Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “DNA sequencing”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Derivatization of Mirror-Image L -Nucleic Acids with 2'-OMe Modification for Thermal and Structural Stabilization

To further expand the functionality and enhance the stability of mirror-image nucleic acids as advanced agents for basic research and therapeutic design, we have synthesized 2'-deoxy-2'-methoxy- L -uridine phosphoramidite and incorporated it into L -DNA and L -RNA by solid-phase synthesis quantitatively. We found that the thermostability of L -nucleic acids is dramatically improved after introducing the modifications. Moreover, we successfully crystallized both L -DNA and L -RNA duplexes containing the 2'-OMe modifications and sharing identical sequences. Crystal structure determination and analysis revealed the overall structures of the mirror-image nucleic acids, and for the first time it was possible to interpret the structural deviations caused by 2'-OMe and 2'-OH groups in the oligonucleotides, which are very similar. In conclusion, this novel chemical nucleic acid modification has the potential to be used to design nucleic acid-based therapeutics and materials in the future.

2'-OMe-modifications↗
DOE OSTI2022

PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

Abstract Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute ( Pf Ago)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments. PlasmidMaker should greatly expand the potential of synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system

Bacteriophage exclusion (‘BREX’) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR (‘BREX Regulator’), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1995

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

NASA Discipline Cell Biology↗
DOE OSTI2021

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis

Abstract Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1993

Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots

The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

NASA Discipline Number 40-50↗
NASA NTRS2017

Development of Solid-State Nanopore Technology for Life Detection

Biomarkers for life on Earth are an important starting point to guide the search for life elsewhere. However, the search for life beyond Earth should incorporate technologies capable of recognizing an array of potential biomarkers beyond what we see on Earth, in order to minimize the risk of false negatives from life detection missions. With this in mind, charged linear polymers may be a universal signature for life, due to their ability to store information while also inherently reducing the tendency of complex tertiary structure formation that significantly inhibit replication. Thus, these molecules are attractive targets for biosignature detection as potential "self-sustaining chemical signatures." Examples of charged linear polymers, or polyelectrolytes, include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as synthetic polyelectrolytes that could potentially support life, including threose nucleic acid (TNA) and other xenonucleic acids (XNAs). Nanopore analysis is a novel technology that has been developed for singlemolecule sequencing with exquisite single nucleotide resolution which is also well-suited for analysis of polyelectrolyte molecules. Nanopore analysis has the ability to detect repeating sequences of electrical charges in organic linear polymers, and it is not molecule- specific (i.e. it is not restricted to only DNA or RNA). In this sense, it is a better life detection technique than approaches that are based on specific molecules, such as the polymerase chain reaction (PCR), which requires that the molecule being detected be composed of DNA.

Bywaters, K. B.↗
NASA NTRS2002

AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development

The full-length sequence and developmental expression of amphioxus AmphiFoxE4 are described. Transcripts of the gene are first detected in the pharyngeal endoderm, where the club-shaped gland is forming and subsequently in the definitive gland itself. AmphiFoxE4 is closely related to vertebrate genes encoding the thyroid-specific transcription factor-2 (TTF2), which plays an early developmental role in the morphogenesis of the thyroid gland and a later role in hormone-mediated control of thyroid function. In amphioxus, AmphiFoxE4 expression is not thyroid specific because the club-shaped gland, the only structure expressing the gene, is not homologous to the vertebrate thyroid; instead, the thyroid homologue of amphioxus is a specialized region of the pharyngeal endoderm called the endostyle. We propose that (a) the pharynx of an amphioxus-like ancestor of the vertebrates included a club-shaped gland that expressed FoxE4 as well as an endostyle that did not, and (b) the club-shaped gland soon disappeared in the vertebrate line of descent but (c) not before there was a homeogenetic transfer of FoxE4 expression from the club-shaped gland to the nearby endostyle. Such a transfer could have provided part of the genetic program enabling the endostyle to separate from the pharyngeal endoderm and migrate away as the rudiment of the thyroid gland.

Non-NASA Center↗
NASA NTRS1992

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

NASA Discipline Plant Biology↗
DOE OSTI2020

A genetic toolbox for metabolic engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. Yet, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As evidence of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Full-Length ASFV B646L Gene Sequencing by Nanopore Offers a Simple and Rapid Approach for Identifying ASFV Genotypes

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time.

