Rna-Dependent Dna-Polymerase Activity in Human Milk

Abstract

PARTICLES resembling type B mouse mammary tumourvirus have been detected by electron microscopy in human milk (Feller and Chopra, 1969;Moore et al., 1969Moore et al., , 1971 Sarkar and Moore, 1972). Their frequency was found to be much higher in women with a family history of breast cancer than in women without such a history (Moore et al., 1971). Schlom, Spiegelman and Moore (1971) were able to correlate the presence of the particles with RNAdependent DNA-polymerase activity, now known to be associated with oncornaviruses, in the milk. On the other hand, Calafat and Hageman (1973) failed to find either type B or type C-like particles in human milk.We have applied a simple and reliable test for RNA-dependent DNA-polymerase activity to determine indirectly the presence of oncornavirus particles in milk samples obtained from nursing mothers. The test examined (1) the ability of the pellet obtained by centrifuging milk at 145,000g to incorporate radioactive thymidine triphosphate into DNA in the presence of all four unlabelled deoxyribonucleoside triphosphates, and (2) the sensitivity of this incorporation to inhibition by ribonuclease, as a check on the RNA-dependent nature of the enzyme reaction.MATERIALS AND METHODS Ten-to 30-ml samples' of milk were collected from mothers at the Grace Maternity Hospital, Halifax, NS., on the 5th or 6th day post-partum. Each sample was taken from a single breast. The women were questioned at the same time about the family history of breast cancer. The migratory nature of the population made it impossible to check the family history from medical records, but any women who were doubtful on this point were excluded from the study.All samples were tested within 1 h of collection. The method was a modification of the technique of Schlom, Spiegelman and Moore (1972). The milk, diluted with an equal volume of 0 . 1 5~ EDTA (disodium ethylenediaminetetraacetic acid) was centrifuged in a refrigerated International Centrifuge, model B-20, for 10 min. at 3000g. The clarified fluid between the surface layer and the casein deposit was removed and centrifuged again for 10 min. at 3000g to separate out the final content of lipid. This was carefully discarded, and the sample was incubated at 30°C for 30 min. with trypsin, added to a h a l concentration of 1 mg per ml. Lima-bean trypsin-inhibitor (Sigma Chemical Co., St Louis, Miss., USA) was then added to a final concentration of 0.5 mg per ml, and the treated material was layered over 10 ml of 20% glycerol and centrifuged at 145,000 g for 60 min. in a model B-60 refrigerated centrifuge. The resulting pellet was suspended in 0.5 ml of 0 . 0 1~ trishydroxymethyl aminomethane (pH 8.3) containing 0-33 % (v/v) "-40 detergent and 0 -l~ dithiothreitol, and kept at 4°C for 30 min.The suspension was added to a standard reverse-transcriptase reaction-mixture containing 10 p.mole of tris-hydroxymethyl aminomethane (PH 8.3), 1 p.mole of magnesium acetate, 12 p.mole of sodium chloride, 4 p.mole of dithiothreitol, 0.2 p.mole each of unlabelled deoxyadenosi...