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NASA NTRS1986

Oxygen and the evolution of metabolic pathways

While a considerable amount of evidence has been accumulated about the history of oxygen on this planet, little is known about the relative amounts to which primitive cells might have been exposed. One clue may be found in the metabolic pathways of extant microorganisms. While eucaryotes are principally aerobic organisms, a number are capable of anaerobic growth by fermentation. One such eucaryotic microorganism, Saccharomyces cerevisiae, will grow in the complete absence of oxygen when supplemented with unsaturated fatty acid and sterol. Oxygen-requiring enzymes are involved in the synthesis of both of these compounds. Studies have demonstrated that the oxidative desaturation of palmitic acid and the conversion of squalene to sterols occur in the range of 10-(3) to 10(-2) PAL. Thus, if the oxygen requirements of these enzymatic processes are an indication, eucaryotes might be more primitive than anticipated from the microfossil record. Results of studies on the oxygen requirements for sterol and unsaturated fatty acid synthesis in a more primitive procaryotic system are also discussed.

Jahnke, L. L.↗

NASA NTRS1989

Model system studies with a phase separated membrane bioreactor

The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described. The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out. Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls. Methods of upgrading the design to eliminate these resistances are proposed. Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestial simulation.

Petersen, G. R.↗

NASA NTRS1989

Phase separated membrane bioreactor - Results from model system studies

Petersen, G. R.↗

NASA NTRS1990

Visualization of yeast chromosomal DNA

The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

Lubega, Seth↗

NASA NTRS1992

Microgravitational effects on chromosome behavior (7-IML-1)

The effects of the two major space-related conditions, microgravity and radiation, on the maintenance and transmission of genetic information have been partially documented in many organisms. Specifically, microgravity acts at the chromosomal level, primarily on the structure and segregation of chromosomes, in producing major abberations such as deletions, breaks, nondisjunction, and chromosome loss, and to a lesser degree, cosmic radiation appears to affect the genic level, producing point mutations and DNA damage. To distinguish between the effects from microgravity and from radiation, it is necessary to monitor both mitotic and meiotic genetic damage in the same organism. The yeast Saccharomyces cerevisiae is used to monitor at high resolution the frequency of chromosome loss, nondisjunction, intergenic recombination, and gene mutation in mitotic and meiotic cells, to a degree impossible in other organisms. Because the yeast chromosomes are small, sensitive measurements can be made that can be extrapolated to higher organisms and man. The objectives of the research are: (1) to quantitate the effects of microgravity and its synergism with cosmic radiation on chromosomal integrity and transmission during mitosis and meiosis; (2) to discriminate between chromosomal processes sensitive to microgravity and/or radiation during mitosis and meiosis; and (3) to relate these findings to anomalous mitotic mating type switching and ascosporogenesis following meiosis.

Bruschi, Carlo↗

NASA NTRS1976

Stability Characteristics of "Aerobic" Acetyl-CoA Synthetase of Yeast

During the purification of the "aerobic" acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae, strain LK2Gl2, it was noted that stronge at 4 C resulted in the loss of enzyme activity within 24 hr. Similar losses were observed during column chromatography. Addition of boiled extracts from either aerobic or anerobic cells completely prevents this. The stabilizing factor (SF) in these extracts is non-dialyzable and organic in nature. SF is excluded on G-25 and G-50 Sephadex columns and is slightly retarded on G-75 columns. On G-100 columns, SF elutes as a peak exactly coincident with that of cytochrome c, indicating a molecular weight of 13,000. SF activity was not destroyed by Pronase treatment, was adsorbed onto Norite, and absorbed in the UV with a single maximum at 260 nm. The action of SF could be replaced by a number of nucleotides. At 0.01 M, the order of effectiveness was: ATP>ADP>AMP>GTP>CTP>/=UTP>XTP. Even at 2 x 10(exp -4) M, ATP and ADP, but not AMP, cyclic AMP, adenosine or adenine, were effective in stabilizing this ACS. The mechanism of stabilization by ATP and AMP appears to be the same, since AMP competitively inhibited the ACS with respect to ATP in in vitro assays, while ADP gave a mixed type of inhibition, thus indicating a different mechanism. ACS from nonaerobic cells is also unstable in the absence of SF but, unlike aerobic ACS, is not affected by ATP or other nucleotides.

Satyanarayana, T.↗

NASA NTRS2001

Monitoring of bacteria growth using a wireless, remote query resonant-circuit sensor: application to environmental sensing

A new technique is presented for in-vivo remote query measurement of the complex permittivity spectra of a biological culture solution. A sensor comprised of a printed inductor-capacitor resonant-circuit is placed within the culture solution of interest, with the impedance spectrum of the sensor measured using a remotely located loop antenna; the complex permittivity spectra of the culture is calculated from the measured impedance spectrum. The remote query nature of the sensor platform enables, for example, the in-vivo real-time monitoring of bacteria or yeast growth from within sealed opaque containers. The wireless monitoring technique does not require a specific alignment between sensor and antenna. Results are presented for studies conducted on laboratory strains of Bacillus subtilis, Escherichia coli JM109, Pseudomonas putida and Saccharomyces cerevisiae.

Non-NASA Center↗

NASA NTRS1998

The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Non-NASA Center↗

NASA NTRS1996

Yeast respond to hypotonic shock with a calcium pulse

We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

NASA Discipline Plant Biology↗

NASA NTRS1996

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Non-NASA Center↗

NASA NTRS1993

Diploid yeast cells yield homozygous spontaneous mutations

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

Non-NASA Center↗

NASA NTRS1991

The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

Non-NASA Center↗

NASA NTRS2009

Life Science Research in Outer Space: New Platform Technologies for Low-Cost, Autonomous Small Satellite Missions

We develop integrated instruments and platforms suitable for economical, frequent space access for autonomous life science experiments and processes in outer space. The technologies represented by three of our recent free-flyer small-satellite missions are the basis of a rapidly growing toolbox of miniaturized biologically/biochemically-oriented instrumentation now enabling a new generation of in-situ space experiments. Autonomous small satellites (~ 1 50 kg) are less expensive to develop and build than fullsize spacecraft and not subject to the comparatively high costs and scheduling challenges of human-tended experimentation on the International Space Station, Space Shuttle, and comparable platforms. A growing number of commercial, government, military, and civilian space launches now carry small secondary science payloads at far lower cost than dedicated missions; the number of opportunities is particularly large for so-called cube-sat and multicube satellites in the 1 10 kg range. The recent explosion in nano-, micro-, and miniature technologies, spanning fields from telecommunications to materials to bio/chemical analysis, enables development of remarkably capable autonomous miniaturized instruments to accomplish remote biological experimentation. High-throughput drug discovery, point-of-care medical diagnostics, and genetic analysis are applications driving rapid progress in autonomous bioanalytical technology. Three of our recent missions exemplify the development of miniaturized analytical payload instrumentation: GeneSat-1 (launched: December 2006), PharmaSat (launched: May 2009), and O/OREOS (organism/organics exposure to orbital stresses; scheduled launch: May 2010). We will highlight the overall architecture and integration of fluidic, optical, sensor, thermal, and electronic technologies and subsystems to support and monitor the growth of microorganisms in culture in these small autonomous space satellites, including real-time tracking of their culture density, gene expression, and metabolic activity while in the space environment. Flight data and results will be presented from GeneSat-1, which tracked gene expression levels of GFP-labeled E. coli and from PharmaSat, which monitored the dose dependency of an antifungal agent against S. cerevisiae. The O/OREOS SESLO instrument, which will study the effects of radiation and microgravity upon the viability and growth characteristics of B. subtilis and the halophile Halorubrum chaoviatoris for periods of 0 - 6 months in space, will be described as well. The ongoing expansion of the small satellite toolbox of biological technologies will be summarized.

Ricco, Antonio J.↗

NASA NTRS2014

Biosentinel: Developing a Space Radiation Biosensor

Ionizing radiation presents a major challenge to human exploration and long-term residence in space. The deep-space radiation spectrum includes highly energetic particles that generate double strand breaks (DSBs), deleterious DNA lesions that are usually repaired without errors via homologous recombination (HR), a conserved pathway in all eukaryotes. While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no terrestrial source duplicates the unique space radiation environment.We are developing a biosensor-based nanosatellite to fly aboard NASAs Space Launch System Exploration Mission 1, expected to launch in 2017 and reach a 1AU (astronomic unit) heliocentric orbit. Our biosensor (called BioSentinel) uses the yeast S. cerevisiae to measure DSBs in response to ambient space radiation. The BioSentinel strain contains engineered genetic defects that prevent growth until and unless a radiation-induced DSB near a reporter gene activates the yeasts HR repair mechanisms. Thus, culture growth and metabolic activity directly indicate a successful DSB-and-repair event. In parallel, HR-defective and wild type strains will provide survival data. Desiccated cells will be carried within independent culture microwells, built into 96-well microfluidic cards. Each microwell set will be activated by media addition at different time points over 18 months, and cell growth will be tracked continuously via optical density. One reserve set will be activated only in the occurrence of a solar particle event. Biological measurements will be compared to data provided by onboard physical dosimeters and to Earth-based experiments.BioSentinel will conduct the first study of biological response to space radiation outside Low Earth Orbit in over 40 years. BioSentinel will thus address strategic knowledge gaps related to the biological effects of space radiation and will provide an adaptable platform to perform human-relevant measurements in multiple space environments. We hope that it can therefore be used on the ISS, on and around other planetary bodies as well as other exploration platforms as a self-contained system that will allow us to compare and calibrate different radiation environments.BioSentinels results will be critical for improving interpretation of the effects of space radiation exposure, and for reducing the risk associated with long-term human exploration.

space radiation↗

NASA NTRS2015

International Internship Report for Asher Williams

For the 2015 NASA I (sup 2) Internship Program, I was selected to work in Dr. John Hogan's laboratory on a Human Nutrient Production in Space (Bio-Nutrients) Project involving Research & Development in advanced microbial strategies for the production of nutrients within crewed spacecraft and habitats. Long-term space missions encounter the hurdle of substantial degradation of certain nutrients in food and supplements with time, potentially resulting in nutrient deficiency and serious health problems. The goal of the Bio-Nutrients Project is to enable rapid, safe, and reliable in situ production of needed nutrients using minimal mass, power, and volume. A platform technology is being developed to employ hydratable single-use packets that contain an edible growth medium and a food microbe engineered to produce target human nutrients. In particular, we examined the production of the carotenoids lutein and zeaxanthin in a spore-forming strain of the yeast Saccharomyces cerevisiae. Carotenoids are important antioxidants required for ocular health, a problematic area for some astronauts on long-duration ISS missions...To meet the first-year milestones for the Bio-Nutrients project, my specific task was to design and run preliminary tests on a disposable bioreactor for in situ production of human nutrients in space.

yeast↗

NASA NTRS2016

The BioSentinel Bioanalytical Microsystem: Characterizing DNA Radiation Damage in Living Organisms Beyond Earth Orbit

We will present details and initial lab test results from an integrated bioanalytical microsystem designed to conduct the first biology experiments beyond low Earth orbit (LEO) since Apollo 17 (1972). The 14-kg, 12x24x37-cm BioSentinel spacecraft (Figure 1) assays radiation-responsive yeast in its science payload by measuring DNA double-strand breaks (DSBs) repaired via homologous recombination, a mechanism common to all eukaryotes including humans. S. cerevisiae (brewer's yeast) in 288 microwells are provided with nutrient and optically assayed for growth and metabolism via 3-color absorptimetry monthly during the 18-month mission. BioSentinel is one of several secondary payloads to be deployed by NASA's Exploration Mission 1 (EM-1) launch vehicle into approximately 0.95 AU heliocentric orbit in July 2018; it will communicate with Earth from up to 100 million km.

BioSentinel↗

NASA NTRS2016

Multi Spectural Flourescence Imager (MSFI)

Multi-Spectural↗

NASA NTRS2016

Multi Spectral Fluorescence Imager (MSFI)

Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

Fluorescence↗