Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions.

The cdc2/cdk2 protein kinases play key roles in the cell cycle at two control points: the G1/S transition and the entry into mitosis. Olomoucine, a specific inhibitor of these kinases, was tested in two plant cell systems: Petunia mesophyll protoplasts induced to divide and Arabidopsis thaliana cell suspension cultures. The cell cycle status was analysed from DNA histograms or through continuous labelling of cells with 5-bromodeoxyuridine (BrdUrd) followed by double staining with bis-benzimide (Hoechst 33258) and propidium iodide (PI). Such analyses resolve cells from several generations according to the extent of their DNA replication. Olomoucine was shown to reversibly arrest differentiated Petunia cells induced to divide at G1 phase and cycling Arabidopsis cells in late G1 and G2. A comparison of the effects of aphidicolin, oryzalin and olomoucine suggests that in the Arabidopsis cell suspension culture, a cdc2/cdk2-like kinase is activated at a restriction point in late G1.

Distribution of rDNA and 28S, 18S, and 5S rRNA in micronuclei containing a single chromosome.

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S-28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S-28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a-e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S-28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S-28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.

Three-dimensional organization of micronuclei induced by colchicine in PtK1 cells.

In PtK1 cells micronucleated by colchicine, we previously demonstrated that some micronuclei contain a single chromosome. Here, we investigated interphase chromosome organization in micronucleated PtK1 cells using conventional electron microscopy and three-dimensional computer reconstruction. The distribution of micronuclei was not always polarized, but in some cells they formed a ring. When this occurred, centrioles and Golgi apparatus were located inside the ring. On freeze-fracture replicas, we observed that nuclear pore distribution among the micronuclei was heterogeneous, and on thin sections some micronuclei displayed an incomplete nuclear envelope, with gaps in the double membrane and areas without lamina or condensed chromatin. By autoradiography, we showed that the fibrillar dots were not sites of active transcription. We applied three-dimensional reconstruction to one micronucleated cell containing 22 micronuclei whose size indicated that each micronucleus probably contained one chromosome. In this cell we demonstrated that only the smallest micronuclei had an incomplete nuclear envelope. The presence in micronuclei of either nucleoli or fibrillar dots was found to be mutually exclusive. These dots might constitute stores of nucleolar proteins which migrate into micronuclei possessing no ribosomal genes. In NOR-bearing micronuclei, the structural organization was similar to that of diploid nuclei: the nucleoli were attached to the nuclear membrane and a nucleolar canal was seen, even in single-chromosome spherical micronuclei. Taken together, these findings indicate that in the diploid nuclei of PtK1 cells, the three-dimensional organization of the nucleolar domain seems to be directly controlled by the X-chromosome.

Identification and sorting of micronuclei containing individual chromosomes.

Micronuclei are diploid genome subfractions which can be induced by colchicine treatment. The purpose of the present study was to identify the chromosome content of micronuclei. Accordingly, PtK1 cells were micronucleated with colchicine and their DNA was labeled by the Hoechst 33342 fluorochrome. The fluorescence intensity of the micronuclei was measured by flow cytometry and compared to that of metaphase chromosomes of the same species. We observed histograms exhibiting five distinct peaks for both micronuclei and chromosomes. Each class of micronuclei corresponded to a peak in the flow karyotype with the same values. As the smallest micronuclei contained the same amount of DNA as one chromosome, it seemed possible to identify the DNA content of the micronuclei containing a single chromosome. This identification was confirmed by fluorescence microscope observations of the micronuclei and chromosomes sorted from their respective peaks. This was clear for peak number 3, which exhibited fluorescent markers both in micronuclei and in chromosomes. These markers, called chromocenters, were connected with the nucleoli in micronuclei and were specific for the nucleolar organizer regions of the X chromosomes. As we paid particular attention to maintaining the transcriptional activity of the micronuclei throughout the procedure used to sort and identify them, it is concluded that such an approach might be very suitable for investigating the expression of reduced nuclear domains corresponding to individual chromosomes.

RNA polymerase activity in PtK1 micronuclei containing individual chromosomes. An in vitro and in situ study.

Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.

Sorting of micronuclei from PtK1 cells.

We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells.

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK1 cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In 'early restoration' cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK1 cells in G1 and those in G2/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1-2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.

Procedure for isolating micronuclei from rat kangaroo cultured cells containing individualized chromosomes.

Micronuclei are small interphase nuclei containing part of the genome; the DNA content of the smallest micronuclei is equivalent to one chromosome. For analysis by biochemical method and by cytofluorometry of interphase micronuclei containing a single chromosome, several isolation and purification procedures were tested and checked by fluorescent microscopy using the DNA dye Hoechst 33 342 and electron microscopy. Micronucleation of rat kangaroo epithelial cells was induced by colchicine treatment for three days. Micronuclei were isolated in a low ionic strength buffer containing collagenase, with concomitant mechanical shocks. Eighty % of the micronuclei were released after 3 to 7 min, with minimum nuclear breakage. Subsequent filtration through several polycarbonate filters 12, 8 and 5 micron in diameter enabled purification of the smallest micronuclei without aggregates or debris. Micronuclear morphology was well preserved, as shown by electron microscope observations. Therefore, we established the optimal conditions allowing gentle mass isolation of individual micronuclei of cultured PtK1 cells, compatible with flow cytometry analysis.