Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis

Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development.     

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts

Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes.     

Maltose uptake and its regulation in Bacillus subtilis

Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity. There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities. Maltose was accumulated by whole cells of B. subtilis by an energy-dependent mechanism. This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsI6 null-mutation. A strain carrying the temperature-sensitive ptsI1 mutation accumulated maltose normally at 37 degrees C but, in contrast to the wild-type, was devoid of maltose transport activity at 47 degrees C. The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.     

Crystal protein synthesis is dependent on early sporulation gene expression in Bacillus sphaericus

Insertional mutations in the spo0A and spoIIAC genes of Bacillus sphaericus 2362 were prepared by conjugation with Escherichia coli using a suicide plasmid containing cloned portions of the target genes. The mutants resembled their Bacillus subtilis counterparts phenotypically and were devoid of crystal proteins as determined by electron microscopy, SDS-PAGE and Western blots. The mutants had greatly reduced toxicity to anopheline mosquito larvae compared to the parental strain. We conclude that crystal protein synthesis in this bacterium is dependent on expression of early sporulation genes.