Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage and polyadenylation factor

Recognition of poly(A) sites in yeast pre-mRNAs is poorly understood. Employing an in vitro cleavage system with cleavage and polyadenylation factor (CPF) and cleavage factor IA we show that the efficiency and positioning elements are dispensable for poly(A)-site recognition within a short CYC1 substrate in vitro. Instead, U-rich elements immediately upstream and downstream of the poly(A) site mediate cleavage-site recognition within CYC1 and ADH1 pre-mRNAs. These elements act in concert with the poly(A) site to produce multiple recognition sites for the processing machinery, since combinations of mutations within these elements were most effective in cleavage inhibition. Intriguingly, introduction of a U-rich element downstream of the GAL7 poly(A) site strongly enhanced cleavage, underscoring the importance of downstream sequences in general. RNA- binding analyses demonstrate that cleavage depends on the recognition of the poly(A)-site region by CPF. Consistent with in vitro results, mutation of sequences upstream and downstream of the poly(A) site affected 3'-end formation in vivo. A model for yeast pre-mRNA cleavage-site recognition outlines an unanticipated high conservation of yeast and mammalian 3'-end processing mechanisms.

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.

Selection of roost sites by the lesser long-eared bat (Nyctophilus geoffroyi) and Gould`s wattled bat (Chalinolobus gouldii) in south-eastern Australia

Physical characteristics of roost sites used by the lesser long-eared bat Nyctophilus geoffroyi and Gould's wattled bat Chalinolobus gouldii were investigated in a farmland–remnant vegetation mosaic and adjacent forested floodplain in south-eastern Australia. A total of 45 individuals of N. geoffroyi and 27 C. gouldii were fitted with radio-transmitters, resulting in the location of 139 and 89 roosts, respectively. Male N. geoffroyi roosted in trees, fallen and decayed timber and artificial structures. These roosts were low to the ground, mainly under bark and in cracks in timber. Roosts of female N. geoffroyi were located higher above ground, and all within trees. Maternity roosts were predominantly located in large dead trees, approximately twice the diameter of roost trees used by females outside the breeding season. No maternity roosts were found under bark, despite half the roosts used by non-breeding females being located in these situations. Both sexes roosted primarily in dead timber and used cavities where the narrowest dimension of the entrance was 2.5 cm. Most roosts of C. gouldii were in dead spouts on large, live river red gum Eucalyptus camaldulensis trees. Intraspecific differences in roost characteristics were less pronounced for this species. Despite access to the same roosting opportunities, there were marked differences in roost selection between N. geoffroyi and C. gouldii. Both species favoured large diameter trees, but differed significantly for all other measured variables: type of roost structure, condition of roost tree (live or dead), height of roost tree, height of roost, and entrance dimensions. Although these species are among the most widespread bats in Australia and are often considered to be habitat 'generalists', both displayed a high level of discrimination in the roosts used. Clearly, roosting requirements are a complex and important issue in the conservation of even the most common species of bats.

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10–5-10–6 M for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.

Are the earliest field monuments of the pacific landscape serial sites?

Explanations of the origin and genesis of Pacific field monuments commonly assume they reflect local social change in islands or island groups which were increasingly isolated following colonization. A recent review of early West Polynesian archaeology suggests that the penecontemporaneous appearance of various kinds of field monuments from eastern Melanesia to Polynesia may be better explained as evidence of interaction and the movement of people and/or ideas, possibly associated with the colonization of East Polynesia.

Development of high quality university web sites: benefits to users and the learning organisation

In recent years, the online delivery of educational material has received much attention by researchers. University web sites are being used to supplement the delivery of knowledge in traditional bricks and mortar environments or as stand-alone external courses. The function of a university web site also has broader applications that can provide strategic benefits to the organisation as a whole. An Internet presence based on a high quality, easy to use web site can provide universities with a credible, professional image. This is particularly important as students and other users of university web sites expect the same high level of quality in these sites as they do from the sites of commercial organisations. This paper explores the various roles that a web site can provide to benefit higher education organisations and their customers. In addition, the paper outlines the results of a study of university web site users that identifies specific web site design features that are considered essential for a high quality university site that will meet the needs of students and other stakeholders in a university environment. By adopting these design principles, universities will be able to improve the quality of their web sites and ultimately, increase user satisfaction. Moreover, an understanding of the various roles that a web site can perform will allow universities to fully utilise the benefits of developing and maintaining an online presence while retaining their traditional role as knowledge managers.

Calcium- and vitamin D3 - fortified milk reduces bone loss at clinically relevant skeletal sites on llder men: a 2-year randomised controlled trial

In this 2-year randomized controlled study of 167 men >50 years of age, supplementation with calcium-vitamin D3-fortified milk providing an additional 1000 mg of calcium and 800 IU of vitamin D3 per day was effective for suppressing PTH and stopping or slowing bone loss at several clinically important skeletal sites at risk for fracture.

Introduction: Low dietary calcium and inadequate vitamin D stores have long been implicated in age-related bone loss and osteoporosis. The aim of this study was to assess the effects of calcium and vitamin D3 fortified milk on BMD in community living men >50 years of age.

Materials and Methods: This was a 2-year randomized controlled study in which 167 men (mean age ± SD, 61.9 ± 7.7 years) were assigned to receive either 400 ml/day of reduced fat (1%) ultra-high temperature (UHT) milk containing 1000 mg of calcium plus 800 IU of vitamin D3 or to a control group receiving no additional milk. Primary endpoints were changes in BMD, serum 25(OH)D, and PTH.

Results: One hundred forty-nine men completed the study. Baseline characteristics between the groups were not different; mean dietary calcium and serum 25(OH)D levels were 941 ± 387 mg/day and 77 ± 23 nM, respectively. After 2 years, the mean percent change in BMD was 0.9-1.6% less in the milk supplementation compared with control group at the femoral neck, total hip, and ultradistal radius (range, p < 0.08 to p < 0.001 after adjusting for covariates). There was a greater increase in lumbar spine BMD in the milk supplementation group after 12 and 18 months (0.8-1.0%, p ≤ 0.05), but the between-group difference was not significant after 2 years (0.7%; 95% CI, −0.3, 1.7). Serum 25(OH)D increased and PTH decreased in the milk supplementation relative to control group after the first year (31% and −18%, respectively; both p < 0.001), and these differences remained after 2 years. Body weight remained unchanged in both groups at the completion of the study.

Conclusions: Supplementing the diet of men >50 years of age with reduced-fat calcium- and vitamin D3-enriched milk may represent a simple, nutritionally sound and cost-effective strategy to reduce age-related bone loss at several skeletal sites at risk for fracture in the elderly.

The case for selective ICT outsourcing : it`s easier for "green field" sites

This paper examines published research on Information and Communications Technology (ICT) Outsourcing and uses this as a framework through which to examine the delivery of ICT services at the South West Alliance of Rural Hospitals, an environment not without challenges. Right from the beginning the CIO appointed to the Alliance decided to address these challenges by implementing a high speed telecommunications network to reduce the costs of data, voice and video communications within the Alliance. The emergence of the truly virtual health care network in Victoria’s South West supported by external service providers but managed by a small yet powerful central ICT team holds a number of useful lessons for the delivery of services in start up cooperative organizations, particularly regional, rural and remote (R3) organizations.

Orientation and impact of nonprofit museum web sites

Museums face increasing pressure to attract wider audiences. This requires an understanding of visitor expectations and motivations for visiting the ‘real’ museum. Websites can act as an attracting mechanism for ‘real’ museum visits. However, for the most part, museums have concentrated their efforts on attracting visitors to promotional efforts and collecting statistical data on the demographic profiles of visitors. Museum websites as a means of attracting audiences have been ignored in the research literature. This paper researches the role of museum websites in developing audiences with special attention to the attracting power of the websites for visitors. Content analysis of 40 Australian museum websites was conducted using a structured tool, WebNPattract, designed and tested specifically for the purpose. Content analysis results can be used for the purpose of strategic positioning of the museum and cultural tourism. The paper outlines five aspects of museums—museum offer; corporate governance; finding the museum; characteristics; and governance communication—and two approaches to museum websites: the ‘aesthetic’ and the ‘services’ perspectives. Marketing implications can be deduced from website orientation.

Responding to terrorism through networks at sites of critical infrastructure : a case study of Australian airport security networks

The importance of effective multilateral security networks is widely recognised in Australia and internationally as being essential to facilitate the large-scale sharing of information required to respond to the threat of terrorism. Australian national security agencies are currently constructing networks in order to bring the diverse national and international security agencies together to achieve this. This paper examines this process of security network formation in the area of critical infrastructure protection, with particular emphasis on airport security. We address the key issues and factors shaping network formation and the dynamics involved in network practice. These include the need for the networks to extend membership beyond the strictly defined elements of national security; the integration of public and private ‘nodes’ in counter-terrorism ‘networks’; and the broader ‘responsibilisation’ of the private sector and the challenges with ‘enabling’ them in counter-terrorism networks. We argue that the need to integrate public and private agencies in counter-terrorism networks is necessary but faces considerable organisational, cultural, and legal barriers.

Nucleation sites for ultrafine ferrite produced by deformation of austenite during single-pass strip rolling

An austenitic Ni-30 wt pct Fe alloy, with a stacking-fault energy and deformation characteristics similar to those of austenitic low-carbon steel at elevated temperatures, has been used to examine the defect substructure within austenite deformed by single-pass strip rolling and to identify those features most likely to provide sites for intragranular nucleation of ultrafine ferrite in steels. Samples of this alloy and a 0.095 wt pct C-1.58Mn-0.22Si-0.27Mo steel have been hot rolled and cooled under similar conditions, and the resulting microstructures were compared using transmission electron microscopy (TEM), electron diffraction, and X-ray diffraction. Following a single rolling pass of ∼40 pct reduction of a 2mm strip at 800 °C, three microstructural zones were identified throughout its thickness. The surface zone (of 0.1 to 0.4 mm in depth) within the steel comprised a uniform microstructure of ultrafine ferrite, while the equivalent zone of a Ni-30Fe alloy contained a network of dislocation cells, with an average diameter of 0.5 to 1.0 µm. The scale and distribution and, thus, nucleation density of the ferrite grains formed in the steel were consistent with the formation of individual ferrite nuclei on cell boundaries within the austenite. In the transition zone, 0.3 to 0.5 mm below the surface of the steel strip, discrete polygonal ferrite grains were observed to form in parallel, and closely spaced “rafts” traversing individual grains of austenite. Based on observations of the equivalent zone of the rolled Ni-30Fe alloy, the ferrite distribution could be correlated with planar defects in the form of intragranular microshear bands formed within the deformed austenite during rolling. Within the central zone of the steel strip, a bainitic microstructure, typical of that observed after conventional hot rolling of this steel, was observed following air cooling. In this region of the rolled Ni-30Fe alloy, a network of microbands was observed, typical of material deformed under plane-strain conditions.