Sequential steps in axonal targeting are mediated by carbohydrate markers

Mannose and hybrid/complex-type oligosaccharides serve as markers for both the full set of peripheral sensory afferent neurons in the leech and also for disjoint subsets of these neurons. We have shown that these various surface carbohydrates play crucial roles in the multistep process by which afferents meet their synaptic parterns in the central nervous system (CNS). The carbohydrate marker common to all these afferents allows their projections (which are fasciculated as they enter the CNS) to disperse and search out target regions. Carbohydrate markers specific for subsets of these afferents subsequently allow each subset to consolidate the position of its projections in appropriate regions of the CNS where it contacts its synaptic partners. - 1995 John Wiley & Sons, Inc.     

Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes.

Micro-injection of catalytically inactive GST-cdc2-K33R or GST-cdk2-K33R fusion proteins, each of which efficiently titrates cyclin B in oocytes and prevents assembly of cyclin B-cdc2 complexes, readily induces premature DNA replication in starfish oocytes after emission of the first polar body. Moreover, partial ablation of cyclin B mRNA by micro-injection of antisense oligonucleotides facilitates premature DNA replication induced by the dominant-negative cdc2 and cdk2 mutant proteins. We thus propose that enhanced translation of cyclin B after GVBD, a universal feature of oocyte maturation in the animal kingdom, and subsequent assembly of cyclin B-cdc2 complexes, are part of the checkpoint that prevents DNA replication in the oocyte after emission of the first polar body. MAPK inactivation is neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation. However, micro-injection of a N-terminally truncated form of the budding yeast STE11 protein, that constitutively maintains MAPK active after the second meiotic cleavage, prevents fertilized eggs from proceeding into embryogenesis, and arrests them at G2, as is the case in unfertilized eggs that cannot inactivate MAPK after the second meiotic cleavage. We thus propose that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development.     

Cytometric analysis of DNA replication inhibited by emetime and cyclosporin A

Summary DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.     

Cytometric analysis of DNA replication inhibited by emetine and cyclosporin A

DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.     

Control of DNA replication and chromosome ploidy by geminin and cyclin A

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, we used the RNA silencing-interference technique (RNAi) to eliminate the Drosophila geminin homologue from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. We found that the silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. We observed that the silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, our studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells.     

Chromosomal DNA replication initiates at the same origins in meiosis and mitosis.

Autonomously replicating sequence (ARS) elements are identified by their ability to promote high-frequency transformation and extrachromosomal replication of plasmids in the yeast Saccharomyces cerevisiae. Six of the 14 ARS elements present in a 200-kb region of Saccharomyces cerevisiae chromosome III are mitotic chromosomal replication origins. The unexpected observation that eight ARS elements do not function at detectable levels as chromosomal replication origins during mitotic growth suggested that these ARS elements may function as chromosomal origins during premeiotic S phase. Two-dimensional agarose gel electrophoresis was used to map premeiotic replication origins in a 100-kb segment of chromosome III between HML and CEN3. The pattern of origin usage in premeiotic S phase was identical to that in mitotic S phase, with the possible exception of ARS308, which is an inefficient mitotic origin associated with CEN3. CEN3 was found to replicate during premeiotic S phase, demonstrating that the failure of sister chromatids to disjoin during the meiosis I division is not due to unreplicated centromeres. No origins were found in the DNA fragments without ARS function. Thus, in both mitosis and meiosis, chromosomal replication origins are coincident with ARS elements but not all ARS elements have chromosomal origin function. The efficiency of origin use and the patterns of replication termination are similar in meiosis and in mitosis. DNA replication termination occurs over a broad distance between active origins.     

Fast control of DNA replication in response to hypoxia and to inhibited protein synthesis in CCRF-CEM and HeLa cells

In order to elucidate whether data about the fast regulation of DNA replication in dependence on oxygen supply and on a functioning protein synthesis, previously elaborated with Ehrlich ascites cells, are valid for human cells too, we repeated key experiments with CCRF-CEM and HeLa cells. The most important techniques employed were DNA fibre autoradiography and alkaline sedimentation analyses of growing (pulse-labeled) daughter strand DNA. It was found that CCRF-CEM and HeLa cells responded to transient hypoxia and to transient inhibition of protein synthesis in an almost identical fashion. Scheduled replicon initiations were reversibly suppressed and the progress rates of replication forks, which were already active before the respective inhibitory conditions were established, were reversibly slowed down. The inclusion of the fork progress rate in the response differs from Ehrlich ascites cells, which respond only by suppressing initiation. Further circumstances of the fast oxygen dependent response, concerning the behaviour of ribonucleotide reductase and of the dNTP pools, revealed no significant differences among the three cell lines. The striking identity of the response of each of the cell lines to hypoxia and to inhibited protein synthesis prompts the suspicion that converging fast regulatory pathways act on the cellular replication machinery. The phenomena as such seem to be rather widespread among mammalian cells.     

Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells

The replication protein A (RPA)–ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

Two mcm3 mutations affect different steps in the initiation of DNA replication

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.     

The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity

The DNA-replication inhibitors aphidicolin (APC) and hydroxyurea (HU) were tested for their ability to induce effects on DNA in the in vitro alkaline comet assay with V79 cells. APC concentrations up to 15 microM and HU concentrations up to 500 microM did not significantly increase the extent of DNA migration after treatment during 4h. Treatment for 18 h, however, led to inconsistently significant increase in DNA migration. These increases in DNA migration were accompanied by severe cell-cycle disturbances, cytotoxic effects (reduced population doubling and reduced mitotic index) and increased frequencies of cells with chromosome aberrations. The results indicate that substances with such secondary effects on DNA (in contrast to agents that directly damage DNA) only induce effects in the comet assay after prolonged exposure, together with cytotoxic effects. We conclude that slight inhibition of DNA replication and cell-cycle delay per se do not cause significant effects in the in vitro comet assay under standard test conditions. Furthermore, the in vitro comet assay seems to be less sensitive towards this type of secondary DNA effects than the in vitro chromosome aberration test.     

Sequential steps in clathrin-mediated synaptic vesicle endocytosis

Synaptic vesicles are recycled with remarkable speed and precision in nerve terminals. A major recycling pathway involves clathrin-mediated endocytosis at endocytic zones located around sites of release. Different 'accessory' proteins linked to this pathway have been shown to alter the shape and composition of lipid membranes, to modify membrane-coat protein interactions, and to influence actin polymerization. These include the GTPase dynamin, the lysophosphatidic acid acyl transferase endophilin, and the phosphoinositide phosphatase synaptojanin. Protein perturbation studies in living nerve terminals are now beginning to link the actions of these proteins with morphologically defined steps of endocytosis.