Virology↗
DOE OSTI2022

The first crystal structures of hybrid and parallel four-tetrad intramolecular G-quadruplexes

Abstract G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of stabilized G-tetrads. GQs play an important role in a variety of biological processes and may form at telomeres and oncogene promoters among other genomic locations. Here, we investigate nine variants of telomeric DNA from Tetrahymena thermophila with the repeat (TTGGGG)n. Biophysical data indicate that the sequences fold into stable four-tetrad GQs which adopt multiple conformations according to native PAGE. Excitingly, we solved the crystal structure of two variants, TET25 and TET26. The two variants differ by the presence of a 3′-T yet adopt different GQ conformations. TET25 forms a hybrid [3 + 1] GQ and exhibits a rare 5′-top snapback feature. Consequently, TET25 contains four loops: three lateral (TT, TT, and GTT) and one propeller (TT). TET26 folds into a parallel GQ with three TT propeller loops. To the best of our knowledge, TET25 and TET26 are the first reported hybrid and parallel four-tetrad unimolecular GQ structures. The results presented here expand the repertoire of available GQ structures and provide insight into the intricacy and plasticity of the 3D architecture adopted by telomeric repeats from T. thermophila and GQs in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

High-throughput mapping of the phage resistance landscape in E. coli

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Host phylogeny matters: Examining sources of variation in infection risk by blood parasites across a tropical montane bird community in India

Abstract Background Identifying patterns and drivers of infection risk among host communities is crucial to elucidate disease dynamics and predict infectious disease risk in wildlife populations. Blood parasites of the genera Plasmodium and Haemoproteus are a diverse group of vector-borne protozoan parasites that affect bird populations globally. Despite their widespread distribution and exceptional diversity, factors underlying haemosporidian infection risk in wild bird communities remain poorly understood. While some studies have examined variation in avian haemosporidian risk, researchers have primarily focused on host ecological traits without considering host phylogenetic relationships. In this study, we employ a phylogenetically informed approach to examine the association between host ecological traits and haemosporidian infection risk in endemic bird communities in the Western Ghats Sky Islands. Methods We used parasite sequence data based on partial mitochondrial cytochrome b gene, that was amplified from genomic DNA extracted from 1177 birds (28 species) across the Western Ghats to assess infection of birds with haemosporidian parasites. We employed a Bayesian phylogenetic mixed effect modelling approach to test whether haemosporidian infection risk was affected by seven species-specific and four individual-level ecological predictors. We also examined the effect of host phylogenetic relationships on the observed patterns of variation in haemosporidian infection risk by estimating phylogenetic signal. Results Our study shows that host ecological traits and host phylogeny differentially influence infection risk by Plasmodium (generalist parasite) and Haemoproteus (specialist parasite). For Plasmodium , we found that sociality, sexual dimorphism and foraging strata were important ecological predictors. For Haemoproteus , patterns of infection risk among host species were associated with sociality, species elevation and individual body condition. Interestingly, variance in infection risk explained by host phylogeny was higher for Haemoproteus parasites compared to Plasmodium . Conclusions Our study highlights that while host ecological traits promoting parasite exposure and host susceptibility are important determinants of infection risk, host phylogeny also contributes substantially to predicting patterns of haemosporidian infection risk in multi-host communities. Importantly, infection risk is driven by joint contributions of host ecology and host phylogeny and studying these effects together could increase our ability to better understand the drivers of infection risk and predict future disease threats. Graphical abstract

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

PlasmidMaker: a Versatile, Automated, and High Throughput End-to-End Platform for Plasmid Construction

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high-throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute ( Pf Ago)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate ~100 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments within 3 days. PlasmidMaker should greatly expand the potential of synthetic biology.

Enghiad, Behnam↗
NASA NTRS2004

Carnobacterium Pleistocaenium sp. nov.: A Novel Psychrotolerant, Facultative Anaerobe Isolated from Permafrost of the Fox Tunnel in Alaska

A novel, psychrotolerant, facultative anaerobe, strain FTRIT1(sup T), was isolated from Pleistocene ice from the permafrost tunnel in Fox, Alaska. Gram-positive, motile, rod-shaped cells with sizes 0.6-0.7 x 0.9-1.5 micrometers were observed. Growth occurred within the pH range 6.5-9.5 and optimum at pH 7.3-7.5. The temperature range of the new isolate was 0-28 C and optimum growth occurred at 24 C. The novel isolate requires NaCl (growth absent at 0 %) and growth was observed between 0 and 5% NaCl with optimum at 0.5% (w/v). The new isolate was a catalase-negative chemoorganoheterotroph that used as substrates sugars and some products of proteolysis. The metabolic end products were: acetate, ethanol and CO2. Strain FTRl was sensitive to ampicillin, tetracycline, chloramphenicol, rifampin, kanamycin, and gentamycin. The 16S rDNA sequence analysis showed 99.8% similarity of strain FTR1 with Carnobacterium alterfunditum, but the DNA-DNA hybridization between them demonstrated 39 plus or minus 5% homology. On the basis of genotypic and phenotypic characteristics, it is proposed that the strain FTR1(sup T) (= ATCC BAA-754(sup T) = JSM 12174(sup T) is assigned to the new species of the genus Carnobacterium with proposed name Carnobacterium pleistocaenium sp. nov.

Pikuta, Elena V.↗
NASA NTRS1999

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Non-NASA Center↗