural property of matter, but the problem of its origin only arose after early scientists refuted continuous spontaneous generation. There is no chance of life arising ‘all at once’, we need the standard scientific incremental explanation with large numbers of small steps, an approach used in both physical and evolutionary sciences. The necessity for considering both theoretical and experimental approaches is emphasized. After describing basic principles that are available (including the Darwin-Eigen cycle), the search for origins is considered under four main themes. These are the RNA-world hypothesis; potential intermediates between an RNA-world and a modern world via the evolution of protein synthesis and then of DNA; possible alternatives to an RNA-world; and finally the earliest stages from the simple prebiotic systems to RNA. The triplicase/proto-ribosome theory for the origin of the ribosome is discussed where triples of nucleotides are added to a replicating RNA, with the origin of a triplet code well-before protein synthesis begins. The length of the code is suggested to arise from the early development of a ratchet mechanism that overcomes the problem of continued processivity of an RNA-based RNA-polymerase. It is probable that there were precursor stages to RNA with simpler sugars, or just two nucleotides, but we do not yet know of any better alternatives to RNA that were likely to arise naturally. For prebiotic stages (before RNA) a flow-reactor model is suggested to solve metabolism, energy gradients, and compartmentation simultaneously – thus the intense interest in some form of flow reactor. If an autocatalytic cycle could arise in such a system we would be major steps ahead. The most likely physical conditions for the origin of life require further clarification and it is still unclear whether the origin of life is more of an entropy (information) problem (and therefore high temperatures would be detrimental), rather than a kinetic problem (where high temperatures may be advantageous).     

Question 6: Early Steps of Evolution and Some Ideas About a Simplified Translational Machinery

Here, the undisputed steps in the beginning of life on earth are compiled, before a retrograde approach is presented outlining a possible minimal set of components required for protein synthesis, based on our knowledge of modern translational apparatus of Escherichia coli. Presented at: International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Question 7: Comparative Genomics and Early Cell Evolution: A Cautionary Methodological Note

Inventories of the gene content of the last common ancestor (LCA), i.e., the cenancestor, include sequences that may have undergone horizontal transfer events, as well as sequences that have originated in different pre-cenancestral epochs. However, the universal distribution of highly conserved genes involved in RNA metabolism provide insights into early stages of cell evolution during which RNA played a much more conspicuous biological role, and is consistent with the hypothesis that extant living systems were preceded by an RNA/protein world. Insights into the traits of primitive entities from which the LCA evolved may be derived from the analysis of paralogous gene families, including those formed by sequences that resulted from internal elongation events. Three major types of paralogous gene families can be recognized. The importance of this grouping for understanding the traits of early cells is discussed. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

A more detailed seasonal division of the energy balance and the protein balance of Japanese macaques (<Emphasis Type="Italic">Macaca fuscata</Emphasis>) on Kinkazan Island,northern Japan

Nakagawa (Am J Primatol 41:267–288, 1997) reported that both the gross energy and gross protein intakes of an adult female Japanese macaque (Macaca fuscata) on Kinkazan Island, northern Japan, were high in spring (March–May) and fall (September–November) and low in summer (June–August) and winter (December–February), and that these values reflected the seasonal differences in nutritional conditions (defined as whether the intakes of energy and protein satisfy the requirements). We estimated the energy balance (energy intake minus its expenditure) and the protein balance (protein intake minus its requirement) of the monkeys on Kinkazan Island every month over the course of 1 year (2004–2005) in order to verify Nakagawa’s conclusions. Like Nakagawa, we found that the energy balance of the monkeys in the fall was higher than in the summer and winter, whereas the protein balance in the fall was higher than in the winter. However, we did not find that spring energy and protein values were greater than summer and winter values. We also did not find that summer protein values were low. Both the energy balance and the protein balance changed rapidly within the same season. The energy intakes and the energy balances were higher in mid-spring and mid- and late fall and lower in late spring and early summer, whereas the protein intakes and the protein balances were higher in mid-spring and mid-summer and lower in early and mid-winter. Since Japanese macaques respond to seasonal changes in food supply by changing their foods, continuous data collection with short intervals is recommended in order to accurately document the energy and protein balances of the monkeys.     

