Radioactive DNA ([(3)H]cDNA) complementary to the RNA of Mason-Pfizer monkey virus was used in molecular hybridization experiments to demonstrate sequence homology between its viral RNA and RNA of human malignant breast tumors. Hybridization products were analyzed by cesium sulfate equilibrium density centrifugation and hydroxylapatite chromatography. Seven of ten human malignant breast tumors tested contained virus-related RNA when assayed by hydroxylapatite chromatography, whereas no sequence homology was found in RNA from 11 human benign breast tumors or from RNA of normal human spleen, liver, or kidney. Hybridizations between [(3)H]cDNA of the monkey virus and human breast adenocarcinoma RNA showed a C(r)t((1/2)) of about 2 x 10(4). The fidelity of the hybrids formed was indicated by a T(m) of 80.5 degrees .

Adenocarcinoma↗

PubMed1974

Conditions for using DNA polymerase I as an RNA-dependent DNA polymerase.

Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture.

Animals↗

PubMed1974

The presence of unique DNA sequences after viral induction of leukemia in mice. (RNA tumor virus-nucleic acid hybridization-insertion of viral DNA).

From previous studies, lymphocyte DNA from human leukemias and DNA from involved tissues of patients with Hodgkin's disease or Burkitt's lymphoma contain sequences that are absent from their normal counterparts. These sequences are related to those found in particulate elements associated with these neoplasias and possessing biochemical properties characteristic of RNA tumor viruses. Similar observations have been made of unique sequences related to those of the feline virus RD-114 and found in spontaneous mastocytomas in cats. Here we extend these results to the classical murine model of virus-induced leukemias. Splenic DNA from BALB/c mice with leukemia induced by Rauscher leukemia virus (RLV) possess some RLV-related sequences that do not exist in normal BALB/c DNA. Furthermore, these leukemia-specific sequences were absent in all other mouse strains examined, including AKR, a strain with a high incidence of spontaneous leukemia. The DNA of all noninfected mouse strains possesses considerable homology with the RLV genome. Temperature denaturation studies indicate, however, that although the RLV-related sequences found in all normal mice are similar to each other, they are not exactly homologous with RLV sequences. We conclude that RLV-induced leukemia in BALB/c results in the insertion of RLV sequences into cellular DNA that itself possesses only partial homology with the RLV genome.

Animals↗

PubMed1974

Evidence of particle-associated RNA-directed DNA polymerase and high molecular weight RNA in human gastrointestinal and lung malignancies.

Previous communications have demonstrated that neoplastic cells of human breast cancers, leukemias, lymphomas, sarcomas, and brain tumors contain particles with similar diagnostic attributes as those found in RNA oncornaviruses. The present paper concerns malignancies of the gastrointestinal and pulmonary systems for which, like brain tumors, no suitable animal model or corresponding virus exists. By means of the simultaneous detection assay, these tumors have been found to contain 70S RNA and RNA-directed DNA polymerase encapsulated in particulate components possessing densities of 1.16-1.17 g/ml. Twelve out of 17 (70%) colon carcinomas, three out of five (60%) gastric carcinomas, all of three rectal carcinomas, and seven out of ten (70%) lung carcinomas contained detectable levels of these virus-like entities. None of the corresponding normal tissues was positive.

Centrifugation, Density Gradient↗

PubMed1974