Definitions and nomenclature of nucleic acid structure parameters

At an EMBO Workshop on DNA Curvature and Bending, held at Churchill College, Cambridge, on 10-15 September 1988, two sessions were scheduled on definitions of parameters used to describe the geometry of nucleic acid chains and helices, and a common nomenclature for these parameters. The most widely used library of helix analysis programs, HELIB (Fratini et al., 1982; Dickerson, 1985) suffers from the fact that the translations and rotations as defined are not fully independent and depend to a certain extent upon the choice of overall helix axis. Several research groups have been engaged independently in developing alternative programs for the geometrical analysis of polynucleotide chains, but with different definitions of quantities calculated and with widely different nomenclature even when the same parameter was involved.

Studies on 4S and 5S RNA of HeLa cells

SECTION I

Section I is concerned with a partial sequence analysis conducted on 5S RNA from HeLa cells. Analysis of the oligonucleotide pattern after pancreatic ribonuclease digestion of a highly-purified preparation of 5S RNA gave results which were in general agreement with those published for KB cells, both with respect to the identity and the frequency of the partial sequences. However, the presence of a trinucleotide not found in the KB 5S pattern, together with the reproducibly much lower than expected molar yield of the larger oligonucleotides strongly suggested the occurrence of alternate sequences at various sites in the 5S molecules of human cells. The presence of ppGp and pppGp at the 5'-terminus of HeLa 5S RNA was clearly demonstrated. The implications of this finding with regard to the origin of 5S RNA are discussed.

SECTION II

In Section II the proportion of the HeLa cell genome complementary to tRNA was investigated by using RNA- DNA hybridization. The value for saturation of the HeLa DNA by tRNA was found to be 1.1 x 10^-5, which corresponds to about 4900 sites for tRNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized tRNA revealed significant differences from the nucleotide composition of the input tRNA, with the purine to pyrimidine ratio indicating, however, that these differences were not produced by excessive RNase attack of the hybrid. The size of the hybridized tRNA was only moderately smaller than that of the input RNA; the average S value in formaldehyde was 2.7 (corresponding to a length of about 65 nucleotides), suggesting that a relatively small portion near the ends of the hybridized 4S chains had been removed by RNase.

SECTION III

The proportion of the HeLa cell genome complementary to 5S RNA was investigated by using RNA-DNA hybridization. The value for saturation of the HeLa DNA by 5S RNA was found to be 2.3 x 10^-5, which corresponds to about 7,000 sites for 5S RNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized 5S RNA revealed no significant difference from the nucleotide composition of the input RNA. At the RNA to DNA input ratio of 1:1000, the average S value in formaldehyde of the hybridized 5S RNA corresponded to a polynucleotide chain about two-thirds the size of the input RNA.

Studies on rabbit reticulocyte ribosomes and polyribosomes and their relation to hemoglobin synthesis

This dissertation is divided into three parts.

The first section is concerned with protein synthesis in cellfree systems from reticulocytes. The sub-cellular reticulocyte fractions, reagents, etc. have been examined for the presence of traces of ribonuclease, using. an assay based upon the loss of infectivity of RNA fran bacteriophage MS2. This assay is sensitive to 5 x 10^-7 γ RNase/ml. In addition, the loss of synthetic capacity of an 80S ribosome on dissociation has been studied, and can be attributed to loss of messenger RNA when the monomer is separated into subunits. The presence of ribonuclease has been shown to be a major cause of polyribosome disintegration during cell-free protein synthesis.

The second section concerns the changes in ribosomes and polyribosomes which occur during the maturation of a reticulocyte into an erythrocyte. With increasing age, the cells lose a large proportion of the ribonucleoprotein, but the percentage of ribosomes present as polyribosomes is only slightly altered. The loss of hemoglobin synthesis on maturation is probably due to both the loss of total ribosomes and to the lessened specific activity of the polyribosomes.

