Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

The Role of Structural Elements in Dimerization of RNA Fragments in Avian Retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop–loop interaction of two hairpins, or kissing hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

Imaging of left main trauma during diagnostic cardiac catheterization with intravascular ultrasound

In this case report the occurrence of a catheter-induced coronary artery dissection is described. In our patient, angiography showed a mushroom-shaped exudate above the left main coronary artery. Intravascular ultrasound revealed a circular dissection with a huge false lumen connected to the true lumen by a small intimal tear. A brief review of the literature on catheter-induced coronary dissection is included. We believe that this case report provides a good illustration of the need for careful reviewing of indications for angiography. Although procedural risks are low, angiography remains an invasive diagnostic test with the potential to cause severe complications.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

Melting of model HIV-1 stem-loop 1 RNA dimers monitored by 2-aminopurine fluorescence

Viral maturation of HIV-1 involves refolding of its genomic RNA, which is believed to include a rearrangement of the SL1 stem-loop from a metastable conformation called kissing loop dimer (KD) to a stable one termed extended dimer (ED). To investigate this rearrangement in vitro we have studied the thermal melting of the RNA dimers formed by slightly modified 23-nucleotide SL1 RNA of HIV-1 Mal. Local structural changes in the RNA dimers during the melting were monitored by changes in the fluorescence of 2-aminopurine (2AP) incorporated in predetermined positions of RNA. We have shown that the stem regions of both preformed KD and ED melt in the temperature interval from 75 ° C to 90 ° C. Kissing loop interface of the KD RNA is found to be disrupted at lower temperatures from 20 ° C to 55 ° C, at which the stem regions remain intact. Conversion of the preformed KD to ED overcoming the kinetic barrier occurs between 55 ° C and 65 ° C. The melting of "loop-loop" regions in both preformed and newly formed EDs takes place around 70 ° C. Our finding that thermoinduced KD-to-ED conversion is preceded by transient dissociation of loop-loop interface disagrees with a common idea of strand exchange without disruption of loop-loop-contact.     

Dimerization of short RNA fragments of avian retroviruses as revealed using 2-aminopurine fluorescence

Dimerization of retroviral RNA is known in detail for several groups of viruses, especially the human immunodeficiency virus (HIV). The dimerization region seems to involve short RNA sequences directly responsible for the formation of dimers of two types, kissing loop-loop (KD) and linear (LD). The 5′-terminal sequences, where dimers are mostly formed, substantially differ between avian retroviruses and HIV, while the mechanisms of their RNA dimerization are the same. RNA dimerization was studied using the adenine analog 2-aminopurine (2-AP), which was incorporated into the loop sequence of short fragments of the avian leucosis virus (ALV) RNA. A temperature dependence of 2-AP fluorescence was used to study the KD formation under various conditions. Magnesium ions and the aminoglycoside antibiotic paromomycin were tested for the effect on KD stability. The highest KD stability was observed at >1 mM Mg²⁺ and >2.5 μM paromomycin.     

Why Termination tRNAs Have Not Been Found? Because They Are Hidden in the Large Ribosomal RNA (A Hypothesis)

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such recognition to make it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.     

Experimental Evidence for Slipped Loop DNA,a Novel Folding Type for Polynucleotide Chain

Abstract

DNA regions with short direct repeats (5–7bp) with a spacer in between, when under super-helical stress, are known to become susceptible to single-strand specific nuclease S1. This is in accord with formation of two shifted loops protruding from the opposite chains. Such type of folding could have been additionally stabilized by base pairing between the complementary parts of the loops that explains existence of the protected from SI moieties of the loops. To test this possibility we designed and synthesized an oligonucleotide of 56 bases, so that it forms a hairpin with a stem which fails to acquire a traditional helix due to a special sequence but may favor the formation of the proposed Slipped Loop Structure (SLS). The oligonucleotide folding was studied by a chemical modification method at one nucleotide level resolution. Three zones, protected from the used probes were found: the one that forms the stem, and the others that are located within the two by-loops in those moieties which have the base pairing potential. Proceeding from the data obtained and stereochemical analysis a 3-D scheme for the SLS form of DNA is suggested.     

Thermodynamics of B-Z transition of poly[d(G-C)] in a water-ethanol solution. The "tie calorimetry"

Poly[d(G-C)] in a 55% ethanol solution undergoes a transition from the Z form to the B form when the temperature is increased from 20 degrees to 50 degrees C. The enthalpy of the transition, delta HBA = -1.4 kcal/mol, has been determined with a "tie" polyamine which stabilizes the Z conformation. This value has been shown to be practically independent of ionic strength within the range of 5 X 10(-4) M - 2 X 10(-3) M NaCl.