DNA-dependent RNA polymerases from Schneider 2-L cells of Drosophila. I. Preliminary characterization

The DNA-dependent RNA polymerases of Schneider 2-L cells of Drosophila melanogaster are described. These cells contain five readily detectable forms of this enzyme, polymerases I_a, I_b, III_a, II, and III_b, which elute from DEAE-Sephadex at 0.08, 0.12, 0.15, 0.20, and 0.22 m ammonium sulfate, respectively. RNA polymerases III_a and III_b, which each constitute about 5–10% of the total RNA polymerase activity in Drosophila embryos, are found to constitute 30 and 10%, respectively, of the total polymerase activity in cultured cells. The two form III polymerases are further characterized by in vitro response to divalent cations and ionic strength, template utilization, and sensitivity to -amanitin. Verification of the class III designation of these two polymerases is provided by their sensitivity to only very high levels of -amanitin (50% inhibition at approximately 800 µg/ml), their 10-fold greater activity on poly[d(A–T)], and their elution from DEAE-cellulose at lower ionic strengths than from DEAE-Sephadex.This work was supported by the Natural Sciences and Engineering Research Council.     

山西朔县种子植物区系及其生态经济意义

朔县(现朔州市朔城区)植物种属相对丰富,具有维管束植物938种,隶属于420属,105科。其植物区系主要起源于古北大陆;种子植物属的地理成分复杂多样,以温带成分占绝对优势,具有典型的温带草原性质;与相邻地区植物区系的比较分析表明,联系最为密切的是蒙古草原植物区系,其次是黄土高原植物区系。该县植物资源利用应采取的对策是:既要合理开发利用,又要加强物种的保护。     

Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters.

DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water.     

1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562

The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned. The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer. Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein. Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues. Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562. The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562. In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein. The results presented here, together with the evidence obtained with other methods [Feng and Sligar (1991) Biochemistry (submitted)], support the notion that the interior of the protein is fluid and may correspond to a molten globule state.     

Chiral resolution of alpha,alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene, a novel antiplatelet compound.

alpha,alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide, a novel antiplatelet agent, was resolved into three isomers A, B, and C, on a chiral alpha 1-acid glycoprotein analytical column using a mobile phase of 0.025 M phosphate buffer containing 0.025 M tetrabutylammonium hydrogen sulfate, at a pH of 6.5. The effect of molarity, temperature, pH, flow rate, and organic modifiers on the enantioselectivity was examined. Based on circular dichroic spectra at 220 nm, A and C appear to be the (-)- and (+)-enantiomers, respectively, and B the meso diastereomer. Attempts at resolution using Pirkle type columns gave unsatisfactory results. It appears that both hydrophobic and polar interactions between the compound and the stationary phase are important determinants of resolution.     

Spatial patterns and sampling plans for cereal aphids [Hom.: Aphididae] killed by entomophthoralean fungi and hymenopterous parasitoids in spring wheat

Cereal aphids infesting spring wheat in southwestern Idaho were surveyed during 1988 and 1989 for the presence of entomophthoralean fungi and hymenopterous parasitoids. Cereal aphids killed by the fungi (cadavers) and parasitoids (mummies) includedDiuraphis noxia (Mordvilko),Metopolophium dirhodum (Walker),Sitobion avenae (F.), andSchizaphis graminum (Rondani). Taylor's power law was used to describe the relationships between mean densities of cadavers or mummies (number per tiller) and associated variances. Except forS. graminum mummies, which were at low levels throughout the growing seasons and tended to be randomly distributed, Taylor's slopes exceeded 1.0 for all the aphid cadavers and mummies, indicating varying degrees of clumping in spatial pattern. The spatial patterns ofD. noxia andM. dirhodum cadavers were similar, simply reflecting those of their own populations, but more aggregated than were their respective mummies resulting from parasitoid attack. The intercepts and slopes from the power law analysis were used to generate functional relationships between the proportion of wheat tillers bearing cadavers or mummies of each aphid species and the mean density, and develop optimal numerical (direct counting) and binomial (presence or absence) sample size curves for both cadavers and mummies of each aphid species.      

Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.     

Pyrophosphate contamination in crystalline phosphocreatine: a source of error in kinetic studies using coupled enzymes.

Commercial crystalline phosphocreatine generally contains a contaminant, probably pyrophosphate, that can inhibit nucleoside triphosphatases and may therefore cause errors in steady-state kinetic studies of creatine kinase-coupled r reactions. Ion-exchange chromatography reveals the presence of pyrophosphate in some batches of phosphocreatine sufficient to account for the observed inhibition. Chromatographically purified phosphocreatine shows no inhibitory effect.