Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae. III. Nitrous acid.

Nitrous acid (NA) induced mutations efficiently in mitDNA, conferring resistance to erythromycin and weakly induces mit- mutations. In some strains of yeast it also enhanced rho- mutations. The frequencies of nuclear and mitochondrial mutations induced with NA are compared.     

Proximo-distal pattern regulation in deficient avian limb buds

Summary Deficient limb buds composed of prospective stylopod and autopod are able to regulate the missing intercalary zeugopod, the origin of which was investigated by heterospecific quail/chick recombinants. The associations of quail prospective autopod and chick prospective stylopod failed to regulate. The reverse combination of chick prospective autopod grafted onto a quail prospective stylopod gave rise to a three-segmented limb. In 13 out of 16 cases the regulated zeugopod was made up of both chick and quail cells. Chick cells were located predominantly along the postaxial half of the zeugopod, while the quail cells made up most of its preaxial half. In two cases, the intercalary zeugopod consisted exclusively of chick cells originating from the tip and in one case of quail cells originating from the base.These results demonstrate that during the regulative processes, the prospective values of some of the original stylopodial and autopodial cells have been shifted along the proximo-distal axis, towards the expression of more distal as well as of more proximal structures.Heteropolar stylo-autopodial or zeugo-autopodial recombinants, in which the proximo-distal axis of the base was reversed with respect to that of the tip, were unable to regulate the pattern defects and thus revealed the importance of concordant p-d polarity for regulative processes to take place between abutted tissues.     

Plant nutrient changes in a semi-arid Mediterranean shrubland after fire

Abstract. Alkaline xeric soils from SE Spain weathered from kakiritized dolomitic rocks and subjected to recurrent fires, show phosphorus immobilization, low nutrient availability and high cation content, which might affect plant nutrition. Currently, Ulex shrubland, with a variety of dolomitophilous endemics, is colonizing eroded areas and replacing the original shrubland communities. Variation in nutrient content during post-fire regeneration was studied in six species which differ widely in their successional status. All species showed a high consumption of nutrients immediately after the fire. A pattern of decline in internal nutrient levels, especially P, with time since fire was detected in mid-to-late successional shrubs: Juniperus oxycedrus, Genista spartioides, and to a lesser extent in Rosmarinus officinalis, a shrub species of intermediate successional status. This pattern of decline may represent a progressive change towards a metabolism with a low turnover along the age gradient. Early successional shrub species behaved in a different way. Cistus clusii showed a nutrient-cumulative strategy, especially for P, across the fire-age gradient. Ulex rivasgodayanus maximized internal N concentrations due to its symbiotic activity with N₂-fixing bacteria. Cistus and Rosmarinus held high cation levels in their tissues. The nutritional characteristics of these shrub species are seen as advantageous adaptations to the specific soil properties and disturbance regime of the area.     

Electric field promotion of the bacteriorhodopsin BR570 to BR412 photoconversion in films of Halobacterium halobium purple membranes

The combined action of electric field (10⁵–10⁷ V · m^?1) and light (380–580 nm, 80 W · m^?2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR₄₁₂ intermediate accumulation in the normal photochromic cycle of BR₅₇₀ has been observed. The formation of the product BR₄₁₂ is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the field. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis.     

Succession of mycorrhizal fungi in stands of Pinus sylvestris in the Netherlands

The mycorrhizal mycoflora was investigated in 35 stands of Pinus sylvestris in three types of young (4-13 yr) and three of old (50-80 yr) stands in the Netherlands, differing in number of rotations and soil type. A plot of 1050 m² (30 m x 35 m) within each stand was searched for carpophores during the autumns of 1986 and 1987. 10 soil samples per plot were taken in October 1987 in order to assess the mycorrhizal status of the tree roots. The composition of mycorrhizal mycoflora in the different plots was subjected to TWINSPAN cluster analysis and Detrended Correspondence Analysis. Plot groupings generated by these analyses largely parallelled the stand types, indicating that each stand type has its own mycoflora. Differences in myco-floristic composition between stand types were parallelled by differences in the composition of green vegetation. The young stand types had 3.5–27 x more carpophores and 1.4–6.8 x more species than two of the old stand types One old stand type was intermediate. Considerable differences in species composition between the young stand types were observed. It is concluded that the succession of mycorrhizal fungi is not primarily influenced by ageing of the trees, but rather by changes in the soil. The results were compared with data on changes in the occurrence of fruiting species of mycorrhizal fungi in the Netherlands during this century. It appeared that species which have declined according to these data were more frequent in the young plots than in the old plots. However, these species are reported to be frequent in old stands of P. sylvestris in Estonia and Finland. It is argued that this difference is related to the high nitrogen deposition in the Netherlands.     

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Sequential interactions of silver–silica nanocomposite (Ag–SiO2NC) with cell wall,metabolism and genetic stability of Pseudomonas aeruginosa,a multiple antibiotic‐resistant bacterium

The study was carried out to understand the effect of silver–silica nanocomposite (Ag–SiO₂NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drug‐resistant bacterium. Bacterial sensitivity towards antibiotics and Ag–SiO₂NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag–SiO₂NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis, while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. Pseudomonas aeruginosa was found to be resistant to β‐lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μg ml^?1 concentration of Ag–SiO₂NC. The cell wall integrity reduced with increasing time and reached a plateau of 70% in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μg ml^?1 Ag–SiO₂NC, followed by DNA breakage. The study thus demonstrates that Ag–SiO₂NC invades the cytoplasm of the multiple drug‐resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability.

Significance and Impact of Study

Although the synthesis, structural characteristics and biofunction of silver nanoparticles are well understood, their application in antimicrobial therapy is still at its infancy as only a small number of microorganisms are tested to be sensitive to nanoparticles. A thorough knowledge of the mode of interaction of nanoparticles with bacteria at subcellular level is mandatory for any clinical application. The present study deals with the interactions of Ag–SiO2NC with the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, which would contribute substantially in strengthening the therapeutic applications of silver nanoparticles.