scholarly journals Dynamic Distribution of an Antigen Involved in Differentiation of Quail Myoblasts Transformed with Rous Sarcoma Virus: I. Requirement of its Quantitative Expression on the Cell Surface for Myoblast Fusion.

1996 ◽  
Vol 21 (6) ◽  
pp. 515-524 ◽  
Author(s):  
Noriko Inoue-Hyodo ◽  
Jeman Kim
Keyword(s):  
Cell Surface ◽  
Rous Sarcoma Virus ◽  
Myoblast Fusion ◽  
Dynamic Distribution ◽  
Quantitative Expression ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes.

1980 ◽  
Vol 85 (2) ◽  
pp. 481-488 ◽  
Author(s):  
Y Mikuni-Takagaki ◽  
B P Toole
Keyword(s):  
Cell Surface ◽  
Rous Sarcoma Virus ◽  
Solution Treatment ◽  
Cell Detachment ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Complete Cell ◽  
Two Populations ◽  
Testicular Hyaluronidase

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.

scholarly journals Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Nuclear Membrane ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.

New procedure for DNA transfection with polycation and dimethyl sulfoxide

1984 ◽  
Vol 4 (6) ◽  
pp. 1172-1174
Author(s):  
S Kawai ◽  
M Nishizawa
Keyword(s):  
Cell Surface ◽  
Dimethyl Sulfoxide ◽  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Chicken Embryo Fibroblast ◽  
Fibroblast Cells ◽  
Dna Transfection ◽  
Transformed Cell ◽  
Rous Sarcoma ◽  
Sarcoma Virus

A new procedure for DNA transfection has been developed in a system of chicken embryo fibroblast cells and cloned Rous sarcoma virus DNA by using a polycation reagent as a mediator to adsorb DNA to the cell surface and dimethyl sulfoxide as an agent to facilitate the uptake of adsorbed DNA by the cells. In this new, simple, and convenient polycation-dimethyl sulfoxide transfection, which requires no carrier DNA even with small amounts of DNA, the number of transformed cell foci induced by Rous sarcoma virus DNA was proportional to the dose of the transfecting DNA, and chicken embryo fibroblast cells were successfully transformed by v-src-containing subgenomic DNA as well.

scholarly journals Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

1983 ◽  
Vol 3 (4) ◽  
pp. 731-746 ◽  
Author(s):  
L Rohrschneider ◽  
M J Rosok
Keyword(s):  
Cell Surface ◽  
Rous Sarcoma Virus ◽  
Soft Agar ◽  
Partial Transformation ◽  
Rous Sarcoma ◽  
Independent Functions ◽  
Sarcoma Virus ◽  
Transformation Parameters ◽  
Adhesion Plaques ◽  
Adhesion Plaque

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

scholarly journals Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport.

1986 ◽  
Vol 103 (3) ◽  
pp. 829-838 ◽  
Author(s):  
J M Hardwick ◽  
K E Shaw ◽  
J W Wills ◽  
E Hunter
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Signal Peptide ◽  
Rous Sarcoma Virus ◽  
Cleavage Site ◽  
Intracellular Transport ◽  
Virus Glycoprotein ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus

Protein sequence requirements for cleavage of the signal peptide from the Rous sarcoma virus glycoprotein have been investigated through the use of deletion mutagenesis. The phenotypes of these mutants have been characterized by expression of the cloned, mutated env genes in CV-1 cells using a late replacement SV40 vector. The deletion mutations were generated by Ba131 digestion at the XhoI site located near the 5' end of the coding sequence for the structural protein gp85, which is found at the amino terminus of the precursor glycoprotein, Pr95. The results of experiments with three mutants (X1, X2, and X3) are presented. Mutant X1 has a 14 amino acid deletion encompassing amino acids 4-17 of gp85, which results in the loss of one potential glycosylation site. In mutants X2 and X3 the amino terminal nine and six amino acids, respectively, of gp85 are deleted. During the biosynthesis of all three mutant polypeptides, the signal peptide is efficiently and accurately cleaved from the nascent protein, even though in mutants X2 and X3 the cleavage site itself has been altered. In these mutants the alanine/aspartic acid cleavage site has been mutated to alanine/asparagine and alanine/glutamine, respectively. These results are consistent with the concept that sequences C-terminal to the signal peptidase site are unimportant in defining the site of cleavage in eucaryotes. Mutants X2 and X3 behave like wild-type with respect to protein glycosylation, palmitic acid addition, cleavage to gp85 and gp37, and expression on the cell surface. Mutant X1, on the other hand, is defective in intracellular transport. Although it is translocated across the rough endoplasmic reticulum and core-glycosylated, its transport appears to be blocked at an early Golgi compartment. No terminal glycosylation of the protein, cleavage of the precursor protein to the mature products, or expression on the cell surface is observed. The deletion in X1 thus appears to destroy signals required for export to the cell surface.

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