Kinetochore inactivation by expression of a repressive mRNA.

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. Meiosis requires transient removal of the outer kinetochore, the complex that connects microtubules to chromosomes. How the meiotic gene expression program temporally restricts kinetochore function is unknown. We discovered that in budding yeast, kinetochore inactivation occurs by reducing the abundance of a limiting subunit, Ndc80. Furthermore, we uncovered an integrated mechanism that acts at the transcriptional and translational level to repress NDC80 expression. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform, which itself cannot produce protein due to regulatory upstream ORFs in its extended 5' leader. Instead, transcription of this isoform represses the canonical NDC80 mRNA expression in cis, thereby inhibiting Ndc80 protein synthesis. This model of gene regulation raises the intriguing notion that transcription of an mRNA, despite carrying a canonical coding sequence, can directly cause gene repression

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 193 (2011): 1065-1081, doi:10.1083/jcb.201012143.The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.This work was supported by the National Science Foundation under grant No. MCB-0719126 to A.S. Gladfelter, the National Institute of Biomedical Imaging and Bioengineering under grant No. EB002583 to R. Oldenbourg, a Drexel CURE grant from the State of Pennsylvania Tobacco Settlement Fund, and National Institute of Neurological Disorders and Stroke grant NS48090- 06A to E.T. Spiliotis

Methods for Obtaining and Analyzing Whole Chloroplast Genome Sequences

During the past decade there has been a rapid increase in our understanding of plastid genome organization and evolution due to the availability of many new completely sequenced genomes. Currently there are 43 complete genomes published and ongoing projects are likely to increase this sampling to nearly 200 genomes during the next five years. Several groups of researchers including ours have been developing new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this chapter. The most important recent developments that enhance our ability to generate whole chloroplast genome sequences involve the generation of pure fractions of chloroplast genomes by whole genome amplification using rolling circular amplification, cloning genomes into Fosmid or BAC vectors, and the development of an organellar annotation program (DOGMA). In addition to providing details of these methods, we provide an overview of methods for analyzing complete plastid genome sequences for repeats and gene content, as well as approaches for using gene order and sequence data for phylogeny reconstruction. This explosive increase in the number of sequenced plastid genomes and improved computational tools will provide many insights into the evolution of these genomes and much new data for assessing relationships at deep nodes in plants and other photosynthetic organisms

SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair

The research leading to these results is supported by Cancer Research UK (XYG, RAB, EG, PM, PE, SG, C Santos, AJR, NM, PAB, AS and C Swanton), Breast Cancer Research Foundation (C Swanton and NK), Medical Research Council (ID: G0902275 to MG and C Santos; ID: G0701935/2 to AJR and C Swanton), the Danish Cancer Society (AMM, J Bartkova and J Bartek), the Lundbeck Foundation (R93-A8990 to J Bartek), the Ministry of the interior of the Czech Republic (grant VG20102014001 to MM and J Bartek), the National Program of Sustainability (grant LO1304 to MM and J Bartek), the Danish Council for Independent Research (grant DFF-1331-00262 to J Bartek), NIHR RMH/ICR Biomedical Research Centre for Cancer (JL), the EC Framework 7 (PREDICT 259303 to XYG, EG, PM, MG, TJ and C Swanton; DDResponse 259892 to J Bartek and J Bartkova and RESPONSIFY ID:259303 to C Swanton), UCL Overseas Research Scholarship (SG). C Swanton is also supported by the European Research Council, Rosetrees Trust and The Prostate Cancer Foundation. This research is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre

A New Highly Conserved Antibiotic Sensing/Resistance Pathway in Firmicutes Involves an ABC Transporter Interplaying with a Signal Transduction System

Signal transduction systems and ABC transporters often contribute jointly to adaptive bacterial responses to environmental changes. In Bacillus subtilis, three such pairs are involved in responses to antibiotics: BceRSAB, YvcPQRS and YxdJKLM. They are characterized by a histidine kinase belonging to the intramembrane sensing kinase family and by a translocator possessing an unusually large extracytoplasmic loop. It was established here using a phylogenomic approach that systems of this kind are specific but widespread in Firmicutes, where they originated. The present phylogenetic analyses brought to light a highly dynamic evolutionary history involving numerous horizontal gene transfers, duplications and lost events, leading to a great variety of Bce-like repertories in members of this bacterial phylum. Based on these phylogenetic analyses, it was proposed to subdivide the Bce-like modules into six well-defined subfamilies. Functional studies were performed on members of subfamily IV comprising BceRSAB from B. subtilis, the expression of which was found to require the signal transduction system as well as the ABC transporter itself. The present results suggest, for the members of this subfamily, the occurrence of interactions between one component of each partner, the kinase and the corresponding translocator. At functional and/or structural levels, bacitracin dependent expression of bceAB and bacitracin resistance processes require the presence of the BceB translocator loop. Some other members of subfamily IV were also found to participate in bacitracin resistance processes. Taken together our study suggests that this regulatory mechanism might constitute an important common antibiotic resistance mechanism in Firmicutes. [Supplemental material is available online at http://www.genome.org.

Bioengineering Strategies for Protein-Based Nanoparticles.

