To determine whether genes retain ancestral functions over a billion years of evolution and to identify principles of deep evolutionary divergence, we replaced 414 essential yeast genes with their human orthologs, assaying for complementation of lethal growth defects upon loss of the yeast genes. Nearly half (47%) of the yeast genes could be successfully humanized. Sequence similarity and expression only partly predicted replaceability. Instead, replaceability depended strongly on gene modules: genes in the same process tended to be similarly replaceable (e.g., sterol biosynthesis) or not (e.g., DNA replication initiation). Simulations confirmed selection for specific function can maintain replaceability despite extensive sequence divergence. Critical ancestral functions of many essential genes are thus retained in a pathway-specific manner, robust to drift in sequences, splicing, and protein interfaces.

Background: Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina's platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs.

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete. INTRODUCTIONThe critical task of mitosis is equal segregation of sister chromatids to daughter cells. Faithful chromosome transmission is required for maintenance of genomic integrity, and failures in this process lead to aneuploid states characteristic of cancer (Nowak et al., 2002). Checkpoints are mechanisms that inhibit cell division in the presence of damage, allowing time for repair and subsequent reentry into the cell cycle (Hartwell and Weinert, 1989). The spindle checkpoint arrests cells in mitosis when spindle function is inadequate for faithful chromosome segregation (Lew and Burke, 2003). This checkpoint can be induced by benzimidazole drugs, which cause microtubule depolymerization and compromised attachment to kinetochores. To arrest in mitosis, cells must stabilize sister chromatid cohesion to prevent premature separation of chromosomes and the mitotic cyclin Clb2 to maintain mitotic cyclin-dependent kinase activity (Lew and Burke, 2003). Maintenance of both cohesion and mitotic cyclin-dependent kinase (CDK) activity under these conditions requires the cell to stabilize proteins whose destruction is normally initiated by an E3 ubiquitin ligase known as the anaphase-promoting complex (APC) (Harper et al., 2002;Vodermaier, 2004).The activity and substrate specificity of the APC is a key point of regulation in the cell cycle. Two distinct APC activators, Cdc20 and Cdh1, act at metaphase and mitotic exit respectively (Visintin et al., 1997). These proteins direct degradation of different, but partially overlapping, substrates to promote the irreversible passage through these cell cycle stages. APC Cdc20 targets Pds1 "securin", a protein required for metaphase sister chromatid cohesion, and the S-phase cyclin Clb5, while APC Cdh1 targets Ase1, Cdc20 and others. Degradation of Clb2 is biphasic, with a subset of the protein being degraded by APC Cdc20 , and the remainder by APCCdh...

SUMMARY We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing of synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with efficiencies >40% without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous β-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.

Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin-like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin-like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitinlike domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin-like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation.