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At least 1,171 records · Page 65
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae

Promoters play an essential role in the regulation of gene expression for fine-tuning genetic circuits and metabolic pathways in Saccharomyces cerevisiae (S. cerevisiae). However, native promoters in S. cerevisiae have several limitations which hinder their applications in metabolic engineering. These limitations include an inadequate number of well-characterized promoters, poor dynamic range, and insufficient orthogonality to endogenous regulations. Therefore, it is necessary to perform promoter engineering to create synthetic promoters with better properties. Here, we review recent advances related to promoter architecture, promoter engineering and synthetic promoter applications in S. cerevisiae. We also provide a perspective of future directions in this field with an emphasis on the recent advances of machine learning based promoter designs.

machine learning↗
DOE OSTI2021

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors including histone PTMs are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for engineering solutions to increase the resilience of crops to climate change. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed as their intact forms by top-down mass spectrometry (MS) coupled to online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow thus yielding the global PTM profile of core histones (H4, H2A, H2B, H3). By comparing the PTM profile among samples corresponding to different conditions (e.g. drought vs. control), potential epigenetic marks can be discovered as targets for further characterization using approaches such as chromatin immunoprecipitation – sequencing (ChIP-seq).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm

Neither the disease mechanism nor treatments for COVID-19 are currently known. Here, we present a novel molecular mechanism for COVID-19 that provides therapeutic intervention points that can be addressed with existing FDA-approved pharmaceuticals. The entry point for the virus is ACE2, which is a component of the counteracting hypotensive axis of RAS. Bradykinin is a potent part of the vasopressor system that induces hypotension and vasodilation and is degraded by ACE and enhanced by the angiotensin 1-9 produced by ACE2. Here, we perform a new analysis on gene expression data from cells in bronchoalveolar lavage fluid (BALF) from COVID-19 patients that were used to sequence the virus. Comparison with BALF from controls identifies a critical imbalance in RAS represented by decreased expression of ACE in combination with increases in ACE2, renin, angiotensin, key RAS receptors, kinogen and many kallikrein enzymes that activate it, and both bradykinin receptors. This very atypical pattern of the RAS is predicted to elevate bradykinin levels in multiple tissues and systems that will likely cause increases in vascular dilation, vascular permeability and hypotension. These bradykinin-driven outcomes explain many of the symptoms being observed in COVID-19.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

To investigate the effects of stabilizing G-quadruplexes on Escherichia coli, cells were grown in the presence of the G-quadruplex stabilizing compound, NMM, and RNA-seq was performed. This was done for the control cell strain (MG1655 dtolC) as well as a strain with a decrease in a translation elongation factor (MG1655 dtolC tufA::kan). TPM values were used for differential gene expression analysis between each growth condition. All samples were grown and sequenced in triplicate.

Keck, James L.↗
DOE OSTI2023

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae

Past work from our lab, focused on engineering the budding yeast Saccharomyces cerevisiae for fermentation of the non-native pentose sugar xylose, discovered that hyperactivation of the RAS/Protein Kinase A (PKA) pathway was needed for rapid anaerobic xylose fermentation. Interestingly, the mechanism of PKA hyperactivation has a dramatic impact on growth and metabolism on xylose; deletion of the RAS inhibitor IRA2 permits rapid growth and fermentation, while deletion of the PKA regulatory subunit BCY1 allows for fermentation without growth on xylose. To understand how a single deletion in the PKA pathway can decouple growth and metabolism, we performed transcriptomic analysis of these strains, predicting that altered PKA activity would impact global gene expression and identify pathways important for growth and metabolism coordination.

Gasch, Audrey↗
NASA NTRS1983

DNA fusion product of phage P2 with plasmid pBR322 - A new phasmid

The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

Nicoletti, M.↗
NASA NTRS1990

The mGA1.0: A common LISP implementation of a messy genetic algorithm

Genetic algorithms (GAs) are finding increased application in difficult search, optimization, and machine learning problems in science and engineering. Increasing demands are being placed on algorithm performance, and the remaining challenges of genetic algorithm theory and practice are becoming increasingly unavoidable. Perhaps the most difficult of these challenges is the so-called linkage problem. Messy GAs were created to overcome the linkage problem of simple genetic algorithms by combining variable-length strings, gene expression, messy operators, and a nonhomogeneous phasing of evolutionary processing. Results on a number of difficult deceptive test functions are encouraging with the mGA always finding global optima in a polynomial number of function evaluations. Theoretical and empirical studies are continuing, and a first version of a messy GA is ready for testing by others. A Common LISP implementation called mGA1.0 is documented and related to the basic principles and operators developed by Goldberg et. al. (1989, 1990). Although the code was prepared with care, it is not a general-purpose code, only a research version. Important data structures and global variations are described. Thereafter brief function descriptions are given, and sample input data are presented together with sample program output. A source listing with comments is also included.

Goldberg, David E.↗
NASA NTRS1992

The neurochemical basis of photic entrainment of the circadian pacemaker

Circadian rhythmicity in mammals is controlled by the action of a light-entrainable hypothalamus, in association with two cell clusters known as the supra chiasmatic nuclei (SCN). In the absence of temporal environmental clues, this pacemaker continues to measure time by an endogenous mechanism (clock), driving biochemical, physiological, and behavioral rhythms that reflect the natural period of the pacemaker oscillation. This endogenous period usually differs slightly from 24 hours (i.e., circadian). When mammals are maintained under a 24 hour light-dark (LD) cycle, the pacemaker becomes entrained such that the period of the pacemaker oscillation matches that of the LD cycle. Potentially entraining photic information is conveyed to the SCN via a direct retinal projection, the retinohypothalamic tract (RHT). RHT neurotransmission is thought to be mediated by the release of excitatory amino acids (EAA) in the SCN. In support of this hypothesis, recent experiments using nocturnal rodents have shown that EAA antagonists block the effects of light on pacemaker-driven behavioral rhythms, and attenuate light induced gene expression in SCN cells. An understanding of the neurochemical basis of the photic entrainment process would facilitate the development of pharmacological strategies for maintaining synchrony among shift workers in environments, such as the Space Station, which provide unreliable or conflicting temporal photic clues.

Rea, Michael A.↗
NASA NTRS1990

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Kumei, Yasuhiro↗
NASA NTRS1993

Microgravity research in plant biological systems: Realizing the potential of molecular biology

The sole all-pervasive feature of the environment that has helped shape, through evolution, all life on Earth is gravity. The near weightlessness of the Space Station Freedom space environment allows gravitational effects to be essentially uncoupled, thus providing an unprecedented opportunity to manipulate, systematically dissect, study, and exploit the role of gravity in the growth and development of all life forms. New and exciting opportunities are now available to utilize molecular biological and biochemical approaches to study the effects of microgravity on living organisms. By careful experimentation, we can determine how gravity perception occurs, how the resulting signals are produced and transduced, and how or if tissue-specific differences in gene expression occur. Microgravity research can provide unique new approaches to further our basic understanding of development and metabolic processes of cells and organisms, and to further the application of this new knowledge for the betterment of humankind.

Lewis, Norman G.↗
NASA NTRS1995

Multirepressor Control Systems For Efficient Promoter Regulation

Report presents numerical-simulation study, based on molecular-level mathematical models, to evaluate effectiveness of eight different configurations of repressor synthesis control of cloned-product-gene expression initiated from promoter/operator genetic sequence. In study, both single- and dual-repressor situations considered, using genetically structured molecules for lac and IPR promotor/operator in example calculations.

Bailey, James E.↗
NASA NTRS1996

[Vestibular compensation studies]

The following topics are reported: neurophysiological studies on MVN neurons during vestibular compensation; effects of spinal cord lesions on VNC neurons during compensation; a closed-loop vestibular compensation model for horizontally canal-related MVN neurons; spatiotemporal convergence in VNC neurons; contributions of irregularly firing vestibular afferents to linear and angular VOR's; application to flight studies; metabolic measures in vestibular neurons; immediate early gene expression following vestibular stimulation; morphological studies on primary afferents, central vestibular pathways, vestibular efferent projection to the vestibular end organs, and three-dimensional morphometry and imaging.

Perachio, Adrian A.↗
NASA NTRS1997

Influence of Zero-Shear on Yeast Development

The objective of the research was to begin evaluating the effect of zero-shear on the development of the cell wall of Saccharomyces cerevisiae employing the High Aspect Rotating-Wall Vessel (HARV) NASA bioreactor. This particular yeast has enormous potential for research as a model eukaryotic system on the International Space Station, as well as the production of food stuffs' at the future lunar colony. Because the cell wall is the barrier between the cell and the environment, its form and function as influenced by microgravity is of great importance. Morphologic studies revealed that the circularity and total area of the individual yeast cells were essentially the same in both the control and test HARV's. The growth rates were also essentially the same. In zero-shear, the yeast grew in clumps consisting of rudimentary pseudohyphae in contrast to solitary budding cells in the control. Based upon mechanical and sonic shear applied to the yeast cells, those grown in zero-shear had stronger cell walls and septa. This suggests that there are structural differences, most likely related to the chitin skeleton of the cell wall. From this research further NASA support was obtained to continue the work. Investigations will deal with gene expression and ultrastructure. These will lead to a clearer assessment of the value of S. cerevisiae eukaryotic as a model for space station research.

McGinnis, Michael R.↗
NASA NTRS1999

The Effect of Spaceflight on Bone Cell Cultures

Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

Landis, William J.↗
NASA NTRS1999

Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

Young, R. B.↗
NASA NTRS1999

Molecular Signaling in Muscle Plasticity

Extended spaceflight under microgravity conditions leads to significant atrophy of weight-bearing muscles. Atrophy and hypertrophy are the extreme outcomes of the high degree of plasticity exhibited by skeletal muscle. Stimuli which control muscle plasticity include neuronal, hormonal, nutritional, and mechanical inputs. The mechanical stimulus for muscle is directly related to the work or exercise against a load performed. Little or no work is performed by weight-bearing muscles under microgravity conditions. A major hypothesis is that focal adhesion kinase (FAK) which is associated with integrin at the adherens junctions and costa meres of all skeletal muscles is an integral part of the major mechanism for molecular signaling upon mechanical stimulation in all muscle fibers. Additionally, we propose that myotonic protein kinase (DMPK) and dystrophin (DYSTR) also participate in distinct mechanically stimulated molecular signaling pathways that are most critical in type I and type II muscle fibers, respectively. To test these hypotheses, we will use the paradigms of hindlimb unloading and overloading in mice as models for microgravity conditions and a potential exercise countermeasure, respectively, in mice. We expect that FAK loss-of-function will impair hypertrophy and enhance atrophy in all skeletal muscle fibers whereas DYSTR and DMPK loss-of-function will have similar but more selective effects on Type IT and Type I fibers, respectively. Gene expression will be monitored by muscle-specific creatine kinase M promoter-reporter construct activity and specific MRNA and protein accumulation in the soleus (type I primarily) and plantaris (type 11 primarily) muscles. With these paradigms and assays, the following Specific Project Aims will be tested in genetically altered mice: 1) identify the roles of DYSTR and its pathway; 2) evaluate the roles of the DMPK and its pathway; 3) characterize the roles of FAK and its pathway and 4) genetically analyze the mechanisms and interactions between the FAK, DYSTR, and DMPK-associated pathways in single and specific combinations of mutants. The identification of potential signaling mechanisms may permit future development of pharmacological countermeasures for amelioration and prevention of the microgravity-induced atrophy in extended spaceflight, and the analysis of both overloading and unloading paradigms may provide further support for development of exercise-based countermeasures. Understanding the basic mechanisms of molecular signaling in muscle plasticity may aid our understanding and treatment of skeletal muscle atrophy not only in spaceflight but in similar problems of the aging population, in prolonged bed rest, and in cachexia associated with chronic disease.

Epstein, Henry F.↗