Regional distribution of Sindbis virus glycoproteins on the plasma membrane of infected baby hamster kidney cells

Sindbis virus-infected baby hamster kidney (BHK) cells were analysed in surface replicas or conventional thin sections after specific immunolabelling with antiviral glycoprotein antibodies in conjunction with colloidal gold-conjugated protein A. Newly synthesized viral glycoproteins were detected, beginning 1 1/2 h after infection, while the virus maturation started 3 h after infection. The glycoproteins appeared to be inserted on the plasma membrane in large spots located mainly in the central area of the cells: no clustering of the labelling was detected. Later, the glycoproteins appeared to arrange linearly in regions in the medial portion of the cells. No labelling was found in the peripheral area or on the cell edges. A drastic change in the surface labelling was detected following the commencement of virus maturation: gold particles were organized mostly in small clusters, each labelling a budding virus. Very few glycoproteins appeared not to be involved in budding figures. The maturation of the virus was clearly regionalized, but during this time it also involved the peripheral area and the cell edges; preferential budding in narrow cellular processes was often observed. It appeared thus that either isolated glycoproteins soon after infection, or clustered glycoproteins at later times, are strictly regionalized on the plasma membrane: however, the early post-infection distribution is clearly different from that seen later during virus maturation. Our experiments support the concept of discrete plasma membrane domains even in cells that do not display distinct specialization of their surface.     

Evolutionary Origin of Nonuniversal Cug(ser) Codon in Some Candida Species as Inferred from a Molecular Phylogeny

CUG, a universal leucine codon, has been reported to be read as serine in various yeast species belonging to the genus Candida. To gain a deeper insight into the origin of this deviation from the universal genetic code, we carried out a phylogenetic analysis based on the small-subunit ribosomal RNA genes from some Candida and other related Hemiascomycetes. Furthermore, we determined the phylogenetic relationships between the tRNA(Ser)CAG, responsible for the translation of CUG, from some Candida species and the other serine and leucine isoacceptor tRNAs in C. cylindracea. We demonstrate that the group of Candida showing the genetic code deviation is monophiletic and that this deviation could have been originated more than 150 million years ago. We also describe how phylogenetic analysis can be used for genetic code predictions.     

Characterization of beta-adrenergic receptors in the rat vas deferens using [3H]-dihydroalprenolol binding

The beta-adrenergic antagonist, [3H]-dihydroalprenolol ([3H] DHA), binds to membranes prepared from the rat vas deferens in a specific and saturable manner. Scatchard and Hill plot analysis indicates a single class of binding sites with no evidence of cooperative interactions. The specific binding sites have a high affinity (Kd = 0.3 nM) and a maximal occupancy estimated to be 460 fmoles [3H]-DHA bound/g wet tissue weight. Beta-adrenergic agonists and/or antagonists inhibit [3H]-DHA binding to rat vas deferens membranes in a stereospecific manner and with a relative order of potency expected for beta-adrenergic receptors of the beta2 subtype. The receptor affinities of various beta-adrenergic antagonists in the rat vas deferens determined using inhibition of [3H]-DHA binding correlated with their receptor affinities determined physiologically using antagonism of isoproterenol-induced inhibition of neurogenic contractions in-vitro.     

Defining Structural Domains of an Intrinsically Disordered Protein: Sic1, the Cyclin-Dependent Kinase Inhibitor of <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis>

The cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) involved in cell–cycle regulation in the yeast Saccharomyces cerevisiae. Notwithstanding many studies on its biological function, structural characterization has been attempted only recently, fostering the development of production and purification protocols suitable to yield large amounts of this weakly expressed protein. In this study, we describe the identification of protein domains by the heterologous expression, purification, and characterization of Sic1-derived fragment. Four C-terminal fragments (Sic1^C-ter) were produced based on functional studies and limited-proteolysis results. The N-terminal fragment (Sic1^1–186) was complementary to the most stable C-terminal fragments (Sic1^Δ186). Both Sic1^1–186 and Sic1^C-ter fragments were, in general, less susceptible to spontaneous proteolysis than the full-length protein. The boundaries of the C-terminal fragments turned out to be crucial for integrity of the recombinant proteins and required two rounds of design and production. Sic1 fragments were purified by a simple procedure, based on their resistance to heat treatment, at the amount and purity required for structural characterization. Circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectra of N- and C-terminal fragments confirm their disordered nature but reveal minor structural differences that may reflect their distinct functional roles.