Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

Fibrinogen--proteolysis in acute myelogenous leukemia (AML)

Fibrinopeptides were measured as direct indices of thrombin, plasmin and elastase in plasma samples obtained from patients with AML. Peptide patterns observed were consistent with spontaneous or drug induced plasmin-specific fibrinogenolysis (AML FAB M 1/3), elastase mediated proteolysis (AML FAB M 3/4) or DIC (AML FAB 4/5). DIC was also observed in septic, agranulocytotic patients.     

Isolation and characterization of a family of alpha-D-galactosyl-containing glycopeptides from Ehrlich ascites tumor cells

A family of glycopeptides that contain nonreducing terminal alpha-D-galactosyl residues has been isolated from Pronase digests of delipidated Ehrlich ascites tumor cells. The glycopeptides, which comprise 17.2% of the total plasma membrane hexose, have an average molecular weight of 7500 and are precipitated by Griffonia simplicifolia B4 isolectin, wheat germ agglutinin, and Ricinus communis lectin. Exo- and endoglycosidase digestion, periodate oxidation, permethylation analysis, and lectin reactivity provided evidence for a tentative carbohydrate structure for the glycopeptide mixture. The glycopeptides possess tetraantennary branched structures containing a trimannosyl core N-glycosidically linked via an N,N'-diacetylchitobiosyl unit to an asparagine residue. Each branch contained repeating leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units resulting in a keratan-like structure, terminated with alpha-D-Galp-(1 leads to 3)-[alpha-D-Galp-(1 leads to 6)]-beta-D-Galp-units. The variation in the molecular weight observed for the glycopeptide mixture can be attributed to the variable amounts of leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units found in the branch chains.     

Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12.

We compare the nucleotide sequences of the regulatory regions of five genes or groups of genes of the arginine regulon of Escherichia coli K-12: argF, argI, argR, the bipolar argECBH operon and the carAB operon. All these regions harbour one or two copies of a conserved 18 bp sequence which appears to constitute the basic arginine operator sequence (ARG box). We discuss the influence of ARG box copy number, degree of dyad symmetry, base composition, and position relative to the cognate promoter site on the derepression-repression ratios of the genes of the regulon. A novel hypothesis, based on structural considerations, is also put forward to account for the absence ot attenuation control.     

Autocatalytic polysialylation of polysialyltransferase-1.

Polysialic acid (PSA) is a specific and highly regulated post-translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase-1 (PST-1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST-1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST-1. The synthesis of PSA chains, which involved N-glycosylation sites, occurred immediately after contact with the activated sugar donor CMP-Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo-PST-1 isolated from CHO cells of the Lec2 complementation group. Pre-formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto-PST-1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST-1.     

The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities.

ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre-mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre-mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high-affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing-deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers.