Faculty Bibliography

LINE retrotransposons actively shape mammalian genomes. Denli et al. reveal a new open reading frame, ORF0, on the antisense strand of human LINE-1 encoding a small regulatory protein. This finding may represent the birth of an emerging retrotransposon gene that can adopt various fates, as it can be fused to adjacent host sequences.

Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed after the disease has metastasized; it is among the most lethal forms of cancer. We recently described aberrant expression of an open reading frame 1 protein, ORF1p, encoded by long interspersed element-1 (LINE-1; L1) retrotransposon, in PDAC. To test whether LINE-1 expression leads to somatic insertions of this mobile DNA, we used a targeted method to sequence LINE-1 insertion sites in matched PDAC and normal samples. We found evidence of 465 somatic LINE-1 insertions in 20 PDAC genomes, which were absent from corresponding normal samples. In cases in which matched normal tissue, primary PDAC and metastatic disease sites were available, insertions were found in primary and metastatic tissues in differing proportions. Two adenocarcinomas secondarily involving the pancreas, but originating in the stomach and duodenum, acquired insertions with a similar discordance between primary and metastatic sites. Together, our findings show that LINE-1 contributes to the genetic evolution of PDAC and suggest that somatic insertions are acquired discontinuously in gastrointestinal neoplasms.

First introduced in 2011, the Synthetic Yeast Genome (Sc2.0) PROJECT is a large international synthetic genomics project that will culminate in the first eukaryotic cell (Saccharomyces cerevisiae) with a fully synthetic genome. With collaborators from across the globe and from a range of institutions spanning from do-it-yourself biology (DIYbio) to commercial enterprises, it is important that all scientists working on this project are cognizant of the ethical and policy issues associated with this field of research and operate under a common set of principles. In this commentary, we survey the current ethics and regulatory landscape of synthetic biology and present the Sc2.0 Statement of Ethics and Governance to which all members of the project adhere. This statement focuses on four aspects of the Sc2.0 PROJECT: societal benefit, intellectual property, safety, and self-governance. We propose that such project-level agreements are an important, valuable, and flexible model of self-regulation for similar global, large-scale synthetic biology projects in order to maximize the benefits and minimize potential harms.

We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing beta-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.

Here we describe the engineering of a distant homolog of the Tet repressor, CamR, isolated from Pseudomonas putida, that is regulated by camphor, a very inexpensive small molecule, at micromolar concentrations, for use in Saccharomyces cerevisiae. The repressor was engineered by expression from a constitutive yeast promoter, fusion to a viral activator protein cassette, and codon optimization. A suitable promoter responsive to the CamR fusion protein was engineered by embedding a P. putida operator binding sequence within an upstream activating sequence (UAS)-less CYC1 promoter from S. cerevisiae. The switch, named the Camphor-Off switch, activates expression of a reporter gene in camphor-free media and represses it with micromolar concentrations of camphor.

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs, or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists an efficient new means by which to engineer S. cerevisiae.

Epigenetic mechanisms, including histone post-translational modifications, control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging phenomenon of shortened life span, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a life span screen in Saccharomyces cerevisiae that is designed to identify amino acid residues of histones that regulate yeast replicative aging. Our results reveal that lack of sustained histone H3K36 methylation is commensurate with increased cryptic transcription in a subset of genes in old cells and with shorter life span. In contrast, deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes, suppresses cryptic transcript initiation, and extends life span. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to loss of transcriptional precision that is detrimental to life span, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.