3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.

Variable effects of the conserved RNA hairpin element upon 3' end processing of histone pre-mRNA in vitro.

We have studied the requirements for efficient histone-specific RNA 3' processing in nuclear extract from mammalian tissue culture cells. Processing is strongly impaired by mutations in the pre-mRNA spacer element that reduce the base-pairing potential with U7 RNA. Moreover, by exchanging the hairpin and spacer elements of two differently processed H4 genes, we find that this difference is exclusively due to the spacer element. Finally, processing is inhibited by the addition of competitor RNAs, if these contain a wild-type spacer sequence, but not if their spacer element is mutated. Conversely, the importance of the hairpin for histone RNA 3' processing is highly variable: A hairpin mutant of the H4-12 gene is processed with almost wild-type efficiency in extract from K21 mouse mastocytoma cells but is strongly affected in HeLa cell extract, whereas an identical hairpin mutant of the H4-1 gene is affected in both extracts. The hairpin defect of H4-12-specific RNA in HeLa cells can be overcome by a compensatory mutation that increases the base complementarity to U7 snRNA. Very similar results were also obtained in RNA competition experiments: processing of H4-12-specific RNA can be competed by RNA carrying a wild-type hairpin element in extract from HeLa, but not K21 cells, whereas processing of H4-1-specific RNA can be competed in both extracts. With two additional histone genes we obtained results that were in one case intermediate and in the other similar to those obtained with H4-1. These results suggest that hairpin binding factor(s) can cooperatively support the ability of U7 snRNPs to form an active processing complex, but is(are) not directly involved in the processing mechanism.

Histone-specific RNA 3' processing in nuclear extracts from mammalian cells.

The mature 3' ends of histone mRNAs are formed by endonucleolytic cleavage of longer precursor transcripts. This process occurs in the nucleus and can be regarded as the equivalent of the polyadenylation reaction involved in 3′-end-generation of all other mRNAs. A sea urchin H3 gene that failed to be properly processed in the Xenopus oocyte system proved particularly useful, because it allowed the identification of a processing component from sea urchins by a complementation assay. Nuclear extracts prepared from cells under various growth conditions have helped to reveal proliferation-dependent changes in the efficiency of histone RNA 3′ processing. RNA substrates for in vitro processing are best prepared by runoff transcription of specific DNA templates with bacterial or phage RNA polymerases. For this purpose, a restriction fragment containing the 3′-terminal region of a histone gene and including the conserved palindrome and spacer motifs is cloned into a polylinker sequence downstream of a strong promoter.

Effect of an intervertebral disk spacer on stiffness after monocortical screw/polymethylmethacrylate fixation in simulated and cadaveric canine cervical vertebral columns.

OBJECTIVE To determine the biomechanical effect of an intervertebral spacer on construct stiffness in a PVC model and cadaveric canine cervical vertebral columns stabilized with monocortical screws/polymethylmethacrylate (PMMA). STUDY DESIGN Biomechanical study. SAMPLE POPULATION PVC pipe; cadaveric canine vertebral columns. METHODS PVC model-PVC pipe was used to create a gap model mimicking vertebral endplate orientation and disk space width of large-breed canine cervical vertebrae; 6 models had a 4-mm gap with no spacer (PVC group 1); 6 had a PVC pipe ring spacer filling the gap (PCV group 2). Animals-large breed cadaveric canine cervical vertebral columns (C2-C7) from skeletally mature dogs without (cadaveric group 1, n = 6, historical data) and with an intervertebral disk spacer (cadaveric group 2, n = 6) were used. All PVC models and cadaver specimens were instrumented with monocortical titanium screws/PMMA. Stiffness of the 2 PVC groups was compared in extension, flexion, and lateral bending using non-destructive 4-point bend testing. Stiffness testing in all 3 directions was performed of the unaltered C4-C5 vertebral motion unit in cadaveric spines and repeated after placement of an intervertebral cortical allograft ring and instrumentation. Data were compared using a linear mixed model approach that also incorporated data from previously tested spines with the same screw/PMMA construct but without disk spacer (cadaveric group 1). RESULTS Addition of a spacer increased construct stiffness in both the PVC model (P < .001) and cadaveric vertebral columns (P < .001) compared to fixation without a spacer. CONCLUSIONS Addition of an intervertebral spacer significantly increased construct stiffness of monocortical screw/PMMA fixation.

Structure Variation And Dynamics of the Nuclear Ribosomal Intergenic Spacer Region in Key Gibberella Fujikuroi Complex Species

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium , a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai , these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events.

Technical note: Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) to assess bacterial diversity in the rumen of sheep

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Decolorization and mineralization of an azo reactive dye using loaded nano-photocatalysts on spacer fabric: kinetic study and operational factors

In this study, the photocatalytic decolorization and mineralization of Remazol Black B (RBB), an azo reactive dye, in aqueous solutions was investigated using UV/H2O2/ZnO, UV/H2O2/TiO2 and UV/H2O2/ZnO:TiO2 systems. ZnO and TiO2 nanoparticles were loaded on 3-dimensional polyethylene terephthalate fabrics (spacer fabrics). Morphology of the spacer fabrics and the presence of the nanoparticles were studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), respectively. Furthermore, the effects of key operational parameters on the efficiency of the decolorization were investigated. These parameters included initial pH value, initial hydrogen peroxide concentration, initial dye concentration, the loaded nanoparticle ratio and the presence of anions (sulfate, chloride and bicarbonate). Zero-, first- and second-order reaction kinetics were evaluated. Complete decolorization and high efficient mineralization with 90% total organic carbon (TOC) reduction were achieved at 120min treatment in the case of ZnO:TiO2 under optimum condition. The results proved that the novel heterogeneous photocatalytic process is capable of decolorizing and mineralizing azo reactive dyes in textile wastewater.

A new low temperature synthesis route of fraipontite (Zn,Al)3(Si,Al)2O5(OH)4

A low temperature synthesis method based on the decomposition of urea at 90°C in water has been developed to synthesise fraipontite. This material is characterised by a basal reflection 001 at 7.44 Å. The trioctahedral nature of the fraipontite is shown by the presence of a 06l band around 1.54 Å, while a minor band around 1.51 Å indicates some cation ordering between Zn and Al resulting in Al-rich areas with a more dioctahedral nature. TEM and IR indicate that no separate kaolinite phase is present. An increase in the Al content however, did result in the formation of some SiO2 in the form of quartz. Minor impurities of carbonate salts were observed during the synthesis caused by to the formation of CO32- during the decomposition of urea.