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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
NASA NTRS1990

Evolutionary tree reconstruction

It is described how Minimum Description Length (MDL) can be applied to the problem of DNA and protein evolutionary tree reconstruction. If there is a set of mutations that transform a common ancestor into a set of the known sequences, and this description is shorter than the information to encode the known sequences directly, then strong evidence for an evolutionary relationship has been found. A heuristic algorithm is described that searches for the simplest tree (smallest MDL) that finds close to optimal trees on the test data. Various ways of extending the MDL theory to more complex evolutionary relationships are discussed.

Cheeseman, Peter↗
NASA NTRS1999

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

NASA Discipline Plant Biology↗
NASA NTRS2001

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

NASA Discipline Plant Biology↗
NASA NTRS2000

Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

NASA Program Fundamental Space Biology↗
NASA NTRS1980

How many theories for the origin of /proto/life

The sequence of primordial chemical events leading to contemporary metabolism is considered, taking into account primordial reactants, amino acids, proteinoid, protocells, ATP, polynucleotides, and protein. The right kind of matter, thermal copolyamino acids, can organize itself into cell-like structures, in the absence of discrete lipid, when triggered to do so by water. Another unpredicted result of examination of the microsystems formed was the step-by-step realization that the component processes of a primitive form of replication were latent in the proteinoid microsystems. At the present time, four modes of primitive replication of proteinoid microsystems have been identified, plus one that has the appearance of protosexual reproduction. Two main conceptual pathways have received attention. One is the proteinoid theory, derived from experiments. The other is the DNA-first theory, for which attempts at conceptual construction and experimental support continue to be sought.

Fox, S. W.↗
NASA NTRS1992

Characterization of photosystem 1 chlorophyll a/b-binding apoprotein accumulation in developing soybean using type-specific antibodies

The structure and supramolecular assembly of the soybean photosystem 1 (PS 1) chlorophyll a/b-binding antenna (LHC 1) was examined. We identified the subunit composition of LHC 1 in soybean and followed the accumulation of individual subunits during light-induced assembly. We observed four LHC 1 subunits, at 23, 22, 21 and 20.5 kDa, obtained partial sequence information by amino-terminal sequence analysis, and classified the 20.5, 22, and 21 kDa subunits as being encoded by type I, II, and IV chlorophyll a/b binding protein genes, respectively. Antisera against LHC 1 subunits were used to follow the accumulation of individual subunits during the light-initiated transition from etioplast to chloroplast. Several points are noteworthy. First, monospecific antibody against the 22 kDa subunit decorated a 25 kDa peptide in etiolated tissue, which declined during maturation. This decline correlated with the light-induced appearance of mature 22 kDa peptide, suggesting a precursor/product relationship. Second, the same antibody identified a 22 kDa protein in mature corn, but not a larger band in etiolated corn, suggesting that LHC 1 accumulation is regulated differently between species before the onset of chlorophyll biosynthesis. Third, the mature 22 kDa subunit appeared somewhat later than the other LHC 1 peptides during greening, implying that this subunit is less intimately associated with the PS1 core than are the subunits appearing earlier in development.

Non-NASA Center↗
NASA NTRS1999

Left-handed Z-DNA: structure and function

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Review↗
NASA NTRS1992

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

NASA Program NSCORT↗
NASA NTRS2000

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

NASA Discipline Musculoskeletal↗
NASA NTRS1986

Investigations with methanobacteria and with evolution of the genetic code

Mycoplasma capricolum was found by Osawa et al. to use UGA as the code of tryptophan and to contain 75% A + T in its DNA. This change could have been from evolutionary pressure to replace C + G by A + T. Numerous studies have been reported of evolution of proteins as measured by amino acid replacements that are observed when homologus proteins, such as hemoglobins from various vertebrates, are compared. These replacements result from nucleotide substitutions in amino acid codons in the corresponding genes. Simultaneously, silent nucleotide substitutions take place that can be studied when sequences of the genes are compared. These silent evolutionary changes take place mostly in third positions of codons. Two types of nucleotide substitutions are recognized: pyrimidine-pyrimidine and purine-purine interchanges (transitions) and pyriidine-purine interchanges (transversions). Silent transitions are favored when a corresponding transversion would produce an amino acid replacement. Conversely, silent transversions are favored by probability when transitions and transversions will both be silent. Extensive examples of these situations have been found in protein genes, and it is evident that transversions in silent positions predominate in family boxes in most of the examples studied. In associated research a streptomycete from cow manure was found to produce an extracellular enzyme capable of lysing the pseudomurein-contining methanogen Methanobacterium formicicum.