In recent years, the practical application of protein-based nanoparticles (PNPs) has expanded rapidly into areas like drug delivery, vaccine development, and biocatalysis. PNPs possess unique features that make them attractive as potential platforms for a variety of nanobiotechnological applications. They self-assemble from multiple protein subunits into hollow monodisperse structures; they are highly stable, biocompatible, and biodegradable; and their external components and encapsulation properties can be readily manipulated by chemical or genetic strategies. Moreover, their complex and perfect symmetry have motivated researchers to mimic their properties in order to create de novo protein assemblies. This review focuses on recent advances in the bioengineering and bioconjugation of PNPs and the implementation of synthetic biology concepts to exploit and enhance PNP's intrinsic properties and to impart them with novel functionalities

Doctor of Philosophy

dissertationEukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, and associate with each other in some of their biologically important contexts. Spt6 functions at several important regulatory steps in transcription, including nucleosome reassembly, transcription elongation, and mRNA processing and export. As a histone chaperone, Spt6 is important for reassembly of nucleosomes in the wake of elongating RNA polymerase II, a process that is required to regulate transcription initiation and prevent inappropriate transcription from repressed promoters as well as cryptic intragenic transcription start sites. In conjunction with Spt6, Spn1 coordinates the recruitment of mRNA processing and export factors, such as Yra1 and the exosome, thereby enabling biogenesis of mature and export competent mRNA molecules. The functional relevance of Spn1 and Spt6 in chromatin organization and mRNA maturation is well established, although mechanistic details of how these processes are performed are poorly understood. In order to enhance our understanding of the molecular details of Spn1 and Spt6 functions, this thesis has focused on structural, biochemical and functional studies of Spn1 and Spt6 from Saccharomyces cerevisiae. iv The work presented in this dissertation establishes the structures of the entire ordered region of the Spt6 protein, the ordered core of the Spn1 protein, and the Spn1 core in complex with the binding determinant of Spt6. Additionally, we demonstrate the capacity of Spt6 to interact with factors that very likely influence Spt6 function, including histones and nucleosomes. The structures and the functional data described in this dissertation have enhanced our understanding of how Spt6 binds and chaperones histones, and describes a novel role for Spn1 in regulating the histone chaperone activity of Spt6. The Spt6 and Spn1 structures and the biochemical assays developed in this work will aid in future functional and mechanistic studies that will aim to develop a complete molecular and mechanistic model for each Spt6 and Spn1 function

Engineering protein biosynthesis apparatus, advanced design and screening strategies for small and fluorinated substrates in orthogonal translation

Protein engineering is a comprehensive toolbox for the chemical modification of enzymes in particular, and for the expansion of molecular functional diversity in general. In recent decades, two different categories have become established for the engineering of proteins. These include the approach of directed evolution approaches on the one hand and the strategies of rational protein design on the other hand. In particular, the use of noncanonical amino acids to introduce new functionalities has gained importance in the engineers’ toolbox. These include isostructural analogues of canonical amino acids as well as molecules with reactivities that can provide sites for further protein modifications. In this study, we have presented a strategy for manipulating the protein biosynthesis machinery towards the incorporation of noncognate fluorinated substrates. In general, fluorinated amino acids are not genetically encoded. These mainly synthetic building block are valuable for the design of particularly stable protein folds and for targeting highly specific protein-protein interactions. Fluorine is small and has a very low polarizability and the strongest inductive effect among the chemical elements found on earth. Due to these unique stereoelectronic properties, fluorine substitution is advantageously used in protein and peptide design. In this context, the strategy of directed evolution was applied to construct isoleucyl-transfer ribonucleic acid synthetase libraries for the isoleucine AUA rare codon reassignment with small aliphatic fluorinated amino acids, such as L-trifluoroethylglycine, by random mutagenesis. A suitable screening plasmid containing a mutant of superfolder green fluorescent protein (sfGFP) as reporter protein and a modified isoleucine transfer ribonucleic acid (tRNA_UAU) from Escherichia coli was produced to create an enhanced molecular adaptor level for gene expression. However, the required selection strain could not be constructed by genome editing due to the complexity of essential gene modification. In the second part of the study, different reporter proteins were used in advanced design with noncanonical amino acids for improvement of their biophysical, chemical, and biological properties. A robust alkene-tagged sfGFP variant was obtained, which is a valuable target in medicinal chemistry. In addition, the residue-specific incorporation of proline analogues into green fluorescent protein (GFP) derivates ─ enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange ─ enables the study of the role of prolines in the typical β-barrel structure organization

Characterization of the RpoN Regulon Reveals Differential Regulation of T6SS and New Flagellar Operons in Vibrio cholerae O37 Strain V52

The alternative sigma factor RpoN is an essential colonization factor of Vibrio cholerae and controls important cellular functions including motility and type VI secretion (T6SS). The RpoN regulon has yet to be clearly defined in T6SS-active V. cholerae isolates, which use T6SS to target both bacterial competitors and eukaryotic cells. We hypothesize that T6SS-dependent secreted effectors are co-regulated by RpoN. To systemically identify RpoN-controlled genes, we used chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to determine RpoN-binding sites and RpoN-controlled gene expression. There were 68 RpoN-binding sites and 82 operons positively controlled by RpoN, among which 37 operons had ChIP-identified binding sites. A consensus RpoN-binding motif was identified with a highly conserved thymine (−14) and an AT-rich region in the middle between the hallmark RpoN-recognized motif GG(−24)/GC(−12). There were seven new RpoN-dependent promoters in the flagellar regions. We identified a small RNA, flaX, downstream of the major flagellin gene flaA. Mutation of flaX substantially reduced motility. In contrast to previous results, we report that RpoN positively regulates the expression of hcp operons and vgrG3 that encode T6SS secreted proteins but has no effect on the expression of the main T6SS cluster encoding sheath and other structural components