Question 2: Relation of Panspermia-Hypothesis to Astrobiology

In the answer to major questions of astrobiology and chirality, the panspermia-hypothesis is often discussed as the only proposal of transportation of life to the Earth. On the basis of the known presence of ionizing radiation in the space, assumed on the level calculated by Clark (Orig Life Evol Biosph 31:185–197, 2001), the hypothesis is rejected as the explanation of origins of life on Earth. In fact, comparatively low doses of radiation sterilize irreversibly all biological material. Sufficiently long sojourn in space of objects containing prebiotic chemical blocks also does not contribute to the origins of life on Earth, because of elimination of homochirality, if any, and of radiation induced reactions of dehydrogenation, decarboxylation and deamination of chemical compounds closing with complete decomposition of organics, leaving elementary nano-carbon and/or minerals like calcium carbonate. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Question 5: On the Chemical Reality of the RNA World

The discovery of catalytic RNA has revolutionised modern molecular biology and bears important implications for the origin of Life research. Catalytic RNA, in particular self-replicating RNA, prompted the hypothesis of an early “RNA world” where RNA molecules played all major roles such information storage and catalysis. The actual role of RNA as primary actor in the origin of life has been under debate for a long time, with a particular emphasis on possible pathways to the prebiotic synthesis of mononucleotides; their polymerization and the possibility of spontaneous emergence of catalytic RNAs synthesised under plausible prebiotic conditions. However, little emphasis has been put on the chemical reality of an RNA world; in particular concerning the chemical constrains that such scenario should have met to be feasible. This paper intends to address those concerns with regard to the achievement of high local RNA molecules concentration and the aetiology of unique sequence under plausible prebiotic conditions. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Question 7: Modelling Minimal ‘Lipid-Peptide’ Cells

This contribution is aimed to give support to ‘bottom-up’ approaches to the minimal or early cell research project. Even if, from this perspective, the most simple living cell still seems very far away, the analysis of less complex, infrabiological cellular systems (some of which could be relatively soon implemented in the lab) probably holds the key, or one of the fundamental keys, to the problem of origins of life. On these lines, we propose a simulation model to study the transition from proto-metabolic ‘lipid’ cells to ‘lipid-peptide’ cells, as a critical step in which self-reproducing vesicles could develop into more functionalized supramolecular systems Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Developmental Plasticity of Postweanling Cotton Rats (Sigmodon hispidus) as an Adaptation to Nutritionally Stochastic Environments

Elucidating interrelationships between rate of growth and sexual maturation in unpredictable or stochastic environments could increase our understanding of life-history strategies of small mammals. It has been hypothesized that species living in environments where food availability is unpredictable might become sexually mature at smaller sizes and channel excess energy into reproduction rather than into compensatory growth. We explored this hypothesis in female cotton rats (Sigmodon hispidus) by feeding variable levels of dietary protein during early postweanling development (14–45days of age) and monitoring compensatory growth and fitness after nutritional rehabilitation (45–100days of age). Growth was optimum in females fed diets containing 16% protein, with minimal requirements estimated to be 12%. Females fed diets containing <12% protein exhibited suppressed development, including delayed puberty. However, these nulliparous females demonstrated compensatory growth during the early period of nutritional rehabilitation, regardless of the severity of previous restrictions in protein. No long-lasting fitness consequences from postweanling nutritional restrictions were apparent as we observed no difference in date of conception, body mass of dams at parturition, litter size, or rate of growth of neonates. We offer a possible adaptive explanation for this observed plasticity in growth and development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Question 7: The First Units of Life Were Not Simple Cells

Five common assumptions about the first cells are challenged by the pre-biotic ecology model and are replaced by the following propositions: firstly, early cells were more complex, more varied and had a greater diversity of constituents than modern cells; secondly, the complexity of a cell is not related to the number of genes it contains, indeed, modern bacteria are as complex as eukaryotes; thirdly, the unit of early life was an ‘ecosystem’ rather than a ‘cell’; fourthly, the early cell needed no genes at all; fifthly, early life depended on non-covalent associations and on catalysts that were not confined to specific reactions. We present here the outlines of a theory that connects findings about modern bacteria with speculations about their origins. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Necessity,Futility and the Possibility of Defining Life are all Embedded in its Origin as a Punctuated-gradualism

The criteria used for defining life are influenced by various philosophical visions about life, ranging from holism to reductionism and from mechanistic-reductionism to vitalism. Using different scenarios about the origin and evolution of life as well as properties of energy-dissipative systems, artificial life simulations and basic tenets of xenobiology, guidelines can be established for formulating a definition of life. A definition of life is proposed that is parametric, non-Earth-centric, quantitative and capable of discriminating ‘living entities’ from ‘life’. Living entities are defined as self-maintained systems, capable of adaptive evolution individually, collectively or as a line of descend. Life is a broader concept indicating that the capacity to express these attributes is either virtual or actual. At least four major phase transitions can be recognized during the origin of life (reflexive activity; self-regulated homeostasis; the advent of informatons and the origin of adaptive evolution); these make the origin and evolution of early life an example of ‘punctuated gradualism’. Such phase transitions can be used to identify a boundary in early evolution where life began. This contribution identifies the step in the evolution of a dynamic system when digital control of the system’s state becomes dominant over analogical control, and genetic information is irreversibly used for adaptive evolution, as the boundary between non-living and living systems.     

