Pellicular fractions, rich in basal bodies, have been obtained from Paramecium aurelia after the animals were homogenized in the presence of buffered raffinose. DNA was extracted from these fractions. This DNA was examined with respect to density and to its susceptibility to digestion by DNase while still associated with pellicles, and was found to be of two kinds: (a) Most resembles nuclear DNA in density, is completely removed from pellicles by pancreatic DNase and forms a broad band when centrifuged to equilibrium in cesium chloride. This DNA may be of nuclear origin, although a pellicular source cannot be ruled out. (b) Several minor species of DNA with densities of 1698, 1.703-1.704 and 1.717-1.718 g/cm3 (1.698 x 103, 1.703-1.704 x 103 and 1.717-1.718 x 103 kg m-3, respectively) are also present. These species were not digested when pellicles were treated with DNase. None of these DNA species occurs consistently in all preparations of pellicular DNA and the results indicate that they are probably attributable to bacteria present during culture of the animals in non-axenic medium.
To test the ability of pellicles to become associated with exogenous DNA during isolation, DNA of Escherichia coli, labelled with [3H]thymidine, was added to whole cell homogenate before isolation of pellicles. Comparison of the specific activities of DNA extracted from the homogenate with that from isolated pellicles provided evidence that exogenous E. coli DNA can bind to pellicles.
These observations suggest that pellicles or basal bodies may become contaminated with nuclear DNA during isolation. This would create a serious problem, which must be rigorously dealt with in experiments designed to demonstrate the existence of basal-body DNA in isolated pellicles by cytochemical, biochemical or autoradiographic methods. The results indicate that it has not been established beyond reasonable doubt that basal bodies or centrioles contain DNA. In addition, the results suggest that further verification of existing evidence for basalbody DNA may require further sophistication of existing analyses or a novel approach, involving different biological material or new techniques.
- Received March 3, 1969.
- Copyright © 1969 The Company of Biologists Ltd.