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All sourcesNASA NTRSDOE OSTI
NASA NTRS1972

Informational biopolymer structure in early living forms.

Some studies devoted to the search in various organisms for 'relics' of the biochemical nature of ancient organisms, preserved by the conservative nature of the evolutionary process in all living species, are reviewed. Investigations of five families of informational molecules constituting such 'relics' in very diverse organisms are reported. They include: cytochrome c, ferredoxin, trypsin, transfer ribonucleic acid (RNA), and 5S ribosomal RNA. It is shown that, even from these few informational molecules, some interesting inferences about early living organisms can be drawn.

Dayhoff, M. O.↗
NASA NTRS1984

Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

Gaynor, J. J.↗
NASA NTRS2017

Novel End-to-End Molecular Biology Approach for Direct Nanopore 1D cDNA Sequencing of Reverse Transcribed mRNAs Purified from Cell Cultures by the NASA ISS WetLab2 SPM

Continued space bioscience research onboard the International Space Station (ISS) and future long-duration flight missions to the Moon or Mars will require the ability to conduct on-orbit molecular analysis of biological samples independently from Earth. In the last year two new molecular analytic technologies have been installed and the technologies demonstrated onboard the ISS: The Sample Prep Module (SPM) WetLab-2 (WL2) qRT-PCR toolbox and the Oxford Nanopore MinIon Biomolecule Sequencer. Here we describe protocol development and integration into existing ISS technology for end-to-end on-orbit biological sample processing and molecular analysis with real time results generated utilizing only field offline analytic software. For this experiment we isolated primary cells from bone marrow flushes of wild type B6129SF2 mice (Jackson Labs) long bones. The cell isolate was then processed using the SPM to produce total 147nanograms of RNA. The total RNA was purified to only messenger RNA (mRNA) and transferred to Smartcycler Thermocycle ISS kit consumable tube using Eppendorf gel loading pipette tips for further processing. Complementary first strand cDNA was synthesized using OLIGO dT priming followed by addition of SuperScript II Reverse Transcriptase and thermal cycling as per manufacturers instruction. All thermal cycling was conducted using the ISS WetLab-2 Cephid Smarcycler real time thermal cycler. Our protocol takes advantage of mRNAs native poly(A) tail, synthesized in vivo to protect the mRNA from degradation by endonucleases, to eliminate end-prep for adapter ligation. The adapted library is purified using MyOne C1 Streptavidin beads before elution in buffer. The pre-sequencing library is diluted in the loading buffer and injected into the MinIon sample port, drawn into the nanopore window by capillary action, and sequenced using the MinKnown software with local basecalling. The sequencing read produced 34.5 million events and local basecalling produced 117,301 successful reads. NCBI Blast of the data for the mouse genome resulted in 2,462 successful nucleotide collection matches (gene sequences) exceeding 70 homology. These results demonstrate the viability of this novel flight ready end-to-end sample analytic methodology and provide a real time homolog for flight experimentation utilizing supply kits and technologies that have already been demonstrated on ISS.

MinIon↗
NASA NTRS2000

In vitro evolution of a ribozyme that contains 5-bromouridine

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

NASA Discipline Exobiology↗
NASA NTRS1992

Directed evolution of an RNA enzyme

An in vitro evolution procedures was used to obtain RNA enzymes with a particular catalytic function. A population of 10 exp 13 variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce 'progeny' ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

Beaudry, Amber A.↗
NASA NTRS2000

Interpreting the universal phylogenetic tree

The universal phylogenetic tree not only spans all extant life, but its root and earliest branchings represent stages in the evolutionary process before modern cell types had come into being. The evolution of the cell is an interplay between vertically derived and horizontally acquired variation. Primitive cellular entities were necessarily simpler and more modular in design than are modern cells. Consequently, horizontal gene transfer early on was pervasive, dominating the evolutionary dynamic. The root of the universal phylogenetic tree represents the first stage in cellular evolution when the evolving cell became sufficiently integrated and stable to the erosive effects of horizontal gene transfer that true organismal lineages could exist.

Non-NASA Center↗
NASA NTRS2023

ChIPPS: Charged Information-storage Polymer Preparation System

Technological advances are required to support principal science objectives of missions to the solar system’s icy worlds to seek biosignatures of past/extant life. Sensitivity and reliability are key concerns due to small sample sizes (µL – mL) and the extraordinary import of the results. The preparation and processing of small samples can constrain limits of detection (LoDs); therefore, the Charged Information-storage Polymer Preparation System (ChIPPS) project is advancing the technologies of autonomous sample preparation and processing to add a new class of reliably detectable biosignatures: charged polymers and particles, which can be the information storage-and-transmission means for life. Specifically, we are developing an integrated microfluidic sample-processing unit to prepare icy-world samples to support complementary solid-state nanopore-based analyses: 1) charged-polymer analysis, to characterize variations in polymer chain size, shape, and charge vs. position along the chain; 2) polymer and nanoparticle sizing-and-counting, to characterize the relative abundance of polymer chains, as well as small (virus-sized) particles, by their dimensions and charge. Although no such autonomous system presently exists, such measurements can reveal the nature and abundance of charged polymers that could be used by biological systems to store and transfer information—as DNA and RNA are used terrestrially—without limitation to terrestrial nucleic acids, given that life elsewhere may utilize different information store-and-transfer moieties. Key system components include (a) a lysis unit for mechanical sample disruption; b) an ion-exchange column for charged macromolecule/nanoparticle purification; c) dialyzer to remove excess salt; d) concentrator to enhance signal; e) supporting pumps, valves, bubble traps, connectors, filters, etc.; f) interface to nanopore detection instruments.

Space Biology↗
NASA NTRS1996

Chance and necessity in the selection of nucleic acid catalysts

In Tom Stoppard's famous play [Rosencrantz and Guildenstern are Dead], the ill-fated heroes toss a coin 101 times. The first 100 times they do so the coin lands heads up. The chance of this happening is approximately 1 in 10(30), a sequence of events so rare that one might argue that it could only happen in such a delightful fiction. Similarly rare events, however, may underlie the origins of biological catalysis. What is the probability that an RNA, DNA, or protein molecule of a given random sequence will display a particular catalytic activity? The answer to this question determines whether a collection of such sequences, such as might result from prebiotic chemistry on the early earth, is extremely likely or unlikely to contain catalytically active molecules, and hence whether the origin of life itself is a virtually inevitable consequence of chemical laws or merely a bizarre fluke. The fact that a priori estimates of this probability, given by otherwise informed chemists and biologists, ranged from 10(-5) to 10(-50), inspired us to begin to address the question experimentally. As it turns out, the chance that a given random sequence RNA molecule will be able to catalyze an RNA polymerase-like phosphoryl transfer reaction is close to 1 in 10(13), rare enough, to be sure, but nevertheless in a range that is comfortably accessible by experiment. It is the purpose of this Account to describe the recent advances in combinatorial biochemistry that have made it possible for us to explore the abundance and diversity of catalysts existing in nucleic acid sequence space.

Review, Tutorial↗
NASA NTRS2014

Rodent Research on the International Space Station - A Look Forward

Rodent Research on the International Space Station (ISS) is one of the highest priority science activities being supported by NASA and is planned for up to two flights per year. The first Rodent Research flight, Rodent Research-1 (RR-1) validates the hardware and basic science operations (dissections and tissue preservation). Subsequent flights will add new capabilities to support rodent research on the ISS. RR-1 will validate the following capabilities: animal husbandry for up to 30 days, video downlink to support animal health checks and scientific analysis, on-orbit dissections, sample preservation in RNA. Later and formalin, sample transfer from formalin to ethanol (hindlimbs), rapid cool-down and subsequent freezing at -80 of tissues and carcasses, sample return and recovery. RR-2, scheduled for SpX-6 (Winter 20142015) will add the following capabilities: animal husbandry for up to 60 days, RFID chip reader for individual animal identification, water refill and food replenishment, anesthesia and recovery, bone densitometry, blood collection (via cardiac puncture), blood separation via centrifugation, soft tissue fixation in formalin with transfer to ethanol, and delivery of injectable drugs that require frozen storage prior to use. Additional capabilities are also planned for future flights and these include but are not limited to male mice, live animal return, and the development of experiment unique equipment to support science requirements for principal investigators that are selected for flight. In addition to the hardware capabilities to support rodent research the Crew Office has implemented a training program in generic rodent skills for all USOS crew members during their pre-assignment training rotation. This class includes training in general animal handling, euthanasia, injections, and dissections. The dissection portion of this training focuses on the dissection of the spleen, liver, kidney with adrenals, brain, eyes, and hindlimbs. By achieving and maintaining proficiency in these basic skills as part of the nominal astronaut training curriculum this allows the rodent research program to focus the mission specific crew training on scientific requirements of research and operations flow.

Experimental capabilities↗
NASA NTRS2014

Detection of Nitric Oxide by the Sample Analysis at Mars (SAM) Instrument Implications for the Presence of Nitrates

One of the main goals of the Mars Science Laboratory is to determine whether the planet ever had environmental conditions able to support microbial life. Nitrogen is a fundamental element for life, and is present in structural (e.g., proteins), catalytic (e.g., enzymes and ribozymes), energy transfer (e.g., ATP) and information storage (RNA and DNA) biomolecules. Planetary models suggest that molecular nitrogen was abundant in the early Martian atmosphere, but was rapidly lost to space by photochemistry, sputtering impact erosion, and oxidized and deposited to the surface as nitrate. Nitrates are a fundamental source for nitrogen to terrestrial microorganisms. Therefore, the detection of nitrates in soils and rocks is important to assess the habitability of a Martian environment. SAM is capable of detecting nitrates by their thermal decomposition into nitric oxide, NO. Here we analyze the release of NO from soils and rocks examined by the SAM instrument at Gale crater, and discuss its origin.

Navarro-Gonzalez, R.↗
NASA NTRS1999

In vitro selection of functional nucleic acids

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

Review↗
NASA NTRS1991

Controlled evolution of an RNA enzyme

It is generally thought that prior to the origin of protein synthesis, life on earth was based on self-replicating RNA molecules. This idea has become especially popular recently due to the discovery of catalytic RNA (ribozymes). RNA has both genotypic and phenotypic properties, suggesting that it is capable of undergoing Darwinian evolution. RNA evolution is likely to have played a critical role in the early history of life on earth, and thus is important in considering the possibility of life elsewhere in the solar system. We have constructed an RNA-based evolving system in the laboratory, combining amplification and mutation of an RNA genotype with selection of a corresponding RNA phenotype. This system serves as a functional model of a primitive organism. It can also be used as a tool to explore the catalytic potential of RNA. By altering the selection constraints, we are attempting to modify the substrate specificity of an existing ribozyme in order to develop ribozymes with novel catalytic function. In this way, we hope to gain a better understanding of RNA's catalytic versatility and to assess its suitability for the role of primordial catalyst. All of the RNA enzymes that are known to exist in contemporary biology carry out cleavage/ligation reactions involving RNA substrates. The Tetrahymena ribozyme, for example, catalyzes phosphoester transfer between a guanosine containing and an oligopyrimidine containing substrate. We tested the ability of mutant forms of the Tetrahymena ribozyme to carry out a comparable reaction using DNA, rather than RNA substrate. An ensemble of structural variants of the ribozyme was prepared and tested for their ability to specifically cleave d(GGCCCTCT-A3TA3TA) at the phosphodiester bond following the sequence CCCTCT. We recovered a mutant form of the enzyme that cleaves DNA more efficiently than does the wild-type. Beginning with this selected mutant we have now scattered random mutations throughout the ribozyme and have begun an evolutionary search to further expand the catalytic repertoire of RNA.

Joyce, G. F.↗
NASA NTRS2003

Transcriptional regulation of IGF-I expression in skeletal muscle

The present study investigated the role of transcription in the regulation of insulin-like growth factor (IGF)-I expression in skeletal muscle. RT-PCR was used to determine endogenous expression of IGF-I pre-mRNA and mRNA in control (Con) and functionally overloaded (FO) rat plantaris. The transcriptional activities of five different-length IGF-I promoter fragments controlling transcription of a firefly luciferase (FLuc) reporter gene were tested in vitro by transfection of myoblasts or in vivo during FO by direct gene transfer into the plantaris. Increased endogenous IGF-I gene transcription during 7 days of plantaris FO was evidenced by an approximately 140-160% increase (P < 0.0001) in IGF-I pre-mRNA (a transcriptional marker). IGF-I mRNA expression also increased by approximately 90% (P < 0.0001), and it was correlated (R = 0.93; P < 0.0001) with the pre-mRNA increases. The three longest IGF-I exon 1 promoters induced reporter gene expression in proliferating C2C12 and L6E9 myoblasts. In differentiated L6E9 myotubes, promoter activity increased approximately two- to threefold over myoblasts. Overexpression of calcineurin and MyoD increased the activity of the -852/+192 promoter in C2C12 myotubes by approximately 5- and approximately 18-fold, respectively. However, FO did not induce these exogenous promoter fragments. Nevertheless, the present findings are consistent with the hypothesis that the IGF-I gene is transcriptionally regulated during muscle hypertrophy in vivo as evidenced by the induction of the endogenous IGF-I pre-mRNA during plantaris FO. The exon 1 promoter region of the IGF-I gene is sufficient to direct inducible expression in vitro; however, an in vivo response to FO may require elements outside the -852/+346 region of the exon 1 IGF-I promoter or features inherent to the endogenous IGF-I gene.

Non-NASA Center↗
NASA NTRS1997

Continuous in vitro evolution of catalytic function

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Non-NASA Center↗
NASA NTRS1986

Molecular replication

The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

Orgel, L. E.↗
NASA NTRS2010

Response of Staphylococcus Aureus to a Spaceflight Analogue

The decreased gravity of the spaceflight environment creates quiescent, low fluid shear conditions. This environment can impart considerable effects on the physiology of microorganisms as well as their interactions with potential hosts. Using the rotating wall vessel (RWV), as a spaceflight analogue, the consequence of low fluid shear culture on microbial pathogenesis has provided a better understanding of the risks to the astronaut crew from infectious microorganisms. While the outcome of low fluid shear culture has been investigated for several bacterial pathogens, little has been done to understand how this environmental factor affects Staphylococcus aureus. S. aureus is an opportunistic human pathogen which presents a high level of infection risk to the crew, as it has been isolated from both the space shuttle and International Space Station. Given that approximately forty percent of the population are carriers of the bacteria, eradication of this organism from in flight environments is impractical. These reasons have lead to us to assess the response of S. aureus to a reduced fluid shear environment. Culture in the RWV demonstrated that S. aureus grown under the low-shear condition had lower cell concentrations after 10 hours when compared to the control culture. Furthermore, the low-shear cultured bacteria displayed a reduction in carotenoid production, pigments responsible for their yellow/gold coloration. When exposed to various environmental stressors, post low-shear culture, a decrease in the ability to survive oxidative assault was observed compared to control cultures. The low fluid shear environment also resulted in a decrease in hemolysin secretion, a staphylococcal toxin responsible for red blood cell lysis. When challenged by the immune components present in human whole blood, low-shear cultured S. aureus demonstrated significantly reduced survival rates as compared to the control culture. Assays to determine the duration of these alterations demonstrated that the low-shear response could be lost in as few as 2.5 hours. These changes in phenotypic properties prompted investigation into variations occurring at the genetic level. Microarray analysis of low-shear cultured S. aureus revealed the differential regulation of genes involved in metabolism, stress response, and phosphate transfer. Additional genetic analysis with quantitative real-time PCR revealed alterations in the expression of Hfq, the conserved RNA chaperone protein involved in global gene regulation. Hfq has been connected to the regulation of a spaceflight microgravity response in S. typhimurium. These findings in S. aureus suggest an evolutionary conserved response to spaceflight conditions among structurally-diverse microorganisms. Furthermore, the reduction in pigmentation, hemolysin secretion, and survival against oxidative stress and immunologically active whole blood demonstrate an overall decrease in the virulence factors of S. aureus in response to spaceflight-like conditions.

Castro, S. L.↗
NASA NTRS2003

Resistance of virus to extinction on bottleneck passages: study of a decaying and fluctuating pattern of fitness loss

RNA viruses display high mutation rates and their populations replicate as dynamic and complex mutant distributions, termed viral quasispecies. Repeated genetic bottlenecks, which experimentally are carried out through serial plaque-to-plaque transfers of the virus, lead to fitness decrease (measured here as diminished capacity to produce infectious progeny). Here we report an analysis of fitness evolution of several low fitness foot-and-mouth disease virus clones subjected to 50 plaque-to-plaque transfers. Unexpectedly, fitness decrease, rather than being continuous and monotonic, displayed a fluctuating pattern, which was influenced by both the virus and the state of the host cell as shown by effects of recent cell passage history. The amplitude of the fluctuations increased as fitness decreased, resulting in a remarkable resistance of virus to extinction. Whereas the frequency distribution of fitness in control (independent) experiments follows a log-normal distribution, the probability of fitness values in the evolving bottlenecked populations fitted a Weibull distribution. We suggest that multiple functions of viral genomic RNA and its encoded proteins, subjected to high mutational pressure, interact with cellular components to produce this nontrivial, fluctuating pattern.

Serial Passage↗
NASA NTRS2011

Stress-Driven Selection of Novel Phenotypes

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Fox, George E.↗