Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgagras protein.
Simultaneous replication of murine mammary tumor virus (type B) and murine leukemia virus (type C) was demonstrated electron microscopically along continuous stretches of the plasma membrane of single cells in cultures ofthe Mm5mt/c1 cell line. Types B and C virus buds were discriminated in thin sections with the aid of a tannic acid fixative that revealed the type B surface spikes as a homogeneous band of intermediate density and constant width on the surfaces of some buds (type B), whereas others (type C) remained with relatively smooth envelopes. Both types B and C buds may contain morphologically identical horseshoe-shaped nucleoids. Therefore, their identify (type B or C) could be ascertained in thin sections only on the basis of recognition of surface spikes.
The sensitivity and specificity of the unlabeled antibody-hemocyanin bridge method for immunoelectron microscopic localization of virion and cell surface antigens has been demonstrated using a hyperimmune serum to the Rauscher murine leukemia virus structural envelope glycoprotein (gp 70). The technique localized the gp70 on the viral envelope and on the infected cell surface at dilutions of greater than 10(-3) for the primary antiviral serum. Little or no nonspecific binding of hemocyanin was detected in control experiments using high concentrations of normal nonreacting or adequately absorbed antiviral primary sera and excess antibody bridge and marker; thus, the system is highly specific. Furthermore, sensitivity can be increased approximately fivefold by marked amplification steps whereby a specimen fixing only a slight amount of hemocyanin can be subsequently treated with antihemocyanin antibody, followed by hemocyanin.
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