Question 9: Prospects for the Construction of Artificial Cells or Protocells

The construction of artificial cells or protocells that are a simplified version of contemporary cells will have implications for both the understanding of the origins of cellular Life and the design of “cell-like” chemical factories. In this short communication, we discuss the progress and remaining issues related to the construction of protocells from metabolic products. We further outline the de novo design of a simple chemical system that mimics the functional properties of a living cell without being composed of molecules of biological origin, thereby addressing issues related to Life’s origins. Presented at: International School of Complexity—4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Relics from the RNA World

An RNA world is widely accepted as a probable stage in the early evolution of life. Two implications are that proteins have gradually replaced RNA as the main biological catalysts and that RNA has not taken on any major de novo catalytic function after the evolution of protein synthesis, that is, there is an essentially irreversible series of steps RNA → RNP → protein. This transition, as expected from a consideration of catalytic perfection, is essentially complete for reactions when the substrates are small molecules. Based on these principles we derive criteria for identifying RNAs in modern organisms that are relics from the RNA world and then examine the function and phylogenetic distribution of RNA for such remnants of the RNA world. This allows an estimate of the minimum complexity of the last ribo-organism—the stage just preceding the advent of genetically encoded protein synthesis. Despite the constraints placed on its size by a low fidelity of replication (the Eigen limit), we conclude that the genome of this organism reached a considerable level of complexity that included several RNA-processing steps. It would include a large protoribosome with many smaller RNAs involved in its assembly, pre-tRNAs and tRNA processing, an ability for recombination of RNA, some RNA editing, an ability to copy to the end of each RNA strand, and some transport functions. It is harder to recognize specific metabolic reactions that must have existed but synthetic and bio-energetic functions would be necessary. Overall, this requires that such an organism maintained a multiple copy, double-stranded linear RNA genome capable of recombination and splicing. The genome was most likely fragmented, allowing each ``chromosome' to be replicated with minimum error, that is, within the Eigen limit. The model as developed serves as an outgroup to root the tree of life and is an alternative to using sequence data for inferring properties of the earliest cells. Received: 14 January 1997 / Accepted: 19 May 1997     

Question 5: Does the RNA-World Still Retain its Appeal After 40 Years of Research?

Forty years after its formulation, the hypothesis of the RNA-World remains rather controversial even though studies of RNA catalysis in cellular processes (for example, in the ubiquitous ribosomal peptide-bond formation) have clearly lent increased plausibility to the idea that an RNA-World existed at some point in the evolution leading to the emergence of cellular life. Indeed, several issues remain that weaken the concept: the synthesis of the RNA monomers under prebiotic conditions, their subsequent, efficient polymerization to yield ribozymes that specifically catalyze their own replication. This communication summarizes existing studies of the RNA polymerization from monomers. In our opinion, the recent developments show that given time plausible answers to some of the issues facing the RNA-World hypothesis will be found. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

The Viability of a Nonenzymatic Reductive Citric Acid Cycle – Kinetics and Thermochemistry

The likelihood of a functioning nonenzymatic reductive citric acid cycle, recently proposed as the precursor to biosynthesis on early Earth, is examined on the basis of the kinetics and thermochemistry of the acetate → pyruvate → oxaloacetate → malate sequence. Using data derived from studies of the Pd-catalyzed phosphinate reduction of carbonyl functions it is shown that the rate of conversion of pyruvate to malate with that system would have been much too slow to have played a role in the early chemistry of life, while naturally occurring reduction systems such as the fayalite–magnetite–quartz and pyrrhotite–pyrite–magnetite mineral assemblages would have provided even slower conversions. It is also shown that the production of pyruvate from acetate is too highly endoergic to be driven by a naturally occurring energy source such as pyrophosphate. It is thus highly doubtful that the cycle can operate at suitable rates without enzymes, and most unlikely that it could have participated in the chemistry leading to life.     

A method to calculate the cumulative energy demand (CED) of lignite extraction

For the utilisation of an energy carrier such as lignite, the whole life cycle including necessary energy supply processes have to be considered. Therefore using the ‘Cumulative Energy Demand’ (CED) is especially suited to determine and compare the energy intensity of processes. The goal of the CED is to calculate the total primary energy input for the generation of a product, taking into account the pertinent front-end process chains. So the CED is in many steps similar to the LCA, especially in the ‘inventory analysis step’. The statements of the CED for energy supply-systems are concerned with the (primary) energy-efficiency of the energy supply and pointing out the life cycle steps with high energy-resources demand. Due to the great environmental impacts of energy supply and use which have to be laboriously assessed in LCA, the CED provides a useful, additional, energy-related ‘screening-indicator’ to LCA. This case study analyses the extraction of lignite in an opencast mine in West-Germany as the first step of energy carrier provision. Our data for the inventory analysis arise from a measuring campaign about the period of one year. The results underline the great energy demand of lignite extraction in West-Germany. With reference to the energy contents of lignite, the fraction of primary energy demands for its’ mining amounts to about 6.2%. This accounts to 93.8 % of the lignite energy content being available as usable energy for further processes, which is obviously worse than other studies have shown.