Genome-wide mapping of allele-specific protein-DNA interactions in human cells

We describe a high-throughput method, named ChIP-SNP, for the identification of allele-specific protein-DNA interactions throughout the human genome. ChIP-SNP combines chromatin immunoprecipitation (ChIP) with whole-genome single nucleotide polymorphism (SNP) genotyping microarray analysis. We demonstrated that it can be used to accurately identify allele-specific binding of RNA polymerase II (RNAP) in the human fibroblast genome, uncovering imprinted genes and other allele-specific binding events. ChIP-SNP will facilitate the study of mechanisms of allele-specific gene expression.     

Recruitment of doctors to non-standard grades in the NHS: analysis of job advertisements and survey of advertisers

Objectives To estimate the proportion of advertised non-consultant hospital posts that do not conform to nationally recognised terms and conditions of service and to investigate why these posts exist, who fills them, and what the doctors in such jobs do.Design Analysis of job advertisements and a cross sectional survey of advertisers.Setting Job advertisements in one of the leading UK publications listing hospital doctor vacancies (BMJ Careers).Results Nearly a quarter of non-consultant posts advertised in the two study periods (23% and 21%) were for non-standard grade posts. A questionnaire was sent to the medical staffing officer for each post. Of 430 questionnaires sent out 192 (45%) were returned. 98 trusts said they advertised non-standard grades because there was no more funding from the deanery for approved posts and 75 because service needs could not be met by doctors in training grades. In 132 posts (69%) the post holder would be required to do on-call work, and 50 advertisers (26%) required on-call duty for 1 in 5 or more frequently, which would conflict with the European Working Time Directive. 131 advertisers (68%) expected the posts to be filled by doctors from outside the European Economic Area.Conclusions Non-standard grade posts are mostly being created to meet service requirements when there is no more funding for standard training posts and are expected to be filled by doctors from overseas. Doctors in such posts can be more easily exploited and their careers hindered. The Department of Health''s annual census should include non-standard grade doctors.     

Temperature-Dependent Modulation of Chromosome Segregation in msh4 Mutants of Budding Yeast

Background

In many organisms, homologous chromosomes rely upon recombination-mediated linkages, termed crossovers, to promote their accurate segregation at meiosis I. In budding yeast, the evolutionarily conserved mismatch-repair paralogues, Msh4 and Msh5, promote crossover formation in conjunction with several other proteins, collectively termed the Synapsis Initiation Complex (SIC) proteins or ‘ZMM’s (Zip1-Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3). zmm mutants show decreased levels of crossovers and increased chromosome missegregation, which is thought to cause decreased spore viability.

Principal Findings

In contrast to other ZMM mutants, msh4 and msh5 mutants show improved spore viability and chromosome segregation in response to elevated temperature (23°C versus 33°C). Crossover frequencies in the population of viable spores in msh4 and msh5 mutants are similar at both temperatures, suggesting that temperature-mediated chromosome segregation does not occur by increasing crossover frequencies. Furthermore, meiotic progression defects at elevated temperature do not select for a subpopulation of cells with improved segregation. Instead, another ZMM protein, Zip1, is important for the temperature-dependent improvement in spore viability.

Conclusions

Our data demonstrate interactions between genetic (zmm status) and environmental factors in determining chromosome segregation.     

Membrane environment exerts an important influence on rac-mediated activation of phospholipase Cγ2

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.     

A novel method for measuring medial compartment pressures within the knee joint in-vivo

A novel method for the measurement of knee joint forces in-vivo is described. A thin (0.2mm) flexible electronic pressure sensor was inserted through a narrow arthroscopic portal into the osteoarthritic medial compartment of the knee joint. The sensor partially covered the load bearing area. The surgery was performed under local anaesthetic during normal arthroscopic examination following patient consent. Results are presented for 11 patients. The method was used in a pilot study to assess the effects of four valgus knee braces on medial compartment forces. An analysis of variance could not detect un-loading by any brace although there were large variations in force output. These variations may be attributable to shifts in the sensor position. In-vivo measurement of joint force is technically feasible.     

The Effect of Inhibitors on Sulphate Reducing Bacteria: a Compilation

Summary: The effects of about 200 bacteriostatic and bactericidal agents on sulphate reducing bacteria, including some hitherto unpublished results, are tabulated.     

Stability of the Human Fragile X (CGG)n Triplet Repeat Array in Saccharomyces cerevisiae Deficient in Aspects of DNA Metabolism

Expanded trinucleotide repeats underlie a growing number of human diseases. The human FMR1 (CGG)(n) array can exhibit genetic instability characterized by progressive expansion over several generations leading to gene silencing and the development of the fragile X syndrome. While expansion is dependent upon the length of uninterrupted (CGG)(n), instability occurs in a limited germ line and early developmental window, suggesting that lineage-specific expression of other factors determines the cellular environment permissive for expansion. To identify these factors, we have established normal- and premutation-length human FMR1 (CGG)(n) arrays in the yeast Saccharomyces cerevisiae and assessed the frequency of length changes greater than 5 triplets in cells deficient in various DNA repair and replication functions. In contrast to previous studies with Escherichia coli, we observed a low frequency of orientation-dependent large expansions in arrays carrying long uninterrupted (CGG)(n) arrays in a wild-type background. This frequency was unaffected by deletion of several DNA mismatch repair genes or deletion of the EXO1 and DIN7 genes and was not enhanced through meiosis in a wild-type background. Array contraction occurred in an orientation-dependent manner in most mutant backgrounds, but loss of the Sgs1p resulted in a generalized increase in array stability in both orientations. In contrast, FMR1 arrays had a 10-fold-elevated frequency of expansion in a rad27 background, providing evidence for a role in lagging-strand Okazaki fragment processing in (CGG)(n) triplet repeat expansion.     

Plant-associated fungi support bacterial resilience following water limitation

Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H₂¹⁸O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages (Rhizophagus irregularis and Serendipita bescii) grown with the bioenergy model grass Panicum hallii. In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii-inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H₂¹⁸O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal–bacterial synergies may support bacterial resilience to moisture limitation.Subject terms: DNA sequencing, Fungal ecology, Stable isotope analysis, Climate-change ecology, Microbial ecology