Analysis of the Essential Functions of the C-terminal Protein/Protein Interaction Domain of Saccharomyces cerevisiae pol epsilon and Its Unexpected Ability to Support Growth in the Absence of the DNA Polymerase Domain

As first observed by Wittenberg (Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenburg, C. (1999) Mol. Cell 3, 679-685), we find that deletion mutants lacking the entire N-terminal DNA polymerase domain of yeast pol epsilon are viable. However, we now show that point mutations in DNA polymerase catalytic residues of pol epsilon are lethal. Taken together, the phenotypes of the deletion and the point mutants suggest that the polymerase of pol epsilon may normally participate in DNA replication but that another polymerase can substitute in its complete absence. Substitution is inefficient because the deletion mutants have serious defects in DNA replication. This observation raises the question of what is the essential function of the C-terminal half of pol epsilon . We show that the ability of the C-terminal half of the polymerase to support growth is disrupted by mutations in the cysteine-rich region, which disrupts both dimerization of the POL2 gene product and interaction with the essential DPB2 subunit, suggesting that this region plays an important architectural role at the replication fork even in the absence of the polymerase function. Finally, the S phase checkpoint, with respect to both induction of RNR3 transcription and cell cycle arrest, is intact in cells where replication is supported only by the C-terminal half of pol epsilon , but it is disrupted in mutants affecting the cysteine-rich region, suggesting that this domain directly affects the checkpoint rather than acting through the N-terminal polymerase active site

Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase epsilon in DNA Replication and the S/M Checkpoint Pathway

It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) epsilon (POL2) couple DNA replication to the S/M checkpoint (Navas, T. A., Zheng, Z., and Elledge, S. J. (1995) Cell 80, 29-39). Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint. All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response. The mutations affect the assembly of the pol epsilon holoenzyme. Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol epsilon . The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol epsilon , but not for interaction with DPB3. Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase ε (pol ε) complex - Demonstration of a dimeric pol ε

Saccharomyces cerevisiae DNA polymerase epsilon (pol ε) is essential for chromosomal replication. A major form of pol ε purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2pzDpb2p and Dpb3pzDpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol ε may be dimeric in vivo. Gel filtration of the Pol2pzDpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2pzDpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication

Financing Climate Resilience: Mobilizing Resources and Incentives to Protect Boston from Climate Risks

This report, sponsored by the Boston Green Ribbon Commission with the generous support of the Barr Foundation, looks at different financial mechanisms for climate resilience. It provides recommendations for the City of Boston and the region on how to pay for climate adaptation investments

On the Oscillation of Combustion of a Laminar Spray

A spray combustor, with flow velocities in the laminar range, exhibits a unique operating mode where large amplitude, self-induced oscillations of the flame shape occur. The phenomenon, not previously encountered, only occurs when fuel is supplied in the form of fine liquid droplets and does not occur when fuel is supplied in gaseous form. Several flow mechanisms are coupled in such a fashion as to trigger and maintain the oscillatory motion of the flame. These mechanisms include heat transfer and evaporation processes, dynamics of two-phase flows, and effects of gravity (buoyancy forces). An interface volume, lying between the fuel nozzle and the flame was found to be the most susceptible to gravity effects, and postulated to be responsible for inducing the oscillatory motion. Heptane fuel was used in the majority of the tests

Measurements and predictions of a liquid spray from an air-assist nozzle

Droplet size and gas velocity were measured in a water spray using a two-component Phase/Doppler Particle Analyzer. A complete set of measurements was obtained at axial locations from 5 to 50 cm downstream of the nozzle. The nozzle used was a simple axisymmetric air-assist nozzle. The sprays produced, using the atomizer, were extremely fine. Sauter mean diameters were less than 20 microns at all locations. Measurements were obtained for droplets ranging from 1 to 50 microns. The gas phase was seeded with micron sized droplets, and droplets having diameters of 1.4 microns and less were used to represent gas-phase properties. Measurements were compared with predictions from a multi-phase computer model. Initial conditions for the model were taken from measurements at 5 cm downstream. Predictions for both the gas phase and the droplets showed relatively good agreement with the measurements

