Specific polyadenylation and purification of total messenger RNA from Escherichia coli

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages, We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese, The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA, Withdrawal of Mg2+ from the polyadenylation reaction rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes, Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species, Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis, Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Light- and cytokinin-induced changes in the levels of leucine and tyrosine isoaccepting tRNA species and modified nucleotide contents of total tRNA in cucumber seedlings

We have determined relative levels of chloroplast leucine and tyrosine isoaccepting tRNAs and modified nucleotide contents from total tRNAs isolated from dark-grown, light-grown, N6-isopentenyladenine (i6A)-treated dark-grown and i6A-treated light-grown cucumber seedlings. Significant increases in the relative amounts of tRNA(Leu)2 and tRNA(Leu)3 were observed in the i6A-treated dark-grown seedlings compared to dark-grown, light-grown and i6A-treated light-grown seedlings. On the other hand, i6A-treated light-grown seedlings tRNA(Tyr)1 increased to 85% of total tRNAs(Tyr) from about 9% in light-grown seedlings and tRNA(Tyr)2 decreased to 15% compared with 91% in light-grown seedlings. Analysis of modified nucleotide of total tRNAs indicated that pT, pI, pm1A, pm5C, pGm, pm1G, pm2G and pm7G contents were significantly higher in the total tRNA of i6A-treated dark-grown seedlings than those from untreated dark-grown seedlings. Illumination of 8-day-old dark-grown seedlings for 12 h increased the contents of pT, pI, pGm and pm1G when compared to 8-day-old dark-grown seedlings with extended growth for 12 h in dark. On the contrary, i6A had no stimulatory effect in the contents of modified nucleotide in the light-grown seedlings.

Intensity Oscillation in the Corona as Observed during the Total Solar Eclipse of 29 March 2006

We obtained the images of the eastern part of the solar corona in the Fe xiv 530.3 nm (green) and Fe x 637.4 nm (red) coronal emission lines during the total solar eclipse of 29 March 2006 at Manavgat, Antalya, Turkey. The images were obtained using a 35 cm Meade telescope equipped with a Peltier-cooled 2k x 2k CCD and 0.3 nm pass-band interference filters at the rates of 2.95 s (exposure times of 100 ms) and 2.0 s (exposure times of 300 ms) in the Fe xiv and Fe x emission lines,respectively. The analysis of the data indicates intensity variations at some locations with period of strongest power around 27 s for the green line and 20 s for the red line. These results confirm earlier findings of variations in the continuum intensity with periods in the range of 5 to 56 s by Singh et al. (Solar Phys. 170, 235, 1997). The wavelet analysis has been used to identify significant intensity oscillations at all pixels within our field of view. Significant oscillations with high probability estimates were detected for some locations only. These locations seem to follow the boundary of an active region and in the neighborhood, rather than within the loops themselves. These intensity oscillations may be caused by fast magneto-sonic waves in the solar corona and partly account for heating of the plasma in the corona.

Stereoselective Formal Total Synthesis of (-)-Didemniserinolipid B

A formal total synthesis of (-)-didemniserinolipid B from L-(+)-tartaric acid is presented. Key features of the synthesis include construction of the bicyclic acetal core from bisdimethyl amide of tartaric acid and further elaboration by cross metathesis.

Differences in the levels of aminoacylation and contents of modified nucleotides between total tRNAs from N2- and NH4+-grown Azospirillum lipoferum cells

Total tRNAs isolated from N2- and NH4(+)-grown Azospirillum lipoferum cells were compared with respect to amino acid acceptance, isoacceptor tRNA species levels and extent of nucleotide modifications. Amino-acylation of these two tRNA preparations with ten different amino acids indicated differences in the relative acceptor activities. Comparison of aminoacyl-tRNA patterns by RPC-5 column chromatography revealed no qualitative differences in the elution profiles. However, quantitative differences in the relative amounts of some isoacceptors were observed. These results indicate that alterations of relative amounts of functional tRNA species occur to match cellular requirements of the bacterial cells using N2 or NH4+ as nitrogen source. In addition, the content of modified nucleotides in total tRNAs of N2- and NH4(+)-grown cells was determined. In the NH4(+)-grown cells, content of most of the modified nucleotides decreased significantly. Based upon these results, the relationship of chargeability of tRNAs to base modifications is discussed.

Identification and characterization of thionucleosides in the total tRNA of cucumber cotyledons

Total tRNA isolated from cucumber cotyledons grown in the presence of radioactive sulfur was analyzed for the occurrence of thionucleosides. The analysis revealed the presence of at least five thionucleosides which were identified as 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-methylthio-N6-isopentenyladenosine (ms2i6A), 2-methylthio-N6-hydroxyisopentenyladenosine (ms2io6A), 5-methyl-2-thiouridine (m5s2U) and N-[(9-beta-ribofuranosyl-2- methylthiopurine-2-yl)-carbamoyl]-threonine (ms2t6A). A comparison of relative amounts of these thionucleosides in the total tRNAs of dark-, and light-grown cotyledons shows that the relative amounts of ms2i6A, ms2io6A and ms2t6A remain unchanged whereas mnm5s2U increases with a concomitant decrease in the relative amounts of m5s2U after light treatment of dark-grown cotyledons.

Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.

Asymmetric total synthesis of erythromycin. 2. Synthesis of an erythronolide A lactone system

In reporting a total synthesis of erythromycin (la) we described in the preceding paper1 the synthesis of the erythronolide A seco acid derivative 2 in optically active form. In this paper we wish to report a successful transformation of 2 to 12 (synthetically equivalent to erythronolide A) via lactonization and also demonstrate that the proper functionalization of a substrate is critical for the successful lactonization.

Asymmetric total synthesis of erythromycin. 3. Total synthesis of erythromycin

In the preceding paper' we described the preparation of the key lactone intermediate la in optically active form. In this paper we report the synthesis of erythromycin (2) from la. In essence,this transformation involves the glycosidation of a suitable derivative of la with L-cladinose and D-desosamine and the generation of the C-9 ketone functionality.

A stereoselective total synthesis of (−)-seychellene

A stereoselective total synthesis of the tricyclic sesquiterpene (−)-seychellene, starting from (R)-carvone via (R)-3-methylcarvone has been accomplished, employing a combination of intermolecular Michael addition–intramolecular Michael addition sequence, a stereoselective hydrogenation, and an intramolecular alkylation reaction.

QCD and total cross-sections : photons and hadrons

In this contribution, we discuss a total cross-section model which can be applied to both photon and purely hadronic processes. We find that the model can reproduce photo-production cross-sections, as well as extrapolation of gamma*p processes to gamma p using Vector Meson Dominance models, with minimal modifications from the proton case.

QCD and total cross-sections : photons and hadrons

In this contribution, we discuss a total cross-section model which can be applied to both photon and purely hadronic processes. We find that the model can reproduce photo-production cross-sections, as well as extrapolation of gamma*p processes to gamma p using Vector Meson Dominance models, with minimal modifications from the proton case.

Total cross-section at LHC from minijets and soft gluon resummation in the infrared region

A model for total cross-sections incorporating QCD jet cross-sections and soft gluon resummation is described and compared with present data on pp and pp cross-sections. Predictions for LHC are presented for different parameter sets. It is shown that they differ according to the small x-behaviour of available parton density functions.