Faculty Bibliography

Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.

Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chip's usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.

The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.

BRISC (Brcc36-containing isopeptidase complex) is a four-subunit deubiquitinating (DUB) enzyme that has a catalytic subunit, called Brcc36, that is a member of the JAMM/MPN(+) family of zinc metalloproteases. A notable feature of BRISC is its high specificity for cleaving Lys(63)-linked polyubiquitin. Here, we show that BRISC selectivity is not due to preferential binding to Lys(63)-linked polyubiquitin but is instead dictated by how the substrate isopeptide linkage is oriented within the enzyme active site. BRISC possesses a high affinity binding site for the ubiquitin hydrophobic surface patch that accounts for the bulk of the affinity between enzyme and substrate. Although BRISC can interact with either subunit of a diubiquitin conjugate, substrate cleavage occurs only when BRISC is bound to the hydrophobic patch of the distal (i.e. the "S1") ubiquitin at a ubiquitin-ubiquitin cleavage site. The importance of the Lys(63)-linked proximal (S1') ubiquitin was underscored by our finding that BRISC could not cleave the isopeptide bond joining a ubiquitin to a non-ubiquitin substrate. Finally, we also show that Abro1, another BRISC subunit, binds directly to Brcc36 and that the Brcc36-Abro1 heterodimer includes a minimal complex with Lys(63)-specific DUB activity.

In budding yeast, silent chromatin is defined at the region of telomeres, rDNA loci, and silent mating loci. Although the silent chromatin at different loci shows structural similarity, the underlying mechanism to establish, maintain, and inherit these structures may be fundamentally different. In this study, we found two arginine residues within histone H2B, which are specifically required to maintain either the telomeric or the rDNA silenct chromatin. Arginine 95 (R95) plays a specific role at telomeres, whereas arginine 102 (R102) is required to maintain the silent chromatin at rDNA and to ensure the integrity of rDNA loci by suppressing recombination between rDNA repeats. R95 mutants show enhanced rDNA silencing but a paradoxically low Sir2 protein abundance. Furthermore weakened silencing at telomeres in R95 mutants can be suppressed by a specific SIR3 allele, SIR3-D205N, which increases the affinity of Sir proteins to telomeres, suggesting H2B-R95 may directly mediate telomeric Sir protein-nucleosome interactions. Double mutations of R95 and R102 lead to desilencing of both rDNA and telomeres, indicating both arginines are necessary to ensure integrity of silent chromatin at these loci. Furthermore, mutations of R102 cause accumulation of extrachromosomal rDNA circles and reduce life span, suggesting that histone H2B contributes to longevity.

Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser(3), an unusual posttranslational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases.

The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.

A recent explosion of work surrounds the interactions between Sir3p (Silent Information Regulator 3) and chromatin. We review here the Sir3p functions related to its role in silencing in Saccharomyces cerevisiae. This unusual protein, which is absolutely required for silencing, is distantly related to the highly conserved replication initiator Orc1p, but is itself phylogenetically limited to "post-genome-duplicated" budding yeasts. Several recent studies revise earlier models for Sir3p action. Specifically, the N-terminal bromo-adjacent homology (BAH) domain plays a now well-defined role in silencing, and a picture is emerging in which both termini of Sir3p bind two locations on the nucleosome: (1) the loss of ribosomal DNA silencing (LRS) surface in the nucleosome core, and (2) the N-terminal histone tails for effective silencing at telomeres. We relate Sir3p structure and function, and summarize recent molecular studies of Sir3p/chromatin binding, Sir3p/Dot1p competition, and the possible role of O-Acetyl ADP ribose (O-AADPR) in Sir3p/chromatin binding. We emphasize recent genetic data that provide important new insights and settle controversies created by in vitro work. Finally, we synthesize these ideas to revise the model for how Sir3p mediates silent chromatin formation in yeast, in part through its affinity for the LRS region of the nucleosome, which must be "just right."

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA "bar-code" sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Delta mutant and exacerbated by a short-lived atg16Delta autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process.