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At least 271 records · Page 15

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NASA NTRS1971

Comparisons of the polypeptide chains of globins.

Discussion of the amino acid differences and minimum base differences per codon due to mutations which took place during divergent evolution of vertebrates from a common ancestral gene. The ?random mutation model' of evolution is examined by comparing a carp alpha Hb chain and six mammalian alpha Hb chains in terms of the genetic code. The occurrence of recognizable three-base changes is analyzed and a summary of the distribution of changes in the hemoglobin and myoglobin chains is given for 148 sites.

Jukes, T. H.↗

NASA NTRS1972

Selective condensation of aminoacyl adenylates by nucleoproteinoid microparticles.

Microparticles composed of each of four enzymically synthesized homopolynucleotides and the same lysine-rich proteinoid have been found to influence the condensation of the AMP-anhydrides of each of four amino acids. The conditions of preparation of the particles and other variables of the experiments control the types of reaction. When a period set of conditions was identified empirically, the incorporation favored the amino acids whose present-day codons are related to the homopolynucleotide in the particle.

Nakashima, T.↗

NASA NTRS1978

The REH theory of protein and nucleic acid divergence - A retrospective update

The random evolutionary hits (REH) theory of evolutionary divergence, originally proposed in 1972, is restated with attention to certain aspects of the theory that have caused confusion. The theory assumes that natural selection and stochastic processes interact and that natural selection restricts those codon sites which may fix mutations. The predicted total number of fixed nucleotide replacements agrees with data for cytochrome c, a-hemoglobin, beta-hemoglobin, and myoglobin. The restatement analyzes the magnitude of possible sources of errors and simplifies calculational methodology by supplying polynomial expressions to replace tables and graphs.

Holmquist, R.↗

NASA NTRS1978

The augmentation algorithm and molecular phylogenetic trees

Moore's (1977) augmentation procedure is discussed, and it is concluded that the procedure is valid for obtaining estimates of the total number of fixed nucleotide substitutions both theoretically and in practice, for both simulated and real data, and in agreement, for experimentally dense data sets, with stochastic estimates of the divergence, provided the restrictions on codon mutability resulting from natural selection are explicitly allowed for. Tateno and Nei's (1978) critique that the augmentation procedure has a systematic bias toward overestimation of the total number of nucleotide replacements is disputed, and a data analysis suggests that ancestral sequences inferred by the method of parsimony contain a large number of incorrectly assigned nucleotides.

Holmquist, R.↗

NASA NTRS1981

Enhancement of peptide bond formation by polyribonucleotides on clay surfaces in fluctuating environments

The selective effects of polyribonucleotides on the formation of glycine peptide bonds in glycine on clay surfaces are investigated as a model for a template mechanism for the effects of polynucleotides on peptide bond formation. Free oligoglycine yields were determined for the cycling reaction of glycine in the presence and absence of clay and polyribonucleotides or polydeoxyribonucleotides. The polyribonucleotides are observed to lead to increases of up to fourfold increases in oligoglycine formed, with greater enhancements for poly-G nucleotides than for poly-A, poly-U and poly-C, indicating a codonic bias. Polydeoxyribonucleotides are found to provide no enhancement in peptide formation rates, and yields were also greatly reduced in the absence of clay. A mechanism for peptide synthesis is proposed which involves the activation of glycine on the clay surface, followed by the formation of esters between glycine and the 2-prime OH groups of the polyribonucleotide and peptide bonds between adjacent amino acyl esters. It is pointed out that if this mechanism is correct, it may provide a basis for a direct template translation process, which would produce a singlet genetic code.

