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Transbilayer diffusion of Mn2+ ions occurred in liposomes formed from dipalmitoyl-phosphatidylcholine or egg-yolk phosphatidylcholine and egg-yolk phosphatidate (molar ratio 2:1) containing DNA and DNase I within their aqueous compartments. Cation diffusion was demonstrated by the hydrolytic activity of DNase I, activated by the Mn2+ ions that diffused into the vesicles, and this was confirmed by light scattering. Phosphatidate, a cone-shaped lipid which has been synthesized under simulated prebiotic conditions, was necessary for cation diffusion across the liposome membranes. Such liposomes represent a simple precellular system that interchanges cations with the surroundings and provides a microenvironment for enzymatic reactions, as evidenced by the hydrolysis of DNA by DNase I inside these closed lipid compartments.

When an aqueous solution (pH 7.0) of deoxythymidine 5'-phosphate, 4-amino-5-imidazolecarboxamide and cyanamide was dried and heated for 18 h at 60 degrees C, P1, P2-dideoxythymidine 5'-pyrophosphate (I) was formed in a 58% yield. Oligonucleotides were not detected in the reaction product. Under conditions employed in the above reaction, (I) was shown to be stable. In prebiotic polymerization reactions employing deoxythymidine 5'-triphosphate as the polymerizing species, (I) could therefore function as a primer and minimize the formation of cyclic nucleotides.

In this paper we summarize some of the most salient observations made recently on the organic molecules and other compounds of the biogenic elements present in the interstellar medium and in the primitive bodies of the solar system. They include the discovery of the first phosphorus molecular species in dense interstellar clouds, the presence of complex organic ions in the dust and gas phase of Halley's coma, the finding of unusual, probably presolar, deuterium-hydrogen ratios in the amino acids of carbonaceous chondrites, and new developments on the chemical evolution of Titan, the primitive Earth, and early Mars. Some of the outstanding problems concerning the synthesis of organic molecules on different cosmic bodies are also discussed from an exobiological perspective.

During the past years we have explored most of the bodies of the solar system by means of the Apollo, Venera, Viking, Voyager, and other space missions. We are now in a better position to be able to compare the conditions of other planets and satellites with those of the Earth in order to determine what is unique about our planet which permitted the emergence and evolution of life on it. On the basis of this and other available scientific information we have arrived at the conclusion that there are at least some twentyfive specific conditions or requirements which have to be fulfilled in order for life as we know it to appear and evolve in a planetary system such as ours. Most of these necessary conditions or requirements are mutually interdependent, but in order to discuss their role in depth they have been divided into five major general areas which are discussed in some detail herein. Planetary criteria, which relate to the physical properties of the planet as it is formed and as it becomes a differentiated cosmic body and potential abode of life. The mass, orbital characteristics and energetic relationships with the central star as well as the discrete separation of gas, liquid and solid phases of the planet are of utmost importance. Chemical criteria, which are concerned with the composition, availability of effective energy sources, and chemical constraints (solvent, pH range, redox potential) of the environment(s) where reactions take place for the prebiological formation of biochemical compounds. Protobiological criteria, which relate to the prebiologically synthesized oligomeric and polymeric biomolecules, how they interact cooperatively to form protobiological structures and functions (replication, catalysis, information transfer, etc.) and self-assemble to give rise to a living system. Evolutionary criteria, which are concerned with the processes responsible for the increase in complexity of organisms by genomic multiplication, symbiotic integration and cellular differentiation, as well as with the negentropic ability of organisms to continuously recycle all the volatile biogenic elements. Altogether these processes made possible the development and evolution of life from the simplest prokaryotic cell ancestor to a cognitive and manipulative multicellular organism (man). In order to extend this inquiry to other systems beyond our solar system a fifth set of requirements based on astronomical observations is also discussed, namely, the Stellar criteria, which relate to the elemental composition mass, lifetime, and other features of Main Sequence stars which may be surrounded by planetary systems similar to our own. Finally, a brief review is made on the probability of the existence of extraterrestrial life as well as of civilizations capable of interstellar communication in our Galaxy.

