An analysis of histone modifications in relation to sex-specific chromatin organization in the mealybug Maconellicoccus hirsutus.

The male-specific heterochromatization of the paternal genome, observed in coccids, is an example of both genomic imprinting and differential regulation of homologous chromosomes. We observed a highly nuclease-resistant chromatin (NRC) organization of a part of the paternal genome in males of Maconellicoccus hirsutus as reported earlier in Planococcus lilacinus. The nuclease resistance of NRC is correlated with nuclear matrix association and is lost when NRC is dissociated from the matrix. We carried out a comparative analysis of epigenetic modifications of histones in matrix associated chromatin of male and female mealybugs by ELISA. We detected H3K27me3, H4K20me3, H3K9me2 and H3K4me3 in both males and females and observed significant enrichment of H3K27me3 in the nuclear matrix of males compared to that of females. To further examine the presence of NRC in the germ line, nuclei were sorted based on chromatin compaction. The analysis of sorted nuclei indicates the presence of NRC in nuclei with different DNA content including the haploid nuclei from males. We discuss these results in the light of the presence of NRC exclusively in male nuclei and the retention of the maternal genome in sperm nuclei of mealybugs.

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum.

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.

Stimulation by DIF causes an increase of intracellular Ca2+ in Dictyostelium discoideum.

Using fluorescence-activated cell sorting (FACS), we have studied the effect of the differentiation-inducing factor (DIF) on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or postaggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or "low Ca2+" class exhibits a prespore tendency and the H or "high Ca2+" class exhibits a prestalk tendency. Upon adding DIF, within 2 min there is an approximately twofold increase in the relative fraction of amoebae falling in the H class. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with the cellular heterogeneity in respect of Ca2+ content, there is a heterogeneity in the response to DIF, which appears to be restricted to L cells.

A Ca(2+)-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry.

When freshly starved amoebae of Dictyostelium discoideum are loaded with the Ca(2+)-specific dye indo-1/ AM and analyzed in a fluorescence-activated cell sorter, they exhibit a quasi-bimodal distribution of fluorescence. This permits a separation of the population into two classes: H, or "high Ca(2+)-indo-1 fluorescence," and L, or "low Ca(2+)-indo-1 fluorescence." Simultaneous monitoring of Ca(2+)-indo-1 and Ca(2+)-chlortetracycline fluorescence shows that by and large the same cells tend to have high (or low) levels of both cytoplasmic and sequestered Ca2+. Next we label H cells with tetramethylrhodamine isothiocyanate (TRITC) and mix them in a 1:4 ratio with L cells. In the slugs that result, TRITC fluorescence is confined mainly to the anterior prestalk region. This implies that amoebae with relatively high Ca2+ at the vegetative stage tend to develop into prestalk cells and those with low Ca2+ into prespores. Polysphondylium violaceum, a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca(2+)-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azhar et al., Curr. Sci. 68, 337-342 (1995)), a prestalk-prespore difference in cellular Ca2+ is present in the cells of the slug in vivo. These findings are discussed in light of the possible roles of Ca2+ for cell differentiation in D. discoideum.

Incomplete splicing of simian virus 40 large T antigen transcripts in a transformed mouse cell line.

The SV40 large T antigen transcripts from an SV40-transformed mouse embryo fibroblast cell line (215) have been analyzed by Northern blots and by mapping and sequencing of the corresponding cDNAs. We have observed that T antigen mRNA is highly overexpressed in the 215 cells, and is mostly unspliced, but there are no sequence changes at the splice sites. However, normal amounts of wild-type T antigen are produced, suggesting that the splicing and translation of T antigen RNA is tightly controlled in these cells.

Nuclear transport of proteins translated in vitro from SP6 plasmid-generated mRNAs.

A sensitive and versatile assay is described for the nuclear transport of 35S-labeled proteins obtained by the in vitro translation of SP6 plasmid-generated mRNAs. A specific nuclear accumulation of greater than 20-fold is observed for the transformation-related nuclear proteins, p53 and E1b, and the nuclear enzyme, thymidine kinase, whereas transport of the nonnuclear proteins, dihydrofolate reductase and simian virus 40 small t antigen, is negligible within 30 min.