Serge Bauwens

Plant CDC2 is not only targeted to the pre‐prophase band, but also co‐localizes with the spindle, phragmoplast, and chromosomes

A polyclonal antiserum against the p34cdc2 homologue of Arabidopsis thaliana, CDC2aAt, was used in parallel with a polyclonal antiserum against the PSTAIRE motif to study the subcellular localization of CDC2 during the cell cycle of isolated root tip cells of Medicago sativa. During interphase, CDC2 was located in the nucleus and in the cytoplasm. The cytoplasmic localization persisted during the complete cell cycle, whereas the nuclear signal disappeared at nuclear envelope breakdown. At the beginning of anaphase, the anti‐CDC2aAt antibody transiently co‐localized with condensed chromosomes. The chromosomal co‐localization disappeared as anaphase continued and remained excluded from the separated chromosomes until cytokinesis, when CDC2 re‐located to the newly forming nuclei. We also observed a co‐localization of CDC2 with three microtubular structures, the pre‐prophase band, the spindle, and the phragmoplast.

Distribution of the rDNA and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana

The distribution of the ribosomal RNA (rRNA) genes and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana was studied for the first time through non-isotopic in situ hybridization and luminescence digital imaging microscopy. Each of the three classes of highly repetitive DNA exhibited a characteristic hybridization pattern, and one class was seen to be primarily localized on two chromocentres, which would allow it to distinguish a particular chromosome. The rDNA was consistently localized on the two largest chromocentres and on one or two smaller chromocentres. A limited number of nuclei exhibited more than four labelled chromocentres, indicative of either polypoidy or differential amplification of the rDNA. In nuclei where the nucleolus could be clearly observed, the nucleolar associated chromocentres (NACs) were seen to be labelled by the ribosomal DNA (rDNA) probe.

Quantitative fluorescence in confocal microscopy : the effect of the detection pinhole aperture on the re-absorption and inner filter phenomena

The relationship between integrated fluorescence intensity and integrated absorbance was measured in Feulgen‐stained pigeon erythrocyte nuclei hydrolysed for different periods of time and stained at different dye concentrations.

Detection of transcripts via fluorescencein-situ hybridization and confocal microscopy in whole mounts of roots ofArabidopsis thaliana

We report a modification of a previously published procedure (de Almedia Engler et al., 1994) aimed at the fluorescent detection of RNA-RNA hybrids. Transcripts ofARAth;Cyc1;1 were located in whole mounts of roots ofArabidopsis thaliana afterin-situ hybridization with digoxigenin-and biotin-labeled antisense probes. Digoxigenin- and biotin-labeled hybrids were detected by FISH with an AP-conjugated antibody against digoxigenin or AP-conjugated streptavidin for detection of biotin, with the UV-excitable dye ELF™ as a substrate. Alternatively, biotin-labeled hybrids were detected with HRP-conjugated streptavidin, with fluorescein tyramine as a substrate. Images were recorded via confocal microscopy. We report on the different fixation and reaction conditions needed for obtaining the highest signal-to-noise ratio, as well as on the requirements for mounting media, filter sets, excitation wavelengths, and recording modes used for recording confocal data sets.