Tuning Molecular Recognition of RNA and DNA via Sugar Modification

This minireview highlights three aspects of our recent work in the area of sugar modified oligonucleotide analogues. It provides an overview over recent results on the conformationally constrained analogue tricyclo-DNA with special emphasis of its antisense properties, it summarizes results on triple-helix forming oligodeoxynucleotides containing pyrrolidino-nucleosides with respect to DNA recognition via the dual recognition mode, and it highlights the advantageous application of the orthogonal oligonucleotidic pairing system homo-DNA in molecular beacons for DNA diagnostics

Revealing the elusive molecular biology of the vault RNA

Recently several novel and previously reported non-protein-coding RNAs (ncRNAs) have been identified to be upregulated upon Epstein-Barr virus (EBV) infection in human B-lymphocytes. A group of these significantly upregulated ncRNAs are called vault RNAs (vtRNAs). ,b Only about 5% of the total cellular vtRNAs are connected to the vault particle, the largest known ribonucleoprotein particle (RNP) in eukaryotic cells. However the function of this ncRNA family and moreover of the vault particle remains still rather unclear. Our previous findings suggest a link between EBV infection and vtRNA expression. Consequently we are interested which part of the viral genome is responsible for the upregulation and moreover which function the vtRNAs might possess during virus propagation. To address this question we have separately overexpressed specific EBV-encoded, latently expressed proteins in BL2-cells to determine the influence on the vault RNA levels. Thereby we identified one EBV-encoded protein, called Latent Membrane Protein 1 (LMP1), which significantly contributes to the vtRNA upregulation. We used LMP1 mutants to characterize the region of the protein and the responsible pathway for triggering the elevated vtRNA expression. Our results suggest that the NFkB- pathway might be involved in this process. To investigate a possible functional connection between the vtRNA and EBV infection, we have overexpressed vtRNA1-1 in BL41, a cell line usually not expressing this vault RNA. We show that overexpression of vtRNA1-1 leads to a better viral establishment and markedly protects cells from undergoing apoptosis. Knock-down of the major vault protein, the main component of the vault particle, had no effect on EBV infection and apoptosis resistance. Thus these results support the view that the observed phenotype is caused by the vtRNA rather than the vault particle.

Molecular cloning, expression analysis and assignment of the porcine tumor necrosis factor superfamily member 10 gene (TNFSF10) to SSC13q34-->q36 by fluorescence in situ hybridization and radiation hybrid mapping

We have cloned the complete coding region of the porcine TNFSF10 gene. The porcine TNFSF10 cDNA has an ORF of 870 nucleotides and shares 85% identity with human TNFSF10, and 75% and 72% identity with rat and mouse Tnfsf10 coding sequences, respectively. The deduced porcine TNFSF10 protein consists of 289 amino acids with the calculated molecular mass of 33.5 kDa and a predicted pI of 8.15. The amino acid sequence similarities correspond to 86, 72 and 70% when compared with human, rat and mouse sequences, respectively. Northern blot analysis detected TNFSF10-specific transcripts (approximately 1.7 kb) in various organs of a 10-week-old pig, suggesting ubiquitous expression. Real-time RT-PCR studies of various organs from fetal (days 73 and 98) and postnatal stages (two weeks, eight months) demonstrated developmental and tissue-specific regulation of TNFSF10 mRNA abundance. The chromosomal location of the porcine TNFSF10 gene was determined by FISH of a specific BAC clone to metaphase chromosomes. This TNFSF10 BAC clone has been assigned to SSC13q34-->q36. Additionally, the localization of the TNFSF10 gene was verified by RH mapping on the porcine IMpRH panel.

