Nuclear magnetic resonance study of the impact of ribose 2′-O-methylation on the aqueous solution conformation of cytidylyl-(3′ → 5′)-cytidine

In order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′-O-methylated region of t-RNA, as well as in the bizarre 5′-terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′-O-methyl-cytidylyl-(3′ → 5′)-cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D₂O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo-nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories. It is shown that the ribofuranose ring exists at a ²E ? ³E equilibrium with clear preference [(75–80)%] for the ³E mode. The C(4′)-C(5′) and C(5′)-O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ₁, Ψ₂ ? 60° and ?₂ ? 180°. The orientation of the 3′-phosphate and 2′-O-methyl groups is such that ?₁′ ? 210° and ?″ ? 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right-handed stack (g^?g^?), left-handed stack (g⁺g⁺), and unstacked arrays (tg^? and tg⁺). Elevation of temperature perturbs the ²E ? ³E equilibrium accompanied with modest depopulation of ψ₁, ψ₂ ? 60° and ?₂ ? 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g^?g^? and g⁺g⁺ conformers. The shift trend of Cmp-H(3′) with temperature shows that torsional variation about O(3′)-P is facilitated by increase in temperature and the preferred rotamer about O(3′)-P in the unstacked form is t (ω₁′ = 180°). A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′-O-methylation causes: (i) a reduction in the magnitude of _χ1; (ii) an increase in the population of ³E pucker at the 3′-nucleotidyl unit; and (iii) modest perturbations in the O(3′)-P and P-O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine-pyrimidine and purine-purine dimers which carry a 2′-O-methylated 3′-nucleotidyl unit are significantly different.     

Endogene Synthese kurzlebiger Messenger-RNS in der Oocyte vonDysdercus intermedius Dist. nach Abschluß der Vitellogenese

Zusammenfassung In den Oocyten des telotroph-meroistischen Ovars vonDysdercus intermedius Dist. findet während der Endphase der Oogenese, 4–14 h vor der Eiablage, eine Synthese von nichtribosomaler RNS statt. Eine in vivo-Markierung dieser RNS läßt sich erreichen, wenn radioaktive RNS-Vorstufen einem Nucleotidpool zugeführt werden, der im Ooplasma vor der Chorionbildung angelegt wird.Diese vor der Eiablage gebildete RNS zeichnet sich durch einen hohen Turnover aus. Sie erscheint zunächst in Form einer hochmolekularen Vorstufe und wird im Verlauf weniger Stunden in kleinere, nichtribosomale Moleküle mit S-Werten zwischen 30 und 5 umgewandelt. Im frisch abgelegten Ei fehlen RNS-Spezies, die dieser endogenen Oocytensynthese entstammen; es sind nur noch ihre Degradationsprodukte, die sich innerhalb der Nucleotidfraktion ansammeln, nachweisbar. Die endogen synthetisierte RNS wird demnach im Gegensatz zu der in den Nährzellen synthetisierten und im Ei in stabiler Form gespeicherten RNS nicht für einen Bedarf während der Embryogenese konserviert.Die endogen synthetisierte RNS zeichnet sich durch einen hohen Poly (A)-Gehalt aus; 57% hybridisieren mit an Glasfaserfiltern immobilisiertem Poly(U). Wenige Stunden vor der Eiablage findet man kurzlebige oocytäre RNS-Moleküle an Polysomen assoziiert. Die Inkubation dieser Polysomen in einem in vitro-Proteinsynthese-System liefert Polypeptide, deren Auftrennung am SDS-Polyacrylamid-Gel ein charakteristisches Bandenspektrum ergibt. Die Molekulargewichte der 4 Hauptbanden liegen bei 65000, 48000, 44000, und 40000. Keines dieser Proteine ist mit einem Chorionprotein identisch.Die Kurzlebigkeit, der relativ hohe Poly (A)-Gehalt sowie die Fähigkeit, die Proteinsynthese sowohl in vivo als auch in vitro zu aktivieren, spricht dafür, daß die spät-oocytär gebildete heterogene Population von RNS-Molekülen mRNS-Komponenten enthält.Bei Frl. Heidrun Greipel bedanken wir uns für die ausgezeichnète Assistenz.     

Localization of vimentin,the nonspecific intermediate filament protein,in embryonal glia and in early differentiating neurons: In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera

Antisera raised against vimentin, the protein subunit of nonspecific intermediate-sized filaments (IFs), were used in conjunction with neurofilament (NF) antisera to study the early development of neurons and glia in the rat embryo. Vimentin-positive fibers spanning the entire thickness of the neural tube including the cerebral vesicles were first observed on Day 12, concomitant with the appearance of NF protein in more confined areas (anterolateral regions of spinal cord and brain stem; motor roots emerging from the NF-positive areas). From Day 15 onwards vimentin and NF antisera selectively decorated glia and neurons, respectively, both in vivo and in vitro. Before Day 15 it appeared that NF-positive structures also stained with antivimentin in cryostat sections. In vitro experiments confirmed the presence of vimentin in early differentiating neurons. NF-positive cells were observed which also reacted with antivimentin in cultures obtained from 13- and 14-day embryos, but not later in development. Most neurons in these cultures became vimentin negative after 2–3 days in vitro.     

The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.     

The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn-glycerophosphoryl diester phosphodiesterase.

The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', and insertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphism among different staphylococcal isolates. We sequenced and characterized the hypervariable region of the methicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bp hypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNA sequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. The dru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open reading frame that codes for 145 amino acids (ORF145), whose deduced amino acid sequence showed 57% amino acid sequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) of Escherichia coli.     

Specific gangliosides increase rapidly in rat liver following partial hepatectomy

Rat liver gangliosides (sialic acid containing glycosphingolipids) were analyzed by HPTLC and HPLC following either partial hepatectomy or sham operation. Analysis of whole liver gangliosides by HPTLC demonstrated that within 6 h after partial (68%) hepatectomy, there was a significant increase in GM1 compared to both sham and control animals. By 48 h, GM1 was further increased and the polysialylgangliosides GD1a, GD1b and GT1b had also risen significantly, whereas changes in GM3 were negligible. Gangliosides associated with the plasma membrane were increased up to 3.5-fold in regenerating liver compared to sham-hepatectomized controls as assessed by HPLC. Although elevations in membrane gangliosides were associated with hepatocyte proliferation, they did not closely follow the growth curve. The time course of changes in ganglioside biosynthesis suggests differential upregulation of GM3 synthase and GD3 synthase in regenerating livers.     

Incorporation of influenza virus M-protein into liposomes.

M-protein from influenza virus vaccine was purified by sodium dodecyl sulfate-gel chromatography and incorporated into liposomes by solubilization with octylglucoside and subsequent dialysis. Liposomes containing M-protein formed a distinct population with a density of 1.22 g/ml on sucrose-gradient centrifugation, regardless of the net charge on the liposomes. Treatment of the liposomes by freeze-fracture followed by electron microscopic examination showed multilamellar structures in those liposomes without M-protein; liposomes containing M-protein were mulberry-like structures which appeared unilamellar. These studies show incorporation of M-protein into the lipid bilayer.