Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

Evidence of a Monoaminergic-Cholinergic Imbalance Related to Visual Hallucinations in Lewy Body Dementia

Senile dementia of Lewy body type is characterized clinically by a relatively acute onset of fluctuating memory loss and confusion, frequently accompanied by visual hallucinations. Neurochemical analyses of temporal cortex has revealed a distinction between hallucinating and nonhallucinating patients in both cholinergic and monaminergic transmitter activities. In contrast with the cholinergic enzyme choline acetyltransferase, which was more extensively reduced in hallucinating individuals, serotonergic S2 receptor binding and both dopamine and serotonin metabolites were significantly decreased in nonhallucinating cases. These results suggest that an imbalance between monaminergic and cholinergic transmitters is involved in hallucinogenesis in the human brain.     

Characterization of Anaplasma marginale isolated from North American bison

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present study was undertaken to confirm A. marginale infection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of this A. marginale bison isolate for cattle. The results of this study showed that these A. marginale isolates obtained from bison were similar to ones from naturally infected cattle.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.     

Determinants of Swe1p degradation in Saccharomyces cerevisiae

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.     

Daphnia magna microRNAs respond to nutritional stress and ageing but are not transgenerational

Maternal effects, where the performance of offspring is determined by the condition of their mother, are widespread and may in some cases be adaptive. The crustacean Daphnia magna shows strong maternal effects: offspring size at birth and other proxies for fitness are altered when their mothers are older or when mothers have experienced dietary restriction. The mechanisms for this transgenerational transmission of maternal experience are unknown, but could include changes in epigenetic patterning. MicroRNAs (miRNAs) are regulators of gene expression that have been shown to play roles in intergenerational information transfer, and here, we test whether miRNAs are involved in D. magna maternal effects. We found that miRNAs were differentially expressed in mothers of different ages or nutritional state. We then examined miRNA expression in their eggs, their adult daughters and great granddaughters, which did not experience any treatments. The maternal (treatment) generation exhibited differential expression of miRNAs, as did their eggs, but this was reduced in adult daughters and lost by great granddaughters. Thus, miRNAs are a component of maternal provisioning, but do not appear to be the cause of transgenerational responses under these experimental conditions. MicroRNAs may act in tandem with egg provisioning (e.g., with carbohydrates or fats), and possibly other small RNAs or epigenetic modifications.     

CENP-C functions in centromere assembly,the maintenance of CENP-A asymmetry and epigenetic age in Drosophila germline stem cells

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)—the epigenetic determinant of centromere identity—is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G₂ phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of ‘mitotic drive’ that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G₂ phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.     

Ancestral polymorphism and recent invasion of transposable elements in Drosophila species

ABSTRACT: BACKGROUND: During the evolutionary history of transposable elements, some processes, such as ancestral polymorphisms and horizontal transfer of sequences between species, can produce incongruences in phylogenies. We investigated the evolutionary history of the transposable elements Bari and 412 in the sequenced genomes of the Drosophila melanogaster group and in the sibling species D. melanogaster and D. simulans using traditional phylogenetic and network approaches. RESULTS: The maximum likelihood (ML) phylogenetic analyses revealed incongruences and unresolved relationships for both the Bari and 412 elements. The DNA transposon Bari within the D. ananassae genome is more closely related to the element of the melanogaster complex than to the sequence in D. erecta, which is inconsistent with the species phylogeny. Divergence analysis and the comparison of the rate of synonymous substitutions per synonymous site of the Bari and host gene sequences explain the incongruence as an ancestral polymorphism inherited stochastically by the derived species. Unresolved relationships were observed in the ML phylogeny of both elements involving D. melanogaster, D. simulans and D. sechellia. A network approach was used to attempt to resolve these relationships. The resulting tree suggests recent transfers of both elements between D. melanogaster and D. simulans. The divergence values of the elements between these species support this conclusion. CONCLUSIONS: We showed that an ancestral polymorphism and recent invasion of genomes due to introgression or horizontal transfer between species occurred during the evolutionary history of the Bari and 412 elements in the melanogaster group. These invasions likely occurred in Africa during the Pleistocene, before the worldwide expansion of D. melanogaster and D. simulans.