We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

The bacterioplankton assemblage in Crater Lake, Oregon (U.S.A.), is different from communities found in other oxygenated lakes, as demonstrated by four small subunit ribosomal ribonucleic acid (SSU rRNA) gene clone libraries and oligonucleotide probe hybridization to RNA from lake water. Populations in the euphotic zone of this deep (589 m), oligotrophic caldera lake are dominated by two phylogenetic clusters of currently uncultivated bacteria: CL120-10, a newly identified cluster in the verrucomicrobiales, and ACK4 actinomycetes, known as a minor constituent of bacterioplankton in other lakes. Deep-water populations at 300 and 500 m are dominated by a different pair of uncultivated taxa: CL500-11, a novel cluster in the green nonsulfur bacteria, and group I marine crenarchaeota. ␤-Proteobacteria, dominant in most other freshwater environments, are relatively rare in Crater Lake (Յ16% of nonchloroplast bacterial rRNA at all depths). Other taxa identified in Crater Lake libraries include a newly identified candidate bacterial division, ABY1, and a newly identified subcluster, CL0-1, within candidate division OP10. Probe analyses confirmed vertical stratification of several microbial groups, similar to patterns observed in open-ocean systems. Additional similarities between Crater Lake and ocean microbial populations include aphotic zone dominance of group I marine crenarchaeota and green nonsulfur bacteria. Comparison of Crater Lake to other lakes studied by rRNA methods suggests that selective factors structuring Crater Lake bacterioplankton populations may include low concentrations of available trace metals and dissolved organic matter, chemistry of infiltrating hydrothermal waters, and irradiation by high levels of ultraviolet light.

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A,-3B, and-3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed. (53). Recent serologic evidence has also revealed differences between EBV-1 and EBV-2 in their EBNA-3 proteins (48). EBV-1 has three distantly related EBNA-3 proteins, EBNA-4084 Vol. 64, No. 9