A study of the encapsulation of poly(U) and poly(C) within liposomes made from dipalmitoylphosphatidyl choline (DPPC), from egg yolk phosphatidyl choline (PC), and from PC with cholesterol (CHOL) was made. The liposomes were prepared under anoxic conditions following the reverse-phase evaporation method. Determinations showed that 36 to 70% of the available lipids form liposomes and 2 to 5% of the polyribonucleotides can be entrapped by liposomes. The encapsulation of polyribonucleotides has also been measured in the presence of urea, cyanamide and Zn++, condensing agents in prebiotic polymerization reactions. DPPC and PC:CHOL liposomes were formed in the presence of 1.0 M urea, although no PC liposomes were formed. The three types of liposomes were readily formed at 0.01 M urea, but in no case an enhancement of encapsulation efficiency of poly(U) was observed due to the presence of urea. Similar results were obtained with cyanamide. An enhanced encapsulation of poly(U) by the three types of liposomes was observed when Zn++ was in the range of 0.001 to 0.01 M. Poly(U) encapsulation was 15 to 25 times higher when liposomes were prepared from DPPC at 0.01 M Zn++. Similar results were obtained with poly(C). The advantages of DPPC-polyribonucleotide liposomes as precellular systems are discussed.

The lines of evidence suggesting that RNA preceded double-stranded DNA as an informational macromolecule are briefly reviewed. RNA polymerase is hypothesized to have been one of the earliest proteins to appear. It is argued that an important vestige of the original enzyme is found in the contemporary eubacterial beta' subunit of DNA-dependent RNA polymerase and its homologues among the archaebacterial and eukaryotic enzymes. The evidence that supports a catalytic role in replicase activity of this polypeptide is reviewed. It is suggested that several characteristics of the Escherichia coli transcriptional apparatus are relatively recent evolutionary developments. The phylogenetic importance of the eubacterial beta' subunit from RNA polymerase and its homologues is emphasized, because it allows the study of the evolutionary relationships of the major cellular lines (eubacteria, archaebacteria, and eukaryotes) as well as of some viral lineages.

Using a primitive Earth evaporating pond model, the synthesis of phosphatidylcholine was accomplished when a reaction mixture of choline chloride and disodium phosphatidate, in the presence of cyanamide and traces of acid, was evaporated and heated at temperatures ranging from 25 degrees to 100 degrees C for 7 hours. Optimum yields of about 15% were obtained at 80 degrees C. Phosphatidylcholine was identified by chromatographic, chemical and enzymatic degradation methods. On enzymatic hydrolysis with phospholipase A2 and phospholipase C, lysophosphatidylcholine and phosphorylcholine were formed, respectively. Alkaline hydrolysis gave glycerophosphorylcholine. The synthesis of phosphatidylcholine as the major compound was accompanied by the formation of lysophosphatidylcholine in smaller amounts. Cyanamide was found to be essential for the formation of phosphatidylcholine, and only traces of HCl, of the order of that required to convert the disodium phosphatidate to free phosphatidic acid were found necessary for the synthesis. This work suggests that phosphatidylcholine, which is an essential component of most biological membranes, could have been synthesized on the primitive Earth.

The prebiotic synthesis of imidazole-4-acetaldehyde and imidazole-4-glycol from erythrose and formamidine has been demonstrated as well as the prebiotic synthesis of imidazole-4-ethanol and imidazole-4-glycol from erythrose, formaldehyde and ammonia. The products were identified by TLC, HPLC, and LC-MS by comparison with authentic samples. The maximum yields of imidazole-4-acetaldehyde, imidazole-4-ethanol, and imidazole-4-glycol obtained in these reactions are 1.6, 5.4, 6.8% respectively, based on the erythrose. Imidazole-4-acetaldehyde would have been converted to histidine on the primitive earth by a Strecker synthesis, and several prebiotic reactions would convert imidazole-4-glycol and imidazole-4-ethanol to imidazole-4-acetaldehyde.

Liposomes are 5 to 50 micron vesicles with an internal aqueous environment, whose amphiphilic lipidic components self-assemble into systems with at least one double-layered membrane. Liposomes have been suggested as possible models of precellular systems formed in the early Archean Earth from lipids of non-enzymatic origin. Since it is generally accepted that RNA molecules preceded double-stranded DNA molecules as genetic material, we have studied the encapsulation of polyribonucleotides within liposomes made from dipalmitoyl phosphatidylcholine, and from egg yolk phosphatidylcholine to which cholesterol was added in some cases. The liposomes were prepared under anoxic conditions following the reverse phase evaporation method described by Szoka and Papahadjopoulos. Quantitative determinations show that approximately 50% of the available lipids form liposomes, and that up to 5% of the polyribonucleotides can be entrapped by them. We have also studied the encapsulation of polyribonucleotides in the presence of 1) urea and cyanamide, two non-electrolytes that have been used as prebiotic condensing agents, and 2) of Zn++ and Pb++, two cations employed in the non-enzymatic template-directed synthesis of polyribonucleotides from activated nucleotides.

The work done in many laboratories during the last two decades has confirmed that hydrogen cyanide and cyanoacetylene are the two major precursors for the prebiotic synthesis of purines and pyrimidines, respectively. Although several different pathways for the synthesis of purines have been described, they are all variations of the initial mechanism proposed by Oró and Kimball, where hydrogen cyanide leads first to the formation of a 4,5-di-substituted imidazole derivative, and then to the closing of the purine ring with a C1 compound. A number of experiments have shown that purines and pyrimidines can also be obtained from methane, ammonia (nitrogen), and water mixtures, provided an activating source of energy (radiation, electric discharges, etc.) is available. However, in this case the yields are lower by about two orders of magnitude because of the intermediate formation of hydrogen cyanide and cyanoacetylene. The latter two compounds have been found in interstellar space, Titan and other bodies of the solar system. They were probably present in the primordial parent bodies from the solar nebula in concentrations of 10(-2) to 10(-3) M as inferred from recent calculations by Miller and coworkers obtained for the Murchison meteorite. These concentrations should have been sufficient to generate relatively large amounts of purine and pyrimidine bases on the primitive Earth.

Some of the chemical species which have been detected in comets include H2O, HCN, CH3CN, CO, CO2, NH3, CS, C2 and C3. All of these have also been detected in the interstellar medium, indicating a probable relationship between interstellar dust and gas clouds and comets. Laboratory experiments carried out with different mixtures of these molecules give rise to the formation of the biochemical compounds which are necessary for life, such as amino acids, purines, pyrimidines, monosaccharides, etc. However, in spite of suggestions to the contrary, the presence of life in comets is unlikely. On the other hand, the capture of cometary matter by the primitive Earth is considered essential for the development of life on this planet. The amount of cometary carbon-containing matter captured by the Earth, as calculated by different authors, is several times larger than the total amount of organic matter present in the biosphere (10(18)g). The major classes of reactions which were probably involved in the formation of key biochemical compounds are discussed. Our tentative conclusions are that: 1) comets played a predominant role in the emergence of life on our planet, and 2) they are the cosmic connection with extraterrestrial life.

A variety of methods have been investigated in different laboratories for the polymerization of amino acids and nucleotides under abiotic conditions. They include (1) thermal polymerization, (2) direct polymerization of certain amino acid nitriles, amides or esters, (3) pi, Tokyo 194, Japan.

The evolution of genetic material can be divided into at least three major phases: first, genomes of "nucleic acid-like" molecules; secondly, genomes of RNA; and finally, double-stranded DNA genomes such as those present in all contemporary cells. Using properties of nucleic acid molecules, we attempt to explain the evolutionary transition from RNA alone as a cellular informational macromolecule prior to the evolution of cell systems based on double-stranded DNA. The idea that ribonucleic acid-based cellular genomes preceded DNA is based on the following: (1) protein synthesis can occur in the absence of DNA but not of RNA; (2) RNA molecules have some catalytic properties; (3) the ubiquity of purine and pyridine nucleotide coenzymes as well as other similar ribonucleotide cofactors in metabolic pathways; and (4) the fact that the biosynthesis of deoxyribonucleotides always proceeds via the enzymatic reduction of ribonucleotides. The "RNA prior to DNA" hypothesis can be further developed by understanding the selective pressures that led to the biosynthesis of deoxyribose, thymine, and proofreading DNA polymerases. Taken together these observations suggest to us that DNA was selected as an informational molecule in cells to stabilize earlier RNA-protein replicating systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Based on our current knowledge of molecular biology a living entity is a negentropic system, made of specific catalytic and informational macromolecules, which has the ability to reproduce itself, selectively interact with the environment, increase complexity, undergo mutation and evolve by natural selection. At the cellular level the essential molecular attributes of a minimal living system are considered to be the following: (a) Linear informational molecules, protoRNA (protoDNA) at least 10 mononucleotides long, capable to code for at least 2 amino acids; (b) Code-translating molecules, aminoacyl-proto tRNAs at least 5 mononucleotides long; (c) Protenzymes, oligopeptides of at least 2 amino acids, with measurable catalytic activity for phosphodiester, pyrophosphate and peptide bond formation; (d) Protoribosomes, RNA-peptide complexes which facilitate the interaction of the above three types of molecules, and (e) Protomembranes, liposomes made of amphiphilic lipids and peptides which can provide the semipermeability and the proton gradient necessary for the synthesis of pyrophosphate, ATP and other biochemical compounds. The transition from subcellular Lamarckian evolution to cellular Darwinian evolution required the cooperative interaction, within an internal microenvironment, of the above five types of molecular species.

