Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment

The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1^ATM and Rad3^ATR kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Δ rad3Δ cells. Thus, Tel1^ATM and Rad3^ATR are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1^ATM/Rad3^ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.     

Formation of a Tap/NXF1 homotypic complex is mediated through the amino-terminal domain of Tap and enhances interaction with nucleoporins

Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.     

Ectodomain shedding of furin: kinetics and role of the cysteine-rich region

Furin, a member of the subtilisin-like pro-protein convertase family, is a type I membrane protein that undergoes ectodomain shedding. Metabolic labeling of cells stably expressing furin demonstrated that the shed form of furin is detected after 30 min. Moreover, sequence analysis revealed that specific residues of the cysteine-rich region of furin aligned with those of tumor necrosis factor receptor, which is also shed. Introduction within furin's cysteine-rich region of mutations that impair TNFR1 shedding also abolished furin shedding. Our results show that shedding of furin occurs rapidly and further suggest that specific cysteine residues may impart a conformation to the enzyme, thereby affecting its susceptibility to proteolysis.     

Innate immune defences in the human endometrium

The human endometrium is an important site of innate immune defence, giving protection against uterine infection. Such protection is critical to successful implantation and pregnancy. Infection is a major cause of preterm birth and can also cause infertility and ectopic pregnancy. Natural anti-microbial peptides are key mediators of the innate immune system. These peptides, between them, have anti-bacterial, anti-fungal and anti-viral activity and are expressed at epithelial surfaces throughout the female genital tract. Two families of natural anti-microbials, the defensins and the whey acidic protein (WAP) motif proteins, appear to be prominent in endometrium. The human endometrial epithelium expresses beta-defensins 1–4 and the WAP motif protein, secretory leukocyte protease inhibitor. Each beta-defensin has a different expression profile in relation to the stage of the menstrual cycle, providing potential protection throughout the cycle. Secretory leukocyte protease inhibitor is expressed during the secretory phase of the cycle and has a range of possible roles including anti-protease and anti-microbial activity as well as having effects on epithelial cell growth. The leukocyte populations in the endometrium are also a source of anti-microbial production. Neutrophils are a particularly rich source of alpha-defensins, lactoferrin, lysozyme and the WAP motif protein, elafin. The presence of neutrophils during menstruation will enhance anti-microbial protection at a time when the epithelial barrier is disrupted. Several other anti-microbials including the natural killer cell product, granulysin, are likely to have a role in endometrium. The sequential production of natural anti-microbial peptides by the endometrium throughout the menstrual cycle and at other sites in the female genital tract will offer protection from many pathogens, including those that are sexually transmitted.     

Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.     

Impact of Fertilizing with Raw or Anaerobically Digested Sewage Sludge on the Abundance of Antibiotic-Resistant Coliforms,Antibiotic Resistance Genes,and Pathogenic Bacteria in Soil and on Vegetables at Harvest

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.     

Biogeographical structure and affinities of the marine demersal ichthyofauna of Australia

Aim To investigate the biogeographical structure and affinities of the Australian marine demersal ichthyofauna at the scale of provinces and bathomes for the purposes of regional marine planning. Location Australia. Methods Patterns of distribution in the Australian fish fauna, at both intra‐regional and global scales, were examined using a science‐based, management framework dividing Australia’s marine biodiversity into 16 province‐level biogeographical units. Occurrences of 3734 species in eight depth‐stratified bathomes (from the coast to the mid‐continental slope) within each province were analysed to determine the structure and local affinities of their assemblages and their association with faunas of nearby regions and oceans basins. Results Strong geographic and depth‐related structure was evident. Fish assemblages in each province, and in each bathome of each province, were distinct, with the shelf‐break bathome more similar to the adjacent continental shelf bathome than to the upper slope bathome. Data based only on endemic species performed well as a surrogate of the entire dataset, yielding comparable patterns of similarity between provinces and bathomes. Tropical and temperate elements were better discriminated than elements of the Pacific and Indian oceans, with the central western province more similar to the tropical provinces (including those in the east), and the eastern province closer to southern temperate provinces. The fauna shares the closest regional affinities with those of the adjacent south‐west Pacific, western Pacific Rim, and elements of wide‐ranging Indo‐Pacific components. Elements unique to the Pacific and Indian oceans are poorly represented. Main conclusions The complex nature of Australia’s marine ichthyofauna is confirmed. A hierarchy of provinces and bathomes, used to ensure that Australia’s developing marine reserve network is both representative and comprehensive, is equally robust when based on all known Australian fish species or on only those species endemic to this continent. Latitude and depth are more important than oceanic influences on the composition of this fauna at these scales.