The third section contains analytical ultracentrifugation data on 80S ribosomes, polyribosomes, and ribosomal RNA from reticulocytes. The 60s and 40s subunits, obtained by dissociation of the 80s particle with inorganic pyrophosphate, were also studied. The RNA from reticulocyte ribosomes has been examined under a variety of denaturing conditions, including dimethyl sulfoxide treatment, formaldehyde reaction and thermal denaturation. From these studies we can conclude that the 28S and 16S RNA's are single polynucleotide chains and are not made up of smaller RNA subunits hydrogen-bonded together.

Novel DNA sensor for guanine content.

The bifunctional complex [Ru(TAP)(2)POQ-Nmet](2+), 1, formed with a [Ru(TAP)(2)Phen](2+) metallic unit linked to a quinoline moiety, and [Ru(TAP)(2)Phen](2+), 2, as reference, have been tested as photoprobes of DNA. Interestingly, 1 exhibits an emission enhancement of a factor of 16-17 upon binding to calf thymus DNA. Moreover, this emission is modulated by the nucleic base content of the polynucleotide. It varies by almost an order of magnitude from a polynucleotide containing 100% of G-C to a guanine-free nucleic acid where the excited-state lifetime reaches about 2 micros. The origins of these interesting properties are analyzed by comparing 1 with reference 2 in the presence of different polynucleotides.

Interplay of Exoribonucleases, Hfq and Small RNAs Structural Determinants in the Control of Gene Expression

Dissertation presented to obtain the Ph.D degree in Biology

Le motif d’empaquetage le long du sillon: une nouvelle entité structurale récurrente dans les ARN ribosomiques

La plupart des molécules d’ARN doivent se replier en structure tertiaire complexe afin d’accomplir leurs fonctions biologiques. Cependant, les déterminants d’une chaîne de polynucléotides qui sont nécessaires à son repliement et à ses interactions avec d’autres éléments sont essentiellement inconnus. L’établissement des relations structure-fonction dans les grandes molécules d’ARN passe inévitablement par l’analyse de chaque élément de leur structure de façon individuelle et en contexte avec d’autres éléments. À l’image d’une construction d’immeuble, une structure d’ARN est composée d’unités répétitives assemblées de façon spécifique. Les motifs récurrents d’ARN sont des arrangements de nucléotides retrouvés à différents endroits d’une structure tertiaire et possèdent des conformations identiques ou très similaires. Ainsi, une des étapes nécessaires à la compréhension de la structure et de la fonction des molécules d’ARN consiste à identifier de façon systématique les motifs récurrents et d’en effectuer une analyse comparative afin d’établir la séquence consensus. L’analyse de tous les cas d’empaquetage de doubles hélices dans la structure du ribosome a permis l’identification d’un nouvel arrangement nommé motif d’empaquetage le long du sillon (AGPM) (along-groove packing motif). Ce motif est retrouvé à 14 endroits dans la structure du ribosome de même qu’entre l’ARN ribosomique 23S et les molécules d’ARN de transfert liées aux sites ribosomaux P et E. Le motif se forme par l’empaquetage de deux doubles hélices via leur sillon mineur. Le squelette sucre-phosphate d’une hélice voyage le long du sillon mineur de l’autre hélice et vice versa. Dans chacune des hélices, la région de contact comprend quatre paires de bases. L’empaquetage le plus serré est retrouvé au centre de l’arrangement où l’on retrouve souvent une paire de bases GU dans une hélice interagissant avec une paire de bases Watson-Crick (WC) dans l’autre hélice. Même si la présence des paires de bases centrales GU versus WC au centre du motif augmente sa stabilité, d’autres alternatives existent pour différents représentants du motif. L’analyse comparative de trois librairies combinatoires de gènes d’AGPM, où les paires de bases centrales ont été variées de manière complètement aléatoire, a montré que le contexte structural influence l’étendue de la variabilité des séquences de nucléotides formant les paires de bases centrales. Le fait que l’identité des paires de bases centrales puisse varier suggérait la présence d’autres déterminants responsables au maintien de l’intégrité du motif. L’analyse de tous les contacts entre les hélices a révélé qu’en dehors du centre du motif, les interactions entre les squelettes sucre-phosphate s’effectuent via trois contacts ribose-ribose. Pour chacun de ces contacts, les riboses des nucléotides qui interagissent ensemble doivent adopter des positions particulières afin d’éviter qu’ils entrent en collision. Nous montrons que la position de ces riboses est modulée par des conformations spécifiques des paires de bases auxquelles ils appartiennent. Finalement, un autre motif récurrent identifié à l’intérieur même de la structure de trois cas d’AGPM a été nommé « adenosine-wedge ». Son analyse a révélé que ce dernier est lui-même composé d’un autre arrangement, nommé motif triangle-NAG (NAG-triangle). Nous montrons que le motif « adenosine-wedge » représente un arrangement complexe d’ARN composé de quatre éléments répétitifs, c’est-à-dire des motifs AGPM, « hook-turn », « A-minor » et triangle-NAG. Ceci illustre clairement l’arrangement hiérarchique des structures d’ARN qui peut aussi être observé pour d’autres motifs d’ARN. D’un point de vue plus global, mes résultats enrichissent notre compréhension générale du rôle des différents types d’interactions tertiaires dans la formation des molécules d’ARN complexes.

Structural aspects of the ribosome evolution and function

Les résultats ont été obtenus avec le logiciel "Insight-2" de Accelris (San Diego, CA)

Method for the production of cotton somatic embryos. United States Patent Application

The present invention provides Inter alia, a method for the production of cotton somatic embryos comprising (a) isolating a totipotent stomatal cell-containing epidermal explant from leaf material excised from a cotton plant; and (b) culturing said explant in a basal medium which comprises an embryogenic callus-inducing quantity of an auxin and a cytokinin under an embryogenic callus inducing intensity of light until embryogenic callus is formed; and (c) sub-culturing said embryogenic callus onto a somatic embryo differentiation media to produce said somatic embryos. Plants may be regenerated from the somatic embryos and in a particular embodiment of the invention said totipotent stomatal cell is transformed, prior to the inducement of embryogenic callus, with a polynucleotide that provides for a desired agronomic trait.

In situ evaluation of anticancer drug methotrexate-DNA interaction using a DNA-electrochemical biosensor and AFM characterization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

THE B-FORM TO Z-FORM TRANSITION OF POLY(DG-M(5)DC) IS SENSITIVE TO NEUTRAL SOLUTES THROUGH AN OSMOTIC-STRESS

Several neutral solutes, ranging in size from methanol to a tetrasaccharide, stachyose, are shown to stabilize the left-handed Z form of the methylated polynucleotide poly(dG-m(5)dC). The action of these solutes is consistent with an osmotic stress, that is, with their effect on water chemical potentials coupled to a difference in the number of-associated water molecules between the B and Z conformations. The apparent difference in hydration between the two forms is, however, dependent on the particular solute used to probe the reaction. The effect of solutes is not consistent either with a direct binding of solute or with an indirect effect on electrostatics or ion binding through changes in the solution dielectric constant. The interplay of NaCl and neutral solute in modulating the B-Z transition suggests that salt also could be stabilizing the Z form through an osmotic stress.

Identification of a nucleotide binding site in HIV-1 integrase

HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3′-azido-3′-deoxythymidine (AZT) analog 3′,5-diazido-2′,3′-dideoxyuridine 5′-monophosphate (5N3-AZTMP) and proteolytic mapping identified the amino acid 153–167 region of integrase as the site of photocrosslinking. Docking of 5N3-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.

