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HOMOLOGY AND HOMOPHONY IN BIOLOGICAL SYSTEMS. I. CONCURRENT COMMON COMPLEXING PROTEINS IN MURINE TUMORS: A DOUBLY BLIND STUDY ROBERT R. PASCAL* Production of homologous proteins by viral RNA in different strains of infected mice has been described previously [I]. However, the characterization of the template RNA has not been attempted. The combined use of computerized ultracentrifugation and immunofluorescence has now made such characterization possible. The induction of a soft tissue tumor in mice by a virus extracted from sausage has been described in an earlier report [2]. This Baloney virus sarcoma, originally induced in NZB mice, has since been transplanted to a new hybrid of Michigan-Indiana curly with Kentucky-English yellow mouse. The MIc/KEy mouse possesses the ability to grow large-sized tumors with minimal systemic effects and is now exclusively used. Baloney virus sarcomas in MIc/KEy mice were homogenized using standard techniques [3], and initial attempts were made to isolate viral RNA by a single-extraction method with the aid of a computer-assisted ultracentrifuge. The failure of the single-extraction method to isolate specific RNA led to the transcribing ofmultiple-extraction programs in a linear fashion on a computer tape. Direct playback of several transcriptions also failed to yield specific RNA. Inadvertendy, one tape was fed retrograde into the computer and led to the present, successful extraction . This method of tape reversal we have since adopted regularly and refer to as reverse transcription. The method now used to identify specific viral RNA is as follows: Cellular RNA is separated into 11 fractions, from 2 to 12 Svedberg units. An initial extraction of either 7S or 1 IS RNA is considered a positive result. An initial extraction of 2S or 12S is considered a negative * Department of Pathology, Hahnemann Medical College, 230 North Broad Street, Philadelphia, Pennsylvania 19102.© 1984 by The University of Chicago. All rights reserved. 0031-5982/84/2702-0384$01.00 276 I Robert R. Pascal · A Doubly Blind Study result. Extraction points of 3S, 4S, 5S, 6S, 8S, 9S, or 1OS RNA must be confirmed by repeated extractions. In the event that a 7S or HS RNA appears in the subsequent extractions before the confirmation of the initial point RNA the result is considered negative and a new sample must be introduced. This is, of course, a modification of thé method of Rowle dem Beaunes et al. [4]. By this technique, a hybrid 7-1 IS RNA as well as a 7S globulin has been isolated from B-V infected MIc/KEy mouse sarcoma cells. Previous work from this laboratory [5] has demonstrated the elaboration of a similar 7-1 IS RNA by B-V infected fibroblasts ofJAX mice in vitro. It was decided to ascertain ifthe B-V sarcoma 7S globulin would react with nuclear antigens of B-V infectedJAX fibroblasts. Because oflow titers of reactive 7S globulin, a multiple "sandwich" immunofluorescence technique utilizing a porcine antibody (PA) to albumin-conjugated fluorescein as a chromogen (C) and the specific 7S murine antibody (MA) was used. These two antibodies and the chromogen were linked to a nuclease (N) which allowed the complex with its specific combining site to enter the nuclei ofJAX fibroblasts and react with intranuclear antigenic material (fig. 1). Murine Antibody Nuclease Fig. 1.—Schematic representation of PA-C-MA-N complex (see text for explanation) Perspectives inBiology and Mediane, 27, 2 ¦ Winter 1984 \ 277 The multiple-sandwich technique was originally described by Supen [6], but the present modification using porcine in place ofhuman serum was found to be less cosdy. Our modified Supen sandwich technique allowed us to demonstrate a low but significant [7] reaction between B-V produced 7S globulin and B-V infected JAX fibroblasts in vitro. The increased awareness of abnormal nucleotide production in conjunction with auto-immunological depression phenomena in viral replicative trans-synthesis of neo-proteins in transformed organelles emphasizes the relevance of these findings. Further tests are necessary to establish the relationship of this work to the cure of cancer. REFERENCES 1.Marx, G.; Marx, H.; and Marx, C. Production of homologous proteins by viral RNA in different strains of mice: a previous report. Nature, Old Biol 56:223, 1957. 2.Meyer, O...


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