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At least 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Distributed Berkeley Efficient Long-Read to Long-Read Aligner and Overlapper (DiBELLA) v1.0.0

We present a parallel algorithm and scalable implementation for genome analysis, specifically the problem of finding overlaps and alignments for data from "third generation" long read sequencers. While long sequences of DNA offer enormous advantages for biological analysis and insight, current long read sequencing instruments have high error rates and therefore require different approaches to analysis than their short read counterparts. Our work focuses on an efficient distributed-memory parallelization of an accurate single-node algorithm for overlapping and aligning long reads. We achieve scalability of this irregular algorithm by addressing the competing issues of increasing parallelism, minimizing communication, constraining the memory footprint, and ensuring good load balance. The resulting application, DiBELLA, is the first distributed memory overlapper and aligner specifically designed for long reads and parallel scalability.

Ellis, Marquita↗
DOE OSTI2025

GenomeDepot v1.0

GenomeDepot is a web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of web-sites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, BLAST search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools.

Kazakov, Alexey [Lawrence Berkeley National Labora↗
DOE OSTI2022

Structural variant analysis of a cancer reference cell line sample using multiple sequencing technologies

The cancer genome is commonly altered with thousands of structural rearrangements including insertions, deletions, translocation, inversions, duplications, and copy number variations. Thus, structural variant (SV) characterization plays a paramount role in cancer target identification, oncology diagnostics, and personalized medicine. As part of the SEQC2 Consortium effort, the present study established and evaluated a consensus SV call set using a breast cancer reference cell line and matched normal control derived from the same donor, which were used in our companion benchmarking studies as reference samples. We systematically investigated somatic SVs in the reference cancer cell line by comparing to a matched normal cell line using multiple NGS platforms including Illumina short-read, 10X Genomics linked reads, PacBio long reads, Oxford Nanopore long reads, and high-throughput chromosome conformation capture (Hi-C). We established a consensus SV call set of a total of 1788 SVs including 717 deletions, 230 duplications, 551 insertions, 133 inversions, 146 translocations, and 11 breakends for the reference cancer cell line. To independently evaluate and cross-validate the accuracy of our consensus SV call set, we used orthogonal methods including PCR-based validation, Affymetrix arrays, Bionano optical mapping, and identification of fusion genes detected from RNA-seq. We evaluated the strengths and weaknesses of each NGS technology for SV determination, and our findings provide an actionable guide to improve cancer genome SV detection sensitivity and accuracy. A high-confidence consensus SV call set was established for the reference cancer cell line. A large subset of the variants identified was validated by multiple orthogonal methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity

The newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known. Results Here, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of >600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (~ 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Effect of fiber reinforcement on the process parameters of injection molding manufacturing process

Use of fillers with the resin is common in injection molding manufacturing technique to enhance the stiffness and strength of the manufactured components. However, the addition of fillers changes the rheological and mechanical properties of the material, required adjustments in the process parameters used for the manufacturing. The present study aims to investigate this impact of fillers on the process parameters of the injection molding manufacturing process. Neat Acrylonitrile Butadiene Styrene (ABS) and ABS reinforced with short glass fibers are used as the materials to perform the comprehensive numerical analysis including complete process sequence, namely filling, packing, cooling and warpage. Parametric studies are conducted to optimize the manufacturing process for minimal warpage (or shrinkage) in the produced part. The selected process parameters for the parametric study include cooling time, packing pressure, and mold temperature, all of which directly impact the quality of the manufactured parts. The optimal values obtained for these parameters are compared to examine the effect fiber reinforcement has on the manufacturing process.

Garg, Nikhil↗
DOE OSTI2020

Real-time structural motif searching in proteins using an inverted index strategy

Biochemical and biological functions of proteins are the product of both the overall fold of the polypeptide chain, and, typically, structural motifs made up of smaller numbers of amino acids constituting a catalytic center or a binding site that may be remote from one another in amino acid sequence. Detection of such structural motifs can provide valuable insights into the function(s) of previously uncharacterized proteins. Technically, this remains an extremely challenging problem because of the size of the Protein Data Bank (PDB) archive. Existing methods depend on a clustering by sequence similarity and can be computationally slow. We have developed a new approach that uses an inverted index strategy capable of analyzing >170,000 PDB structures with unmatched speed. The efficiency of the inverted index method depends critically on identifying the small number of structures containing the query motif and ignoring most of the structures that are irrelevant. Our approach (implemented at motif.rcsb.org ) enables real-time retrieval and superposition of structural motifs, either extracted from a reference structure or uploaded by the user. Herein, we describe the method and present five case studies that exemplify its efficacy and speed for analyzing 3D structures of both proteins and nucleic acids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates

The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Bayesian Analysis of Congruence of Core Genes in Prochlorococcus and Synechococcus and Implications on Horizontal Gene Transfer

It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. “Universal proteins” have been used to infer the organismal phylogeny, but have been criticized as being only the “tree of one percent.” Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees – a “treecloud” – for each, as well as for a concatenated dataset based on putative “universal proteins.” We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS). We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the “tree of one percent” is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Deep RNA Sequencing Reveals Hidden Features and Dynamics of Early Gene Transcription in Paramecium bursaria Chlorella Virus 1

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of the genus Chlorovirus (family Phycodnaviridae) that infects the unicellular, eukaryotic green alga Chlorella variabilis NC64A. The 331-kb PBCV-1 genome contains 416 major open reading frames. A mRNA-seq approach was used to analyze PBCV-1 transcriptomes at 6 progressive times during the first hour of infection. The alignment of 17 million reads to the PBCV-1 genome allowed the construction of single-base transcriptome maps. Significant transcription was detected for a subset of 50 viral genes as soon as 7 min after infection. By 20 min post infection (p.i.), transcripts were detected for most PBCV-1 genes and transcript levels continued to increase globally up to 60 min p.i., at which time 41% or the poly (A+)-containing RNAs in the infected cells mapped to the PBCV-1 genome. For some viral genes, the number of transcripts in the latter time points (20 to 60 min p.i.) was much higher than that of the most highly expressed host genes. RNA-seq data revealed putative polyadenylation signal sequences in PBCV-1 genes that were identical to the polyadenylation signal AAUAAA of green algae. Several transcripts have an RNA fragment excised. However, the frequency of excision and the resulting putative shortened protein products suggest that most of these excision events have no functional role but are probably the result of the activity of misled splicesomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ 70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Comparing residue clusters from thermophilic and mesophilic enzymes reveals adaptive mechanisms

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. As a result, the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.

09 BIOMASS FUELS↗
DOE OSTI2016

Rapid discovery and functional characterization of terpene synthases from four endophytic xylariaceae

Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Lastly, out of the 26 TPS’s profiled, 12 TPS’s were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65 Åand 2.75 Å respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Crystal structure of a Thermus aquaticus diversity-generating retroelement variable protein

Diversity-generating retroelements (DGRs) are widely distributed in bacteria, archaea, and microbial viruses, and bring about unparalleled levels of sequence variation in target proteins. While DGR variable proteins share low sequence identity, the structures of several such proteins have revealed the C-type lectin (CLec)-fold as a conserved scaffold for accommodating massive sequence variation. This conservation has led to the suggestion that the CLec-fold may be useful in molecular surface display applications. Thermostability is an attractive feature in such applications, and thus we studied the variable protein of a DGR encoded by a prophage of the thermophile Thermus aquaticus. We report here the 2.8 Å resolution crystal structure of the variable protein from the T. aquaticus DGR, called TaqVP, and confirm that it has a CLec-fold. Remarkably, its variable region is nearly identical in structure to those of several other CLec-fold DGR variable proteins despite low sequence identity among these. TaqVP was found to be thermostable, which appears to be a property shared by several CLec-fold DGR variable proteins. These results provide impetus for the pursuit of the DGR variable protein CLec-fold in molecular display applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor.

59 BASIC BIOLOGICAL SCIENCES↗