FFAME.org :: Notable Publications

Expanded Genetic Alphabets in the Polymerase Chain Reaction
Yang, ZY Chen, F Chamberlin, SG Benner, SA
Angew. Chem. Int. Ed. 49 (1) 177-180 (2010)

Artificial Genetic Systems: Self-Avoiding DNA in PCR and Multiplexed PCR
Hoshika, S Chen, F Leal, NA Benner, SA
Angew. Chem. Int. Ed. 49 (32) 5554-5557 (2010)

Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection
Chen, F Gaucher, EA Leal, NA Hutter, D Havemann, SA Govindarajan, S Ortlund, EA Benner, SA
Proc. Natl. Acad. Sci. USA 107 (5) 1948-1953 (2010)

2'-Deoxy-1-methylpseudocytidine, a stable analog of 2'-deoxy-5-methylisocytidine
Kim, HJ Leal, NA Benner, SA
Bioorg. Med. Chem. 17 (10) 3728-3732 (2009)
<Abstract>

2 '-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2'-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2 '-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2 '-deoxycytidine and 2 '-deoxyguanosine pair. (C) 2009 Elsevier Ltd. All rights reserved.

A Convenient Synthesis of N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside and 1-Methyl-2'-Deoxypseudoisocytidine
Wellington, KW Ooi, HC Benner, SA
Nuc. Nuc. Nuc. acids 28 (4) 275-291 (2009)
<Abstract>

The syntheses of N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside and 1-methyl-2'-deoxypseudoisocytidine via Heck coupling are described. A survey of the attempts to use the Heck coupling to synthesize N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside is provided, indicating a remarkable diversity in outcome depending on the specific heterocyclic partner used.

Signatures of a Shadow Biosphere
Davies, PCW Benner, SA Cleland, CE Lineweaver, CH McKay, CP Wolfe-Simon, F
Astrobiology 9 (2) 241-249 (2009)
<Abstract>

Astrobiologists are aware that extraterrestrial life might differ from known life, and considerable thought has been given to possible signatures associated with weird forms of life on other planets. So far, however, very little attention has been paid to the possibility that our own planet might also host communities of weird life. If life arises readily in Earth-like conditions, as many astrobiologists contend, then it may well have formed many times on Earth itself, which raises the question whether one or more shadow biospheres have existed in the past or still exist today. In this paper, we discuss possible signatures of weird life and outline some simple strategies for seeking evidence of a shadow biosphere.

The challenges of sequencing by synthesis
Fuller, CW Middendorf, LR Benner, SA Church, GM Harris, T Huang, XH Jovanovich, SB Nelson, JR Schloss, JA Schwartz, DC Vezenov, DV
Nat. Biotechnol. 27 (11) 1013-1023 (2009)
<Abstract>

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $ 100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

Design of a novel molecular beacon: modification of the stem with artificially genetic alphabet
Sheng, PP Yang, ZY Kim, YM Wu, YR Tan, WH Benner, SA
Chem. Comm. (41) 5128-5130 (2008)
<Abstract>

A molecular beacon that incorporates components of an artificially expanded genetic information system (AEGIS) in its stem is shown not to be opened by unwanted stem invasion by adventitious standard DNA; this should improve the "darkness" of the beacon in real-world applications.

The planetary biology of ascorbate and uric acid and their relationship with the epidemic of obesity and cardiovascular disease
Johnson, RJ Gaucher, EA Sautin, YY Henderson, GN Angerhofer, AJ Benner, SA
Medical Hypotheses 71 (1) 22-31 (2008)
<Abstract>

Humans have relatively low plasma ascorbate levels and high serum uric acid levels compared to most mammals due to the presence of genetic mutations in L-gulonotactone oxidase and uricase, respectively. We review the major hypotheses for why these mutations may have occurred. In particular, we suggest that both mutations may have provided a survival advantage to early primates by helping maintain blood pressure during periods of dietary change and environmental stress. We further propose that these mutations have the inadvertent disadvantage of increasing our risk for hypertension and cardiovascular disease in today's society characterized by Western diet and increasing physical inactivity. Finally, we suggest that a "planetary biology" approach in which genetic changes are analyzed in relation to their biological action and historical context may provide the ideal approach towards understanding the biology of the past, present and future. (c) 2008 Elsevier Ltd. All rights reserved.

