The Elg1 Clamp Loader Plays a Role in Sister Chromatid Cohesion

Mutations in the ELG1 gene of yeast lead to genomic instability, manifested in high levels of genetic recombination, chromosome loss, and gross chromosomal rearrangements. Elg1 shows similarity to the large subunit of the Replication Factor C clamp loader, and forms a RFC-like (RLC) complex in conjunction with the 4 small RFC subunits. Two additional RLCs exist in yeast: in one of them the large subunit is Ctf18, and in the other, Rad24. Ctf18 has been characterized as the RLC that functions in sister chromatid cohesion. Here we present evidence that the Elg1 RLC (but not Rad24) also plays an important role in this process. A genetic screen identified the cohesin subunit Mcd1/Scc1 and its loader Scc2 as suppressors of the synthetic lethality between elg1 and ctf4. We describe genetic interactions between ELG1 and genes encoding cohesin subunits and their accessory proteins. We also show that defects in Elg1 lead to higher precocious sister chromatid separation, and that Ctf18 and Elg1 affect cohesion via a joint pathway. Finally, we localize both Ctf18 and Elg1 to chromatin and show that Elg1 plays a role in the recruitment of Ctf18. Our results suggest that Elg1, Ctf4, and Ctf18 may coordinate the relative movement of the replication fork with respect to the cohesin ring.     

Regulation of fructosyltransferase activity by carbohydrates, in solution and immobilized on hydroxyapatite surfaces

We tested the effect of several carbohydrates on the activity of cell-free fructosyltransferases (FTF) in solution and immobilized onto hydroxyapatite (HA) and found an inhibitory dose-dependent effect of glucose on FTF activity, both on the surface and in solution. Glucose at 160 mM inhibits FTF activity by 75% both on HA and in solution. Fructose at 160 mM inhibited FTF activity by 25% in solution and by 15% on HA. Levan inhibited FTF activity by 30% in solution, while dextrans and inulin had a limited effect on FTF activity. Circular dichroism and infrared analysis demonstrated no major changes in the chemical structure of fructans synthesized by cell-free FTF on HA and in solution, in the presence or absence of glucose. However, as verified by size-exclusion chromatography, glucose inhibited the synthesis of high molecular-weight fructans. The results indicate that glucose, a byproduct of the FTF enzymatic reaction, is the main carbohydrate affecting FTF activity. Selective inhibition of high molecular-weight fructan production by glucose, may indicate that two mechanisms are involved in the synthesis of fructans, both in solution and on the surface.     

Blocking of Pseudomonas aeruginosa lectins by human milk glycans

The opportunistic human pathogen Pseudomonas aeruginosa produces a D-galactophilic (PA-IL) lectin and another lectin (PA-IIL) that binds L-fucose > D-arabinose > D-mannose in close association with its host-attacking factors. These lectins contribute to the virulence of P. aeruginosa by their involvement in the production, adhesion, and pathogenic effects of its biofilm on host cells. Therefore, they are considered targets for anti-Pseudomonas therapy. The present study compares their blocking by human milk samples with that of the plant lectin Con A. It demonstrates that human milk inhibits the hemagglutinating activities of the three lectins, with PA-IIL much more strongly inhibited than PA-IL or Con A. Using these lectins, Western blots of the milk samples accord with the hemagglutination inhibition data and disclose the distribution of the human milk glycoproteins that inhibit each lectin. The data of this paper reveal the high efficiency of human milk components in blocking the P. aeruginosa lectins and the usefulness of these lectins for detecting milk glycoprotein saccharides, which may protect the infant against infections.     

Combined use of micro-preparative gel electrophoresis and reversed-phase high-performance liquid chromatography for purification of amyloid beta peptides deposited in brains of Alzheimer's disease patients

A new micro-technique is developed for purification of amyloid beta peptides (A beta) extracted from brain tissues of patients with Alzheimer's disease (AD). It includes SDS-polyacrylamide gel electrophoresis of the extracted brain tissue material, electroblotting onto supporting membranes, and reversed-phase HPLC of the proteins eluted from membranes. By this technique, the extracted A beta are first separated electrophoretically from the higher and lower molecular mass tissue components, and then purified by reversed-phase HPLC from the contaminants having similar molecular masses, but different retention times on the column. In contrast to the common large-scale isolation procedures employing density gradient centrifugation, enzymatic digestions and size-exclusion chromatography, the developed micro-technique might be applied for biochemical analysis of A beta contained in small AD brain tissue specimens.     

Correlated effects of the response to conditioned medium in the flour beetle,Tribolium castaneum

Dispersal rates were measured in lines selected for high and low response of egg production to conditioned media, and responsivness of egg production was measured in lines selected for high and low dispersal. A positive correlation was found between these two traits, each of which had previously been found to have a simple genetic basis. It is suggested that in Tribolium castaneum the sensitivity to environmental conditions is mediated through a Sensor gene, which can activate either response, according to circumstances.     

Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae

The Saccharomyces cerevisiae gene deletion project revealed that approximately 20% of yeast genes are required for viability. The analysis of essential genes traditionally relies on conditional mutants, typically temperature-sensitive (ts) alleles. We developed a systematic approach (termed "diploid shuffle") useful for generating a ts allele for each essential gene in S. cerevisiae and for improved genetic manipulation of mutant alleles and gene constructs in general. Importantly, each ts allele resides at its normal genomic locus, flanked by specific cognate UPTAG and DNTAG bar codes. A subset of 250 ts mutants, including ts alleles for all uncharacterized essential genes and prioritized for genes with human counterparts, is now ready for distribution. The importance of this collection is demonstrated by biochemical and genetic screens that reveal essential genes involved in RNA processing and maintenance of chromosomal stability.     

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.