The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

Ciliary receptors associated with the mouth and pharynx of Acoela (Acoelomorpha): a comparative ultrastructural study

The ultrastructure and distribution of receptor cells near the mouth and (where present) the pharynx of Hofstenia miamia, Proporus bermudensis, Conaperta thela, and Convoluta convoluta (Acoela) were investigated by transmission electron microscopy and confocal laser scanning microscopy of specimens stained with a fluorescence marker for actin. Five types of monociliary receptors were identified: (1) non‐collared receptors with a single long and narrow ciliary rootlet; (2) non‐collared receptors with a wide main ciliary rootlet and a smaller posterior rootlet; (3) non‐collared receptors with a single wide and hollow ciliary rootlet with a granulated core; (4) Collar (?) receptors with obliquely radial filament bundles in the cell apex and with a single hollow ciliary rootlet composed of numerous strand‐like elements; and (5) Collar receptors lacking a striated rootlet but with a granular body (swallow's nest rootlet). While H. miamia bears the first two receptor types, P. bermudensis has receptors of type 1, 3 and 5, and Cona. thela and Conv. convoluta have receptors of type 3, 4 and 5. The density of receptors is generally highest at the anterior body tip, regardless of where the mouth is located. Most receptor types occur scattered over the whole body but type 2 receptors of H. miamia are restricted to the pharynx and mouth region. The lack of a common receptor type specific for the mouth and pharynx of the investigated species points to an independent origin of the pharynges in Hofsteniidae and in Proporidae and of the mouth tube in Convolutidae. Moreover, the homology of the so‐called collar receptors in Acoela with typical collar receptors in other invertebrates is questioned.     

A second type of protein methylation reaction in bacterial chemotaxis

CheZ is the product of one of six genes required for sensory processing in Escherichia coli and Salmonella typhimurium chemotaxis. This 24-kDa cytoplasmic protein is modified by a posttranslational methylation reaction. The modified residue has been identified by analysis of radioactively labeled protein from two-dimensional electrophoretograms and Edman degradation of CheZ protein isolated by immunoaffinity chromatography using anti-CheZ monoclonal antibodies. The methylated group is an N-monomethylmethionine residue at the amino terminus of CheZ. L16, a ribosomal protein that is required for peptidyltransferase activity during protein synthesis, is also methylated at its amino-terminal methionine (Chen, R., Brosius, J., and Wittmann-Liebold, B. (1977) J. Mol. Biol. 111, 173-181). Homologous sequences at the amino termini of L16 and CheZ raise the possibility that a single S-adenosylmethionine-dependent methyltransferase modifies both proteins.     

Interaction of the 4 S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene

The induction of cytochrome P-450c, the isozyme most closely associated with aryl hydrocarbon hydroxylase activity in the rat, is mediated through a cytosolic polycyclic aromatic hydrocarbon (PAH)-binding protein(s). We have reported on the purification and characterization of a 4 S protein that interacts in a specific and saturable manner with [3H]benzo[a]pyrene and other PAHs. (W. H. Houser et al. (1985) Biochemistry 24, 7839-7845). We have also reported on the specific and saturable interaction of the 4 S protein with a plasmid containing 1.9 kbp of cloned rat P-450c sequence including exon 1, the 5' half of intron 1, and approximately 882 bp upstream information. Our investigations now show that incubation of this protein with a portion of the rat P-450c gene, followed by digestion with either lambda exonuclease or exonuclease III, tentatively identified two protected regions at -225 and -455 bp 5' from exon 1. To further study the significance of these protected regions, a 3.4-kbp fragment containing cytochrome P-450c promoter and 5'-upstream sequences (-882 to +2545) was fused to the chloramphenicol acetyl transferase (CAT) reporter gene and transfected into either rat epithelial RL-PR-C cells or rat hepatoma H-4-II-E cells. Both cell lines expressed CAT activity in response to induction by 3-methylcholanthrene (3MC), indicating that important regulatory regions responsive to 3MC are present in these constructs. However, neither cell line expressed CAT activity in response to 3MC when transfected with plasmids containing deletions (-95 to -724 or -240 to -720) in the regions shown to be protected by our footprinting studies. These results corroborate previous studies which indicated that the 4 S PAH-binding protein interacts in a specific manner with regions of the rat cytochrome P-450c gene. We conclude that the 4 S protein may play an important role in the regulation of expression of cytochrome P-450c in the rat.     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Seasonal and inter-annual variations of nitrogen diagenesis in the sediments of a recently impounded basin

The Méry-sur-Oise (France) storage reservoir is an artificial basin of 9 m average depth, fed by water from the river Oise with a mean residence time of about 4 days. Sediments are accumulating at a rate of about 0.7 cm/month. In the sediments, two fractions of organic nitrogen with different rates of bacterial degradation could be distinguished, one associated with fresh phytoplankton, the other made of detrital and more refractory compounds. The fluxes of oxygen, nitrate and ammonium across the sediment-water interface were measured with a bell-jar system at different seasons during a 3 year period following flooding of the basin. The measurements show clear seasonal variations in relation with the variations of temperature and input of fresh phytoplanktonic material to the sediment. In addition, a long term trend of increasing ammonium was observed. Measurements were also carried out after dredging of all accumulated sediments of the basin. They showed a considerable reduction of the flux of nitrate to the sediments and a significant reduction of the flux of ammonium to the water column.These results are interpreted in the light of a non stationary model of N diagenesis in accumulating sediments. This model is able to predict at least the general trends of benthic N cycling of basins during the early stage of their ecological succession.     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

Extraction of monocrotaline from Crotalaria spectabilis using supercritical carbon dioxide and carbon dioxide-ethanol mixtures

Monocrotaline, a pyrrolizidine alkaloid of chemotherapeutic interest, was successfully extracted from the crushed seeds of Crotalaria spectabilis using supercritical carbon dioxide and ethanol mixtures. Overall solubilities of the plant material in the supercritical fluid phase were as high as 1.1 mass percent, and monocrotaline solubilities were as high as 0.07 mass percent. The solubility of monocrotaline in the presence of other plant material was smaller by 50 to 98% compared with the solubility of pure monocrotaline in the supercritical fluid. Also, it was found that the extraction of the complex plant material was time-dependent after approximately one percent of the original mass of the material had been extracted.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.