Mechanistic studies of ribonucleic acid renaturation by a helix-destabilizing protein

The ability of a nucleic acid helix-destabilizing protein from calf thymus, UP1, to facilitate renaturation of yeast tRNALeu3 and Escherichia coli 5S RNA is shown to be a consequence of the protein's ability to bind stoichiometrically to single-stranded polynucleotide regions. A comparison of the inhibitory effect of different homopolymers on UP1-induced renaturation of tRNALeu3 does not indicate significant base specificity in UP1 binding, and a 3'-5' ribose phosphate polymer devoid of heterocyclic bases inhibits as well as the homopolynucleotides. These inhibition studies also show that UP1 requires polynucleotide segments of at least three phosphate residues to bind. Mg2+ (which is required for the stabilization of native tRNALeu3) dissociates complexes of UP1 with inactive tRNA, and since the RNAs in those complexes lack a substantial amount of secondary structure, it can upon dissociation readily refold into the native structure. A semiquantitative treatment of UP1-RNA interaction is developed that suggests that only a small number (approximately six) of protein molecules are bound to tRNALeu3 in the complex while analysis of the inhibition studies suggests that these UP1 molecules are not bound in a highly cooperative manner.     

Vegetation change in a man‐made salt marsh affected by a reduction in both grazing and drainage

Abstract. In order to restore natural salt marsh in a 460‐ha nature reserve established in man‐made salt marsh in the Dollard estuary, The Netherlands, the artificial drainage system was neglected and cattle grazing reduced. Vegetation changes were traced through two vegetation surveys and monitoring of permanent plots over 15 yr after the management had been changed. Exclosure experiments were started to distinguish grazing effects from effects of increased soil waterlogging caused by the neglect of the drainage system. Both vegetation surveys and permanent plots demonstrated a dichotomy in vegetation succession. The incidence of secondary pioneer vegetation dominated by Salicornia spp. and Suaeda maritima increased from 0 to 20%, whereas the late‐successional (Phragmites australis) vegetation from 10 to 15%. Grazing intensity decreased towards the sea. The grazed area contracted landward, which allowed vegetation dominated by tall species to increase seaward. Grazing and increased waterlogging interacted in several ways. The impact of trampling increased, and in the intensively grazed parts soil salinity increased. This can probably be explained by low vegetation cover in spring. Framework Ordination, an indirect‐gradient‐analysis technique, was used to infer the importance of environmental factors in influencing changes in species composition. Many changes were positively or negatively correlated with soil aeration and soil salinity, whereas elevation was of minor importance. Grazing accounted for only a few changes in species frequency. Changes in permanent plots were greater during the first than during the second half of the study period. In exclosures that were installed halfway through the study period, there was a relatively rapid recovery of previously dominant species that had decreased during the first half of the study period. Species richness per unit area in the reserve increased. At the seaward side of the marsh, the altered management allowed succession to proceed leading to establishment of stands of Phragmites australis, whereas on the landward side, the combination of moderate grazing with neglect of the drainage system appeared an effective measure in maintaining habitats for a wider range of halophytic species.     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".     

The effect of fluctuations in tidal inundation frequency on a salt-marsh vegetation

Over a period of 15 years recordings were made of the species cover in permanent plots on the salt marsh of one of the West Frisian Islands, Schiermonnikoog (The Netherlands). Correlations between annual changes in the cover of the major species, and fluctuations in the monthly frequency of inundation by seawater were studied. First, a spectral analysis was carried out on the inundation frequency data to look for predictable patterns. Subsequently, fluctuations were defined as deviations from these predictable patterns. In a repeated multiple regression model, the effects of the season in which the fluctuations occurred, and the elevational position of the plots on the salt marsh were studied as factors influencing the correlation patterns. The behaviour of various species is discussed in relation to their seed bank characteristics and their salt tolerance.     

Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.     

Effects of restoration measures on plant communities of wet heathland ecosystems

Question: Which are the success and failure of restoration measures, particularly sod‐cutting and hydrological measures, in small wetlands on mineral soils in The Netherlands. Location: Twente, in the eastern part of The Netherlands. Methods: Success or failure of restoration measures has been assessed by comparing experimental plots with that in reference plots for (1) species richness, (2) re‐establishment of endangered species and (3) species composition, including life forms and Red List species. In total 119 samples were taken in 42 permanent plots in fen meadows, small‐sedge marshes, wet heathlands, and soft‐water pools. Topsoil samples were analysed for pH, Ca, Mg, Na, K and Cation Exchange Capacity. Gradient analysis was carried out by means of Canonical Correspondence Analysis. Results: Sod‐cutting, in combination with re‐wetting measures (infilling of ditches and drains), led to restoration of communities of soft‐water pools and small sedge marshes within five years. This rapid recovery is probably related to the presence of persistent seed banks of the component species of these communities. Complete restoration of fen meadows took longer (10–15 yr). Apparently, many species of fen meadows have short‐lived seed banks. Sod‐cutting of a degraded wet heathland and a soft‐water pool was only successful temporarily, probably as a consequence of low water tables. Conclusions: To prevent depletion of (persistent) soil seed banks, sod‐cutting in nutrient‐poor wetlands is not recommended in areas where the groundwater regime and the base status of the soil can not be restored to levels required by plant communities of wet heathlands.     

Rabbit grazing and species diversity in a dune area

Summary The relation between density of rabbit populations and plant species diversity is discussed. In the dunes of the Dutch Frisian Island of Schiermonnikoog, the rabbit grazing pressure has been quantified on the basis of traces of recent grazing activity. Moderate grazing turned out to bring about maximal species richness. Current changes in grazing pressure (either decrease or increase) can result in plant species diversity that is lower than in other places with equal but stabilized grazing pressure.Nomenclature follows Heukels-van Ooststroom, Flora van Nederland, 18e druk, 1975.The work has been done by Mrs Ada Zeevalking-van Y peren and Mr Hein Zeevalking as part of their study for a doctoral degree.     

Effect of the 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole residue on the stability of DNA duplexes and triplexes.

3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.     

Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei

A methodology has been developed for binding oligodeoxyribonucleotide ’third strands’ to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique multicopy target sequence in human chromosome 17 α-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin. Received: 1 October 1998; in revised form: 22 December 1998 / Accepted: 12 February 1999