The formation of glycerol occurs when a solution of DL-glyceraldehyde is heated in the presence of hydrogen sulfide at room temperature. DL-glyceraldehyde and dihydroxyacetone treated with hydrazine, as well as DL-glyceraldehyde incubated with formaldehyde are also partially converted to glycerol. The yields of the above reactions are from approximately 1% to about 3%. The formation of glycerophosphates occurs when glycerol is heated with ammonium dihydrogen phosphate and either urea or cyanamide. The yield of glycerophosphates is about 30%, most of which is sn-glycero-1 (3)-phosphate. These findings indicate that glycerol and sn-glycero-3-phosphate, which are moieties of glycerolipids, could have been formed under conditions which may have prevailed on the primitive Earth.

Cosmochemical considerations suggest various potential sources for the accumulation of organic matter on Mars. However the Viking Molecular Analysis did not indicate any indigenous organic compounds on the surface of Mars. Their disappearance from the top layer is most likely caused by the combined action of the high solar radiation flux and various oxidizing species in the substances and a sample of the Murchison meteorite was tested under simulated Martian conditions. After adsorption on powdered quartz, samples of adenine, glycine and naphthalene were irradiated with UV light at various oxygen concentrations and exposure times. In the absence of oxygen, adenine and glycine appeared stable over the given irradiation period, whereas a definite loss was observed in the case of naphthalene, as well as in the volatilizable and pyrozable content of the Murchison meteroite. The presence of oxygen during UV exposure caused a significant increase in the degradation rate of all samples. It is likely that similar processes have led to the destruction of organic materials on the surface of Mars.

In the past decade significant advances have been made in the synthesis of oligonucleotides and other polymers by means of imidazoles and other condensing agents. In spite of the current knowledge of the chemistry of imidazoles and their importance as prebiotic catalysts, their formation under primitive earth conditions has not been properly demonstrated. We have now been able to synthesize imidazole as well as its 2-methyl and 4-methyl derivatives under plausible prebiotic conditions. One method utilizes an aldehyde (formaldehyde or acetaldehyde), glyoxal and ammonia as the starting materials for the formation of imidazole and 2-methylimidazole. The other method uses a carbohydrate and ammonia as the key reagents for the synthesis of 4-methylimidazole. The importance of imidazole and related compounds (e.g., cyanamide) in the synthesis of oligonucleotides has been studied by us as well as others. Apparently the charge relay group (-N-C-N-) present in imidazoles, carbodiimides, cyanamide, or the histidine and arginine of enzyme active centers is essential for the synthesis of phosphodiester and pyrophosphate bonds.

A mixture of ammonium palmitate, 14C-sn-glycero-1(3)-phosphate, cyanimide and imidazole when heated for several hours formed significant quantities of phospholipids. These reaction products were shown by chromatographic, chemical and enzymatic procedures to be monopalmitoylglycerophosphate (MPGP), dipalmitoylglycerophosphate (DPGP) and monopalmitoyl cyclic glycerophosphate (cMPGP). A portion of the MPGP and DPGP possessed the same steric configuration as naturally occurring lysophosphatidic acid and phosphatidic acid. The yield of total phospholipid was maximal at temperatures between 60 degrees and 90 degrees after 8 h. When ratios of reactants were varied, up to 45% of radioactive glycerophosphate was converted into phospholipids. The average proportions of individual phosphatidic acids were: 60% MPGP, 27% DPGP and 13% cMPGP. Evidence was obtained for a synergistic relationship between cyanamide and imidazole in promoting the formation of phosphatidic acids. These results suggest that phosphatidic acids, which are essential precursors for the biochemical synthesis of more complex membrane phospholipids, could have been produced on the primitive Earth.

The impact of cigarette smoking on the distribution of organic substances in ambient air has been determined for the intermediate volatility range. A simple sampling procedure was employed, involving gas-solid adsorption onto an organic polymer followed by direct thermal elution onto a glass capillary column. Aliphatic and substituted aromatic hydrocarbons are predominant in urban atmospheres. Depending on location and weather conditions the total concentration of such volatiles can differ by as much as a factor of 20. This high background variation makes it difficult to analyze for trace substances with low odor threshold values, such as encountered in cigarette smoke. Standard cigarettes were smoked in a relatively small room, having no air filtration system. Air samples of approximately 3.51 were taken. The amount of volatiles added to air by cigarette smoking is unsignificant. Substances were analyzed and identified by gas chromatography and gas chromatography-mass spectrometry with glass capillary columns. Many compounds reported in cigarette smoke condensate have been confirmed.

Sarcina lutea was grown in Trypticase Soy Broth, Nutrient Broth, and a chemically defined medium. Gas chromatographic analysis of lipid components demonstrated that the composition of the medium had an effect on the relative per cent composition of the aliphatic hydrocarbons and fatty acids present in the cells. The branched olefinic hydrocarbons from the organisms grown in Trypticase Soy Broth showed no predominance or only a slight predominance of odd-numbered carbon chains, whereas the hydrocarbons from cells grown in the other two media showed an obvious predominance of odd-numbered carbon chains. The monocarboxylic fatty acid content and distribution showed only minor differences, with all normal saturated fatty acids present in relatively small quantities for cells grown in Nutrient Broth and in a chemically defined medium.

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.

An initial investigation into the mechanism of hydrocarbon biosynthesis in Sarcina lutea was performed by measuring the amounts of (14)C incorporated into the hydrocarbons and fatty acids by use of a combination gas chromatograph and high-temperature gas-flow ionization apparatus. Uniformly labeled l-isoleucine-(14)C was predominantly incorporated into the anteiso-branched chains. Palmitate-16-(14)C gave evidence that a direct correlation may exist between the nonpolar end of the palmitate and the biosynthesis of hydrocarbons and carotenoids. The label from palmitate-1-(14)C was incorporated into the various hydrocarbon groups as a compound, derived from the polar end of the palmitate, consisting of more than two carbon atoms. Palmitate-16-(14)C and -1-(14)C gave no detectable evidence that transformed products were incorporated into other fatty acids. Sodium acetate-2-(14)C and uniformly labeled l-leucine-(14)C gave evidence of a nonspecific incorporation into both the aliphatic hydrocarbons and fatty acids of Sarcina lutea.

The composition and nature of the fatty acids and hydrocarbons of Sarcina lutea were elucidated by gas chromatography and by combined gas chromatography-mass spectrometry. The distribution of fatty acids found in S. lutea showed two families of pairs, or dyads, of saturated monocarboxylic acids (C12-C18) with and without methyl branching. These pairs of fatty acids showed a pattern of iso and anteiso structures for C13, C15, and C17, and iso and normal structures for C12, C14, and C16. Only the C18 showed unsaturation. The distribution of hydrocarbons in the range C22-C29 showed two families of tetrads of unsaturated aliphatic hydrocarbons all showing methyl branching. Each tetrad was composed of four isomers identified as two iso olefins and two anteiso olefins. The only difference between the tetrads pertaining to different families was found in the relative gas chromatographic retention times of the last two components of each group.

Organogenic elements, mainly carbon, nitrogen, phosphorus, and sulfur are present in the particulate material and in a breccia rock from Tranquillity Base in amounts ranging from 5 to 4200 parts per million. The major compounds of carbon released by heating are carbon monoxide and carbon dioxide; the former predominates. Small amounts of other compounds of carbon have also been observed. Sulfur can be released as hydrogen sulfide by treatment with acid. The carbon isotopic delta(13L)C values are definitely nonterrestrial (+ 13 to + 18.5 per mil).

Normal paraffins in the C(16) to C(32) range and the saturated isoprenoid hydrocarbons, pristane and phytane, have been found in chert from the Gunflint iron formation (1.9 x 10(9) years old) of the north shore of Lake Superior. The distribution of n-alkanes shows two maxima, one at about C(18) to C(19) and the other at about C(22) with a minimum occurring at C(20) to C(21). No predominance of odd- to even-carbon-number alkanes is observed within the C(16) to C(32) range. The results agree with micropaleontological observations made on the Gunflint chert and provide a chemical characterization of Precambrian life existing about two eons ago.

The chlamydospores of Ustilago maydis, U. nuda, and Sphacelotheca reiliana were analyzed by gas chromatography and mass spectrometry for their hydrocarbon contents. For the first time we observed that they contain paraffinic hydrocarbons; the average contents were 42, 58, and 146 parts per million, respectively. n-Alkanes having odd numbers of carbon atoms predom-inate, with carbon-chain lengths ranging from C(14) to C(37). The major alkanes are n-C(27) in U. maydis, n-C(27) and n-C(35) in U. nuda, and n-C(29) in S. reiliana. Each type of spore carried a distinctly characteristic population of hydrocarbons.