G1 Protein Research Articles

Monoclonal antibodies against La Crosse virus.

Monoclonal antibodies have been used to show that an epitope is present on the G1 glycoprotein of prototype La Crosse virus that is absent or significantly altered on several isolates of La Crosse virus made in New York State, U.S.A. The portion of the G1 protein where this epitope is located plays a role in both virus neutralization and haemagglutination. Additional experiments revealed that under the appropriate assay conditions the monoclonal antibodies permitted discrimination between representatives of the North American members of the California serogroup.

Structural and cell-associated proteins of Lassa virus.

Lassa virus was purified from culture fluids of infected CV-1 monkey kidney cells and its structural proteins analysed by polyacrylamide gel electrophoresis. Stained gels showed a typical arenavirus profile, with a prominent protein of molecular weight 60000, corresponding to the nucleocapsid protein N, and two faint broad bands with molecular weights of 45000 and 38000, the envelope glycoproteins G1 and G2. G1 and G2 were shown to be glycosylated by their ability to bind concanavalin A to nitrocellulose transfers of the separated proteins ('Western blots'). N and G2 bound antibody from guinea-pig or human convalescent sera but G1 was inactive, presumably as a result of denaturation. This technique also revealed other apparently virus-specific minor bands with molecular weights of 76000 and 68000. When Western blots of proteins of infected cells which had been lysed in SDS were probed with anti-Lassa virus serum or stained for glycoproteins, four virus-specific bands were apparent: the N, G1 and G2 proteins seen in purified virus, and a glycoprotein of molecular weight 72000 which probably corresponds to the envelope protein precursor (GPC) seen in other arenavirus systems. Immunoprecipitates from infected CV-1 cells labelled with [35S]methionine contained three major virus-specific proteins: the nucleocapsid protein N and proteins of 36000 and 24000 molecular weight (designated fN1 and fN2). Similar immunoprecipitates from Vero cells contained fN1 and fN2 and only very low levels of N. The polypeptides fN1 and fN2 are most probably fragments of N, since Western blots probed with anti-Lassa virus serum showed that lysis of cells in non-ionic detergent rather than SDS results in the appearance of fN2 with concomitant reduction or disappearance of N. These fragments do not exist in the intact cell, but are found as a consequence of rather specific proteolysis upon disruption under non-denaturing conditions. The proteolytic activity responsible was refractory to inhibition by phenylmethylsulphonyl fluoride, aprotinin, pepstatin A or sodium bisulphite, and was more active in Vero than in CV-1 cells.

Monosaccharide sequence of protein-bound glycans of Uukuniemi virus.

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [(3)H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [(3)H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)(3)(GlcNAc)(2) peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [(3)H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [(35)S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.

Genetic interactions among viruses of the Bunyamwera complex.

Seventy-seven temperature-sensitive (ts) mutants belonging to three antigenically distinct and geographically isolated members of the Bunyamwera complex--Batai virus, Bunyamwera virus, and Maguari virus--have been isolated after 5-fluorouracil treatment. High-frequency recombination was observed, and the mutants of each virus were classified into two groups, which were shown to be equivalent by heterologous recombination experiments. In most combinations heterologous recombination was less efficient than homologous recombination, but all crosses of group I and II mutants yielded viable recombinants. Recombination was an early event. Analysis by polyacrylamide gel electrophoresis of the proteins of the wild-type viruses and recombinant clones obtained from the six possible heterologous combinations of group I and II mutants indicated that recombination occurred by reassortment of genome subunits. Group I appeared to correspond to the genome subunit coding for the N protein, and group II corresponded to the G1/G2 determinant. The G1 (or G2 or both) protein was associated with neutralization specificity and plaque diameter, and the N protein was associated with plaque opacity. Complementation was observed between two nonrecombining mutants of Maguari virus belonging to group I, which may indicate that the N genome subunit codes for an additional protein. There appeared to be no genetic barrier to exchange of genetic material between Batai, Bunyamwera, and Maguari viruses in vitro, and it is concluded that the Bunyamwera complex is potentially a single gene pool if geographical and ecological constraints are discounted.

Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages.

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Clathrin-coated vesicles have been purified from CHO cells infected with vesicular stomatitis virus and shown to contain G protein in amounts nearly stoichiometric with clathrin. Pulse-chase experiments have demonstrated that this G protein is a transit form and have revealed that G is transported to the cell surface in two successive waves of coated vesicles. The oligosaccharides of G1 protein carried in the early wave are of the "high-mannose" variety which can be cleaved by the enzyme endoglycosidase H; the oligosaccharides of G2 protein in the second, later wave are resistant to endoglycosidase H. The early wave is therefore proposed to correspond to transport of G protein in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, where the oligosaccharides are processed and resistance to endoglycosidase H is conferred; the succeeding wave would represent transport from the Golgi apparatus to the plasma membrane.

Messenger RNA for G1 protein of French bean seeds: Cell-free translation and product characterization

The fraction of poly(A)-containing RNA isolated from ripening bean (Phaseolus vulgaris) cotyledons that sedimented at 16 S in linear logarithmic sucrose gradients was at least as active a messenger as viral RNA when added to a cell-free protein-synthesizing system from wheat germ. The major products synthesized in vitro were polypeptides of about 47,000 and 43,000 daltons, corresponding to two of the three subunits of G1 protein, the most abundant bean seed storage protein. No trace of the largest (53,000 daltons) subunit was found among the polypeptides synthesized in vitro. Proof that the 47,000- and 43,000-dalton polypeptides coded by the 16S RNA were indeed subunits of G1 protein was obtained by immunoprecipitation with monovalent antibody to G1 protein and by electrophoretic mapping of peptides on acrylamide gels after digestion of mixtures of authentic protein and radioactive translation products with protease V8, chymotrypsin, and trypsin. The subunits synthesized in vitro were slightly smaller than the native subunits, probably because they lacked the sugar residues present on the holoprotein.