Self-Avoiding Molecular Recognition Systems (SAMRS)
Hoshika, S Chen, F Leal, NA Benner, SA
Nucleic Acids Symp. Ser. 52 (1) 129-130 (2008)

Incorporation of Multiple Sequential Pseudothymidines by DNA Polymerases and Their Impact on DNA Duplex Structure
Havemann, SA Hoshika, S Hutter, D Benner, SA
Nuc. Nuc. Nuc. acids 27 (3) 261-278 (2008)
<Abstract>

In this article, we focus on the synthesis of aryl C-glycosides via Heck coupling. It is organized based on the type of structures used in the assembly of the C-glycosides (also called C-nucleosides) with the following subsections: pyrimidine C-nucleosides, purine C-nucleosides, and monocyclic, bicyclic, and tetracyclic C-nucleosides. The reagents and conditions used for conducting the Heck coupling reactions are discussed. The subsequent conversion of the Heck products to the corresponding target molecules and the application of the target molecules are also described.

Synthesis of pyrophosphates for in vitro selection of catalytic RNA molecules
Kim, HJ Kim, MJ Karalkar, N Hutter, D Benner, SA
Nuc. Nuc. Nuc. acids 27 43-56 (2008)

Computational reconstruction of ancestral genomic regions from evolutionarily conserved gene clusters
Danchin, EGJ Gaucher, EA Pontarotti, P
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 139-150 (2007)

Experimental resurrection of ancient biomolecules: gene synthesis, heterologous protein expression, and functional assays
Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 153-163 (2007)

A thermophilic last universal ancestor inferred from its estimated amino acid composition
Brooks, DJ Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 200-207 (2007)

Ancestral sequence reconstruction as a tool to understand natural history and guide synthetic biology: realizing and extending the vision of Zuckerkandl and Pauling
Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 20-33 (2007)

The resurrection of ribonucleases from mammals: from ecology to medicine
Sassi, SO Benner, SA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 208-224 (2007)

The early days of paleogenetics: connecting molecules to the planet
Benner, SA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 3-19 (2007)

Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis
Ruhland, A Leal, N Kima, PE
Cell Microbiol. 9 (1) 84-96 (2007)
<Abstract>

Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NF kappa B and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NF kappa B signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

PduL is an evolutionarily distinct phosphotransacylase involved in B-12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2
Liu, Y Leal, NA Sampson, EM Johnson, CLV Havemann, GD Bobik, TA
J. Bacteriol. 189 (5) 1589-1596 (2007)
<Abstract>

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B-12-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V-max was 51.7 +/- 7.6 mu mol min(-1) mg(-1), and the K-m values for propionyl-PO42- and acetyl-PO42- were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.

In vivo expression of human ATP : cob(I)atamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8
Erger, KE Conlon, TJ Leal, NA Zori, R Bobik, TA Flotte, TR
J. Gene Med. 9 (6) 462-469 (2007)
<Abstract>

Background Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B-12, adenosylcobalamin (AdoCbl). AdoCbl is. a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Methods The human ATR cDNA was cloned into a recombinant adenoassociated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/B16 mice at a dose of 1 x 10(10) and 1 x 10(11), particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Results Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken P-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. Conclusions These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments. Copyright (C) 2007 John Wiley & Sons, Ltd.

The evolution of seminal ribonuclease: Pseudogene reactivation or multiple gene inactivation events?
Sassi, SO Braun, EL Benner, SA
Mol. Biol. Evol. 24 (4) 1012-1024 (2007)
<Abstract>

Two approaches, one novel, are applied to analyze the divergent evolution of ruminant seminal ribonucleases (RNases), paralogs of the well-known pancreatic RNases of mammals. Here, the goal was to identify periods of divergence of seminal RNase under functional constraints, periods of divergence as a pseudogene, and periods of divergence driven by positive selection pressures. The classical approach involves the analysis of nonsynonymous to synonymous replacements ratios (omega) for the branches of the seminal RNase evolutionary tree. The novel approach coupled these analyses with the mapping of substitutions on the folded structure of the protein. These analyses suggest that seminal RNase diverged during much of its history after divergence from pancreatic RNase as a functioning protein, followed by homoplastic inactivations to create pseudogenes in multiple ruminant lineages. Further, they are consistent with adaptive evolution only in the most recent episode leading to the gene in modern oxen. These conclusions contrast sharply with the view, cited widely in the literature, that seminal RNase decayed after its formation by gene duplication into an inactive pseudogene, whose lesions were repaired in a reactivation event. Further, the 2 approaches, omega estimation and mapping of replacements on the protein structure, were compared by examining their utility for establishing the functional status of the seminal RNase genes in 2 deer species. Hog and roe deer share common lesions, which strongly suggests that the gene was inactive in their last common ancestor. In this specific example, the crystallographic approach made the correct implication more strongly than the omega approach. Studies of this type should contribute to an integrated framework of tools to assign functional and nonfunctional episodes to recently created gene duplicates and to understand more broadly how gene duplication leads to the emergence of proteins with novel functions.

Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Yang, ZY Sismour, AM Benner, SA
Nucl. Acids Res. 35 (9) 3118-3127 (2007)
<Abstract>

The use of DNA polymerases to incorporate phosphorothioate linkages into DNA, and the use of exonuclease III to determine where those linkages have been incorporated, are re- examined in this work. The results presented here show that exonuclease III degrades single- stranded DNA as a substrate and digests through phosphorothioate linkages having one absolute stereochemistry, assigned ( assuming inversion in the polymerase reaction) as S, but not the other absolute stereochemistry. This contrasts with a general view in the literature that exonuclease III favors double-stranded nucleic acid as a substrate and stops completely at phosphorothioate linkages. Furthermore, not all DNA polymerases appear to accept exclusively the ( R) stereoisomer of nucleoside alpha- thiotriphosphates [ and not the ( S) diastereomer], a conclusion inferred two decades ago by examination of five Family- A polymerases and a reverse transcriptase. This suggests that caution is appropriate when extrapolating the detailed behavior of one polymerase from the behaviors of other polymerases. Furthermore, these results provide constraints on how exonuclease III - thiotriphosphate - polymerase combinations can be used to analyze the behavior of the components of a synthetic biology.

Enzymatic incorporation of a third nucleobase pair
Yang, ZY Sismour, AM Sheng, PP Puskar, NL Benner, SA
Nucl. Acids Res. 35 (13) 4238-4249 (2007)
<Abstract>

DNA polymerases are identified that copy a nonstandard nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro3-(l'-p-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-standard 'small' donordonor-acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1-beta-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4 (8H)-one [dP) implements the 'large' acceptor-acceptor-donor (puAAD) pattern. These nucleobases were designed to present electron density to the minor groove, density hypothesized to help determine specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo(-)) and Deep Vent (exo-) polymerases, with 94.4%, 97.5% and 97.5%, respectively, retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson-Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, molecular-, systems- and synthetic-biology.

The origin of proteins and nucleic acids
Ricardo, A Benner, SA
Planets and Life: The Emerging Science of Astrobiology, ed. Woodruff T. Sullivan and John A. Baross, Cambridge University Press 154-173 (2007)

Alien biochemistries
Ward, PD Benner, SA
Planets and Life: The Emerging Science of Astrobiology, ed. Woodruff T. Sullivan and John A. Baross, Cambridge University Press 537-544 (2007)

Molecular Paleoscience: Systems Biology from the Past
Benner, SA Sassi, SO Gaucher, EA
Adv. Enzymol. Relat. Areas Mol. Biol. 75 (2006)

Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins
Bradley, ME Benner, SA
BMC Bioinformatics 7 89 (2006)
<Abstract>

Background: When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results: The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1) multiple sequence alignments, 2) mapping of alignment sites to crystal structure sites, 3) phylogenetic trees, 4) inferred ancestral sequences at internal tree nodes, and 5) amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion: We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural bioinformatics resources that are useful for identifying experimentally testable hypotheses about the molecular basis of protein behaviors and functions, as illustrated with the examples from the cellular retinoid binding proteins.

Application of DETECTER, an Evolutionary Genomic Tool to Analyze Genetic Variation, to the Cystic Fibrosis Gene Family
Gaucher, EA DeKee, DW Benner, SA
BMC Genomics 7 44 (2006)
<Abstract>

Background: The medical community requires computational tools that distinguish genetic differences having phenotypic impact within the vast number of mutations that do not. Tools that do this will become increasingly important for those seeking to use human genome sequence data to predict disease, make prognoses, and customize therapy to individual patients.
Results: An approach, termed DETECTER, is proposed to identify sites in a protein sequence where amino acid replacements are likely to have a significant effect on phenotype, including causing genetic disease. This approach uses a model-dependent tool to estimate the normalized replacement rate at individual sites in a protein sequence, based on a history of those sites extracted from an evolutionary analysis of the corresponding protein family. This tool identifies sites that have higher-than-average, average, or lower- than-average rates of change in the lineage leading to the sequence in the population of interest. The rates are then combined with sequence data to determine the likelihoods that particular amino acids were present at individual sites in the evolutionary history of the gene family. These likelihoods are used to predict whether any specific amino acid replacements, if introduced at the site in a modern human population, would have a significant impact on fitness. The DETECTER tool is used to analyze the cystic fibrosis transmembrane conductance regulator (CFTR) gene family.
Conclusions: In this system, DETECTER retrodicts amino acid replacements associated with the cystic fibrosis disease with greater accuracy than alternative approaches. While this result validates this approach for this particular family of proteins only, the approach may be applicable to the analysis of polymorphisms generally, including SNPs in a human population.

