Construction and utilisation of a bidirectional reporter vector in the analysis of two nod-boxes in of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in Molecular Genetics at Massey University

The nod-box is a 47bp cis-acting regulatory region which has been conserved amongst every species of Rhizobium studied to date. In species such as R. meliloti and R. leguminosarum, the nod-box has been shown to promote constitutive activity towards the regulatory nodD gene, and flavonoid-inducible expression towards the divergently-transcribed nodABCIJ operon. This bidirectional regulation of the so-called common nod genes was not observed in R. loti. A previous analysis of this species had shown that its nod-box promoted inducible activity towards the truncated 'nodD' gene, as well as the nodACIJ operon. It was the unusual arrangement of these R. loti nod genes that had initially aroused interest in this bacteria. To further investigate the role of the nod-box in the regulation of the R. loti common nod genes, a bidirectional reporter vector (pSPV4) was constructed. This novel vector allowed the promoter activity of a cloned nod-box-containing fragment to be concurrently measured in either direction using the same culture of cells. To achieve this construct, the gusA gene from pRAJ260 was blunt-end ligated into pUC21. An in-frame ribosome binding site (rbs) was cloned upstream of the gusA coding sequence to facilitate transcriptional fusions. The rbs and gusA gene were later excised as a functional unit and blunt-end ligated into pMP220 alongside the B-galactosidase reporter gene but in the opposite orientation. Hence, both reporter genes could be divergently transcribed from a common regulatory region cloned into the multiple cloning site that separated the genes. The fragments of DNA that were eventually cloned into the bidirectional vector were generated through the polymerase chain reaction. Each DNA insert contained the nod-box bracketed by differing lengths of flanking region. Once these PCR-generated fragments had been sequenced in pUC118 and subcloned into pSPV4, the resulting constructs were transformed into R. loti cells by electroporation. As the electroporation of these cells had not previously been reported, the conditions for this procedure were established and optimised. The results obtained from the bidirectional reporter assays disagreed with those observed in the earlier assays by Teo (1990). Neither the nodACIJ nod-box of NZP2037 nor the nodB nod-box of NZP2213, showed bidirectional inducible expression. In fact, both nod-boxes showed constitutive expression in the 'nodD' direction and inducible expression in the opposite direction. This indicates that the control of the nod genes in R. loti is fundamentally the same as that seen in other fast-growing Rhizobium species. Three regulatory elements affecting the levels of nod gene expression have tentatively been identified outside the nod-box sequence, though the results indicating their presence may simply be·due to spacing differences between the nod-box and the reporter gene

Hydrological summary for the United Kingdom: February 2012

The monthly summary of hydrological conditions in the United Kingdom is compiled as part of the National Hydrological Monitoring Programme (a joint CEH and BGS enterprise). The report features contemporary data for rainfall, river flow, reservoir and groundwater levels in the form of maps and graphs. A commentary is provided on the status of the nation's water resources and any notable hydrological events during the month. The National River Flow and National Groundwater Level Archives help provide an historical context for these contemporary assessments. Financial support for the production of the Hydrological Summaries is provided by Defra, the Environment Agency, the Scottish Environment Protection Agency, the Rivers Agency in Northern Ireland and the Office of Water Services

Hydrological summary for the United Kingdom: January 2012

The monthly summary of hydrological conditions in the United Kingdom is compiled as part of the National Hydrological Monitoring Programme (a joint CEH and BGS enterprise). The report features contemporary data for rainfall, river flow, reservoir and groundwater levels in the form of maps and graphs. A commentary is provided on the status of the nation's water resources and any notable hydrological events during the month. The National River Flow and National Groundwater Level Archives help provide an historical context for these contemporary assessments. Financial support for the production of the Hydrological Summaries is provided by Defra, the Environment Agency, the Scottish Environment Protection Agency, the Rivers Agency in Northern Ireland and the Office of Water Services

Hydrological summary for the United Kingdom: June 2013

The monthly summary of hydrological conditions in the United Kingdom is compiled as part of the National Hydrological Monitoring Programme (a joint CEH and BGS enterprise). The report features contemporary data for rainfall, river flow, reservoir and groundwater levels in the form of maps and graphs. A commentary is provided on the status of the nation’s water resources and any notable hydrological events during the month. The National River Flow and National Groundwater Level Archives help provide an historical context for these contemporary assessments. Financial support for the production of the Hydrological Summaries is provided by Defra, the Environment Agency, the Scottish Environment Protection Agency, the Rivers Agency in Northern Ireland and the Office of Water Services

