Genome-wide analysis of poly(A) site selection in schizosaccharomyces pombe,

Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3′ UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3′RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A)

Decline of meticillin-resistant Staphylococcus aureus in Oxfordshire hospitals is strain-specific and preceded infection-control intensification

Background In the past, strains of Staphylococcus aureus have evolved, expanded, made a marked clinical impact and then disappeared over several years. Faced with rising meticillin-resistant S aureus (MRSA) rates, UK government-supported infection control interventions were rolled out in Oxford Radcliffe Hospitals NHS Trust from 2006 onwards. Methods Using an electronic Database, the authors identified isolation of MRS among 611 434 hospital inpatients admitted to acute hospitals in Oxford, UK, 1 April 1998 to 30 June 2010. Isolation rates were modelled using segmented negative binomial regression for three groups of isolates: from blood cultures, from samples suggesting invasion (eg, cerebrospinal fluid, joint fluid, pus samples) and from surface swabs (eg, from wounds). Findings MRSA isolation rates rose rapidly from 1998 to the end of 2003 (annual increase from blood cultures 23%, 95% CI 16% to 30%), and then declined. The decline accelerated from mid-2006 onwards (annual decrease post-2006 38% from blood cultures, 95% CI 29% to 45%, p=0.003 vs previous decline). Rates of meticillin-sensitive S aureus changed little by comparison, with no evidence for declines 2006 onward (p=0.40); by 2010, sensitive S aureus was far more common than MRSA (blood cultures: 2.9 vs 0.25; invasive samples 14.7 vs 2.0 per 10 000 bedstays). Interestingly, trends in isolation of erythromycin-sensitive and resistant MRSA differed. Erythromycin-sensitive strains rose significantly faster (eg, from blood cultures p=0.002), and declined significantly more slowly (p=0.002), than erythromycin-resistant strains (global p<0.0001). Bacterial typing suggests this reflects differential spread of two major UK MRSA strains (ST22/36), ST36 having declined markedly 2006-2010, with ST22 becoming the dominant MRSA strain. Conclusions MRSA isolation rates were falling before recent intensification of infection-control measures. This, together with strain-specific changes in MRSA isolation, strongly suggests that incompletely understood biological factors are responsible for the much recent variation in MRSA isolation. A major, mainly meticillin-sensitive, S aureus burden remains

PomBase – the scientific resource for fission yeast

The fission yeast Schizosaccharomyces pombe has become well established as a model species for studying conserved cell-level biological processes, especially the mechanics and regulation of cell division. PomBase integrates the S. pombe genome sequence with traditional genetic, molecular and cell biological experimental data as well as the growing body of large datasets generated by emerging high-throughput methods. This chapter provides insight into the curation philosophy and data organization at PomBase, and provides a guide to using PomBase for infrequent visitors and anyone considering exploring S. pombe in their research

Long-term monitoring in primary care for chronic kidney disease and chronic heart failure: a multi-method research programme

Background: Long-term monitoring is important in chronic condition management. Despite considerable costs of monitoring, there is no or poor evidence on how, what and when to monitor. The aim of this study was to improve understanding, methods, evidence base and practice of clinical monitoring in primary care, focusing on two areas: chronic kidney disease and chronic heart failure. Objectives: The research questions were as follows: does the choice of test affect better care while being affordable to the NHS? Can the number of tests used to manage individuals with early-stage kidney disease, and hence the costs, be reduced? Is it possible to monitor heart failure using a simple blood test? Can this be done using a rapid test in a general practitioner consultation? Would changes in the management of these conditions be acceptable to patients and carers? Design: Various study designs were employed, including cohort, feasibility study, Clinical Practice Research Datalink analysis, seven systematic reviews, two qualitative studies, one cost-effectiveness analysis and one cost recommendation. Setting: This study was set in UK primary care. Data sources: Data were collected from study participants and sourced from UK general practice and hospital electronic health records, and worldwide literature. Participant: The participants were NHS patients (Clinical Practice Research Datalink: 4.5 million patients), chronic kidney disease and chronic heart failure patients managed in primary care (including 750 participants in the cohort study) and primary care health professionals. Interventions: The interventions were monitoring with blood and urine tests (for chronic kidney disease) and monitoring with blood tests and weight measurement (for chronic heart failure). Main outcome measures: The main outcomes were the frequency, accuracy, utility, acceptability, costs and cost-effectiveness of monitoring. Results: Chronic kidney disease: serum creatinine testing has increased steadily since 1997, with most results being normal (83% in 2013). Increases in tests of creatinine and proteinuria correspond to their introduction as indicators in the Quality and Outcomes Framework. The Chronic Kidney Disease Epidemiology Collaboration equation had 2.7% greater accuracy (95% confidence interval 1.6% to 3.8%) than the Modification of Diet in Renal Disease equation for estimating glomerular filtration rate. Estimated annual transition rates to the next chronic kidney disease stage are ≈ 2% for people with normal urine albumin, 3–5% for people with microalbuminuria (3–30 mg/mmol) and 3–12% for people with macroalbuminuria (> 30 mg/mmol). Variability in estimated glomerular filtration rate-creatinine leads to misclassification of chronic kidney disease stage in 12–15% of tests in primary care. Glycaemic-control and lipid-modifying drugs are associated with a 6% (95% confidence interval 2% to 10%) and 4% (95% confidence interval 0% to 8%) improvement in renal function, respectively. Neither estimated glomerular filtration rate-creatinine nor estimated glomerular filtration rate-Cystatin C have utility in predicting rate of kidney function change. Patients viewed phrases such as ‘kidney damage’ or ‘kidney failure’ as frightening, and the term ‘chronic’ was misinterpreted as serious. Diagnosis of asymptomatic conditions (chronic kidney disease) was difficult to understand, and primary care professionals often did not use ‘chronic kidney disease’ when managing patients at early stages. General practitioners relied on Clinical Commissioning Group or Quality and Outcomes Framework alerts rather than National Institute for Health and Care Excellence guidance for information. Cost-effectiveness modelling did not demonstrate a tangible benefit of monitoring kidney function to guide preventative treatments, except for individuals with an estimated glomerular filtration rate of 60–90 ml/minute/1.73 m2, aged < 70 years and without cardiovascular disease, where monitoring every 3–4 years to guide cardiovascular prevention may be cost-effective. Chronic heart failure: natriuretic peptide-guided treatment could reduce all-cause mortality by 13% and heart failure admission by 20%. Implementing natriuretic peptide-guided treatment is likely to require predefined protocols, stringent natriuretic peptide targets, relative targets and being located in a specialist heart failure setting. Remote monitoring can reduce all-cause mortality and heart failure hospitalisation, and could improve quality of life. Diagnostic accuracy of point-of-care N-terminal prohormone of B-type natriuretic peptide (sensitivity, 0.99; specificity, 0.60) was better than point-of-care B-type natriuretic peptide (sensitivity, 0.95; specificity, 0.57). Within-person variation estimates for B-type natriuretic peptide and weight were as follows: coefficient of variation, 46% and coefficient of variation, 1.2%, respectively. Point-of-care N-terminal prohormone of B-type natriuretic peptide within-person variability over 12 months was 881 pg/ml (95% confidence interval 380 to 1382 pg/ml), whereas between-person variability was 1972 pg/ml (95% confidence interval 1525 to 2791 pg/ml). For individuals, monitoring provided reassurance; future changes, such as increased testing, would be acceptable. Point-of-care testing in general practice surgeries was perceived positively, reducing waiting time and anxiety. Community heart failure nurses had greater knowledge of National Institute for Health and Care Excellence guidance than general practitioners and practice nurses. Health-care professionals believed that the cost of natriuretic peptide tests in routine monitoring would outweigh potential benefits. The review of cost-effectiveness studies suggests that natriuretic peptide-guided treatment is cost-effective in specialist settings, but with no evidence for its value in primary care settings. Limitations: No randomised controlled trial evidence was generated. The pathways to the benefit of monitoring chronic kidney disease were unclear. Conclusions: It is difficult to ascribe quantifiable benefits to monitoring chronic kidney disease, because monitoring is unlikely to change treatment, especially in chronic kidney disease stages G3 and G4. New approaches to monitoring chronic heart failure, such as point-of-care natriuretic peptide tests in general practice, show promise if high within-test variability can be overcome

Understanding non-coding DNA regions in yeast.