On the combustion of a laminar spray

A spray combustor, with flow velocities in the laminar range, exhibits a unique operating mode where large amplitude, self-induced oscillations of the flame shape occur. The phenomenon, not previously encountered, only occurs when fuel is supplied in the form of fine liquid droplets and does not occur when fuel is supplied in gaseous form. Several flow mechanisms are coupled in such a fashion as to trigger and maintain the oscillatory motion of the flame. These mechanisms include heat transfer and evaporation processes, dynamics of two-phase flows, and effects of gravity (buoyancy forces). An interface volume, lying above the fuel nozzle and below the flame was found to be the most susceptible to gravity effects and postulated to be responsible for inducing the oscillatory motion. Heptane fuel was used in the majority of the tests. Tests performed with iso-octane also showed similar results

"On our own, we can't manage": experiences with infant feeding recommendations among Malawian mothers living with HIV

<p>Abstract</p> <p>Background</p> <p>Infant feeding in communities with a high prevalence of HIV and AIDS is a potential challenge for mothers who must ultimately decide how to feed their infants within contexts that constrain their choices.</p> <p>Methods</p> <p>We investigated how infant feeding policy recommendations translate into maternal infant feeding decisions and practices using ethnographic research conducted between August 2004 and June 2005 among women participating in a prevention of mother-to-child transmission (PMTCT) program in Lilongwe, Malawi.</p> <p>Results</p> <p>Qualitative findings are that maternal ability to adhere to recommendations to breastfeed exclusively for the first six months of infant life was constrained by expectations and psycho-social support. The most salient were women's pre-existing views on breastfeeding, their understanding of the medico-scientific information, and the quality of counselling received. In contrast, maternal decisions to wean were largely influenced by household economic factors and food insecurity.</p> <p>Conclusions</p> <p>We conclude that PMTCT programs delivered in ways which "download" the responsibility of adhering to recommendations to women in the absence of adequate psycho-social and livelihood supports contribute to substantial maternal psychosocial distress in this and, likely, similar settings.</p

Consistency of linkage results across exams and methods in the Framingham Heart Study

BACKGROUND: The repeated measures in the Framingham Heart Study in the Genetic Analysis Workshop 13 data set allow us to test for consistency of linkage results within a study across time. We compared regression-based linkage to variance components linkage across time for six quantitative traits in the real data. RESULTS: The variance components approach found 11 significant linkages, the regression-based approach found 4. There was only one region that overlapped. Consistency between exams generally decreased as the time interval between exams increased. The regression-based approach showed higher consistency in linkage results across exams. CONCLUSION: The low consistency between exams and between methods may help explain the lack of replication between studies in this field

The epidemiology of heart failure: The Framingham Study

AbstractCongestive heart failure has become an increasingly frequent reason for hospital admission during the last 2 decades and clearly represents a major health problem. Data from the Framingham Heart Study indicate that the incidence of congestive heart failure increases with age and is higher in men than in women. Hypertension and coronary heart disease are the two most common conditions predating its onset. Diabetes mellitus and electrocardiographic left ventricular hypertrophy are also associated with an increased risk of heart failure. During the 1980s, the annual age-adjusted incidence of congestive heart failure among persons aged ≥ 45 years was 7.2 cases/1,000 in men and 4.7 cases/1,000 in women, whereas the age-adjusted prevalence of overt heart failure was 24/1,000 in men and 25/1,000 in women. Despite improved treatments for ischemic heart disease and hypertension, the age-adjusted incidence of heart failure has declined by only 11%/calendar decade in men and by 17%/calendar decade in women during a 40-year period of observation. In addition., congestive heart failure remains highly lethal, with a median survival time of 1.7 years in men and 3.2 years in women and a 5-year survival rate of 25% in men and 38% in women