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At least 199 records · Page 11

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

A novel, psychrotolerant, facultative anaerobe, strain FTR1T, was isolated from Pleistocene ice from the permafrost tunnel in Fox, Alaska. Gram-positive, motile, rod-shaped cells were observed with sizes 0.6-0.7 x 0.9-1.5 microm. Growth occurred within the pH range 6.5-9.5 with optimum growth at pH 7.3-7.5. The temperature range for growth of the novel isolate was 0-28 degrees C and optimum growth occurred at 24 degrees C. The novel isolate does not require NaCl; growth was observed between 0 and 5 % NaCl with optimum growth at 0.5 % (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 FTR1T was sensitive to ampicillin, tetracycline, chloramphenicol, rifampicin, kanamycin and gentamicin. 16S rRNA gene sequence analysis showed 99.8 % similarity between strain FTR1T and Carnobacterium alterfunditum, but DNA-DNA hybridization between them demonstrated 39+/-1.5 % relatedness. On the basis of genotypic and phenotypic characteristics, it is proposed that strain FTR1T (=ATCC BAA-754T=JCM 12174T=CIP 108033T) be assigned to the novel species Carnobacterium pleistocenium sp. nov.

Gram-Positive Asporogenous Rods/classification/gen↗

Microbial Characterization During the Early Habitation of the International Space Station

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance. Copyright 2004 Springer-Verlag.

Fungi/genetics↗

Microbial structural diversity estimated by dilution-extinction of phenotypic traits and T-RFLP analysis along a land-use intensification gradient

The present work tested whether the relationship between functional traits and inoculum density reflected structural diversity in bacterial communities from a land-use intensification gradient applying a mathematical model. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also performed to provide an independent assessment of species richness. Successive 10-fold dilutions of a soil suspension were inoculated onto Biolog GN(R) microplates. Soil bacterial density was determined by total cell and plate counts. The relationship between phenotypic traits and inoculum density fit the model, allowing the estimation of maximal phenotypic potential (Rmax) and inoculum density (KI) at which Rmax will be half-reduced. Though Rmax decreased with time elapsed since clearing of native vegetation, KI remained high in two of the disturbed sites. The genetic pool of bacterial community did not experience a significant reduction, but the active fraction responding in the Biolog assay was adversely affected, suggesting a reduction in the functional potential. c2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

NASA Center KSC↗

Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation

RNA granules are a macromolecular structure observed in neurons, where they serve as motile units that translocate mRNAs. Isolated RNA granules are highly enriched in Staufen protein and ultrastructurally contain densely packed clusters of ribosomes. With depolarization, many mRNAs, including those involved in plasticity, rapidly shift from the RNA granule fraction to polysomes. Depolarization reorganizes granules and induces a less compact organization of their ribosomes. RNA granules are not translationally competent, as indicated by the failure to incorporate radioactive amino acids and the absence of eIF4E, 4G, and tRNAs. We concluded that RNA granules are a local storage compartment for mRNAs under translational arrest but are poised for release to actively translated pools. Local release of mRNAs and ribosomes from granules may serve as a macromolecular mechanism linking RNA localization to translation and synaptic plasticity.

NASA Discipline Neuroscience↗

Three-dimensional structure-guided evolution of a ribosome with tethered subunits

We report RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Model of metabolism and gene expression predicts proteome allocation in Pseudomonas putida

Abstract The genome-scale model of metabolism and gene expression (ME-model) forPseudomonas putidaKT2440,iPpu1676-ME, provides a comprehensive representation of biosynthetic costs and proteome allocation. Compared to a metabolic-only model,iPpu1676-ME significantly expands on gene expression, macromolecular assembly, and cofactor utilization, enabling accurate growth predictions without additional constraints. Multi-omics analysis using RNA sequencing and ribosomal profiling data revealed translational prioritization inP. putida, with core pathways, such as nicotinamide biosynthesis and queuosine metabolism, exhibiting higher translational efficiency, while secondary pathways displayed lower priority. Notably, the ME-model significantly outperformed the M-model in alignment with multi-omics data, thereby validating its predictive capacity. Thus,iPpu1676-ME offers valuable insights intoP. putida’s proteome allocation and presents a powerful tool for understanding resource allocation in this industrially relevant microorganism.

Mathematical & Computational Biology↗

Structural and cytochemical investigations of the gravity receptor under conditions of relative rest and following exposure to accelerations

When investigating the influence of g-forces on vertebrates, a number of cytochemical and ultrastructural changes are observed: deflection of the stereocilia, change in concentration as well as the localization of a number of chemical biologically active substances, displacement of the mitochondria, their pressure against the membranes, the release into the cytoplasm of the ribosomes of nucleolar RNA, the formation of additional membranes in the cytoplasmatic network, etc. According to preliminary data, it is assumed that similar processes are observed in the gravity receptors of invertebrates as well. It is concluded that gravity receptors in vertebrates and invertebrates are strikingly similar despite different evolutionary paths.

Aronova, M. Z.↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.

Mjolsness, Eric↗

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro . Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC . We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

59 BASIC BIOLOGICAL SCIENCES↗

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

Non-NASA Center↗

Is it Possible to have the Similar Unit Cell in Crystals of Different form from the same Macromolecule? (A Case Study of Ribosome Crystals)

Three different types of ribosome crystals were grown by the vapor diffusion technique in hanging drops as described in (1,2). The ribosome is a large asymmetric RNA-protein complex (2.3 million Da), which is protein syntheses machinery of the cell. In this poster we would like to discuss the features of ribosome crystallization. Ribosomes were purified from the thermophilic bacteria Thermus thermophilus by centrifugation (3). Three types of crystals (needle, flat tetragonal and tetragonal-like pyramid) can be grown from the same solution; furthermore, in the same drop using 10-15% 2-methyl-2,4- pentanediol as a precipitant. The crystals appeared in 5-48 hours. The crystals were stable and can co-exist in solution over long period of time. The kinetics of appearance of different crystal forms was different: first the needle crystals were grown, then the tetragonal, and finally the tetragonal pyramids. Later studies of the process of ribosome crystal growth depending on supersaturation showed that low supersaturation results in the appearance of tetragonal plates or tetragonal-like pyramids. An electron microscopy study, together with computer modeling, has shown that crystals of different forms have a high probability of having the same unit cell parameters. According to these experiments the following conclusion can be dranvn: the level of supersaturation of the macromolecule in a crystallizing solution is one of the major factors for forming three-dimensional crystals convenient for X-rays diffraction analysis. From the same macromolecule solution, crystals of different forms can be grown at approximately the same conditions by varying the concentration of macromolecule in the solution. Ion-macromolecule and water-macromolecule interactions, apparently, play the main role in the formation of the unit cell of the crystals.

Karpova, E. A.↗

Mechanisms of cardiac hypertrophy in canine volume overload

This study tested whether the modest hypertrophy that develops in dogs in response to mitral regurgitation is due to a relatively small change in the rate of protein synthesis or, alternatively, is due to a decreased rate of protein degradation. After 3 mo of severe experimental mitral regurgitation, the left ventricular (LV) mass-to-body weight ratio increased by 23% compared with baseline values. This increase in LV mass occurred with a small, but not statistically significant, increase in the fractional rate of myosin heavy chain (MHC) synthesis (Ks), as measured using continuous infusion with [3H]leucine in dogs at 2 wk, 4 wk, and 3 mo after creation of severe mitral regurgitation. Translational efficiency was unaffected by mitral regurgitation as measured by the distribution of MHC mRNA in polysome gradients. Furthermore, there was no detectable increase in translational capacity as measured by either total RNA content or the rate of ribosome formation. These data indicate that translational mechanisms that accelerate the rate of cardiac protein synthesis are not responsive to the stimulus of mitral regurgitation. Most of the growth after mitral regurgitation was accounted for by a decrease in the fractional rate of protein degradation, calculated by subtracting fractional rates of protein accumulation at each time point from the corresponding Ks values. We conclude that 1) volume overload produced by severe mitral regurgitation does not trigger substantial increases in the rate of protein synthesis and 2) the modest increase in LV mass results primarily from a decrease in the rate of protein degradation.

