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All sourcesNASA NTRSDOE OSTI
NASA NTRS1992

Self-replication of chemical systems based on recognition within a double or a triple helix - A realistic hypothesis

A scenario is proposed for the non-enzymatic self-replication of short RNA molecules. The self-replication of an oligopyrimidine strand is considered and the process of template-directed synthesis based on recognition within a double helix is discussed. Replication mechanisms are suggested for selected oligonucleotides. The mechanisms are based on Watson-Crick base pairing between complementary nucleotides as well as Hoogsteen base pairing between a duplex and the complementary third strand. It is suggested that self-replication based on these mechanisms may be accomplished but may result in a substantial amount of misinformation transfer when mixed oligonucleotides are used.

Kanavarioti, Anastassia↗
NASA NTRS1991

Catalytic RNA and synthesis of the peptide bond

We are studying whether the L-19 IVS ribozyme from Tetrahymena thermophila can catalyze the formation of the peptide bond when it is supplied with synthetic aminoacyl oligonucleotides. If this reaction works, it could give us some insight into the mechanism of peptide bond formation and the origin of coded protein synthesis. Two short oligoribonucleotides, CCCCC and a protected form of CCCCU were prepared; the former was made by the controlled hydrolysis of Poly(C), and the later by multistep chemical synthesis from the protected monomers. The homopentamer was then aminocylated using C-14 labelled Boc-protected glycine imidazolide. This aminoacylated oligo-nucleotide has now been shown to enter the active site of the L-19 IVS, and aminoacyl transfer, and peptide bond formation reactions are being sought. Our synthesis of CCCCU made us aware of the inadequacy of many of the 2'- hydroxyl protecting groups that are in use today and we therefore designed a new 2'- protecting group that is presently being tested.

Usher, D. A.↗
NASA NTRS1992

Origin and early evolution of photosynthesis

Photosynthesis was well-established on the earth at least 3.5 thousand million years ago, and it is widely believed that these ancient organisms had similar metabolic capabilities to modern cyanobacteria. This requires that development of two photosystems and the oxygen evolution capability occurred very early in the earth's history, and that a presumed phase of evolution involving non-oxygen evolving photosynthetic organisms took place even earlier. The evolutionary relationships of the reaction center complexes found in all the classes of currently existing organisms have been analyzed using sequence analysis and biophysical measurements. The results indicate that all reaction centers fall into two basic groups, those with pheophytin and a pair of quinones as early acceptors, and those with iron sulfur clusters as early acceptors. No simple linear branching evolutionary scheme can account for the distribution patterns of reaction centers in existing photosynthetic organisms, and lateral transfer of genetic information is considered as a likely possibility. Possible scenarios for the development of primitive reaction centers into the heterodimeric protein structures found in existing reaction centers and for the development of organisms with two linked photosystems are presented.

NASA Program Exobiology↗
NASA NTRS2006

Chance of Necessity: Modeling Origins of Life

The fundamental nature of processes that led to the emergence of life has been a subject of long-standing debate. One view holds that the origin of life is an event governed by chance, and the result of so many random events is unpredictable. This view was eloquently expressed by Jacques Monod in his book Chance or Necessity. In an alternative view, the origin of life is considered a deterministic event. Its details need not be deterministic in every respect, but the overall behavior is predictable. A corollary to the deterministic view is that the emergence of life must have been determined primarily by universal chemistry and biochemistry rather than by subtle details of environmental conditions. In my lecture I will explore two different paradigms for the emergence of life and discuss their implications for predictability and universality of life-forming processes. The dominant approach is that the origin of life was guided by information stored in nucleic acids (the RNA World hypothesis). In this view, selection of improved combinations of nucleic acids obtained through random mutations drove evolution of biological systems from their conception. An alternative hypothesis states that the formation of protocellular metabolism was driven by non-genomic processes. Even though these processes were highly stochastic the outcome was largely deterministic, strongly constrained by laws of chemistry. I will argue that self-replication of macromolecules was not required at the early stages of evolution; the reproduction of cellular functions alone was sufficient for self-maintenance of protocells. In fact, the precise transfer of information between successive generations of the earliest protocells was unnecessary and could have impeded the discovery of cellular metabolism. I will also show that such concepts as speciation and fitness to the environment, developed in the context of genomic evolution also hold in the absence of a genome.

Pohorille, Andrew↗
NASA NTRS2019

The Omics of Stem Cell Mediated Regeneration: A Pilot Single Cell RNA-Seq Study of Mechanotransduction

Mechanical forces are potent modulators of stem cell based tissue regenerative mechanisms, inducing cell fate decisions and tissue specific commitment. A unique platform for investigating mechanotransduction is spaceflight, where microgravity and altered fluid mechanics provide a loading-null experimental condition. Seminal investigations of regenerative capacity in a wholly regenerative species, the newt model, and in a variety of totipotent and adult stem cell populations have demonstrated the detrimental effects of unloading on maintenance of stem cell based regeneration. Of particular interest is the observation that unloading interferes with the transition of stem cell pools from proliferative state to differentiation commitment. In this work we sought to test the hypothesis that gravity mechanotransduction regulates stem cell tissue regenerative processes by modulating stem cell proliferation and differentiation fates at specific cell cycle stages. To do this, clonally-derived ESCs were plated on a collagen matrix and expanded for 36 hours before re-plating on a non-adherent culture dish in the absence of leukemia inhibitory factor (LIF) to form spheroid aggregate EBs. After formation, the EBs were transferred to a collagen matrix coated culture dishes and given 4 days to allow implantation and outgrowth. In parallel, totipotent ESCs were plated 24 hours before mechanical stimulation on collagen matrix culture dishes in the presence of LIF to maintain totipotency and serve as un-differentiation committed controls. The EBs and ESCs were then subjected to either a 60 minute pulse of gravity (static loading) or 60 minutes of cyclic stretch (dynamic loading) mechanotransduction. Six hours post-stimulation, we used a 10X Genomics Single Cell controller to generate bar-coded single cell Illumina libraries and sequenced expressomes for 5,000 static loaded cells, representative of a change in gravity mechanotransduction, 5,000 dynamic loaded cells, representative of tissue loading associate with physiologic function, and 5,000 unstimulated 1g control cells. The comparison of these 3 libraries by cluster assignment based on like gene expression patterns show substantial alteration in cluster geometry due to mechanical loading. Specifically the mechanically loaded EB outgrowth cells to retain potency markers (PAX6, SOX2, CD34) and suppress early commitment markers (Dhh, VCAN, Igf1). Whereas the EBs cultured under the non-stimulated conditions display clear departure from the ESC expressome with lineage commitment markers upregulated and several tissue specific markers being expressed (BMP "early musculoskeletal development, Mesp1" early cardiovascular cell lineage). These markers are not seen in the mechano-stimulated cultures or the totipotent ESC cultures. Comparison of like clusters between our experimental conditions revealed an array of regenerative and stem cell genes are significantly mechano-regulated. Of particular importance CDKN1a/p21, a gene shown by previous investigation of our research team to be significantly upregulated in unloading, was suppressed in the static and dynamic loaded EBS. In addition to CDKN1a/p21 many genes related to cell cycle and transitory differentiation markers had elevated expression in the mechano-stimulated EBs, but surprisingly these trends were not observed in the ESC cultures. This study is the first of its kind investigating for mechano-signaling and mechano-regulated pathways, and has alre

Single cell RNA-sequecing↗
NASA NTRS2016

Electromagnetic Basis of Metabolism and Heredity

Living organisms control their cellular biological clocks to maintain functional oscillation of the redox cycle, also called the "metabolic cycle" or "respiratory cycle". Organization of cellular processes requires parallel processing on a synchronized time-base. These clocks coordinate the timing of all biochemical processes in the cell, including energy production, DNA replication, and RNA transcription. When this universal time keeping function is perturbed by exogenous induction of reactive oxygen species (ROS), the rate of metabolism changes. This causes oxidative stress, aging and mutations. Therefore, good temporal coordination of the redox cycle not only actively prevents chemical conflict between the reductive and oxidative partial reactions; it also maintains genome integrity and lifespan. Moreover, this universal biochemical rhythm can be disrupted by ROS induction in vivo. This in turn can be achieved by blocking the electron transport chain either endogenously or exogenously by various metabolites, e.g. hydrogen sulfide (H2S), highly diffusible drugs, and carbon monoxide (CO). Alternatively, the electron transport in vivo can be attenuated via a coherent or interfering transfer of energy from exogenous ultralow frequency (ULF) and extremely low frequency (ELF) electromagnetic (EM) fields, suggesting that-on Earth-such ambient fields are an omnipresent (and probably crucially important) factor for the time-setting basis of universal biochemical reactions in living cells. Our work demonstrated previously un-described evidence for quantum effects in biology by electromagnetic coupling below thermal noise at the universal electron transport chain (ETC) in vivo.

Electromagnetic↗
NASA NTRS2020

GeneLab

GeneLab collects and enables analysis of spaceflight and ground-based spaceflight simulation genomic data, RNA and protein expression, and metabolic profiles. It interfaces with other existing databases containing spaceflight omic data. The 2011 National Research Council (NRC) Decadal Survey on NASA Life and Physical Sciences called for increased opportunities for multi-investigator spaceflight opportunities and greater use of genomic approaches to meet the needs of NASA researchers. To address these recommendations of the NRC Decadal Survey, the Space Life and Physical Sciences Research and Applications Division of NASA's Human Exploration and Operations Mission Directorate has initiated a transition to an Open Science architecture to increase research opportunities, and has developed the GeneLab Platform based on highly leveraged and integrated bioinformatics analytics. GeneLab is an interactive, open-access resource where scientists can upload, download, store, search, share, transfer, and analyze omics data from spaceflight and corresponding analogue experiments. Users can explore GeneLab datasets in the Data Repository, analyze data using the Analysis Platform, visualize high-order data and create collaborative projects using the Collaborative Workspace. Our primary goal is to maximize the utilization of the valuable biological research conducted aboard the International Space Station (ISS) by collecting genomic, transcriptomic, proteomic, and metabolomics data known as “omics”. By providing a portal linking processed data to flight parameters, GeneLab enables exploration of the molecular network responses of terrestrial biology to the space environment. This allows researchers to understand the complex responses of biological systems to the space environment. This technology development activity was transferred from the Human Exploration and Operations Mission Directorate to the Science Mission Directorate Division of Biological and Physical Sciences (BPS) in October 2020.

GeneLab↗
NASA NTRS2015

NASA's Rodent Research Project: Validation of Capabilities for Conducting Long Duration Experiments in Space

Research using rodents is an essential tool for advancing biomedical research on Earth and in space. Prior rodent experiments on the Shuttle were limited by the short flight duration. The International Space Station (ISS) provides a new platform for conducting rodent experiments under long duration conditions. Rodent Research (RR)-1 was conducted to validate flight hardware, operations, and science capabilities that were developed at the NASA Ames Research Center. Twenty C57BL6J adult female mice were launched on Sept 21, 2014 in a Dragon Capsule (SpaceX-4), then transferred to the ISS for a total time of 21-22 days (10 commercial mice) or 37 days (10 validation mice). Tissues collected on-orbit were either rapidly frozen or preserved in RNAlater at -80C (n2group) until their return to Earth. Remaining carcasses on-orbit were rapidly frozen for dissection post-flight. The three controls groups at Kennedy Space Center consisted of: Basal mice euthanized at the time of launch, Vivarium controls housed in standard cages, and Ground Controls (GC) housed in flight hardware within an environmental chamber. Upon return to Earth, there were no differences in body weights between Flight (FLT) and GC at the end of the 37 days in space. Liver enzyme activity levels of FLT mice and all control mice were similar in magnitude to those of the samples that were processed under optimal conditions in the laboratory. Liver samples dissected on-orbit yielded high quality RNA (RIN8.99+-0.59, n7). Liver samples dissected post-flight from the intact, frozen FLT carcasses yielded RIN of 7.27 +- 0.52 (n6). Additionally, wet weights of various tissues were measured. Adrenal glands and spleen showed no significant differences in FLT compared to GC although thymus and livers weights were significantly greater in FLT compared to GC. Over 3,000 tissue aliquots collected post-flight from the four groups of mice were deposited into the Ames Life Science Data Archives for future Biospecimen Sharing Program. Together, the RR validation flight successfully demonstrates the capability to support long-duration experimentation on the ISS to achieve both basic science and biomedical objectives.

spaceflight↗
NASA NTRS2015

Inflight Microbial Monitoring-An Alternative Method to Culture Based Detection Currently Used on International Space Station

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS). The potential to introduce new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Previous research has shown that microorganisms introduced to the ISS are readily transferred between crew and subsystems and back (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and a 48-hour incubation time. This increases the microbial load while detecting a limited number of microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification, To identify and enumerate ISS samples requires that samples to be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganism at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗
NASA NTRS2015

Inflight Microbial Monitoring- An Alternative Method to Culture Based Detection Currently Used on the International Space Station

Previous research has shown that potentially destructive microorganisms and human pathogens have been detected on the International Space Station (ISS). The likelihood of introducing new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of the total microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS microbes requires that samples be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗
NASA NTRS2015

In-Flight Microbial Monitor

Previous research has shown that potential human pathogens have been detected on the International Space Station (ISS). New microorganisms are introduced with every exchange of crew and cargo. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e., ECLSS, environmental control and life support systems). Current microbial characterization methods require a culture-based enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of microorganisms. The culture-based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS samples requires that the microbes be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, inflight method of microbial detection, identification, and enumeration is needed. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Technology Portfolio System↗
NASA NTRS1995

Cleavage of an amide bond by a ribozyme

A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

NASA Discipline Exobiology↗
NASA NTRS2014

Chronic Lunar Dust Exposure on Rat Cornea: Evaluation by Gene Expression Profiling

Lunar dust is capable of entering habitats and vehicle compartments by sticking to spacesuits or other objects that are transferred into the spacecraft from the lunar surface and has been reported to cause irritation upon exposure. During the Apollo missions, crewmembers reported irritation specifically to the skin and eyes after contamination of the lunar and service modules. It has since been hypothesized that ocular irritation and abrasion might occur as a result of such exposure, impairing crew vision. Recent work has shown that both ultrafine and unground lunar dust exhibited minimal irritancy of the ocular surface (i.e., cornea); however, the assessment of the severity of ocular damage resulting from contact of lunar dust particles to the cornea has focused only on macroscopic signs of mechanical irritancy and cytotoxicity. Given the chemical reactive properties of lunar dust, exposure of the cornea may contribute to detrimental effects at the molecular level including but not limited to oxidative damage. Additionally, low level chronic exposures may confound any results obtained in previous acute studies. We report here preliminary results from a tissue sharing effort using 10‐week‐old Fischer 344 male rats chronically exposed to filtered air or jet milled lunar dust collected during Apollo 14 using a Jaeger‐NYU nose‐only chamber for a total of 120 hours (6 hours daily, 5 days a week) over a 4‐week period. RNA was isolated from corneas collected from rats at 1 day and 7 days after being exposed to concentrations of 0, 20, and 60 mg/m3 of lunar dust. Microarray analysis was performed using the Affymetrix GeneChip Rat Genome 230 2.0 Array with Affymetrix Expression Console and Transcriptome Analysis Console used for normalization and secondary analysis. An Ingenuity iReport"TM" was then generated for canonical pathway identification. The number of differentially expressed genes identified increases with dose compared to controls suggesting a more severe response to the lunar dust insult at higher levels. Pathways of interests that have been identified in all exposed samples include oxidative stress response, mitochondrial dysfunction, fibrosis, epithelial healing, TGF-Beta signaling, and extracellular matrix remodeling. Several biological processes related to cell migration, cellular proliferation, and eye development were also identified to be altered by exposure to lunar dust. Our preliminary results suggest that even a chronic insult of lunar dust as low as 20 mg/m(exp 3) elicits a molecular response in cornea tissue. Lunar dust on the surface of the moon would have the added properties of ionization and activation potentially leading to further damage to the cornea and greater sensitivity to any other environmental insult such as exposure to radiation. Additional studies are required to fully assess the risk of vision impairment and the mechanistic responses initiated in cornea exposed to lunar dust as well as the potential for long‐term effects to astronaut health

Theriot, C. A.↗
NASA NTRS2003

Non-Genomic Origins of Proteins and Metabolism

It is proposed that evolution of inanimate matter to cells endowed with a nucleic acid- based coding of genetic information was preceded by an evolutionary phase, in which peptides not coded by nucleic acids were able to self-organize into networks capable of evolution towards increasing metabolic complexity. Recent findings that truly different, simple peptides (Keefe and Szostak, 2001) can perform the same function (such as ATP binding) provide experimental support for this mechanism of early protobiological evolution. The central concept underlying this mechanism is that the reproduction of cellular functions alone was sufficient for self-maintenance of protocells, and that self- replication of macromolecules was not required at this stage of evolution. The precise transfer of information between successive generations of the earliest protocells was unnecessary and, possibly, undesirable. The key requirement in the initial stage of protocellular evolution was an ability to rapidly explore a large number of protein sequences in order to discover a set of molecules capable of supporting self- maintenance and growth of protocells. Undoubtedly, the essential protocellular functions were carried out by molecules not nearly as efficient or as specific as contemporary proteins. Many, potentially unrelated sequences could have performed each of these functions at an evolutionarily acceptable level. As evolution progressed, however proteins must have performed their functions with increasing efficiency and specificity. This, in turn, put additional constraints on protein sequences and the fraction of proteins capable of performing their functions at the required level decreased. At some point, the likelihood of generating a sufficiently efficient set of proteins through a non-coded synthesis was so small that further evolution was not possible without storing information about the sequences of these proteins. Beyond this point, further evolution required coupling between proteins and informational polymers that is characteristic to all known forms of life. The emergence of such coupling must be postulated in any scenario of the origin of life, no matter whether it starts with RNA or proteins. To examine the evolutionary potential of non-genomic systems, a simple, computationally tractable model, which is still capable of capturing the essential features of the real system, has been studied computationally. Both constructive and destructive processes have been introduced into the model in a stochastic manner. Instead of assuming random reaction sets, only a suite of protobiologically plausible reactions has been considered. Peptides have been explicitly considered as protoenzymes and their catalytic efficiencies have been assigned on the basis of biochemical principles and experimental estimates. Simulations have been carried out using a novel approach (The Next Reaction Method) that is appropriate even for very low concentrations of reactants. Studies have focused on global autocatalytic processes and their diversity.

Pohorille, Andrew↗
NASA NTRS2015

NASA's Rodent Research Project on ISS: Validation of a New Platform for Conducting Biomedical and Basic Research into the Consequences of Long Duration Habitation in Space

Rodent research has played a key role in advancing biomedical discoveries both on Earth and in space. The National Research Counsel’s Decadal survey(1) emphasized the importance of expanding NASAs life sciences research to perform long duration, rodent experiments on the International Space Station (ISS). To accomplish this objective, flight hardware, operations, and science capabilities were developed at NASA ARC to support both commercial and government-sponsored rodent research.Rodent Research-1 (RR-1) was the first mission in which animals were delivered and maintained in the ISS for a long duration mission in modified Animal Enclosure Module hardware. Both RR validation and commercial science objectives were pursued on the RR-1 mission. Adult female mice (20 total Flight, FLT) were launched Sept 21, 2014 in RR hardware within a Dragon Capsule (SpaceX4), then after 4 days in transit, were transferred for habitation on the ISS for 17 days (commercial) or 33 days (validation), when animals were euthanized and select tissues recovered on orbit. Various controls groups consisted of: 1) Basal mice from the same cohorts as FLT mice, but tissues were recovered at time of launch, 2) Vivarium (VIV) were housed in standard cages 3) Ground Controls (GC) were housed in flight hardware within an environmental chamber at Kennedy Space Center. The health and behavior of all mice on the ISS were monitored by video feed on a daily basis. Mice were euthanized by injection of Euthasol, then either fast frozen intact or dissected to preserve livers (fast frozen) and spleens (RNAlater). Samples were stored at ≤ -80˚C until their return to Earth for later analyses.Hardware performed nominally throughout the mission and the planned in-flight science operations were completed successfully. FLT mice appeared generally more physically active on orbit than respective GC groups. After 33 days on the ISS, mean body weights of FLT mice did not differ from GC, with both groups showing a 6% rise compared to time of launch, while VIV mice showed an 8% rise over the same period. Importantly, there were no significant differences in body weights between groups at the end of 33 days on the ISS, providing an indication that the RR hardware supported the health of the mice both on Earth and in space. Based on the preliminary data obtained from the livers and spleens of mice after 17 days on the ISS, purified RNA was of high quality (RIN values of spleen: FLT=9.48 +0.40, GC=9.28 +0.44, n=5/group); therefore, RNA quality from samples retrieved on orbit was acceptable for even the most demanding transcriptomic analyses. In addition, liver enzyme activity levels (units/mg protein) of FLT mice (after 17d on ISS) and all control mice were similar in magnitude to samples that were optimally prepared by freezing in liquid nitrogen in the laboratory (enzymes analyzed included catalase, glutathione reductase and glyceraldehyde-3-phosphate dehydrogenase). Validation analyses still in progress include behavior and tissue biochemistries, as well as optimization of science return by post-flight recovery of tissues for biospecimen sharing and global expression analyses.Together, these preliminary findings demonstrate new capability for supporting long duration rodent research on the ISS to achieve both basic science and biomedical objectives.

Globus, R. K.↗
NASA NTRS1995

Partial gene sequences for the A subunit of methyl-coenzyme M reductase (mcrI) as a phylogenetic tool for the family Methanosarcinaceae

Representatives of the family Methanosarcinaceae were analyzed phylogenetically by comparing partial sequences of their methyl-coenzyme M reductase (mcrI) genes. A 490-bp fragment from the A subunit of the gene was selected, amplified by the PCR, cloned, and sequenced for each of 25 strains belonging to the Methanosarcinaceae. The sequences obtained were aligned with the corresponding portions of five previously published sequences, and all of the sequences were compared to determine phylogenetic distances by Fitch distance matrix methods. We prepared analogous trees based on 16S rRNA sequences; these trees corresponded closely to the mcrI trees, although the mcrI sequences of pairs of organisms had 3.01 +/- 0.541 times more changes than the respective pairs of 16S rRNA sequences, suggesting that the mcrI fragment evolved about three times more rapidly than the 16S rRNA gene. The qualitative similarity of the mcrI and 16S rRNA trees suggests that transfer of genetic information between dissimilar organisms has not significantly affected these sequences, although we found inconsistencies between some mcrI distances that we measured and and previously published DNA reassociation data. It is unlikely that multiple mcrI isogenes were present in the organisms that we examined, because we found no major discrepancies in multiple determinations of mcrI sequences from the same organism. Our primers for the PCR also match analogous sites in the previously published mcrII sequences, but all of the sequences that we obtained from members of the Methanosarcinaceae were more closely related to mcrI sequences than to mcrII sequences, suggesting that members of the Methanosarcinaceae do not have distinct mcrII genes.

NASA Discipline Number 52-30↗
NASA NTRS2001

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Non-NASA Center↗