The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes
Phillips, SE Vincent, P Rizzieri, KE Schaaf, G Bankaitis, VA Gaucher, EA
Crit. Rev. Biochem. Mol. Biol. 41 (1) 21-49 (2006)
<Abstract>

Phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs) remain largely functionally uncharacterized, despite the fact that they are highly conserved and are found in all eukaryotic cells thus far examined by biochemical or sequence analysis approaches. The available data indicate a role for PITPs in regulating specific interfaces between lipid-signaling and cellular function. In this regard, a role for PITPs in controlling specific membrane trafficking events is emerging as a common functional theme. However, the mechanisms by which PITPs regulate lipid-signaling and membrane-trafficking functions remain unresolved. Specific PITP dysfunctions are now linked to neurodegenerative and intestinal malabsorbtion diseases in mammals, to stress response and developmental regulation in higher plants, and to previously uncharacterized pathways for regulating membrane trafficking in yeast and higher eukaryotes, making it clear that PITPs are integral parts of a highly conserved signal transduction strategy in eukaryotes. Herein, we review recent progress in deciphering the biological functions of PITPs, and discuss some of the open questions that remain.

2-Hydroxymethylboronate as a Reagent To Detect Carbohydrates: Application to the Analysis of the Formose Reaction
Ricardo, A Frye, F Carrigan, MA Tipton, JD Powell, DH Benner, SA
J. Org. Chem. 71 (25) 9503-9505 (2006)
<Abstract>

2-Hydroxymethylphenylboronate is described as a reagent that converts neutral 1,2-diols, as found in simple carbohydrates, into 1:1 anionic complexes that are easily detected by Fourier transform ion cyclotron resonance mass spectrometry. The value of this reagent was demonstrated through its application to analyze complex mixtures of carbohydrates formed in the formose process, often cited as a way that biologically significant carbohydrates might have been generated from formaldehyde under prebiotic conditions. Coupled with isotope studies, the reagent shows that the simplest autocatalytic cycle for the consumption of formaldehyde in this process cannot account for the bulk consumption of formaldehyde.

Dynamic assembly of primers on nucleic acid templates
Leal, NA Sukeda, M Benner, SA
Nucl. Acids Res. 34 4702-4710 (2006)
<Abstract>

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of 1 µM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern
Yang, ZY Hutter, D Sheng, PP Sismour, AM Benner, SA
Nucl. Acids Res. 34 (21) 6095-6101 (2006)
<Abstract>

To support efforts to develop a 'synthetic biology' based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin -4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where 'py' indicates a pyrimidine analog and 'pu' indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

A review: Synthesis of aryl C-glycosides via the heck coupling reaction
Wellington, KW Benner, SA
Nuc. Nuc. Nuc. acids 25 (12) 1309-1333 (2006)
<Abstract>

In this article, we focus on the synthesis of aryl C-glycosides via Heck coupling. It is organized based on the type of structures used in the assembly of the C-glycosides (also called C-nucleosides) with the following subsections: pyrimidine C-nucleosides, purine C-nucleosides, and monocyclic, bicyclic, and tetracyclic C-nucleosides. The reagents and conditions used for conducting the Heck coupling reactions are discussed. The subsequent conversion of the Heck products to the corresponding target molecules and the application of the target molecules are also described.

