Molecular Mechanisms of Translation Initiation in Mammals Studied by in vitroReconstruction of Initiation Complexes

Papers on the mechanisms of translation initiation in mammals studied by reconstruction of initiation complexes from individual components are reviewed. The author points to the constraints of this approach and to the pitfalls ignoring which one might come to erroneous conclusions and even artifacts. In addition, some methods employed in the field as well as some technical problems are discussed in the paper, together with the means of obviating them. The review could be a guidebook for newcomers into this quite labor-consuming field.     

Reactivities of the coordinated organonitriles in molybdenum(0) and tungsten(0) phosphine complexes: protonation of the nitrile carbon and cleavage of the CN triple bond

The molybdenum and tungsten dinitrogen-organonitrile complexes trans-[M(N₂)(NCR)(dppe)₂] (2, M=Mo; 4, M=W; R=Ph, C₆H₄Me-p, C₆H₄OMe-p, Me; dppe=Ph₂PCH₂CH₂PPh₂) underwent double protonation at the nitrile carbon atom with loss of N₂ and a change in oxidation state to +4 on treatment with hydrochloric acid to afford the cationic imido complexes trans-[MCl(NCH₂R)(dppe)₂]⁺. The solid-state structure of trans-[WCl(NCH₂CH₃)(dppe)₂][PF₆]·CH₂Cl₂ was determined by single-crystal X-ray analysis. Protonation of complexes 2 by fluoroboric acid or hydrobromic acid also formed the similar imido complexes trans-[MoX(NCH₂R)(dppe)₂]⁺ (X=F, Br). In contrast, the dinitrogen complex trans-[Mo(N₂)₂(dppe)₂] reacted with two equiv. of benzoylacetonitrile, a nitrile with acidic CH hydrogen atoms, to give the nitrido complex trans-[Mo(N)(NKCCHCOPh)(dppe)₂] (12), which was accompanied by evolution of dinitrogen and the formation of 1-phenyl-2-propen-1-one in high yields. For complex 12, the zwitterionic structure, where the anionic enolate ligand PhC(O⁺)=CHCN coordinates to the cationic Mo(IV) center through its nitrogen atom, was confirmed by spectroscopic measurements and single-crystal X-ray analysis. A unique intermolecular aromatic C---HO hydrogen bonding was observed in that crystal structure. Complex 12 is considered to be formed via the cleavage of the CN triple bond of benzoylacetonitrile on the metal. A reaction mechanism is proposed, which includes the double protonation of the nitrile carbon atom of the ligating benzoylacetonitrile on a low-valent molybdenum center.     

Gradients in maize roots: local elongation and pH

The elongation rate, the gradient of the local elongation rate and the surface pH of maize roots were measured over 12 h. A data bank was constituted by storing these values. By sorting these results on the basis of different elongation rates, different classes of root were obtained. Two classes were chosen: the low-growth roots and the high-growth roots. The mean growth of these two root classes was stable with time and differed significantly from one another. The surface pH of the elongation zone was the same for the roots of these two classes, but the roots selected for their higher growth rate had a larger acid efflux in this zone.     

Radioiodinated surface proteins of rabbit trophectoderm cells

Summary We have previously used surface iodination to discriminate between the protein patterns of epithelial cell surfaces in uteri of rabbits receptive (Day 6.5) or nonreceptive (Day 4) to nidation (Ricketts et al. 1984). In this paper, we describe application of the same technique to the trophoblastic surface of rabbit blastocysts collected on the same days of pregnancy. Analysis of labelled proteins by polyacrylamide-gel electrophoresis under denaturing conditions did not reveal qualitative differences between the two days of pregnancy. Scanning densitometry was used to quantitate the area under each protein peak on an autoradiogram; these areas were used as variables in statistical analysis of the protein pattern of individual animals. Quantitative differences between the protein patterns of the two surfaces were detected by canonical variate analysis of the pattern of relative areas of labelled protein peaks. In proteins separated on 7.5% gels, this statistical analysis correctly assigned blastocysts from 8 out of 10 animals to one of two groups according to day of pregnancy. The discrimination was not statistically significant, however, in protein patterns on 12.5% gels, used to give better separation in the lower range of molecular weights. The same analysis in the uterus unequivocally separated the surface iodination patterns from these same days of pregnancy. Thus the changes detected by surface iodination appear to be less pronounced on the trophectoderm than on the uterine epithelium in relation to the time of ovoimplantation.     

Rare earth thiocyanate complexes with tripiperidinophosphine oxide: synthesis and characterization. Crystal structure of Pr(SCN)3(tpppO)3

The synthesis and characterization of rare-earth (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y) thiocyanate adducts with tripiperidinophosphine oxide (tpppO) with general formula (RE)(SCN)₃(tpppO)₃ are reported. Conductance measurements in acetonitrile indicate the non-electrolytic nature of the complexes. Infrared absorption spectra evidence that the SCN⁻ ion coordinates through the nitrogen atom (isothiocyanate form) and that tpppO coordinates through the phosphoryl oxygen. X-ray powder patterns suggest the existence of three different crystal forms: (1) La; (2) an isomorphous series including Ce, Nd and Pr; and (3) another isomorphous series, including Sm, Gd, Eu, Ho, Er, Tb, Lu and Y. The visible spectra of the Nd adduct and the calculated parameters β = 0.98, b^1/2 = 0.072 and δ = 1.06 indicate that the metal-ligand bonds are essentially electrostactic. The emission spectra of the Eu compound showed ⁵D₀ → ⁷F_J bands (J = 0, 1, 2), suggesting a C_3v symmetry for the coordination polyhedron. The lifetime of the ⁵D₀ state is 1.28 ms. The emission spectra of the Tb complex presented ⁵D₄ → ⁷F_J bands (J = 4, 5, 6) and the Dy complex showed the ⁴F_9/2 → ⁶H_13/2 band. The structure of the Pr complex showed that the coordination polyhedron is a trigonal antiprism, with the isothiocyanate anions in one base and three tpppO ligands in the other. Thermal analyses (TG-DTG) were carried out for the Ce, Nd and Gd adducts. Mass losses start between 250 and 334 °C. The final residues at 1300 °C are the corresponding phosphates.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Protein processing as a regulatory mechanism in the synthesis of the photosynthetic antenna in Chloroflexus

Chloroflexus aurantiacus can be induced to shift from respiratory to photosynthetic energy production by introducing light and/or lowering the oxygen concentration of a culture. After induction, cells synthesize bacteriochlorophyll and proteins for the formation of a functional photosynthetic apparatus. Bacteriochlorophyll is detectable within 2 h after induction. Chlorosome polypeptides are detected after 8–12 h. Two proteins, M_r 60,000 and M_r 47,000, are present in both induced and noninduced cells and react specifically with antibodies against chlorosome polypeptides. Immunological data suggest that these proteins (M_r 60,000 and 47,000) are polyproteins which are transcribed and translated in the dark. When cells are exposed to light or low oxygen tension these proteins are processed into functional polypeptides required in the assembly of the chlorosome. The reaction center polypeptide (M_r 26,000) appears to be part of a separate genetic control system.Dedicated to Prof. G. Drews on occasion of his 60th birthday     

The role of duplication in the expression of a variable surface glycoprotein gene of Trypanosoma brucei

The variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to expression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well-characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accommodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.