INTRODUCTION It has long been an interesting question whether a living cell can be constructed from scratch in the lab, a goal that may not be realized anytime soon. Nonetheless, with advances in DNA synthesis technology, the complete genetic material of an organism can now be synthesized chemically. Hitherto, genomes of several organisms including viruses, phages, and bacteria have been designed and constructed. These synthetic genomes are able to direct all normal biological functions, capable of self-replication and production of offspring. Several years ago, a group of scientists worldwide formed an international consortium to reconstruct the genome of budding yeast, Saccharomyces cerevisiae . RATIONALE The synthetic yeast genome, designated Sc2.0, was designed according to a set of arbitrary rules, including the elimination of transposable elements and incorporation of specific DNA elements to facilitate further genome manipulation. Among the 16 S. cerevisiae chromosomes, chromosome XII is unique as one of the longest yeast chromosomes (~1 million base pairs) and additionally encodes the highly repetitive ribosomal DNA locus, which forms the well-organized nucleolus. We report on the design, construction, and characterization of chromosome XII, the physically largest chromosome in S. cerevisiae. RESULTS A 976,067–base pair linear chromosome, synXII, was designed based on the native chromosome XII sequence of S. cerevisiae , and chemically synthesized. SynXII was assembled using a two-step method involving, successive megachunk integration to produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding a full-length functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII were caused by deletion of tRNA genes and were corrected by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit. The same synthetic rDNA unit was also used to regenerate rDNA at three distinct chromosomal locations. The rDNA signature sequences of the internal transcribed spacer (ITS), often used to determine species identity by standard DNA barcoding procedures, were swapped to generate a Saccharomyces synXII strain that would be identified as S. bayanus. Remarkably, these substantial DNA changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION The rDNA locus of synXII is highly plastic; not only can it be moved to other chromosomal loci, it can also be altered in its ITS region to masquerade as a distinct species as defined by DNA barcoding, used widely in taxonomy. The ability to perform “species morphing” reported here presumably reflects the degree of evolutionary flexibility by which these ITS regions change. However, this barcoding region is clearly not infinitely flexible, as only relatively modest intragenus base changes were tolerated. More severe intergenus differences in ITS sequence did not result in functional rDNAs, probably because of defects in rRNA processing. The ability to design, build, and debug a megabase-sized chromosome, together with the flexibility in rDNA locus position, speaks to the remarkable overall flexibility of the yeast genome. Hierarchical assembly and subsequent restructuring of synXII. SynXII was assembled in two steps: First, six semisynthetic synXII strains were built in which segments of native XII DNA were replaced with the corresponding designer sequences. Next, the semisynthetic strains were combined withmultiple rounds ofmating/sporulation, eventually generating a single strain encoding fulllength synXII.The rDNA repeats were removed, modified, and subsequently regenerated at distinct chromosomal locations for species morphing and genome restructuring.

NuA4 catalyzes the acetylation of nucleosomes at histone H4, which is a well-established epigenetic event, controlling many genomic processes in Saccharomyces cerevisiae. Here we report the crystal structures of the NuA4 core complex and a cryoelectron microscopy structure with the nucleosome. The structures show that the histone-binding pocket of the enzyme is rearranged, suggesting its activation. The enzyme binds the histone tail mainly through the target lysine residue, with a preference for a small residue at the -1 position. The complex engages the nucleosome at the dish face and orients its catalytic pocket close to the H4 tail to achieve selective acetylation. The combined data reveal a space-sequence double recognition mechanism of the histone tails by a modifying enzyme in the context of the nucleosome.

Background Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction. Results We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified. Conclusions We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.