White, D. H.↗

NASA NTRS1981

The estimation of genetic divergence

Consideration is given to the criticism of Nei and Tateno (1978) of the REH (random evolutionary hits) theory of genetic divergence in nucleic acids and proteins, and to their proposed alternative estimator of total fixed mutations designated X2. It is argued that the assumption of nonuniform amino acid or nucleotide substitution will necessarily increase REH estimates relative to those made for a model where each locus has an equal likelihood of fixing mutations, thus the resulting value will not be an overestimation. The relative values of X2 and measures calculated on the basis of the PAM and REH theories for the number of nucleotide substitutions necessary to explain a given number of observed amino acid differences between two homologous proteins are compared, and the smaller values of X2 are attributed to (1) a mathematical model based on the incorrect assumption that an entire structural gene is free to fix mutations and (2) the assumptions of different numbers of variable codons for the X2 and REH calculations. Results of a repeat of the computer simulations of Nei and Tateno are presented which, in contrast to the original results, confirm the REH theory. It is pointed out that while a negative correlation is observed between estimations of the fixation intensity per varion and the number of varions for a given pair of sequences, the correlation between the two fixation intensities and varion numbers of two different pairs of sequences need not be negative. Finally, REH theory is used to resolve a paradox concerning the high rate of covarion turnover and the nature of general function sites as permanent covarions.

Holmquist, R.↗

NASA NTRS1981

Amino acid codes in mitochondria as possible clues to primitive codes

Differences between mitochondrial codes and the universal code indicate that an evolutionary simplification has taken place, rather than a return to a more primitive code. However, these differences make it evident that the universal code is not the only code possible, and therefore earlier codes may have differed markedly from the previous code. The present universal code is probably a 'frozen accident.' The change in CUN codons from leucine to threonine (Neurospora vs. yeast mitochondria) indicates that neutral or near-neutral changes occurred in the corresponding proteins when this code change took place, caused presumably by a mutation in a tRNA gene.

Jukes, T. H.↗

NASA NTRS1981

The current status of REH theory

A response is made to the evaluation of Fitch (1980) of REH (random evolutionary hits) theory for the evolutionary divergence of proteins and nucleic acids. Correct calculations for the beta hemoglobin mRNAs of the human, mouse and rabbit in the absence and presence of selective constraints are summarized, and it is shown that the alternative evolutionary analysis of Fitch underestimates the total fixed mutations. It is further shown that the model used by Fitch to test for the completeness of the count of total base substitutions is in fact a variant of REH theory. Considerations of the variance inherent in evolutionary estimations are also presented which show the REH model to produce no more variance than other evolutionary models. In the reply, it is argued that, despite the objections raised, REH theory applied to proteins gives inaccurate estimates of total gene substitutions. It is further contended that REH theory developed for nucleic sequences suffers from problems relating to the frequency of nucleotide substitutions, the identity of the codons accepting silent and amino acid-changing substitutions, and estimate uncertainties.

Holmquist, R.↗

NASA NTRS1984

Binding constants of phenylalanine for the four mononucleotides

Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: to which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

Khaled, M. A.↗

NASA NTRS1986

Intramolecular interactions in aminoacyl nucleotides: Implications regarding the origin of genetic coding and protein synthesis

Cellular organisms store information as sequences of nucleotides in double stranded DNA. This information is useless unless it can be converted into the active molecular species, protein. This is done in contemporary creatures first by transcription of one strand to give a complementary strand of mRNA. The sequence of nucleotides is then translated into a specific sequence of amino acids in a protein. Translation is made possible by a genetic coding system in which a sequence of three nucleotides codes for a specific amino acid. The origin and evolution of any chemical system can be understood through elucidation of the properties of the chemical entities which make up the system. There is an underlying logic to the coding system revealed by a correlation of the hydrophobicities of amino acids and their anticodonic nucleotides (i.e., the complement of the codon). Its importance lies in the fact that every amino acid going into protein synthesis must first be activated. This is universally accomplished with ATP. Past studies have concentrated on the chemistry of the adenylates, but more recently we have found, through the use of NMR, that we can observe intramolecular interactions even at low concentrations, between amino acid side chains and nucleotide base rings in these adenylates. The use of this type of compound thus affords a novel way of elucidating the manner in which amino acids and nucleotides interact with each other. In aqueous solution, when a hydrophobic amino acid is attached to the most hydrophobic nucleotide, AMP, a hydrophobic interaction takes place between the amino acid side chain and the adenine ring. The studies to be reported concern these hydrophobic interactions.

