The 5'' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.

We have developed techniques for the detailed analysis of cis-acting sequences in the pre-rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5' end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5' to the end of 5.8S(S), and acts as an entry site for 5'-->3' exonuclease digestion which generates the 5' end of 5.8S(S). This pathway is inhibited in strains mutant for XRN1p and RAT1p. Both of these proteins have been reported to have 5'-->3' exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RAT1p and XRN1p in trans, and is kinetically faster than 5.8S(S) synthesis.     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Co-localization of cytokinins with proteins related to cell proliferation in developing somatic embryos of Dactylis glomerata L.

The present report provides evidence for co-localization ofcytokinins with cell proliferation-associated nuclear proteins.Somatic embryos of Dactylis glomerata in two stages of developmentare used as a model system comprising both proliferating andinitially differentiated cells. Cytokinins are localized usingantibodies with marked specificity against isopentenyladenine/adenosine(2iP/2iPA) or zeatin/ riboside (Z/ZR). The proliferation-associatednuclear antigen, mitotin, is analysed using a specific monoclonalantibody. The nuclear protein BM28, required for the onset ofDNA replication and for cell division, is identified by an affinity-purifiedpolyclonal antibody. Using double immunofiuorescence labellingwith the antibodies against cytokinins and against each of thenuclear proteins, immunoreaction is observed generally in thesame nuclei of almost all cells in globular embryos and in thenuclei of cells in meristematic areas of the more developedembryos. Only small numbers of individual nuclei positive forboth type of antibodies were found in the surrounding vacuolatedparenchymatous cells. The occurrence of plant antigens homologousto BM28 and mitotin is confirmed by immunoblotting assay. InSDS-PAGE blots the anti-BM28 antibody reacts with a proteinof 58 kDa. The anti-mitotin antibody recognizes several (160,140, 125, 93, and 80 kDa) polypeptides. The data showing nuclearco-localization of cytokinins and proteins with a suggestedrole in the onset of DNA synthesis and in cell division providea new base for further study on the mode of action of cytokininsin cell cycle regulation. Key words: Immunolocalization, cytokinins, nuclear proteins, mitotin, BM28, cell proliferation, somatic embryo(s), Dactylis glomerata     

Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene

A cosmid gene bank of partially EcoRI-digested genomic DNA from Methylobacterium extorquens IBT no. 6 was screened for DNA fragments restoring polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mutant Alkaligenes eutrophus H16 PHB^–4. The M. extorquens PHA-synthase structural gene phaC _Mex was mapped on a 23-kbp EcoRI fragment by complementation studies, by hybridization experiments with heterologous DNA probes from A. eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis. Evidence for the presence of genes for a -ketothiolase or an acetoacetyl-coenzyme A reductase on this fragment was not obtained. The nucleotide sequence of a 3.7-kbp region was obtained. It contained the entire 1.815-kbp phaC _Mex plus approximately each 900-bp upstream and downstream of phaC _Mex. PhaC _Mex encoded a protein of 605 amino acods with a relative molecular mass (M_r) of 66742, which exhibited 38.1% amino acid identity with the A. eutrophus PHA synthase. Determination of the N-terminal amino acid sequence of an M_r 65 000 protein, which was enriched concomitantly with the purification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviously removed post-translationally. Enzyme analysis, which was done with the native gene and a phaC _Mex -lacZ fusion gene, gave no evidence for expression of phaC _Mex in Escherichia coli.     

The amylase-secreting cells of the stomach of the scallop, Pecten maximus: ultrastructural, immunohistochemical and immunocytochemical characterizations

(1) alpha-amylase was extracted and purified from the stomach/digestive gland complex of the scallop Pecten maximus and an anti-serum was induced against the purified amylase by rabbit immunization. (2) The anti scallop amylase was used to localize the amylase-secreting cells in the stomach of Pecten maximus by immunofluorescence and immunogold labelling. The amylase-secreting cells are glandular cells particularly numerous in the main sorting area of the stomach. Their secretory granules were found strongly positive for anti-amylase. Three types of glandular cells were observed, actually corresponding to the three stages of the glandular-cell activity, synthesis, secretion and excretion. (3) The synthesizing cell shows the characteristic features of a protein-synthesizing cell: a conspicuous nucleolus and abundant granular endoplasmic reticulum. In the secretory cell, the secretory granules are formed by the Golgi apparatus and accumulate in the apical part of the cell. The secretory cell is filled with two types of secretory granules which are released in the stomach lumen by apocrine excretion. (4) The present study brings the first demonstration of the synthesis and extracellular release of amylase by glandular cells of the stomach epithelium of a bivalve.     

Conformational transitions using molecular dynamics with minimum biasing

The molecular dynamics algorithm (MD), which simulates intramolecular motions on the subnanosecond timescale, has been modified to allow the investigation of slow conformational transitions that do not necessarily occur spontaneously in MD simulations. The method is designated CONTRA MD (CONformational TRAnsitions by Molecular Dynamics with minimum biasing). The method requires the prior definition of a single conformational variable that is required to vary monotonically from an initial conformation to a final target conformation. The simulation is broken up into a series of short free MD segments, and we determine, after each segment of MD, whether or not the system has evolved toward the final conformation. Those segments that do not move the system in that direction are deleted. Those that do move it toward the final conformation are patched together sequentially to generate a single representative trajectory along the transition pathway. The CONTRA MD method is demonstrated first by application to the simultaneous C2′-endo to C3′-endo repucker and anti to syn N-glycosidic torsion transitions in 2′-deoxyadenosine and then to the large-scale bending in phenylalanine transfer RNA. © 1993 John Wiley & Sons, Inc.