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 recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.

We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunitsubunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.The initiation of DNA replication in lower eukaryotes is similar to that observed in bacteria in that it occurs at well defined origins of DNA replication that are recognized by specific initiator proteins (1-5). Saccharomyces cerevisiae origins of DNA replication are short, ϳ100-base pair (bp) 1 segments that consist of two essential regions: the A domain, which contains a conserved 11-bp consensus sequence (ACS) that is required for origin function, and the B domain, which contains several stimulatory elements (6, 7). The ACS is recognized specifically by the S. cerevisiae initiator protein, the origin recognition complex (ORC) (8, 9). S. cerevisiae ORC (ScORC) is comprised of six proteins (Orc1-6), each of which is essential for viability in S. cerevisiae (10 -13). A number of lines of evidence indicate that ScORC is required for initiation of DNA replication in vivo and functions in part to recruit other initiation factors, such as Cdc6 and the minichromosome maintenance proteins, to replication origins (13-15).Homologues of ORC subunits have been identified in a variety of other eukaryotic species (2, 16), and ORC has been purified from the fission yeast Schizosaccharomyces pombe and the metazoans Drosophila melanogaster and Xenopus laevis (17)(18)(19). Genetic and biochemical data indicate that specific ORC subunits from t...

Recent scientific advances have significantly contributed to our understanding of the complex connection between the microbiome and cancer. Our bodies are continuously exposed to microbial cells, both resident and transient, as well as their byproducts, including toxic metabolites. Circulation of toxic metabolites may contribute to cancer onset or progression at locations distant from where a particular microbe resides. Moreover, microbes may migrate to other locations in the human body and become associated with tumor development. Several case-control metagenomics studies suggest that dysbiosis in the commensal microbiota is also associated with inflammatory disorders and various cancer types throughout the body. Although the microbiome influences carcinogenesis through mechanisms independent of inflammation and immune system, the most recognizable link is between the microbiome and cancer via the immune system, as the resident microbiota plays an essential role in activating, training, and modulating the host immune response. Immunologic dysregulation is likely to provide mechanistic explanations as to how our microbiome influences cancer development and cancer therapies. In this review, we discuss recent developments in understanding the human gut microbiome's relationship with cancer and the feasibility of developing novel cancer diagnostics based on microbiome profiles. .