Defensive extrusive ectosymbionts of Euplotidium (Ciliophora) that contain microtubule-like structures are bacteria related to Verrucomicrobia

Epixenosomes, ectosymbionts on hypotrich ciliates (genus Euplotidium) defend their host against the ciliate predator Litonotus lamella. Although here only Euplotidium itoi and Euplotidium arenarium from tide pools along a rocky shore near Leghorn (Ligurian sea) were studied in detail, these epibionts are certainly present on specimens of E. itoi and on other Euplotidium species in similar north coastal habitats. The complex life history of epixenosomes has two main stages. In stage I, cells with typical prokaryotic structure divide by binary fission. Stage II cells show complex organization with different cytoplasmic compartments where an extrusive apparatus within a proteinaceous matrix, although not membrane-bounded, differs from the remaining cytoplasm. The ejection process is involved in defense; extrusive apparatus is surrounded by a basket consisting of bundles of tubules. These tubules, 22 ± 3 nm in diameter, delimited by a wall made up of globular structures, are sensitive to inhibitor of tubulin polymerization (nocodazole/4°C temperature) and react positively with different antitubulin antibodies, two of which are monoclonal. The prokaryotic vs. eukaryotic nature of epixenosomes was resolved by comparative sequence analysis of amplified small subunit rRNA genes and in situ hybridization with fluorescently labeled rRNA-targeted polynucleotide probes. These unique ectosymbionts are phylogenetically related to Verrucomicrobia. Epixenosomes represent marine symbionts in this recently discovered division of the Bacteria.

Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection

Translation inhibitors such as chloramphenicol in prokaryotes or cycloheximide in eukaryotes stabilize many or most cellular mRNAs. In Escherichia coli, this stabilization is ascribed generally to the shielding of mRNAs by stalled ribosomes. To evaluate this interpretation, we examine here how inhibitors affect the stabilities of two untranslated RNAs, i.e., an engineered lacZ mRNA lacking a ribosome binding site, and a small regulatory RNA, RNAI. Whether they block elongation or initiation, all translation inhibitors tested stabilized these RNAs, indicating that stabilization does not necessarily reflect changes in packing or activity of translating ribosomes. Moreover, both the initial RNase E-dependent cleavage of RNAI and lacZ mRNA and the subsequent attack of RNAI by polynucleotide phosphorylase and poly(A)-polymerase were slowed. Among various possible mechanisms for this stabilization, we discuss in particular a passive model. When translation is blocked, rRNA synthesis is known to increase severalfold and rRNA becomes unstable. Meanwhile, the pools of RNase E and polynucleotide phosphorylase, which, in growing cells, are limited because these RNases autoregulate their own synthesis, cannot expand. The processing/degradation of newly synthesized rRNA would then titrate these RNases, causing bulk mRNA stabilization.

Radiation target analysis of RNA.

Ribozymes are polynucleotide molecules with intrinsic catalytic activity, capable of cleaving nucleic acid substrates. Large RNA molecules were synthesized containing a hammerhead ribozyme moiety of 52 nucleotides linked to an inactive leader sequence, for total lengths of either 262 or 1226 nucleotides. Frozen RNAs were irradiated with high energy electrons. Surviving ribozyme activity was determined using the ability of the irradiated ribozymes to cleave a labeled substrate. The amount of intact RNA remaining was determined from the same irradiated samples by scanning the RNA band following denaturing gel electrophoresis. Radiation target analyses of these data revealed a structural target size of 80 kDa and a ribozyme activity target size of 15 kDa for the smaller ribozyme, and 319 kDa and 16 kDa, respectively, for the larger ribozyme. The disparity in target size for activity versus structure indicates that, in contrast to proteins, there is no spread of radiation damage far from the primary site of ionization in RNA molecules. The smaller target size for activity indicates that only primary ionizations occurring in the specific active region are effective. This is similar to the case for oligosaccharides. We concluded that the presence of the ribose sugar in the polymer chain restricts radiation damage to a small region and prevents major energy transfer throughout the molecule. Radiation target analysis should be a useful technique for evaluating local RNA:RNA and RNA:protein interactions in vitro.

Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.