Non-NASA Center↗

A potential role for RNA aminoacylation prior to its role in peptide synthesis

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid–specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid–bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid–bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid–specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis.

Science & Technology - Other Topics↗

Structural switching dynamically controls the doubly pseudoknotted Rous sarcoma virus–programmed ribosomal frameshifting element

A hallmark of retrovirus replication is the translation of two different polyproteins from one RNA through programmed –1 frameshifting. This is a mechanism in which the actively translating ribosome is induced to slip in the 5′ direction at a defined codon and then continues translating in the new reading frame. Programmed frameshifting controls the stoichiometry of viral proteins and is therefore under stringent evolutionary selection. Forty years ago, the first frameshifting stimulatory element was discovered in the Rous sarcoma virus. The ~120 nt RNA segment was predicted to contain a pseudoknot, but its 3D structure has remained elusive. Now, we have determined cryoEM and X-ray crystallographic structures of this classic retroviral element, finding that it adopts a butterfly-like double-pseudoknot fold. One “wing” contains a dynamic pyrimidine-rich helix, observed crystallographically in two conformations and in a third conformation via cryoEM. The other wing encompasses the predicted pseudoknot, which interacts with a second unexpected pseudoknot through a toggle residue, A2546. This key purine switches conformations between structural states and tunes the stability of interacting residues in the two wings. We find that its mutation can modulate frameshifting by as much as 50-fold, likely by altering the relative abundance of different structural states in the conformational ensemble of the RNA. Taken together, our structure–function analyses reveal how a dynamic double pseudoknot junction stimulates frameshifting by taking advantage of conformational heterogeneity, supporting a multistate model in which high Shannon entropy enhances frameshifting efficiency.

Science & Technology - Other Topics↗

Combined Imaging and RNA-Seq on a Microfluidic Platform for Viral Infection Studies

The goal of this work was to pioneer a novel, low-overhead protocol for simultaneously assaying cell-surface markers and intracellular gene expression in a single mammalian cell. The purpose of developing such a method is to be able to understand the mechanisms by which pathogens engage with individual mammalian cells, depending on their cell surface proteins, and how both host and pathogen gene expression changes are reflective of these mechanisms. The knowledge gained from such analyses of single cells will ultimately lead to more robust pathogen detection and countermeasures. Our method was aimed at streamlining both the upstream cell sample preparation using microfluidic methods, as well as the actual library making protocol. Specifically, we wanted to implement a random hexamer-based reverse transcription of all RNA within a single cell (as opposed to oligo dT-based which would only capture polyadenylated transcripts), and then use a CRISPR-based method called scDash to deplete ribosomal DNAs (since ribosomal RNAs make up the majority of the RNA in a mammalian cell). After significant troubleshooting, we demonstrate that we are able to prepare cDNA from RNA using the random hexamer primer, and perform the rDNA depletion. We also show that we can visualize individually stained cells, setting up the pipeline for connecting surface markers to RNA-sequencing profiles. Finally, we test a number of devices for various parts of the pipeline, including bead generation, optical barcoding and cell dispensing, and demonstrate that while some of these have potential, more work is needed to optimize this part of the pipeline.

59 BASIC BIOLOGICAL SCIENCES↗

Circularization of 23S rRNA but not 16S rRNA within archaeal ribosomes

Background Processing of archaeal 16S and 23S rRNAs is believed to involve excision of individual rRNAs from polycistronic precursors, circularization of excised rRNAs, and re-linearization before the incorporation into ribosomes. However, all the knowledge is derived from several isolated species, leaving open the possibility that different processes may occur in other archaeal groups. Results Here, we investigate rRNAs from diverse and mostly uncultivated archaea. Sequencing of total cellular RNA from eight phylum-level lineages indicates that archaeal circular 23S rRNA transcript abundances vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggest that ribosomes contain circular 23S rRNAs. Thus, we directly sequence RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirm that the 23S rRNAs in the ribosomes are circular. Structural modeling places the 5′ and 3′ ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their existence as circular molecules. We also confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups, suggesting that certain archaea do not circularize 16S rRNA during processing. Conclusions Our findings uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

Archaea↗