Jukes, T. H.↗
NASA NTRS1998

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Non-NASA Center↗
NASA NTRS1998

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Non-NASA Center↗
NASA NTRS1994

Halococcus salifodinae sp. nov., an Archaeal Isolate from an Austrian Salt Mine

A novel extremely halophilic archaeon (archaebacterium) was isolated from rock salt obtained from an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225 x 106 to 280 x 10(exp 6) years ago). This organism grew over a pH range of 6.8 to 9.5. Electron microscopy revealed cocci in tetrads or larger clusters. The partial 16S rRNA sequences, polar lipid composition, and menaquinone content suggested that this organism was related to members of the genus Halococcus, while the whole-cell protein patterns, the presence of several unknown lipids, and the presence of pink pigmentation indicated that it was different from previously described coccoid halophiles. We propose that this isolate should be recognized as a new species and should be named Halococcus salifodinae. The type strain is Bl(sub p) (= ATCC 51437 = DSM 8989). A chemotaxonomically similar microorganism was isolated from a British salt mine.

Denner, Ewald B. M.↗
NASA NTRS2002

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

NASA Discipline Cell Biology↗
NASA NTRS2001

The Role of Stress Proteins in Cell Stabilization: A Perspective from an Extremophile

The existence of organisms that live at near boiling temperatures is living proof that all of the complex biochemical machinery of life can be adapted to function under these harsh conditions. The purpose of our research is to elucidate the role of a group of proteins known as heat shock proteins or HSP60s in this adaptation to high temperatures. HSP60s are found in all organisms and they are among the most highly conserved proteins known. We are investigating HSP60s in an organism growing at 80 C and pH 2.0 (Sulfolobus shibatae). This organism produces three closely-related HSP60 proteins, referred to as HSP60 alpha, beta, and gamma. Our DOE-funded research during the last two years has focused on clarifying the role of FiSP60 alpha and beta. These are among the two most abundant proteins in S. shibatae grown at high temperatures and significantly increase in abundance when the cells are exposed to near-lethal temperatures. We have demonstrated that these proteins protect the cells from lethal temperatures by stabilizing their membranes. During this last year we have been studying gamma, which was discovered by genome sequence analysis but nothing was known about its function. We have determined that gamma is only expressed at low temperatures. that it interacts with alpha and beta, and that it influences their ability to form higher-order structures critical to their function. We propose that gamma modulates HSP60 function at low temperatures.

Trent, Jonathan↗
NASA NTRS2001

Leiomodins: larger members of the tropomodulin (Tmod) gene family

The 64-kDa autoantigen D1 or 1D, first identified as a potential autoantigen in Graves' disease, is similar to the tropomodulin (Tmod) family of actin filament pointed end-capping proteins. A novel gene with significant similarity to the 64-kDa human autoantigen D1 has been cloned from both humans and mice, and the genomic sequences of both genes have been identified. These genes form a subfamily closely related to the Tmods and are here named the Leiomodins (Lmods). Both Lmod genes display a conserved intron-exon structure, as do three Tmod genes, but the intron-exon structure of the Lmods and the Tmods is divergent. mRNA expression analysis indicates that the gene formerly known as the 64-kDa autoantigen D1 is most highly expressed in a variety of human tissues that contain smooth muscle, earning it the name smooth muscle Leiomodin (SM-Lmod; HGMW-approved symbol LMOD1). Transcripts encoding the novel Lmod gene are present exclusively in fetal and adult heart and adult skeletal muscle, and it is here named cardiac Leiomodin (C-Lmod; HGMW-approved symbol LMOD2). Human C-Lmod is located near the hypertrophic cardiomyopathy locus CMH6 on human chromosome 7q3, potentially implicating it in this disease. Our data demonstrate that the Lmods are evolutionarily related and display tissue-specific patterns of expression distinct from, but overlapping with, the expression of Tmod isoforms. Copyright 2001 Academic Press.

Carrier Proteins/biosynthesis/genetics↗
NASA NTRS2019

Radiation Tolerance of Nanopore Sequencing Technology for Life Detection on Mars and Europa

The search for life beyond Earth is a key motivator in space exploration. Informational polymers, like DNA and RNA, are key biosignatures for life as we know it. The MinION is a miniature DNA sequencer based on versatile nanopore technology that could be implemented on future planetary missions. A critical unanswered question is whether the MinION and its protein-based nanopores can withstand increased radiation exposure outside Earth’s shielding magnetic field. We evaluated the effects of ionizing radiation on the MinION platform – including flow cells, reagents, and hardware – and discovered limited performance loss when exposed to ionizing doses comparable to a mission to Mars. Targets with harsher radiation environments, like Europa, would require improved radiation resistance via additional shielding or design refinements.

Mark A. Sutton↗
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↗