Regulation and transcript analysis of the ceroperon responsible for quorum sensing in Rhodobacter sphaeroides 2.4.1

Rhodobacter sphaeroides 2.4.1 is a Gram negative facultative photoheterotrophic bacterium that has been shown to have an N-acyl homoserine lactone-based quorum sensing system called cer for c&barbelow;ommunity e&barbelow;scape r&barbelow;esponse. The cer ORFs are cerR, the transcriptional regulator, cerI, the autoinducer synthase and cerA , whose function is unknown. The autoinducer molecule, 7,8- cis-N-(tetradecenoyl) homoserine lactone, has been characterized. The objective of this study was to identify an environmental stimulus that influences the regulation of cerRAI and, to characterize transcription of the cer operon. ^ A cerR::lacZ transcriptional fusion was made and β-Galactosidase assays were performed in R. sphaeroides 2.4.1 strains, wild type, AP3 (CerI−) and AP4 (CerR−). The cerR::lacZ β-Galactosidase assays were used as an initial survey of the mode of regulation of the Cer system. A cerA::lacZ translational fusion was created and was used to show that cerA can be translated. The presence of 7,8-cis-N-(tetradecenoyl) homoserine lactone was detected from R. sphaeroides strains wild type and AP4 (CerR−) using a lasR::lacZ translational fusion autoinducer bioassay. The cerR::lacZ transcriptional fusion in R. sphaeroides 2.4.1 wild type was tested under different environmental stimuli, such as various carbon sources, oxygen tensions, light intensities and culture media to determine if they influence transcription of the cer ORFs. Although lacZ assay data implicated high light intensity at 100 W/m2 to stimulate cer transcription, quantitative Northern RNA data of the cerR transcript showed that low light intensity at 3 W/m2 is at least one environmental stimulus that induces cer transcription. This finding was supported by DNA microarray analysis. Northern analysis of the cerRAI transcript provided evidence that the cer ORFs are co-transcribed, and that the cer operon contains two additional genes. Bioinformatics was used to identify genes that may be regulated by the Cer system by identifying putative lux box homologue sequences in the presumed promoter region of these genes. Genes that were identified were fliQ, celB and calsymin, all implicated in interacting with plants. Primer extension was used to help localize cis-elements in the promoter region. The cerR::lacZ transcriptional fusion was monitored in a subset of different global DNA binding transcriptional regulator mutant strains of R. sphaeroides 2.4.1. Those regulators involved in maintaining an anaerobic photosynthetic lifestyle appeared to have an effect. Collectively, the data imply that R. sphaeroides 2.4.1 activates the Cer system when grown anaerobic photosynthetically at low light intensity, 3 W/m2, and it may be involved in an interaction with plants. ^

EGFR and Notch signaling pathways are regulated by distinct isoforms of Drosophila cbl

Activation of cell surface receptors transduces extracellular signals into cellular responses such as proliferation, differentiation and survival. However, the appropriate spatial and temporal down-regulation of signaling receptors is essential for normal development and homeostasis. The Cbl family of E3-ubiquitin ligases plays a major role for the ligand-dependent inactivation of growth factor receptors through ubiquitin-mediated endocytosis and lysosomal degradation. Here, we report the D-cbl mutant phenotypes in the Drosophila eye. D-cbl mutants display overgrowth, inhibition of apoptosis, differentiation defects and increased ommatidial spacing. Many of these phenotypes are caused by lack of down-regulation of the Drosophila EGFR signaling. However, not all D-cbl phenotypes can be explained by inappropriate EGFR activity. We found that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. Strikingly, the long isoform, D-CblL, preferentially regulates the EGFR, whereas the short isoform, D-CblS, preferentially regulates Notch. Taken together, these data suggest that D-Cbl controls at least two signaling pathways, EGFR and Notch, through production of two alternatively spliced isoforms during development in Drosophila.^

Cloning and molecular analysis of paraxis: A {\it trans\/}-acting factor required for somite maturation