Desorption/ionization on porous silicon mass spectrometry studies on pentose-borate complexes
Li, Q Ricardo, A Benner, SA Winefordner, JD Powell, DH
Anal. Chem. 77 (14) 4503-4508 (2005)
<Abstract>

Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) was used to investigate the binding affinities between aldopentose isomers and boron. Boron has been recognized for its importance in pentose synthesis and stabilization in prebiotic conditions. Boron may also account for the fact that ribose, among other aldopentoses, is the favored building block in RNA synthesis. This research started with the detection of aldopentoses in the positive mode through cationization and the aldopentose-borate complexes in the negative mode. Then two competition schemes, one using a pentose structure analogue and the other using C-13-labeled ribose, were designed to compare the relative binding affinities of four aldopentoses (xylose, lyxose, arabinose, and ribose) to boron. Both approaches determined the binding preference to be ribose > lyxose > arabinose > xylose. This work illustrates the potential of DIOS-MS in the analyses of nonvolatile, small molecules in delicate chemical equilibria. Without externally introduced matrices, background signals are not a limiting factor. Furthermore, the possible dramatic change of pH associated with the matrix introduction, which may disturb the equilibria of interest, is avoided.

Synthetic biology
Sismour, AM Benner, SA
Expert Opin. Biol. Ther. 5 (11) 1409-1414 (2005)
<Abstract>

Chemistry is a broadly powerful discipline in contemporary science because it has the ability to create new forms of the matter that it studies. By doing so, chemistry can test models that connect molecular structure to behaviour without having to rely on what nature has provided. This creation, known as synthesis', began to be applied to living systems in the 1980s as recombinant DNA technologies allowed biologists to deliberately change the molecular structure of the microbes that they studied, and automated chemical synthesis of DNA became widely available to support these activities. The impact of the information that has emerged has made biologists aware of a truism that has long been known in chemistry: synthesis drives discovery and understanding in ways that analysis cannot. Synthetic biology is now setting an ambitious goal: to recreate in artificial systems the emergent properties found in natural biology. By doing so, it is advancing our understanding of the molecular basis of genetics in ways that analysis alone cannot. More practically, it has yielded artificial genetic systems that improve the healthcare of some 400,000 Americans annually. Synthetic biology is now set to take the next step, to create artificial Darwinian systems by direct construction. Supported by the National Science Foundation as part of its Chemical Bonding program, this work cannot help but generate clarity in our understanding of how biological systems work.

A call for likelihood phylogenetics even when the process of sequence evolution is heterogeneous
Gaucher, EA Miyamoto, MM
Mol. Phylogenet. Evol. 37 (3) 928-931 (2005)
<Abstract>

All methods of phylogenetic inference make assumptions about the underlying evolutionary process of their characters and it is these assumptions that determine their relative successes and failures in the estimation of the true phylogeny for a group. This dependency of phylogenetic accuracy and robustness on evolutionary assumptions has been most extensively studied for the classic case of Felsenstein (1978) and its four-taxon phylogeny with two long, unrelated, terminal branches interspersed with two short ones. Given this model phylogeny, "long branch attraction" can occur and thereby lead to the convergence of a phylogenetic method onto an incorrect tree with the two long and two short terminal branches directly connected rather than interspersed. The extent to which a particular phylogenetic method is susceptible to this problem depends on what assumptions it makes about the evolution of the characters and data themselves.

The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system
Sismour, AM Benner, SA
Nucl. Acids Res. 33 5640-5646 (2005)
<Abstract>

Synthetic biology based on a six-letter genetic alphabet that includes the two non-standard nucleobases isoguanine (isoG) and isocytosine (isoC), as well as the standard A, T, G and C, is known to suffer as a consequence of a minor tautomeric form of isoguanine that pairs with thymine, and therefore leads to infidelity during repeated cycles of the PCR. Reported here is a solution to this problem. The solution replaces thymidine triphosphate by 2-thiothymidine triphosphate (2-thioTTP). Because of the bulk and hydrogen bonding properties of the thione unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. To test whether this might allow PCR amplification of a six-letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis. The concentrations of isoCTP and 2-thioTTP were optimal to best support PCR amplification using thermostable polymerases of a six-letter alphabet that includes the isoC-isoG pair. The fidelity-per-round of amplification was found to be approximately 98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only approximately 93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.

Resurrecting ancestral alcohol dehydrogenases from yeast
Thomson, JM Gaucher, EA Burgan, MF De Kee, DW Li, T Aris, JP Benner, SA
Nature Genet. 37 (6) 630-635 (2005)
<Abstract>

Modern yeast living in fleshy fruits rapidly convert sugars into bult ethanol through pyruvate. Pyruvate loses carbon dioxide to become acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. Because many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the reconstruction of the last common ancestor of Adh1 and Adh2, called AdhA. The kinetic behavior of AdhA suggests that it was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used AdhA to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

Synthetic Biology
Sismour, AM Benner, SA
Nat. Rev. Genet. 6 533-543 (2005)
<Abstract>

Synthetic biologists come in two broad classes. One uses unnatural molecules to reproduce emergent behaviours from natural biology, with the goal of creating artificial life. The other seeks interchangeable parts from natural biology to assemble into systems that function unnaturally. Either way, a synthetic goal forces scientists to cross uncharted ground to encounter and solve problems that are not easily encountered through analysis. This drives the emergence of new paradigms in ways that analysis cannot easily do. Synthetic biology has generated diagnostic tools that improve the care of patients with infectious diseases, as well as devices that oscillate, creep and play tic-tac-toe.

Inferred thermophily of the Last Universal Ancestor based on estimated amino acid composition
Brooks, DJ Gaucher, EA
(2005) Submitted
<Abstract>

The environmental temperature of the last universal ancestor (LUA) of all extant organisms is the subject of heated debate. Because the amino acid composition of proteins differs between mesophiles and thermophiles, the inferred amino acid composition of proteins in the LUA could be used to classify it as one or the other. We applied expectation maximization (EM) to estimate the amino acid composition of a set of thirty-one proteins in the LUA based on alignments of their modern day descendants, a phylogenetic tree relating those descendants and a model of evolution. Separate estimates of amino acid composition in LUA proteins were derived using modern day sequences of eight mesophilic species, eight thermophilic species or the sixteen species combined. We show that the relative mean Euclidean distance between the amino acid composition in one species and that of a set of mesophiles or thermophiles can be employed as a classifier with 100% accuracy. Applying this classifier to the estimated amino acid composition of the ancestral protein set in the LUA, we find it to be classified as a thermophile even when only the proteins of mesophilic species are used to derive the estimate. Based on the estimated amino acid composition of proteins in the LUA, we infer that it was a thermophile. We discuss our findings in the context of previous data pertaining to the OGT of the LUA, particularly the inferred G + C content of its rRNA. We conclude that the gathering evidence strongly supports a thermophilic LUA.

Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways
West, CM van der Wel, H Sassi, S Gaucher, EA
Biochim. Biophys. Acta 1673 (1-2) 29-44 (2004)
<Abstract>

The Skp1 protein, best known as a subunit of E3(SCF)-ubiquitin ligases, is subject to complex glycosylation in the cytoplasm of the cellular slime mold Dictyostelium. Pro143 of this protein is sequentially modified by a prolyl hydroxylase and five soluble glycosyltransferases (GT), to yield the structure Galalpha1,Galalpha1,3Fucalpha1,2Galbeta1,3GlcNAcalpha1-HyPro143. These enzymes are unusual in that they are expressed in the cytoplasmic compartment of the cell, rather than the secretory pathway where complex glycosylation of proteins usually occurs. The first enzyme in the pathway appears to be related to the soluble animal prolyl 4-hydroxylases (P4H), which modify the transcriptional factor subunit HIF-1alpha in the cytoplasm, and more distantly to the P4Hs that modify collagen and other proteins in the rER, based on biochemical and informatics analyses. The soluble alphaGlcNAc-transferase acting on Skp1 has been cloned and is distantly related to the mucin-type polypeptide N-acetyl-alpha-galactosaminyltransferase in the Golgi of animals. Its characterization has led to the discovery of a family of related polypeptide N-acetyl-alpha-glucosaminyltransferases in the Golgi of selected lower eukaryotes. The Skp1 GlcNAc is extended by a bifunctional diglycosyltransferase that sequentially and apparently processively adds beta1,3Gal and alpha1,2Fuc. Though this structure is also formed in the animal secretory pathway, the GTs involved are dissimilar. Conceptual translation of available genomes suggests the existence of this kind of complex cytoplasmic glycosylation in other eukaryotic microorganisms, including diatoms, oomycetes, and possibly Chlamydomonas and Toxoplasma, and an evolutionary precursor of this pathway may also occur in prokaryotes. (C) 2004 Elsevier B.V. All rights reserved.

The planetary biology of cytochrome P450 aromatases
Gaucher, EA Graddy, LG Li, T Simmen, RC Simmen, FA Schreiber, DR Liberles, DA Janis, CM Benner, SA
BMC Biology 2 (1) 19 (2004)
<Abstract>

BACKGROUND: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.
RESULTS: Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene.
CONCLUSIONS: This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems.