Non-coding transcripts play an important role in gene expression regulation in all species, including budding and fission yeast. Such regulatory transcripts include intergenic ncRNA (non-coding RNA), 5' and 3' UTRs, introns and antisense transcripts. In the present review, we discuss advantages and limitations of recently developed sequencing techniques, such as ESTs, DNA microarrays, RNA-Seq (RNA sequencing), DRS (direct RNA sequencing) and TIF-Seq (transcript isoform sequencing). We provide an overview of methods applied in yeast and how each of them has contributed to our knowledge of gene expression regulation and transcription

Understanding non-coding DNA regions in yeast.

Non-coding transcripts play an important role in gene expression regulation in all species, including budding and fission yeast. Such regulatory transcripts include intergenic ncRNA (non-coding RNA), 5' and 3' UTRs, introns and antisense transcripts. In the present review, we discuss advantages and limitations of recently developed sequencing techniques, such as ESTs, DNA microarrays, RNA-Seq (RNA sequencing), DRS (direct RNA sequencing) and TIF-Seq (transcript isoform sequencing). We provide an overview of methods applied in yeast and how each of them has contributed to our knowledge of gene expression regulation and transcription

Understanding non-coding DNA regions in yeast.

Non-coding transcripts play an important role in gene expression regulation in all species, including budding and fission yeast. Such regulatory transcripts include intergenic ncRNA (non-coding RNA), 5' and 3' UTRs, introns and antisense transcripts. In the present review, we discuss advantages and limitations of recently developed sequencing techniques, such as ESTs, DNA microarrays, RNA-Seq (RNA sequencing), DRS (direct RNA sequencing) and TIF-Seq (transcript isoform sequencing). We provide an overview of methods applied in yeast and how each of them has contributed to our knowledge of gene expression regulation and transcription

Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks

DNA is constantly exposed to endogenous and exogenous damage. Various types of DNA repair counteract highly toxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilizes small RNA species, which are produced as long non-coding (nc)RNA precursors and promote recognition of DSBs. However, regulatory principles that control production of such transcripts remain largely elusive. Here we show that the Abelson tyrosine kinase c-Abl/ABL1 causes formation of RNA polymerase II (RNAPII) foci, predominantly phosphorylated at carboxy-terminal domain (CTD) residue Tyr1, at DSBs. CTD Tyr1-phosphorylated RNAPII (CTD Y1P) synthetizes strand-specific, damage-responsive transcripts (DARTs), which trigger formation of double-stranded (ds)RNA intermediates via DNA–RNA hybrid intermediates to promote recruitment of p53-binding protein 1 (53BP1) and Mediator of DNA damage checkpoint 1 (MDC1) to endogenous DSBs. Interference with transcription, c-Abl activity, DNA–RNA hybrid formation or dsRNA processing impairs CTD Y1P foci formation, attenuates DART synthesis and delays recruitment of DDR factors and DSB signalling. Collectively, our data provide novel insight in RNA-dependent DDR by coupling DSB-induced c-Abl activity on RNAPII to generate DARTs for consequent DSB recognition

Transcription facilitates sister chromatid cohesion on chromosomal arms.

Cohesin is a multi-subunit protein complex essential for sister chromatid cohesion, gene expression and DNA damage repair. Although structurally well studied, the underlying determinant of cohesion establishment on chromosomal arms remains enigmatic. Here, we show two populations of functionally distinct cohesin on chromosomal arms using a combination of genomics and single-locus specific DNA-FISH analysis. Chromatin bound cohesin at the loading sites co-localizes with Pds5 and Eso1 resulting in stable cohesion. In contrast, cohesin independent of its loader is unable to maintain cohesion and associates with chromatin in a dynamic manner. Cohesive sites coincide with highly expressed genes and transcription inhibition leads to destabilization of cohesin on chromatin. Furthermore, induction of transcription results inde novorecruitment of cohesive cohesin. Our data suggest that transcription facilitates cohesin loading onto chromosomal arms and is a key determinant of cohesive sites in fission yeast