During vertebrate embryogenesis, cells from the paraxial mesoderm coalesce in a rostral-to-caudal progression to form the somites. Subsequent compartmentalization of the somites yields the sclerotome, myotome and dermatome, which give rise to the axial skeleton, axial musculature, and dermis, respectively. Recently, we cloned a novel basic-Helix-Loop-Helix (bHLH) protein, called scleraxis, which is expressed in the sclerotome, in mesenchymal precursors of bone and cartilage, and in connective tissues. This dissertation focuses on the cloning, expression and functional analysis of a bHLH protein termed paraxis, which is nearly identical to scleraxis within the bHLH region but diverges in both its amino and carboxyl termini. During the process of mouse embryogenesis, paraxis transcripts are first detected at about day 7.5 post coitum within the primitive mesoderm lying posterior to the head and heart primordia. Subsequently, paraxis expression progresses caudally through the paraxial mesoderm, immediately preceding somite formation. Paraxis is expressed at high levels in newly formed somites before the first detectable expression of the myogenic bHLH genes, and as the somite becomes compartmentalized, paraxis becomes downregulated within the myotome.^ To determine the function of paraxis during mammalian embryogenesis, mice were generated with a null mutation in the paraxis locus. Paraxis null mice survived until birth, but exhibited severe foreshortening along the anteroposterior axis due to the absence of vertebrae caudal to the midthoracic region. The phenotype also included axial skeletal defects, particularly shortened bifurcated ribs which were detached from the vertebral column, fused vertebrae and extensive truncation and disorganization caudal to the hindlimbs. Mutant neonates also lacked normal levels of trunk muscle and exhibited defects in the dermis as well as the stratification of the epidermis. Analysis of paraxis -/- mutant embryos has revealed a failure of the somites to both properly epithelialize and compartmentalize, resulting in defects in somite-derived cell lineages. These results suggest that paraxis is an essential component of the genetic pathway regulating somitogenesis. ^

Diseño y desarrollo de un modelo booleano probabilístico de regulación genética en el simulador de colonias bacterianas GRO

Resulta interesante comprender como microorganismos sencillos como la bacteria Escherichia coli poseen mecanismos no tan simples para responder al entorno en el que está gestionada por complicadas redes de regulación formadas por genes y proteínas, donde cada elemento de la red genética debe tomar parte en armonía, en el momento justo y la cantidad adecuada para dar lugar a la respuesta celular apropiada. La biología sintética es un nuevo área de la biología y la tecnología que fusiona la biolog ía molecular, la ingeniería genética y las herramientas computacionales, para crear sistemas biológicos con funcionalidades novedosas. Los sistemas creados sintéticamente son ya una realidad, y cada vez se acumulan más trabajos alrededor del mundo que muestran su factibilidad. En este campo no solo se hacen pequeñas modificaciones en la información genética, sino que también se diseñan, manipulan e introducen circuitos genéticos a los organismos. Actualmente, se hace un gran esfuerzo para construir circuitos genéticos formados por numerosos genes y caracterizar la interacción de los mismos con otras moléculas, su regulaci ón, expresión y funcionalidad en diferentes organismos. La mayoría de los proyectos de biología sintética que se han desarrollado hasta ahora, se basan en el conocimiento actual del funcionamiento de los organismos vivos. Sin embargo, la información es numerosa y creciente, por lo que se requiere de herramientas computacionales y matem áticas para integrar y hacer manejable esta gran cantidad de información. El simulador de colonias bacterianas GRO posee la capacidad de representar las dinámicas más simples del comportamiento celular, tales como crecimiento, división y comunicación intercelular mediante conjugación, pero carece de la capacidad de simular el comportamiento de la colonia en presencia de un circuito genético. Para ello, se ha creado un nuevo módulo de regulación genética que maneja las interaciones entre genes y proteínas de cada célula ejecutando respuestas celulares específicas. Dado que en la mayoría de los experimentos intervienen colonias del orden de 105 individuos, es necesario un módulo de regulación genética simplificado que permita representar de la forma más precisa posible este proceso en colonias de tales magnitudes. El módulo genético integrado en GRO se basa en una red booleana, en la que un gen puede transitar entre dos estados, on (expresado) o off (reprimido), y cuya transición viene dada por una serie de reglas lógicas.---ABSTRACT---It is interesting to understand how simple organisms such as Escherichia coli do not have simple mechanisms to respond to the environment in which they find themselves. This response is managed by complicated regulatory networks formed by genes and proteins, where each element of the genetic network should take part in harmony, at the right time and with the right amount to give rise to the appropriate cellular response. Synthetic biology is a new area of biology and technology that combines molecular biology, genetic engineering and computational tools to create biological systems with novel features. The synthetically created systems are already a reality, and increasingly accumulate work around the world showing their feasibility. In this field not only minor changes are made in the genetic information but also genetic circuits designed, manipulated and introduced into the organisms. Currently, it takes great effort to build genetic circuits formed by numerous genes and characterize their interaction with other molecules, their regulation, their expression and their function in different organisms. Most synthetic biology projects that have been developed so far are based on the current knowledge of the functioning of living organisms. However, there is a lot of information and it keeps accumulating, so it requires computational and mathematical tools to integrate and manage this wealth of information. The bacterial colonies simulator, GRO, has the ability to represent the simplest dynamics of cell behavior, such as growth, division and intercellular communication by conjugation, but lacks the ability to simulate the behavior of the colony in the presence of a genetic circuit. To this end, a new genetic regulation module that handles interactions between genes and proteins for each cell running specific cellular responses has been created. Since most experiments involve colonies of about 105 individuals, a simplified genetic module which represent cell dynamics as accurately and simply as possible is needed. The integrated genetic GRO module is based on a Boolean network, in which a gene can be in either of two states, on (expressed) or off (repressed), and whose transition is given by a set of logical rules.