Hydrological summary for the United Kingdom: May 2013

The monthly summary of hydrological conditions in the United Kingdom is compiled as part of the National Hydrological Monitoring Programme (a joint CEH and BGS enterprise). The report features contemporary data for rainfall, river flow, reservoir and groundwater levels in the form of maps and graphs. A commentary is provided on the status of the nation’s water resources and any notable hydrological events during the month. The National River Flow and National Groundwater Level Archives help provide an historical context for these contemporary assessments. Financial support for the production of the Hydrological Summaries is provided by Defra, the Environment Agency, the Scottish Environment Protection Agency, the Rivers Agency in Northern Ireland and the Office of Water Services

The 2010-12 drought and subsequent extensive flooding: a remarkable hydrological transformation

Across most of the UK, the 2010‑12 period was remarkable in climatic terms with exceptional departures from normal rainfall, runoff and aquifer recharge patterns. Generalising broadly, drought conditions developed through 2010, intensified during 2011 and were severe across much of England & Wales by the early spring of 2012. Record late spring and summer rainfall then triggered a hydrological transformation that has no close modern parallel. Seasonally extreme river flows were common through the summer, heralding further extensive flooding during the autumn and, particularly, the early winter when record runoff at the national scale provided a culmination to the wettest nine‑month sequence for England & Wales in an instrumental record beginning in 1766. This report provides comprehensive documentation and hydrometeorological appraisals of a three‑year period which incorporated a number of important regional drought episodes as well as the outstanding runoff and recharge patterns which characterised most of 2012. An examination of the wide range of impacts of the drought and flood episodes is included and the extreme hydrometeorological conditions are examined within an extended historical context. Finally, the recent exceptional conditions are reviewed in the light of observational evidence for trends in temperature, rainfall, river flow and aquifer recharge patterns

Hydrological summary for the United Kingdom: August 2013

The monthly summary of hydrological conditions in the United Kingdom is compiled as part of the National Hydrological Monitoring Programme (a joint CEH and BGS enterprise). The report features contemporary data for rainfall, river flow, reservoir and groundwater levels in the form of maps and graphs. A commentary is provided on the status of the nation’s water resources and any notable hydrological events during the month. The National River Flow and National Groundwater Level Archives help provide an historical context for these contemporary assessments. Financial support for the production of the Hydrological Summaries is provided by Defra, the Environment Agency, the Scottish Environment Protection Agency, the Rivers Agency in Northern Ireland and the Office of Water Services

Briefing note. Severity of the December 2015 floods - preliminary analysis

The purpose of this briefing note is to collate some of the key points from the December 2015 Hydrological Summary, and to provide some additional context beyond the space and format constraints of the Summary. Neither the Hydrological Summary nor the briefing note provide a fully comprehensive review of the severity of the December 2015 floods

UK hydrological review 2007

This hydrological review, which also provides an overview of water resources status throughout 2007, was undertaken as part of the National Hydrological Monitoring Programme (NHMP). The NHMP was set up in 1988 to document hydrological and water resources variability across the UK. It is a collaborative programme between the Centre for Ecology & Hydrology, which maintains the National River Flow Archive and the British Geological Survey which maintains the National Groundwater Level Archive. Both organisations are component bodies of the Natural Environment Research Council. This report has been compiled with the active cooperation of the principal measuring authorities in the UK: the Environment Agencya, the Scottish Environment Protection Agency and, in the Northern Ireland, the Rivers Agency. These organisations provided the great majority of the required river and groundwater level data. The Met Office provided almost all of the rainfall and climatological information featured in the report and the reservoir stocks information derive from the Water Service Companies, Scottish Water and Northern Ireland Water. Groundwater level data for Northern Ireland was provided by the Northern Ireland Environment Agency. The provision of the basic data, which provides the foundation both of this report and the wider activities of the NHMP, is gratefully acknowledged. A primary source of information for this review is the series of monthly UK Hydrological Summaries (for further details please visit: http://www.ceh.ac.uk/data/nrfa/water_watch.html. Financial support for the production of the Hydrological Summaries is provided by the Environment Agency, the Scottish Environment Protection Agency, the Rivers Agency (Northern Ireland) and the Office of Water Services (OFWAT)

Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation