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NASA NTRS1992

Crystal structure of an Okazaki fragment at 2-A resolution

In DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type conformation at the junction between RNA.DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.

Non-NASA Center↗

NASA NTRS2005

Carnobacterium pleistocenium sp. nov., a novel psychrotolerant, facultative anaerobe isolated from permafrost of the Fox Tunnel in Alaska

A novel, psychrotolerant, facultative anaerobe, strain FTRl, was isolated from Pleistocene ice from the permafrost tunnel in Fox, Alaska. Gram-positive, motile, rod-shaped cells were observed with sizes 0(raised dot)6-0(raised dot)7 x 0(raised dot)9-1(raised dot)5 microns. Growth occurred within the pH range 6(raised dot)5-9(raised dot)5 with optimum growth at pH 7(raised dot)3-7(raised dot)5. The temperature range for growth of the novel isolate was 0-28 C and optimum growth occurred at 24 C. The novel isolate does not require NaCl; growth was observed between 0 and 5% NaCl with optimum growth at 0(raised dot)5% (w/v). The novel isolate was a catalase-negative chemoorganoheterotroph that used as substrates sugars and some products of proteolysis. The metabolic end products were acetate, ethanol and CO2. Strain FTRl was sensitive to ampicillin, tetracycline, chloramphenicol, rifampicin, kanamycin and gentamicin. 16s rRNA gene sequence analysis showed 99(raised dot)8% similarity between strain FTR1 and Carnobacterium alterfunditum, but DNA-DNA hybridization between them demonstrated 39 plus or minus 1(raised dot)5% relatedness. On the basis of genotypic and phenotypic characteristics, it is proposed that strain FTRl (= ATCC BAA-754T= JCM 12174T=CIP 108033) be assigned to the novel species Carnobacterium pleistocenium sp. nov.

Pilkuta, Elena V.↗

NASA NTRS2014

Characterization of Plastic Degrading Bacteria from Environmental Samples by Genetic and Biochemical Analysis

Plastic is the major waste-product during NASA space missions, recycling this waste-stream to produce other beneficial materials would decrease upmass. Bacterial called plastisomes have been demonstrated to metabolize non-biodegradable plastics such as polyethylene and polystyrene. Characterization and engineering of these bacteria, and their eventual incorporation as life support systems would enable space flight beyond lower earth orbit. We will utilize molecular techniques to identify and isolate the most productive plastisome. Environmental samples obtained from locations known to be rich in plastic will be cultured in a laboratory defined-media supplemented with plastic as the sole carbon source. Cultures will be monitored for growth over time. Ribosomal DNA will be amplified from cultures that exhibit growth using PCR. These amplified fragments will be sequenced to determine the identity of the consortia in the cultures. We will then perform bioinformatics analysis on the data to identify the plastisomes and generate phylogenetic trees. Morphological and physiological profile of the plastisomes will also be conducted by microscopy and biochemical tests. Our results would reveal a bacterial strain that can break down plastics efficiently. The implication for this project would not only benefit space exploration but also make a major impact towards sustainability development on Earth.

plastic conversion↗

NASA NTRSDate not supplied

Simulation of Radiation-Induced DNA Damage With the Code RITRACKS

INTRODUCTION DNA damage is one of the most physiologically important effects of ionizing radiation. Clustered DNA damage events, like double-strand breaks (DSBs), have the most notable biological consequences. DNA damage types depend on both the track structure of the radiation and the spatial organization of the DNA. High linear energy transfer (LET) charged nuclei, found in galactic cosmic rays (GCR), are known to produce large numbers of complex DNA damage events. The human genome is packaged into chromatin, which can take on locus-dependent and cell type-dependent spatial conformations that correspond to epigenetic states, such as more open, extended structures in transcriptionally active chromatin. These epigenetic differences can affect DNA break patterns in response to ionizing radiation, potentially creating distinct DNA repair and signaling outcomes across the genome in different cells. MATERIAL AND METHODS The code RITRACKS (Relativistic Ion Tracks), which simulates stochastic radiation track structures and radiation chemistry, was used to model damage on isolated and histone-bound DNA by various types of ions and photons. The changes made to the code to perform radiation-induced DNA damage, and simulation results on single nucleosomes are given in our recent paper. In this work, the DNA building capabilities of RITRACKS have been extended to simulate more complex DNA structures build on the coarse-grain simulation framework meso-WLCsim. This code can sample generic chromatin fiber conformation ensembles based on the geometry of nucleosomes and mechanical properties of DNA. Using RITRACKS, we simulated the fragment length distributions (FLD) of irradiated DNA structures built using the chromatin conformations of WLCsim and obtained results representative of those obtained with Radiation-Induced Correlated Cleavage with sequencing (RICC-Seq) experiments [6]. We have also performed Fe ion and photon irradiations of K562, IMR90, BJ and RPE-1 cells at NSRL to experimentally validate results. Sample processing and data analysis are in progress and any available preliminary results will be discussed. DISCUSSION The recent updates in the code RITRACKS allow the calculation of several quantities such as the DNA damage yield and the FLD. This approach can be used to model epigenetic state-specific chromatin structure parameters to leverage the epigenetic state data available for many human cell types to infer relative DNA damage sensitivity among genomic loci.

I Plante↗

NASA NTRS2005

Multiple melanocortin receptors are expressed in bone cells

Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

NASA Discipline Musculoskeletal↗

NASA NTRS1992

Crosslinking transcription factors to their recognition sequences with PtII complexes

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

NASA Discipline Exobiology↗

NASA NTRSDate not supplied

Space Algae: Understanding the Genomic Impacts on Microalgae After Growth in the International Space Station

Plants and microbes can be used for biological support of crewed space missions. The radiation and microgravity environment of spaceflight is expected to increase genetic mutation of all organisms. It is essential to understand how spaceflight impacts mutation rates in photosynthetic organisms to enable appropriate countermeasures and ensure productivity during long duration and deep space missions. The Space Algae flight experiments to the International Space Station (ISS) are studying the genomic stability of microalgae that could potentially be used in biological life support systems. Space Algae-1 grew ultraviolet light mutagenized Chlamydomonas reinhardtii in the VEGGIE plant growth chamber for approximately 40 mitotic generations over one month on the ISS. Whole genome sequencing from pooled cell samples every 10 generations revealed that spaceflight cultures had an ~50% increase in DNA polymorphisms relative to ground controls. These mutations had a novel base substitution signature and suggested a risk that microalgae may be unstable for long-term production in space. Space Algae-2 is focusing on the edible cyanobacterium Arthrospira platensis, commonly known as Spirulina. This experiment seeks to grow serial cultures to allow the organism to evolve in long-term spaceflight. Biological responses of the cells to spaceflight will be assessed with multi-omics analyses to determine mutation load, gene/protein expression, metabolic/nutritional composition, and cell morphology.

Algae↗

NASA NTRSDate not supplied

Space Algae: Understanding the Genomic Impacts on Microalgae After Growth in the International Space Station

Algae↗

NASA NTRS2006

Anaerovirgula multivorans gen. nov., sp. nov., a Novel Spore-Forming, Alkaliphilic Anaerobe Isolated from Owens Lake, California, USA

A novel, alkaliphilic, obligately anaerobic bacterium, strain SCAT, was isolated from mud sediments of a soda lake in California, USA. The rod-shaped cells were motile, Gram-positive, formed spores and were 0.4-0.5x2.5-5.0 micrometers in size. Growth occurred within the pH range 6.7-10.0 and was optimal at pH 8.5. The temperature range for growth was 10-45 degrees C, with optimal growth at 35 degrees C. NaCl was required for growth. Growth occurred at 0.5-9.0% (w/v) NaCl and was optimal at 1-2% (w/v). The novel isolate was a catalase-negative chemo-organoheterotroph that fermented sugars, proteolysis products, some organic and amino acids, glycerol, d-cellobiose and cellulose. It was also capable of growth by the Stickland reaction. Strain SCAT was sensitive to tetracycline, chloramphenicol, rifampicin and gentamicin, but it was resistant to ampicillin and kanamycin. The G+C content of the genomic DNA was 34.2 mol%. Major fatty acid components were C14:0, iso-C15:0, C16:1omega9c and C16:0. 16S rRNA gene sequence analysis of strain SCAT showed a similarity of approximately 97% with the type strains of Clostridium formicaceticum and Clostridium aceticum in clostridial cluster XI and a similarity of less than 94.2% to any other recognized Clostridium species and those of related genera in this cluster. Strain SCAT was clearly differentiated from C. formicaceticum and C. aceticum based on comparison of their phenotypic properties and fatty acid profiles, as well as low levels of DNA-DNA relatedness between strain SCAT and the type strains of these two species. Therefore, strain SCAT is considered to represent a novel species of a new genus, Anaerovirgula multivorans gen. nov., sp. nov., in clostridial cluster XI. The type strain is SCAT (=ATCC BAA-1084T=JCM 12857T=DSM 17722T=CIP 107910T).

Pikuta, Elena V.↗

NASA NTRS2000

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗

NASA NTRS1993

Ribosomal RNA: a key to phylogeny

As molecular phylogeny increasingly shapes our understanding of organismal relationships, no molecule has been applied to more questions than have ribosomal RNAs. We review this role of the rRNAs and some of the insights that have been gained from them. We also offer some of the practical considerations in extracting the phylogenetic information from the sequences. Finally, we stress the importance of comparing results from multiple molecules, both as a method for testing the overall reliability of the organismal phylogeny and as a method for more broadly exploring the history of the genome.

NASA Program Exobiology↗

NASA NTRS1992

Identification of a new EF-hand superfamily member from Trypanosoma brucei

We identified several open reading frames between the regions encoding calmodulin and ubiquitin-EP52/1 in the genome of Trypanosoma brucei. One of these, EFH5, encodes a protein 192 amino acids long. The EFH5 transcript is present in poly(A)+ mRNA and is present at similar levels in the mammalian bloodstream form and the insect procyclic form. EFH5 contains four EF-hand homolog domains, two of which are inferred to bind Ca2+ ions. We expressed EFH5 as a fusion protein in Escherichia coli and demonstrated calcium-binding activity of the fusion protein using the 45Ca-overlay technique. The function of EFH5 remains unknown; however, as the fourth EF-hand homolog identified in trypanosomes, it attests to the broad range of functions assumed by calcium functioning as a second messenger. EFH5, which is most closely related to LAV1-2 from Physarum, represents a distinct subfamily among the EF-hand-containing proteins.

Non-NASA Center↗

NASA NTRS1995

Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: effect of caffeine

We examined the effect of caffeine (1,3,7-trimethylxanthine) on the quantity and quality of mutations in cultured mammalian AL human-hamster hybrid cells exposed to 137Cs gamma radiation. At a dose (1.5 mg/ml for 16 h) that reduced the plating efficiency (PE) by 20%, caffeine was not itself a significant mutagen, but it increased by approximately twofold the slope of the dose-response curve for induction of S1- mutants by 137Cs gamma radiation. Molecular analysis of 235 S1- mutants using a series of DNA probes mapped to the human chromosome 11 in the AL hybrid cells revealed that 73 to 85% of the mutations in unexposed cells and in cells treated with caffeine alone, 137Cs gamma rays alone or 137Cs gamma rays plus caffeine were large deletions involving millions of base pairs of DNA. Most of these deletions were contiguous with the region of the MIC1 gene at 11p13 that encodes the S1 cell surface antigen. In other mutants that had suffered multiple marker loss, the deletions were intermittent along chromosome 11. These "complex" mutations were rare for 137Cs gamma irradiation (1/63 = 1.5%) but relatively prevalent (23-50%) for other exposure conditions. Thus caffeine appears to alter both the quantity and quality of mutations induced by 137Cs gamma irradiation.

NASA Discipline Radiation Health↗

NASA NTRS2000

Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

NASA Discipline Cardiopulmonary↗

NASA NTRS1974

Comments on papers by Reichert and Wong

Papers of Reichert and Wong (1971) and Reichert (1972) are referred to as erroneous in stating that in their work with cytochrome c sequences they demonstrated that vertebrates have achieved a higher organized genetic message with a higher fidelity in the DNA-to-protein information processing channel. Arguments are given to contest that statement.

Gatlin, L. L.↗

NASA NTRS1996

An Archaea 5S rRNA analog is stably expressed in Escherichia coli

Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

Non-NASA Center↗

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 NTRS2002

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Non-NASA Center↗