Presence in human breast cancer of RNA homologous to mouse mammary tumour virus RNA.
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Biomedical subjects
Publications and source records attributed to R Axel.
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Human breast cancers contain an RNA related to that of mouse mammary tumor virus. In 79% of the breast malignancies examined, this type of RNA is a 70S-component encapsulated with RNA-instructed DNA polymerase in a particle possessing the density characteristics of RNA tumor viruses. Further, the DNA synthesized by the human RNA enzyme complex hybridizes specifically with the RNA of mouse mammary tumor virus. Thus, four features diagnostic of agents similar to mouse mammary tumor virus are also exhibited by a particle found with high frequency in human breast cancers. The accumulating evidence for the involvement of RNA tumor viruses in at least some human neoplasias is becoming increasingly compelling.
A procedure is described permitting the detection of viral-specific RNA in a mouse mammary tumor. The method involves molecular hybridization with radioactively labeled DNA complementary to the RNA of the mouse mammary-tumor virus. RNA homologous to that of the mammary agent has been found in both the nuclear and polyribosomal fractions of tumor cells. The results imply that the oncogenic information is serving as messenger RNA that directs the synthesis of proteins required for virus production, and perhaps for the maintenance of the neoplastic state. The technology developed is immediately applicable to tumors of human origin.
An experimental procedure is detailed that permits the detection of 70S RNA-directed DNA synthesis in mouse mammary carcinomas. The DNA synthesized is complementary to the RNA of the mouse mammary tumor virus by molecular hybridization, thus, completing the proof that an RNA-instructed DNA polymerase has been identified. Further, RNA-instructed DNA polymerase and its 70S RNA template are physically associated in a particle that has a density characteristic of oncornaviruses. These experiments now provide the technology required to perform similar examinations of human neoplasias.
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After heating at 65 C, ribosomes isolated from Bacillus stearothermophilus were strikingly more heat-stable than comparable preparations from Escherichia coli when tested for ability to support polyuridylic acid-directed phenylalanine incorporation at 37 C. The stability of ribosomes was also determined by measurements of hyperchromicity at 259 mmu while heating them from 25 to 90 C. In standard buffer containing 0.01 m Mg(++), the T(m) (temperature at the midpoint of total hyperchromicity) of E. coli and B. stearothermophilus ribosomes was 71 and 81 C, respectively. In a magnesium-free buffer, the T(m) of E. coli and B. stearothermophilus ribosomes was 44 and 64 C, respectively. Putrescine (0.01 m) was more effective in stabilizing ribosomes from B. stearothermophilus than those from E. coli. Spermidine (0.001 m), on the other hand, was more effective in stabilizing ribosomes from E. coli than those from B. stearothermophilus. Melting curves of total ribosomal ribonucleic acid (rRNA) from E. coli and B. stearothermophilus revealed T(m) values of 50 and 60 C, respectively. Putrescine stabilized thermophile rRNA, but had no effect on E. coli rRNA. Sucrose density gradients demonstrated that thermophile 23S ribonucleic acid was degraded during storage at -20 C; the 23S component from E. coli was stable under these conditions. The results are discussed in terms of the mechanism of ribosome heat stability and the role of the ribosome in governing the temperature limits for bacterial growth.
Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.
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