We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

JCVI-syn3A, a robust minimal cell with a 543 kbp genome and 493 genes, provides a versatile platform to study the basics of life. Using the vast amount of experimental information available on its precursor, Mycoplasma mycoides capri, we assembled a near-complete metabolic network with 98% of enzymatic reactions supported by annotation or experiment. The model agrees well with genome-scale in vivo transposon mutagenesis experiments, showing a Matthews correlation coefficient of 0.59. The genes in the reconstruction have a high in vivo essentiality or quasi-essentiality of 92% (68% essential), compared to 79% in silico essentiality. This coherent model of the minimal metabolism in JCVI-syn3A at the same time also points toward specific open questions regarding the minimal genome of JCVI-syn3A, which still contains many genes of generic or completely unclear function. In particular, the model, its comparison to in vivo essentiality and proteomics data yield specific hypotheses on gene functions and metabolic capabilities; and provide suggestions for several further gene removals. In this way, the model and its accompanying data guide future investigations of the minimal cell. Finally, the identification of 30 essential genes with unclear function will motivate the search for new biological mechanisms beyond metabolism.

Abstract-Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K m values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia. Key Words: nucleoside Ⅲ adenosine Ⅲ transport Ⅲ ischemia Ⅲ pH T he purine nucleoside adenosine is produced by the action of both endo-and ecto-nucleotidases on adenine nucleotides in the heart and plays key roles in the regulation of coronary blood flow and myocardial O 2 supply-demand balance. 1-4 For example, action of adenosine on A 2A receptors on vascular smooth muscle and endothelial cells causes coronary vasodilatation. 1,5 In contrast, the negative inotropic and dromotropic effects of adenosine on the heart are mediated primarily by A 1 receptors. 2 Similarly, the negative chromotropic effect of adenosine involves action of A 1 receptors in the sinoatrial (SA) node on the inwardly rectifying potassium channel current I K-Ado and the hyperpolarization-activated pacemaker current I f . 2,6 Endogenous adenosine, acting on mitochondrial K ATP channels via A 1 and A 3 receptors, also makes a major contribution to the phenomenon of ischemic preconditioning. 5,7 Extracellular adenosine concentrations in the heart are governed both by action of ecto-5Ј-nucleotidase on adenine nucleotides released from cells and by transporter-mediated flux of adenosine across cell membranes. 3,4 Although most adenosine production occurs intracellularly, under normoxic conditions, metabolism maintains a low intracellular concentration and, therefore, the net flux of adenosine is into cardiomyocytes and endothelial cells. Under such conditions, administration of transport inhibitors increases extracellular concentrations of adenosine, leading to vasodilatation. 8 However, increased adenine nucleotide breakdown and inhibition of adenosine kinase duri...