The Molecular Biology Database Collection: an updated compilation of biological database resources

The Molecular Biology Database Collection is an online resource listing key databases of value to the biological community. This Collection is intended to bring fellow scientists’ attention to high-quality databases that are available throughout the world, rather than just be a lengthy listing of all available databases. As such, this up-to-date listing is intended to serve as the initial point from which to find specialized databases that may be of use in biological research. The databases included in this Collection provide new value to the underlying data by virtue of curation, new data connections or other innovative approaches. Short, searchable summaries of each of the databases included in the Collection are available through the Nucleic Acids Research Web site, at http://www.nar.oupjournals.org.

The spinal biology in humans and animals of pain states generated by persistent small afferent input

Behavioral models indicate that persistent small afferent input, as generated by tissue injury, results in a hyperalgesia at the site of injury and a tactile allodynia in areas adjacent to the injury site. Hyperalgesia reflects a sensitization of the peripheral terminal and a central facilitation evoked by the persistent small afferent input. The allodynia reflects a central sensitization. The spinal pharmacology of these pain states has been defined in the unanesthetized rat prepared with spinal catheters for injection and dialysis. After tissue injury, excitatory transmitters (e.g., glutamate and substance P) acting though N-methyl-d-aspartate (NMDA) and neurokinin 1 receptors initiate a cascade that evokes release of (i) NO, (ii) cyclooxygenase products, and (iii) activation of several kinases. Spinal dialysis show amino acid and prostanoid release after cutaneous injury. Spinal neurokinin 1, NMDA, and non-NMDA receptors enhance spinal prostaglandin E2 release. Spinal prostaglandins facilitate release of spinal amino acids and peptides. Activation by intrathecal injection of receptors on spinal C fiber terminals (μ,/∂ opiate, α2 adrenergic, neuropeptide Y) prevents release of primary afferent peptides and spinal amino acids and blocks acute and facilitated pain states. Conversely, consistent with their role in facilitated processing, NMDA, cyclooxygenase 2, and NO synthase inhibitors act to diminish only hyperalgesia. Importantly, spinal delivery of several of these agents diminishes human injury pain states. This efficacy emphasizes (i) the role of facilitated states in humans, (ii) shows the importance of spinal systems in human pain processing, and (iii) indicates that these preclinical mechanisms reflect processes that regulate the human pain experience.

Probing the pore region of recombinant N-methyl-D-aspartate channels using external and internal magnesium block.

Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations of residues five to seven positions downstream of the Q/R/N site do not influence the external Mg2+ block, but clearly influence the block by internal Mg2+. These data add support to the hypothesis that there are two separate binding sites for external and internal Mg2+ block. They also indicate that the C-terminal end of TMII contributes to the inner vestibule of the pore of NMDA channels and thus provide additional evidence that TMII forms a loop that reemerges toward the cytoplasmic side of the membrane.

Molecular biology of retinal ganglion cells.

Retinal ganglion cells are the output neurons that encode and transmit information from the eye to the brain. Their diverse physiologic and anatomic properties have been intensively studied and appear to account well for a number of psychophysical phenomena such as lateral inhibition and chromatic opponency. In this paper, we summarize our current view of retinal ganglion cell properties and pose a number of questions regarding underlying molecular mechanisms. As an example of one approach to understanding molecular mechanisms, we describe recent work on several POU domain transcription factors that are expressed in subsets of retinal ganglion cells and that appear to be involved in ganglion cell development.

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

Molecular biology of bacterial viruses /

Bibliography: p. 429-465.