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Removal of inhibitors against RNA-directed DNA polymerase activity in human milk
Abstract:
Milk from a number of species (e.g., man, mouse, rat, dog, and cow) contains inhibitors of the RNA-directed DNA polymerase. When attempts are made to isolate virions from the milk, part of the inhibitors follow the virions in the purification. The amount of inhibitors varies in different milk samples. These inhibitors can probably account for the large discrepancies reported in studies of the presence of oncornaviruses in human milk. Phosphatases bound to subcellular particles or fragments seem to be the most important inhibitors in the milk interfering with the RNA-directed DNA polymerase assay. It is shown that the inhibitory enzymes can be completely removed by sedimentation of the milk through a Metrizamide gradient.
Insights
Milk contains inhibitors that interfere with RNA-directed DNA polymerase assays. These phosphatases can be removed using Metrizamide gradient sedimentation, clarifying oncornavirus detection in human milk.
Area of Science:
- Biochemistry
- Virology
- Molecular Biology
Background:
- Milk contains inhibitors affecting RNA-directed DNA polymerase assays.
- These inhibitors can lead to discrepancies in oncornavirus detection studies.
- Phosphatases bound to milk particles are key inhibitors.
Purpose of the Study:
- To identify and characterize inhibitors in milk affecting RNA-directed DNA polymerase assays.
- To explain discrepancies in oncornavirus detection in human milk.
- To develop a method for removing inhibitory substances from milk.
Main Methods:
- Isolation of virions from milk of various species.
- Analysis of inhibitors co-purifying with virions.
- Characterization of inhibitory enzymes, particularly phosphatases.
- Metrizamide gradient sedimentation for inhibitor removal.
Main Results:
- Milk from multiple species contains RNA-directed DNA polymerase inhibitors.
- Inhibitors, primarily phosphatases, interfere with viral assays.
- Metrizamide gradient sedimentation effectively removes inhibitory enzymes.
- This purification method can resolve discrepancies in oncornavirus detection.
Conclusions:
- Milk-borne phosphatases are significant inhibitors of RNA-directed DNA polymerase.
- Effective removal of these inhibitors is crucial for accurate oncornavirus detection.
- Metrizamide gradient sedimentation offers a reliable method for purifying milk samples.