Significance of cytoplasmic prolyl hydroxylation and complex glycosylation in the cellular slime mold Dictyostelium
West, CM van der Wel, H Sassi, S Gaucher, E Ercan, A
Glycobiology 14 (11) 1063-1063 (2004)

Empirical analysis of protein insertions and deletions determining parameters for the correct placement of gaps in protein sequence alignments
Chang, MSS Benner, SA
J. Mol. Biol. 341 (2) 617-631 (2004)
<Abstract>

To understand how protein segments are inserted and deleted during divergent evolution, a set of pairwise alignments contained exactly one gap, and therefore arising from the first insertion-deletion (indel) event in the time separating the homologs, was examined. The alignments showed that "structure breaking" amino acids (PGDNS) were preferred within and flanking gapped regions, as are two residues with hydrophilic side-chains (QE) that frequently occur at the surface of protein folds. Conversely, hydrophobic residues (FMILYVW) occur infrequently within and flanking the gapped region. These preferences are modestly different in protein pairs separated by an episode of adaptive evolution, than in pairs diverging under strong functional constraints. Surprisingly, regions near an indel have not evolved more rapidly than the sequence pair overall, showing no evidence that an indel event must be compensated by local amino acid replacement. The gap-lengths are best approximated by a Zipfian distribution, with the probability of a gap of length L decreasing as a function of L-1.8. These features are largely independent of the length of the gap and the extent of divergence (measured by both silent and non-silent sequence changes) separating the two proteins. Surprisingly, amino acid repeats were discovered in more than a third of the polypeptide segments in and around the gap. These correspond to repeats in the DNA sequence. This suggests that a signature of the mechanism by which indels occur in the DNA sequence remains in the encoded protein sequences. These data suggest specific tools to score gap placement in an alignment. They also suggest tools that distinguish true indels from gaps created by mistaken gene finding, including under-predicted and overpredicted introns. By providing mechanisms to identify errors, the tools will enhance the value of genome sequence databases in support of integrated paleogenomics strategies used to extract functional information in a post-genomic environment.

Evolutionary, structural and biochemical evidence for a new interaction site of the leptin obesity protein
Gaucher, EA Miyamoto, MM Benner, SA
Genetics 163 (4) 1549-1553 (2003)
<Abstract>

The Leptin protein is central to the regulation of energy metabolism in mammals. By integrating evolutionary, structural, and biochemical information, a surface segment, outside of its known receptor contacts, is predicted as a second interaction site that may help to further define its roles in energy balance and its functional differences between humans and other mammals.

Initiation of mucin-type O-glycosylation in lower eukaryotes (O-alpha-GlcNAc-type) and higher eukaryotes (O-alpha-GalNAc-type) is homologous
West, CM Wang, F van der Wel, H Gaucher, E Sassi, S Metcalf, T Heise, N Mendonca-Previato, L Previato, JO
Glycobiology 13 (11) 875-876 (2003)

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins
Gaucher, EA Thomson, JM Burgan, MF Benner, SA
Nature 425 (6955) 285-288 (2003)
<Abstract>

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences(1-9) of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65degreesC. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria(10), the distribution of thermophily in derived bacterial lineages(11), the inferred G+C content of ancient ribosomal RNA(12), and the geological record combined with assumptions concerning molecular clocks(13). The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins
West, CM van der Wel, H Gaucher, EA
Glycobiology 12 (2) 17R-27R (2002)
<Abstract>

Recently, complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the eukaryotic microorganism Dirtyostelium. Skp1's glycosylation is mediated by the sequential action of a prolyl hydroxylase and five conventional sugar nucleotide-dependent glycosyltransferase activities that reside in the cytoplasm rather than the secretory compartment. The Skp1-HyPro GlcNAc-Transferase, which adds the first sugar, appears to be related to a lineage of enzymes that originated in the prokaryotic cytoplasm and initiates mucin-type O-linked glycosylation in the lumen of the eukaryotic Golgi apparatus. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc. The architecture of this enzyme resembles that of certain two-domain prokaryotic glycosyl-transferases. The catalytic domains are related to those of a large family of prokaryotic and eukaryotic, cytoplasmic, membrane-bound, inverting glycosyltransferases that modify glycolipids and polysaccharides prior to their translocation across membranes toward the secretory pathway or the cell exterior. The existence of these enzymes in the eukaryotic cytoplasm away from membranes and their ability to modify protein acceptors expose a new set of cytoplasmic and nuclear proteins to potential prolyl bydroxylation and complex O-linked glycosylation.