In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes.

Translation in vitro of the mRNA coding for the vesicular stomatitis virus membrane glycoprotein G in a membrane-free ribosomal extract from HeLa cells allowed the synthesis of only the unglycosylated protein G1 (molecular weight, 63,000). Addition of stripped crude microsomal membranes from HeLa cells resulted in the conversion of G1 to the glycosylated protein G2 (molecular weight, 67,000). The G2 protein synthesized by the reconstructed microsomal membrane/ribosome system was found to be segregated inside the microsomal membrane vesicles and was thus protected from the proteolytic action of trypsin and chymotrypsin. Stripped membranes were required at an early stage of protein synthesis for the synthesized protein to be inserted into the membrane vesicles and to be glycosilated. The segregated protein G2, however, was not completely protected from proteolytic digestion, showing that a portion of the polypeptide chain of about 3000 daltons was present on the cytoplasmic side of the membrane vesicle. Our data thus suggest that, unlike the secretory proteins, the membrane glycoproteins are not completely discharged across the microsomal membranes.

Equal Expression of the Maternal and Paternal Alleles for the Polypeptide Subunits of the Major Storage Protein of the Bean Phaseolus vulgaris L.

Discontinuous sodium dodecyl sulfate slab gel electrophoresis of G1 globulin from several strains of Phaseolus vulgaris L. seed permitted clear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F(1) seed from the cross BBL 240 x PI 229,815 showed four polypeptides on dissociation of the G1 protein; however, the amount of each of the 53,000 and 50,500 subunits was half that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues.

Heritable Variation in a Polypeptide Subunit of the Major Storage Protein of the Bean, Phaseolus vulgaris L.

Electrophoretic analysis of the major seed protein, G1 globulin, from four strains of Phaseolus vulgaris L. revealed a three-banded pattern for two strains having a high methionine content (BBL 240 and PI 302,542). The other two strains (PI 207,227 and PI 229,815) known to have a lower seed methionine content, had a two-banded subunit pattern for the G1 globulin. Analytical ultracentrifugation confirmed that globulin from the two-banded strains underwent pH-dependent reversible dissociation similar to that previously found for a three-banded cultivar; additionally, the protomer molecular weight showed that three subunits of about 50,000 molecular weight each were present in the G1 globulin of the two-banded strain. Gel patterns of G1 globulin from the two strains used as parents, BBL 240 and PI 229,815, showed differences in the largest subunit, which appeared as either a 53,000 molecular weight polypeptide known to be present in the three-banded strain, or as a shorter polypeptide having a molecular weight close to 47,000. Analysis of G1 protein from portions of single hybrid seeds showed a banding pattern intermediate between the two- and three-banded types. The subunit pattern from all seeds with intermediate-banded parents segregated in a manner consistent with that expected for control of the polypeptide by a single Mendelian gene. The remaining portions of the seeds were grown to confirm that they represented true crosses. The procedures used are essentially nondestructive, and can be used as a basis for selecting seeds having different protein characters.

Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.

The disulphide bridges of a mouse immunoglobulin G1 protein.

[(35)S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy-light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab')(2) fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species.

Effects of actinomycin D and puromycin on the cell progress from M to G 1 and S stages in cultured mouse leukemia L5178Y cells.

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G1 stage, but blocked their progress from G1 to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G1 stage. Actinomycin D in this concentration also significantly depressed uridine‐3H uptake into G1 stage cells, but did not suppress leucine‐3H uptake by M and G1 cells. This suggests that some proteins may be synthesized in M and G1 stage cells by messenger RNA left over from the previous cell cycle. However, entry of G1 cells into S stage would require synthesis of new messenger RNA near the beginning of G1 stage. Puromycin (10 μg/ml) did not prevent M cells from entering G1 stage, but blocked their progress from G1 to S stage. The site of blockage was about 0.7 hr before S stage or in the first two‐third of G1 stage. This might be the site where the cells synthesize new G1 proteins necessary for entry to S stage.Comparison of sensitivities of G1 and G2 stages to the two antibiotics reveals that the puromycin sensitivity of G1 cells was similar to that of G2 cells, but the actinomycin D sensitivity of G1 was greater than that of G2 cells.

Characterization of the Serum and Secretory Immune Systems of the Cow and Sheep

Abstract The secretory immune systems of the cow and sheep were found to be strikingly similar. The principal immunoglobulin in the saliva and colostrum of both species is a “fast” 7 S γ G1 protein which is low in carbohydrate. Although suggestive evidence was obtained for a 10 S secretory immunoglobulin, proof of its existence must await isolation and complete separation from the major 7 S γ G1 component. The secretory 7 S γ G1 is immunologically identical to the γ G1 of serum and in this study no evidence of a component analogous to the human secretory “piece” was detected.

Synthesis of G1 protein preceding DNA synthesis in cultured mammalian cells

Synthesis of G1 protein preceding DNA synthesis in cultured mammalian cells