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Results for “gene amplification”

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.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2001

Gene amplification and microsatellite instability induced in tumorigenic human bronchial epithelial cells by alpha particles and heavy ions

Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either alpha particles or (56)Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1-3 x 10(-3) in tumor cell lines, 5-8 x 10(-5) in transformed cells prior to inoculation into nude mice, and less than 10(-7) in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by alpha particles compared to those induced by (56)Fe ions.

Non-NASA Center↗
DOE OSTI2020

Gene Amplification, Laboratory Evolution, and Biosensor Screening Reveal MucK as a Terephthalic Acid Transporter in Acinetobacter baylyi ADP1

Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Natural transformation as a tool in Acinetobacter baylyi : Evolution by amplification of gene copy number

For many years, the natural competency of Acinetobacter baylyi ADP1 facilitated studies of bacterial metabolism, biochemistry, and physiology. With the advent of synthetic biology, new opportunities arise to exploit the remarkable transformability and chromosomal plasticity of this model organism. In this chapter, we describe a recently developed method, “Evolution by Amplification and Synthetic Biology” (EASy). EASy allows the targeted amplification of chromosomal segments that give rise to new phenotypes. Increased gene dosage regulates protein expression in a rudimentary fashion by establishing a chromosomal array in which copy number adjusts via recombination between repeated DNA sequences. Selective conditions enrich for cells within the population that confer a growth advantage. Under continuous selective pressure, beneficial mutations may accumulate in any genomic region. Such mutations favor decreases in the average copy number of the target region. Thus, the genetic flexibility afforded by transient copy number variation helps accelerate the selection of engineered strains with desired traits during laboratory evolution. As a result, thanks to the extremely simple genetic manipulation of Acinetobacter baylyi ADP1, the EASy method can be readily implemented by researchers without the need for advanced instrumentation or complex cloning techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Optimizing neural networks

A system and method design and optimize neural networks. The system and method include a data store that stores a plurality of gene vectors that represent diverse and distinct neural networks and an evaluation queue stored with the plurality of gene vectors. Secondary nodes construct, train, and evaluate the neural network and automatically render a plurality of fitness values asynchronously. A primary node executes a gene amplification on a select plurality of gene vectors, a crossing-over of the amplified gene vectors, and a mutation of the crossing-over gene vectors automatically and asynchronously, which are then transmitted to the evaluation queue. The process continuously repeats itself by processing the gene vectors inserted into the evaluation queue until a fitness level is reached, a network's accuracy level plateaus, a processing time period expires, or when some stopping condition or performance metric is met or exceeded.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2021

Differential structure and functional gene response to geochemistry associated with the suspended and attached shallow aquifer microbiomes from the Illinois Basin, IL

Despite the clear ecological significance of the microbiomes inhabiting groundwater and connected ecosystems, our current understanding of their habitats, functionality, and the ecological processes controlling their assembly have been limited. In this study, an efficient pipeline combining geochemistry, high-throughput Fluidigm TM functional gene amplification and sequencing was developed to analyze the suspended and attached microbial communities inhabiting five groundwater monitoring wells in the Illinois Basin, USA. The dominant taxa in the suspended and the attached microbial communities exhibited significantly different spatial and temporal changes in both alpha- and beta-diversity. Further analyses of representative functional genes affiliated with N 2 fixation (nifH), methane oxidation (pmoA), and sulfate reduction (dsrB, and aprA), suggested functional redundancy within the shallow aquifer microbiomes. While more diversified functional gene taxa were observed for the suspended microbial communities than the attached ones except for pmoA, different levels of changes over time and space were observed between these functional genes. Notably, deterministic and stochastic ecological processes shaped the assembly of microbial communities and functional gene reservoirs differently. While homogenous selection was the prevailing process controlling assembly of microbial communities, the neutral processes (e.g., dispersal limitation, drift and others) were more important for the functional genes. The results suggest complex and changing shallow aquifer microbiomes, whose functionality and assembly vary even between the spatially proximate habitats and fractions. As a result, this research underscored the importance to include all the interface components for a more holistic understanding of the biogeochemical processes in aquifer ecosystems, which is also instructive for practical applications.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Artemether-Lumefantrine Treatment Selects Plasmodium falciparum Multidrug Resistance 1 ( pfmdr1 ) Increased Copy Number Among African Malaria Infections

Abstract Background Decreased efficacy of artemether-lumefantrine, the globally most used antimalarial, has recently emerged in Africa. Methods An efficacy trial was carried out based on directly observed artemether-lumefantrine therapy at Bengo, Northern Angola. One-hundred Plasmodium falciparum uncomplicated malaria patients (2–10 years old) were enrolled, hospitalized for the treatment period, and followed up for 42 days. Polymerase chain reaction (PCR) correction was performed with pfmsp1/2 plus glurp, with analysis considering 2 or 3 coincident markers. Infections were tested by quantitative PCR (qPCR) for pfmdr1 copy number (pfmdr1×N), a potential P. falciparum marker of lumefantrine resistance previously identified in the region. In vitro clone mixtures were built and used to determine the relation between qPCR copy number scores and actual intrainfection quantitative fractions of pfmdr1×N. Results We observed a significant posttreatment selection of gene amplification, suggesting a role in the parasite in vivo response to this drug. pfmdr1×2 qPCR scores of 1.3, 1.4, and 1.5 were determined to correspond to 15%, 25%, and 35% intrainfection rates. Patients carrying infections with a score ≥1.4 at baseline were linked to decreased artemether-lumefantrine day 42 efficacy (79% vs 97% single-copy pfmdr1). All infections were pfmdr1 N86 carriers and no pfk13 mutations were found. Conclusions Our study suggests pfmdr1×N as a marker of P. falciparum in vivo response to lumefantrine in Africa, while indicating patients carrying infections with a pretreatment pfmdr1×N score ≥1.4 before treatment are a group experiencing decreased artemether-lumefantrine performance.

Fançony, Claudia (ORCID:0000000344211769)↗
DOE OSTI2021

Targeting Drugs Against Fibroblast Growth Factor(s)-Induced Cell Signaling

Background:: The fibroblast growth factor (FGF) family is comprised of 23 highly regulatedmonomeric proteins that regulate a plethora of developmental and pathophysiological processes,including tissue repair, wound healing, angiogenesis, and embryonic development. Binding ofFGF to fibroblast growth factor receptor (FGFR), a tyrosine kinase receptor, is facilitated by a glycosaminoglycan,heparin. Activated FGFRs phosphorylate the tyrosine kinase residues that mediateinduction of downstream signaling pathways, such as RAS-MAPK, PI3K-AKT, PLCγ, andSTAT. Dysregulation of the FGF/FGFR signaling occurs frequently in cancer due to gene amplification,FGF activating mutations, chromosomal rearrangements, integration, and oncogenic fusions.Aberrant FGFR signaling also affects organogenesis, embryonic development, tissue homeostasis,and has been associated with cell proliferation, angiogenesis, cancer, and other pathophysiologicalchanges. Objective:: This comprehensive review will discuss the biology, chemistry, and functions of FGFs,and its current applications toward wound healing, diabetes, repair and regeneration of tissues, andfatty liver diseases. In addition, specific aberrations in FGFR signaling and drugs that target FGFRand aid in mitigating various disorders, such as cancer, are also discussed in detail. Conclusion:: Inhibitors of FGFR signaling are promising drugs in the treatment of several types ofcancers. The clinical benefits of FGF/FGFR targeting therapies are impeded due to the activationof other RTK signaling mechanisms or due to the mutations that abolish the drug inhibitory activityon FGFR. Thus, the development of drugs with a different mechanism of action for FGF/FGFRtargeting therapies is the recent focus of several preclinical and clinical studies.

Pharmacology & Pharmacy↗
NASA NTRS1994

Heavy ion induced mutations in mammalian cells: Cross sections and molecular analysis

Our investigations of heavy ion-induced mutations in mammalian cells, which had been begun a few years ago, were systematically continued. For the first time, it was possible to cover a large LET range with a few kinds of ions. To do this, both UNILAC and SIS were used to yield comparable data for a large energy range. This is a necessary condition for a comprehensive description of the influence of such ion parameters as energy and LET. In these experiments, the induced resistance against the poison 6-thioguanin (6-TG), which is linked to the HPRT locus on the genome, is being used as mutation system. In addition to the mutation-induction cross-section measurements, the molecular changes of the DNA are being investigated by means of Multiplex PCR ('Polymerase Chain Reaction') gene amplification. From these experiments we expect further elucidation of the mutation-inducing mechanisms composing the biological action of heavy-ion radiation.

Stoll, U.↗
NASA NTRS2003

Radiation-induced genomic instability and its implications for radiation carcinogenesis

Radiation-induced genomic instability is characterized by an increased rate of genetic alterations including cytogenetic rearrangements, mutations, gene amplifications, transformation and cell death in the progeny of irradiated cells multiple generations after the initial insult. Chromosomal rearrangements are the best-characterized end point of radiation-induced genomic instability, and many of the rearrangements described are similar to those found in human cancers. Chromosome breakage syndromes are defined by chromosome instability, and individuals with these diseases are cancer prone. Consequently, chromosomal instability as a phenotype may underlie some fraction of those changes leading to cancer. Here we attempt to relate current knowledge regarding radiation-induced chromosome instability with the emerging molecular information on the chromosome breakage syndromes. The goal is to understand how genetic and epigenetic factors might influence the onset of chromosome instability and the role of chromosomal instability in carcinogenesis.

Review, Tutorial↗
DOE OSTI2021

Natural variation in the consequences of gene overexpression and its implications for evolutionary trajectories

Copy number variation through gene or chromosome amplification provides a route for rapid phenotypic variation and supports the long-term evolution of gene functions. Although the evolutionary importance of copy-number variation is known, little is understood about how genetic background influences its tolerance. Here, we measured fitness costs of over 4000 overexpressed genes in 15 Saccharomyces cerevisiae strains representing different lineages, to explore natural variation in tolerating gene overexpression (OE). Strain-specific effects dominated the fitness costs of gene OE. We report global differences in the consequences of gene OE, independent of the amplified gene, as well as gene-specific effects that were dependent on the genetic background. Natural variation in the response to gene OE could be explained by several models, including strain-specific physiological differences, resource limitations, and regulatory sensitivities. This work provides new insight on how genetic background influences tolerance to gene amplification and the evolutionary trajectories accessible to different backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Rewiring Aromatic Compound Consumption: Chromosomal Amplification and Evolution of a Foreign Pathway in Acinetobacter baylyi ADP1

Rational engineering strategies that seek to harness the remarkable diversity of microbial metabolism can be limited by incomplete biological knowledge. As described here, a novel approach to address this challenge involved replacing a native pathway for degrading lignin-derived aromatic compounds via ortho cleavage of protocatechuate in Acinetobacter baylyi ADP1 with a foreign meta-cleavage pathway that uses different enzymes, metabolites, and redox carriers. This alteration may improve lignin valorization and coordinate catabolism with bioproduction strategies. When a 14-kbp region of foreign DNA was inserted in the chromosome, the heterologous genes failed to confer growth on target substrates. Regional gene dosage was increased using a synthetic DNA fragment to promote recombination, and higher copy number enabled growth. During adaptive laboratory evolution, compensatory mutations arose that permit growth with one copy of the foreign genes. This complex metabolic remodeling was accomplished without assumptions about the impediments that initially prevented growth. To understand the changes that emerged, a novel transformation assay identified a combination of mutations sufficient for the new phenotype. Three unexpected changes were revealed: loss of one foreign enzyme, loss of one native enzyme, and loss of a two-component transcriptional regulatory system. This study establishes that large multicopy tandem arrays of poorly adapted pathway genes can confer new functions and improve understanding of metabolism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1993

Evolution of catalytic function

An RNA-based evolution system was constructed in the laboratory and used to develop RNA enzymes with novel catalytic function. By controlling the nature of the catalytic task that the molecules must perform in order to survive, it is possible to direct the evolving population toward the expression of some desired catalytic behavior. More recently, this system has been coupled to an in vitro translation procedure, raising the possibility of evolving protein enzymes in the laboratory to produce novel proteins with desired catalytic properties. The aim of this line of research is to reduce darwinian evolution, the fundamental process of biology, to a laboratory procedure that can be made to operate in the service of organic synthesis.

NASA Program Exobiology↗
NASA NTRS1995

Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa

In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

Non-NASA Center↗
DOE OSTI2022

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.

Evaluation Studies↗
NASA NTRS1997

A low molecular weight artificial RNA of unique size with multiple probe target regions

Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

Non-NASA Center↗