Identification of a Golgi-associated UDP-GlcNAc : polypeptide mucin-type alpha-N-acetylglucosaminyltransferase that modifies cell surface proteins in Dictyostelium
West, CM van der Wel, H Metcalf, T Kaplan, L Gaucher, EA
Glycobiology 12 (10) 697-697 (2002)

The crystal structure of eEF1A refines the functional predictions of an evolutionary analysis of rate changes among elongation factors
Gaucher, EA Das, UK Miyamoto, MM Benner, SA
Mol. Biol. Evol. 19 (4) 569-573 (2002)

Evolution - Planetary biology - Paleontological, geological, and molecular histories of life
Benner, SA Caraco, MD Thomson, JM Gaucher, EA
Science 296 (5569) 864-868 (2002)
<Abstract>

The history of life on Earth is chronicled in the geological strata, the fossil record, and the genomes of contemporary organisms. When examined together, these records help identify metabolic and regulatory pathways, annotate protein sequences, and identify animal models to develop new drugs, among other features of scientific and biomedical interest. Together, planetary analysis of genome and proteome databases is providing an enhanced understanding of how life interacts with the biosphere and adapts to global change.

Predicting functional divergence in protein evolution by site-specific rate shifts
Gaucher, EA Gu, X Miyamoto, MM Benner, SA
Trends Biochem. Sci. 27 (6) 315-321 (2002)
<Abstract>

Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the 'non-homogeneous gamma model', which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

A bifunctional diglycosyltransferase forms the Fuca1,2Galb,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium
van der Wel, H Fisher, SZ Gaucher, EA West, CM
Glycobiology 11 (10) 884-884 (2001)

Function-structure analysis of proteins using covarion-based evolutionary approaches: Elongation factors
Gaucher, EA Miyamoto, MM Benner, SA
Proc. Natl. Acad. Sci. USA 98 (2) 548-552 (2001)
<Abstract>

The divergent evolution of protein sequences from genomic databases can be analyzed by the use of different mathematical models. The most common treat all sites in a protein sequence as equally variable. More sophisticated models acknowledge the fact that purifying selection generally tolerates variable amounts of amino acid replacement at different positions in a protein sequence. In their "stationary" versions, such models assume that the replacement rate at individual positions remains constant throughout evolutionary history. "Nonstationary" covarion versions, however, allow the replacement rate at a position to vary in different branches of the evolutionary tree. Recently, statistical methods have been developed that highlight this type of variation in replacement rates. Here, we show how positions that have variable rates of divergence in different regions of a tree ("covarion behavior"), coupled with analyses of experimental three-dimensional structures, can provide experimentally testable hypotheses that relate individual amino acid residues to specific functional differences in those branches. We illustrate this in the elongation factor family of proteins as a paradigm for applications of this type of analysis in functional genomics generally.

Evolution, language and analogy in functional genomics
Benner, SA Gaucher, EA
Trends in Genetics 17 (7) 414-418 (2001)
<Abstract>

Almost a century ago, Wittgenstein pointed out that theory in science is intricately connected to language. This connection is not a frequent topic in the genomics literature. But a case can be made that functional genomics is today hindered by the paradoxes that Wittgenstein identified. If this is true, until these paradoxes are recognized and addressed, functional genomics will continue to be limited in its ability to extrapolate information from genomic sequences.

Functional inferences from reconstructed evolutionary biology involving rectified databases. An evolutionarily-grounded approach to functional genomics.
Benner, SA Chamberlin, SG Liberles, DA Govindarajan, S Knecht, L
Res. MicroBiol. 151 (2) 97-106 (2000)
<Abstract>

If bioinformatics tools are constructed to reproduce the natural, evolutionary history of the biosphere, they offer powerful approaches to some of the most difficult tasks in genomics, including the organization and retrieval of sequence data, the updating of massive genomic databases, the detection of database error, the assignment of introns, the prediction of protein conformation from protein sequences, the detection of distant homologs, the assignment of function to open reading frames, the identification of biochemical pathways from genomic data, and the construction of a comprehensive model correlating the history of biomolecules with the history of planet Earth.

Chance and Necessity in Biomolecular Chemistry - Is Life as We Know It Universal?
Benner, SA Switzer, CY
Simplicity and Complexity in Proteins and Nucleic Acids, ed. H. Frauenfelder, J. Deisenhofer, P.G. Wolynes, Dahlem University Press 339-363 (1999)

How small can a microorganism be?
Benner, SA
Size Limits of Very Small Microorganisms: Proceedings of a Workshop, Steering Group on Astrobiology of the Space Studies Board, National Research Council 126-135 (1999)

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