Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles

The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores. These particles are unusual in that they have variable radif. A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series. Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.     

Chromosomal and genomic organization of Ty1-copia-like retrotransposon sequences in the genus Avena

Regulation of ligand-mediated signal transduction through transmembrane tyrosine kinase growth factor receptors involves phosphorylation of tyrosine residues in the intracellular domain of the receptor. The insulin-like growth factor-I (IGF-I) receptor contains three tyrosine residues in the carboxy-terminal domain at positions 1250, 1251, and 1316. Of these, only the tyrosine at position 1316 is conserved in the homologous position of the insulin receptor. Mutational analysis was used to study the role of these tyrosines in specific outcomes of IGF-I-mediated signal transduction. Mutations in the human IGF-I receptor were either replacement of tyrosines 1250 and 1251 with phenylalanine and histidine (yyFH), respectively, or replacement of the conserved distal tyrosine (position 1316) with phenylalanine (yCF). The yyFH mutation results in an IGF-I receptor with the amino acids found in the homologous position of the human insulin receptor. Cells overexpressing mutated IGF-I receptors were compared with cells expressing only endogenous IGF-I receptors or overexpressing wild-type IGF-I receptors. The ability of yyFH mutant IGF-I receptors to autophosphorylate the beta-subunit or phosphorylate insulin receptor substrate-1 was not significantly different from wild-type type IGF-I receptors. However, one or both of the proximal tyrosine residues (positions 1250 and 1251) in the carboxy-terminus of the IGF-I receptor are essential for IGF-I-stimulation of mitogenic and tumorigenic pathways. IGF-I-induced mitogenesis, measured as thymidine incorporation and cellular proliferation, was abrogated in cells overexpressing mutant IGF-I receptors with replacement of the proximal double tyrosines (positions 1250 and 1251). Fibroblasts expressing this mutant IGF-I receptor formed fewer tumors than the negative control cells, whereas cells expressing wild-type IGF-I receptors formed large tumors in all recipient mice injected. Conversely, cells expressing mutant IGF-I receptors with only the conserved distal tyrosine (position 1316) replaced had slightly reduced IGF-I-stimulated beta-subunit autophosphorylation, thymidine incorporation, and cellular proliferation when compared with cells expressing wild-type receptors. Phosphorylation of insulin receptor substrate-1 by the yCF mutant receptors was not impaired. Despite the ability of these mutant receptors to stimulate mitogenic growth, fibroblasts expressing this mutant receptor were also incapable of forming tumors in recipient nude mice. The distal tyrosine (position 1316) of the IGF-I receptor is crucial for tumor formation but is not essential for IGF-I stimulated mitogenesis. Thus, the tyrosine moieties in the carboxy-terminus of the IGF-I receptor participate in the signal transduction pathways that affect the mitogenic and tumorigenic potentials of cells expressing mutant IGF-I receptors.     

In vitro integration of retrotransposon Ty1: a direct physical assay

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.     

Cell cycle control of chorion gene amplification

Over-replication of two clusters of chorion genes in Drosophila ovarian follicle cells is essential for rapid eggshell biosynthesis. The relationship of this amplification to the follicle cell cycles has remained unclear. To investigate the regulation of amplification, we developed a technique to detect amplifying chorion genes in individual follicle cells using BrdU incorporation and FISH. Amplification occurs in two developmental phases. One of the gene clusters begins to amplify periodically during S phases of follicle cell endocycles. Subsequently, after endocycles have ceased, both clusters amplify continuously during the remainder of oogenesis. In contrast to the early phase, late amplification commences synchronously among follicle cells. The pattern of Cyclin E expression mirrors these two phases. We present evidence that Cyclin E is required positively for amplification. We suggest that Cyclin E also acts negatively to inhibit refiring of most origins within a cycle, and that specific factors at chorion origins allow them to escape this negative rereplication control. Our findings suggest that chorion amplification is a model for understanding metazoan replicons and the controls that restrict replication to once per cell cycle.     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Differentiated cells have been established in monolayer culture from adult rat liver and their ultrastructural and biochemical features characterized after 20-30 generations. Hepatocytes were isolated by enzyme perfusion of the liver followed by treatment with papain, which allowed cultures to be established more readily and to be cloned at an early stage. Ultrastructural studies indicated that the cells were derived largely from hepatic parenchymal cells. The cells showed structural modifications during primary culture but were stable thereafter. The cultured cells retained some differentiated functions unique to liver cells, including the synthesis of ornithine form arginine and the secretion of serum proteins, albumin, chi- and beta-globulins.     

In vitro selection for different mutational patterns in the HIV-1 reverse transcriptase using high and low selective pressure of the nonnucleoside reverse transcriptase inhibitor HBY 097

Measurement of Na-Ca exchange activity was used to examine subsarcolemmal sodium levels ([Na+]s) in single, voltage-clamped guinea-pig cardiac myocytes while Na-K pump activity was modulated pharmacologically. Changes in Nas were evaluated from phase-plane analysis of the changes in intracellular calcium, measured using the fluorescent indicators Fura-red and Fluo-3. Activation of beta-adrenoceptors with 1 microM isoprenaline resulted in activation of the cAMP-dependent chloride current, but had no effect on the calcium transient mediated via the Na-Ca exchanger, regardless of whether the Na-K pump was active or inhibited (with strophanthidin). The ability of Na-Ca exchange activity to report [Na+]s was demonstrated by the effect of changing the extracellular rubidium concentration from 1 to 5.4 mM to modulate Na-K pump activity. We suggest that beta-adrenergic stimulation does not directly affect either the Na-K pump or the Na-Ca exchanger and that the Na-Ca exchanger can be used as a sensitive indicator of changes in [Na+]s and Na-K pump activity.