Absence of DNA in the basal body ofChlamydomonas reinhardtii by fluorimetry using a video-intensified microscope photon-counting system

Abstract

A search was made for DNA in both the basal bodies (BBs) in situ and BBs isolated from cells ofChlamydomonas reinhardtii by high-resolution epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS) and by immunofluorescence microscopy after staining with a monoclonal tubulin-specific antibody. The flagella and intracellular microtubules radiate from the BBs. The BBs in young vegetative cells, gametes and young zygotes do not emit fluorescence after staining with DAPI but the spherical cell nucleus, the ovoid chloroplast nuclei and the tiny mitochondrial nuclei emit bright, blue-white fluorescence. Thus, it appears that BBs do not contain larger amounts of DNA than do the other organelles. To avoid the halation effects of fluorescence from the cell debris and cytoplasm and to measure carefully any extremely low levels of DNA that might be present in the organelles, a complex, composed of two flagella, a pair of BBs and the cell nucleus, was isolated from the gametes by treatment with autolysin and 0.1% Triton X-100. After staining with DAPI, the BBs of such complexes exhibit faint fluorescence while the cell nucleus emits strong fluorescence. The point and total intensities of the fluorescence emitted from each portion of the complex were measured with the VIMPICS. When the fluorescence intensity “T” of T 4 phage is taken as a standard, the fluorescence intensities of the flagella, the pair of BBs, the cell nucleus and the nucleus ofEscherichia coli are respectively 0.2 T, 0.40 T, 1452.2 T and 20.4 T. The slight fluorescence emitted from the BB seems to be due to the halation of the fluorescence emitted from the cell nucleus. The intensity of the fluorescence from the BBs is reduced to the intensity of the fluorescence of the flagella when the cell nucleus is removed from the complex. From these results, we conclude that the BBs do not contain DNA. Discrepancies related to the reported presence of DNA in the BBs are discussed.