Lacey, J. C., Jr.↗

NASA NTRS1990

Was there a universal tRNA before specialized tRNAs came into existence?

It is generally true that evolving systems begin simply and become more complex in the evolutionary process. For those who try to understand the origin of a biochemical system, what is required is the development of an idea as to what simpler system preceded the present one. A hypothesis is presented that a universal tRNA molecule, capable of reading many codons, may have preceded the appearance of individual tRNAs. Evidence seems to suggest that this molecule may have been derived from a common ancestor of the contemporary 5S rRNAs and tRNAs.

Lacey, James C., Jr.↗

NASA NTRS1997

Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

Jackson, Jacquelyn↗

NASA NTRS2003

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

NASA Discipline Musculoskeletal↗

NASA NTRS2002

Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

Case Reports↗

NASA NTRS1995

Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia

Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and > 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA editing leading to excessive Ca2+ permeability, may contribute to neuronal dysfunction in schizophrenia and to neuronal death in Alzheimer's disease and Huntington's disease.

NASA Discipline Neuroscience↗

NASA NTRSDate not supplied

INFLUENCE OF ACTN3 GENE ON MUSCLE HEALTH AND PHYSICAL FITNESS TO INFORM FUTURE INTERVENTIONS IN SPACEFLIGHT MISSIONS

INTRODUCTION: Exposure to reduced gravity environments leads to diminished muscle size, strength, and endurance. Preservation of physical fitness is critical for mission essential tasks such as extravehicular activities or adaptation to changes in gravity loads. Current countermeasures to maintain ISS astronauts’ muscle mass and fitness include dedicated time for a combination of cardiovascular exercise and resistance training. Countermeasure effectiveness is monitored through VO2 max and isometric mid-thigh pulls. It is important to understand both environmental and genetic contributors to astronaut physical fitness. Multiple genes, including ACTN3, are known to correlate with exercise phenotypes and have the potential to help personalize countermeasures based on an astronaut’s genotypic makeup. TOPIC: The ACTN3 gene encodes a protein expressed only in fast-twitch muscles and correlates with sprint and power phenotypes. A common polymorphism in ACTN3 gene is R577X (rs1815739), produced by a C−to−T base substitution resulting in a nonsense mutation from arginine (R) to a premature stop codon (X) present in approximately 18% of the population. This polymorphism causes an absence of α−actinin−3 in type II muscle fibers but does not lead to a disease phenotype. The RR genotype is associated with elite athletes, especially in sprint and power sports. The XX genotype is believed to be more common in endurance athletes. The ACTN3 gene also has potential associations with training adaptation, post-exercise recovery, and exercise-associated injuries. APPLICATION: Understanding how ACTN3 and other genetic determinates of fitness phenotypes affect astronaut physical performance can inform personalized exercise and recovery prescriptions. For example, individuals with the RR or RX genotype may respond better to high-load and low repetition exercise, while XX may benefit from high repetition with low weight exercise. Since the wild-type protein may confer more resistance to muscle damage, those individuals with the RR or RX genotype may benefit from high intensity interval training for improved VO2 max, whereas those with an XX genotype may benefit from low intensity, high volume endurance activity. Additionally, omics data related to health and performance could be used as biomarkers to monitor astronaut fitness and the effectiveness of training regimens during a mission. Learning objectives: • Learn about the phenotypic effects on muscle function and fitness secondary to R577X mutation in the ACTN3 gene • Learn how mutations in the ACTN3 gene may inform future countermeasures for future spaceflight missions

Lynn K Stanwyck↗

DOE OSTI2014

Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

Biopolymers Containing Unnatural Building Blocks

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty amino acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.

60 APPLIED LIFE SCIENCES↗