Biology subjects

Bielinski, V. A.

Publications and source records attributed to Bielinski, V. A..

4 recordsLinked to original sources

bioRxiv2021

LoopViz: A uLoop Assembly Clone Verification Tool for Nanopore Sequencing Reads

Cloning has been an integral part of most laboratory research questions and continues to be an essential tool in defining the genetic elements determining life. Cloning can be difficult and time consuming as each plasmid is unique to a particular project and each sequence must be carefully selected, cloned and sequenced to determine correctness. Loop assembly (uLOOP) is a recursive, Golden Gate-like assembly method that allows rapid cloning of domesticated DNA fragments to robustly refactor novel pathways. With uLOOP methodologies, one can clone several sequences directionally to generate a library of transcriptional units (TUs) in plasmids within a single reaction but analysis of the plasmid population has been impeded by current sequencing and analysis methods. Here we develop LoopViz, a quality control tool that quantifies and visualizes results from assembly reactions using long-read Oxford Nanopore Technologies (ONT) sequencing. LoopViz identifies full length reads originating from a single plasmid in the population, and visualizes them in terms of a user input DNA fragments file, and provides QC statistics. This methodology enables validation and analysis of cloning and sequencing reactions in less than a day, determination of the entire plasmids sequence, and sequencing through repetitive meta-regions that cannot be meaningfully assembled. Finally, LoopViz represents a new paradigm in determining plasmid sequences that is rapid, cost-effective and performed in-lab. LoopViz is made publicly available at https://gitlab.com/marielelensink325/loopseq

bioinformatics↗

bioRxiv2020

The Phaeodactylum tricornutum Diaminopimelate Decarboxylase was Acquired via Horizontal Gene Transfer from Bacteria and Displays Substrate Promiscuity

Diatoms are predicted to synthesize certain amino acids within the chloroplast, including L-lysine via a diaminopimelate-dependent pathway. Herein, we report that the model diatom, Phaeodactylum tricornutum, possesses a chimeric lysine biosynthetic pathway, which coalesces bacterial and plant genes, and is terminated by a chloroplast-localized diaminopimelate decarboxylase (DAPDC, PtLYSA). We show that while RNAi ablation of PtLYSA is either synthetically lethal or concomitant with a slower growth rate, Cas9-mediated mutagenesis of PtLYSA results in recovery of heterozygous cells lines, suggesting that PtLYSA is an essential gene. Previously characterized DAPDCs are unique within the PLP-dependent decarboxylases where catalysis occurs at the D-stereocenter of the substrate and display a strict stereochemical preference for a (D,L)- or meso-substrate and not the D,D- or L,L-isomers of diaminopimelate (DAP) to synthesize L-lysine. Using decarboxylation assays and differential scanning calorimetry analyses, we validate that PtLYSA is a bona fide DAPDC and uncover its unexpected stereopromiscuous behavior in substrate specificity. The crystal structure of PtLYSA confirms the enzyme is an obligate homodimer in which both protomers reciprocally participate in the active site. The structure underscores features unique to the PtLYSA clan of DAPDC and provides structural insight into the determinants responsible for the substrate-promiscuity observed in PtLYSA.

biochemistry↗

bioRxiv2019

Phytotransferrin endocytosis mediates a direct cell surface-to-chloroplast iron trafficking axis in marine diatoms

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed, however proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

cell biology↗

bioRxiv2016

Diatom Centromeres Suggest a Novel Mechanism for Nuclear Gene Acquisition

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. Diatom centromere sequences contain low GC content regions and an abundance of long contiguous AT windows, but lack repeats or other conserved sequence features. Native and foreign sequences of similar GC content can maintain episomes and recruit the diatom centromeric histone protein CENP-A, suggesting non-native sequences can also function as diatom centromeres. Thus, simple sequence requirements enable DNA from foreign sources to incorporate into the nuclear genome repertoire as stable extra-chromosomal episomes, revealing a potential mechanism for bacterial and foreign eukaryotic DNA acquisition.

molecular biology↗