The role of bystin in embryo implantation and in ribosomal biogenesis

Human bystin was identified as a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. Although the trophinin gene is unique to mammals, the bystin gene (BYSL) is conserved across eukaryotes. Recent studies show that bystin plays a key role during the transition from silent trophectoderm to an active trophoblast upon trophinin-mediated cell adhesion. Bystin gene knockout and knockdown experiments demonstrate that bystin is essential for embryonic stem cell survival and trophectoderm development in the mouse. Furthermore, biochemical analysis of bystin in human cancer cells and mouse embryos indicates a function in ribosomal biogenesis, specifically in processing of 18S RNA in the 40S subunit. Strong evidence that BYSL is a target of c-MYC is consistent with a role for bystin in rapid protein synthesis, which is required for actively growing cells. Received 30 June 2007; received after revision 7 August 2007; accepted 29 August 2007     

eIF2A,an initiator tRNA carrier refractory to eIF2α kinases,functions synergistically with eIF5B

The initiator tRNA (Met-tRNA _i ^Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA _i ^Met on the small ribosomal subunit. Eukarya contain the Met-tRNA _i ^Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA _i ^Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA _i ^Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B–eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.     

Ribosomal gene numbers in Microseridinae (Compositae: Cichorieae)

Summary 8 species of the subtribe Microseridinae contain between 1100 and 3400 genes for 25 and 18 S ribosomal RNA. The gene numbers seem to evolve by discrete steps. Their trend follows a general reduction in genome size during the evolution of the annual species ofMicroseris, but numbers remain high in one of them and inAgoseris grandiflora. 2 species ofPyrrhopappus differ by a duplication of the ribosomal gene numbers; 5 S ribosomal RNA genes in 4 species are repeated roughly 10,000 times.We thank Miss S. Werner, Miss A. Roth and Miss U. Krehan for help with some of the experiments. This paper is part of a project supported by grant Ba 536/1-5 from the Deutsche Forschungsgemeinschaft.     

Molecular genetics of the peptidyl transferase center and the unusual Var1 protein in yeast mitochondrial ribosomes

Mitochondria posses their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome. In yeast,Saccharomyces cerevisiae, the two ribosomal RNAs and a single ribosomal protein, Varl, are products of mitochondrial genes, and the remaining approximately 80 ribosomal proteins are encoded in the nucleus. The mitochondrial translation system is dispensable in yeast, providing an excellent experimental model for the molecular genetic analysis of the fundamental properties of ribosomes in general as well as adaptations required for the specialized role of ribosomes in mitochondria. Recent studies of the peptidyl transferase center, one of the most highly conserved functional centers of the ribosome, and the Varl protein, an unusual yet essential protein in the small ribosomal subunit, have provided new insight into conserved and divergent features of the mitochondrial ribosome.     

PCR jumping in clones of 30-million-year-old DNA fragments from amber preserved termites (Mastotermes electrodominicus)

DNA from 30-million-year-old amber preserved termites (Mastotermes electrodominicus) was PCR amplified with nuclear ribosomal RNA small subunit primers and cloned into the TA vector (INVITROGEN). We obtained several classes of recombinant clones as a result. AuthenticMastotermes electrodominicus clones were identified. The source of other classes of clones was identified as contaminants of the ancient DNA template. Several of the clones appeared to be chimeric in structure with half of the clone identical to the termite sequence and the other half identical to contaminant sequences. The phenomenon of PCR jumping was identified as a possible source for the chimeric clones.     

A structural view of translation initiation in bacteria

The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNA^fMet to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation. Received 16 July 2008; received after revision 31 August 2008; accepted 10 September 2008 A. Simonetti, S. Marzid: These authors contributed equally to this work.     

Consensus phylogeny ofDictyostelium

The evolutionary relationship ofDictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics. Previous analyses of five proteins indicated thatDictyostelium diverged after the yeasts but before the metazoan radiation. However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts. We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast. A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishesDictyostelum's place in evolution between the yeastsSaccharomyces cerevisiae andSchizzosaccharomyces pombe and the wormCaenorhabditis elegans.     

RNA editing in plant organelles: machinery, physiological function and evolution

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins. Received 30 September 2005; received after revision 5 November 2005; accepted 28 November 2005     

More than just scanning: the importance of cap-independent mRNA translation initiation for cellular stress response and cancer

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5′ end of the mRNA and scans the 5′ untranslated region (5′UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5′UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N ⁶-methyladenosine (m⁶A) residues in their 5′UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets.     

Denaturation map of the ribosomal DNA ofLytechinus variegatus sperm

Summary Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6±0.2 m, corresponding to a mol.wt of 7.2±0.4×10⁶. Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47±0.11 m (mol.wt 4.9±0.22×10⁶) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16±0.09 m (mol.wt 2.3±0.18×10⁶) is presumed to contain non-transcribed spacer sequences.Acknowledgments. This paper is dedicated to the memory of Dr D. P. Costello whose insprration made this work possible. I gratefully acknowledge the valuable advice of Drs D.W. Stafford and M.A. Bleyman.     

Purification of an L-fucose binding lectin fromUlex europeus by affinity column chromatography

Summary An L-fucose binding lectin fromUlex europeus was purified by affinity column chromatography using an L-fucose-starch complex. The lectin thus purified had a mol.wt of 60,000, and consisted of 2 glycoprotein subunit with mol.wt 29,000 and 31,000, respectively.     

Ancient DNA sequences reveal unsuspected phylogenetic relationships within New Zealand wrens (Acanthisittidae)

Ancient DNA sequences from preserved specimens are increasingly being used for the investigation of Pacific Island ecosystems prior to the large scale modification and extinction of endemic biota associated with human colonization. However, many difficulties are associated with the use of ancient DNA sequences in studies of genetically close taxa. In this paper, these difficulties are discussed as they relate to a study involving extinct and extant members of an ancient New Zealand avian family, the New Zealand wrens (Acanthisittidae).Sequences of the mitochondrial small ribosomal subunit RNA gene (12S) were obtained from museum specimens of several wren taxa in order to investigate their phylogenetic relationships and the taxonomic status of a rock wren (Xenicus gilviventris) subspecies. Limitations due to sample size and 12S sequence variability as well as the difficulties in authenticating ancient DNA sequences prevent firm conclusions but the data suggest unsuspected phylogenetic relationships exist and raise the possibility that conservation management of rock wren populations is required.     

Evidence for the nonhydrophobic interaction of aromatic,nitrogen-containing compounds with the coenzyme binding site of a NAD-linked D-lactate dehydrogenase

Summary Quantitative structure activity relationships suggest that the binding of quinoline and phenanthroline analogs to D-lactate dehydrogenase from the barnacle,Balanus nubilus Darwin, does not involve primarily hydrophobic effects. This phenomenon appears to exist also for other lactate dehydrogenases.We thank C. Hansch for advice regarding QSAR studies. Supported by U.S.P.H.S. (GM-22868-01) to GLL.     

The reduction of genic activity in the tetraploid amphibianOdontophrynus americanus is not due to loss of ribosomal DNA

Summary Genic activity in tetraploid members of the amphibian speciesOdontophrymus americanus is reduced to that of diploid ones. Loss of ribosomal genes, a mechanism suggested by others as a means of decreasing genetic activity, could be ruled out. The diploids and the tetraploids have almost identical proportions of their genomes complementary to (28s+18s) ribosomal RNA.     

Ligand recognition by the I domain-containing integrins

Seven of the integrin α subunits described to date, α ₁ , α ₂ , α _L , α _X , α _d , α _M and α _E , contain a highly conserved I (or A) domain of approximately 200 amino acid residues inserted near the amino-terminus of the subunit. As the result of a variety of independent experimental approaches, a large body of data has recently accumulated that indicates that the I domains are independent, autonomously folding domains capable of directly binding ligands that play a necessary and important role in ligand binding by the intact integrins. Recent crystallographic studies have elucidated the structures of recombinant α _M and α _L I domains and also delineated a novel divalent cation-binding motif within the I domains (metal ion-dependent adhesion site, MIDAS) that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands.     

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

The coordination and function of the redox centres of the membrane-bound nitrate reductases

Under anaerobic conditions and in the presence of nitrate, the facultative anaerobe Escherichia coli synthesises an electron-transport chain comprising a primary dehydrogenase and the terminal membrane-bound nitrate reductase A (NarGHI). This review focuses on recent advances obtained on the structure and function of the three protein subunits of membrane-bound nitrate reductases. We discuss a global architecture for the Mo-bisMGD-containing subunit (NarG) and a coordination model for the four [Fe–S] centres of the electron-transfer subunit (NarH) and for the two b-type haems of the anchor subunit NarI.     

Untersuchungen über stabile und während der frühen Quellungsstadien neu synthetisierte Poly(A)-RNA vonAgrostemma-Samen

Summary RNA isolated from dry embryos ofAgrostemma githago seeds contains poly(A)-sequences, but in very small amounts. In the early phase of imbibition, an intensive synthesis of poly(A)-containing RNA is brought about. The importance of this synthesis of poly(A)-RNA is discussed.

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Herrn Dr.E. Serfling (Gatersleben, DDR) danke ich für methodische Hinweise und für die Überlassung einiger Chemikalien.     

Y chromosome in the sibling speciesAnopheles atroparvus (van Thiel, 1927) andA. labranchiae (Falleroni, 1926) (Diptera: Culicidae): Differential behaviour of the short arm after acid-alkaline treatment and Coriphosphine-O staining

Summary An acid-alkaline treatment followed by Coriphosphine-O staining was used for detecting chromosomal differences between the 2 sibling speciesAnopheles atroparvus (van Thiel) andA. labranchiae (Falleroni) (Diptera: Culicidae). The short arm of the Y chromosome was found to stain differently in the 2 species.The authors wish to thank the entomologist C. Contini for collecting and classifyingA. labranchiae larvae and Prof. U. Bianchi and Prof. U. Laudani for helpful discussions and suggestions on the subject.