Biology subjects

Ong, J. Y.

Publications and source records attributed to Ong, J. Y..

5 recordsLinked to original sources

bioRxiv2024

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in Gram-negative bacteria

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labelling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

microbiology↗

bioRxiv2023

Cul3 substrate adaptor SPOP targets Nup153 for degradation

SPOP is a Cul3 substrate adaptor responsible for degradation of many proteins related to cell growth and proliferation. Because mutation or misregulation of SPOP drives cancer progression, understanding the suite of SPOP substrates is important to understanding regulation of cell proliferation. Here, we identify Nup153, a component of the nuclear basket of the nuclear pore complex, as a novel substrate of SPOP. SPOP and Nup153 bind to each other and colocalize at the nuclear envelope and some nuclear foci in cells. The binding interaction between SPOP and Nup153 is complex and multivalent. Nup153 is ubiquitylated and degraded upon expression of SPOPWT but not its substrate binding-deficient mutant SPOPF102C. Depletion of SPOP via RNAi leads to Nup153 stabilization. Upon loss of SPOP, the nuclear envelope localization of spindle assembly checkpoint protein Mad1, which is tethered to the nuclear envelope by Nup153, is stronger. Altogether, our results demonstrate SPOP regulates Nup153 levels and expands our understanding of the role of SPOP in protein and cellular homeostasis.

cell biology↗

bioRxiv2021

MaveDB v2: a curated community database with over three million variant effects from multiplexed functional assays

A central problem in genomics is understanding the effect of individual DNA variants. Multiplexed Assays of Variant Effect (MAVEs) can help address this challenge by measuring all possible single nucleotide variant effects in a gene or regulatory sequence simultaneously. Here we describe MaveDB v2, which has become the database of record for MAVEs. MaveDB now contains a large fraction of published studies, comprising over two hundred datasets and three million variant effect measurements. We created tools and APIs to streamline data submission and access, transforming MaveDB into a hub for the analysis and dissemination of these impactful datasets.

genomics↗

bioRxiv2021

Synonymous Mutation Generator: a web tool for designing RNAi-resistant sequences

RNA interference (RNAi) is a useful technique for knocking down a protein of interest, allowing for the study of the function of a gene product. However, RNAi techniques are prone to off-target effects, such as non-specific knockdown of genes besides the protein of interest. An important control and companion to RNAi knockdown experiments is the rescue experiment, wherein gene function is restored by expression of an RNAi-resistant construct of the protein of interest. Generating an RNAi-resistant construct of the protein of interest involves generating silent mutations within the coding sequence of the protein so that the resulting amino acid product is the same, but the protein mRNA is no longer a target for the RNAi. Here, Synonymous Mutation Generator, a Python-based web tool that takes an input DNA coding sequence and outputs a synonymous DNA coding sequence that is RNAi-resistant, is described. This web tool should be a useful resource for researchers cloning RNAi-resistant constructs. Synonymous Mutation Generator is easy to use and can be found at jong2.pythonanywhere.com, and the source code is available on GitHub.

molecular biology↗

bioRxiv2020

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of weeks. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methylmethane sulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to MMS demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain but with lower sensitivity than the plate-based assay. In conclusion, we propose yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.View Full Text

pharmacology and toxicology↗