A family of destabilized cyan fluorescent proteins as transcriptional reporters in S. cerevisiae.

The 'programmable' features of the N-end rule degradation pathway and a ubiquitin fusion strategy were exploited to create a family of destabilized cyan fluorescent proteins (CFP) to be used as transcriptional reporters. The N-degron CFP reporters characterized in this report have half-lives of approximately 75, 50 and 5 min, but further modification of the N-degron signal sequences could readily generate additional variants within this range. These destabilized CFP reporters have been engineered into convenient plasmid constructs with features to enable their expression from upstream activating sequences of choice and to facilitate their targeted integration to the URA3-TIM9 intergenic region of chromosome V. The advantages and limitations of these reporters as temporal indicators of gene expression in living cells are illustrated by their application as reporters of galactose- and pheromone-induced transcription. The plasmid design we describe and the range of different stabilities that are theoretically feasible with this strategy make the N-degron CFP reporters easily adapted to a variety of applications.

Persistent activation by constitutive Ste7 promotes Kss1-mediated invasive growth but fails to support Fus3-dependent mating in yeast.

Mitogen-activated protein kinase kinase kinase-Ste11 (MAPKKK-Ste11), MAPKK-Ste7, and MAPK-Kss1 mediate pheromone-induced mating differentiation and nutrient-responsive invasive growth in Saccharomyces cerevisiae. The mating pathway also requires the scaffold-Ste5 and the additional MAPK-Fus3. One contribution to specificity in this system is thought to come from stimulus-dependent recruitment of the MAPK cascade to upstream activators that are unique to one or the other pathway. To test this premise, we asked if stimulus-independent signaling by constitutive Ste7 would lead to a loss of biological specificity. Instead, we found that constitutive Ste7 promotes invasion without supporting mating responses. This specificity occurs because constitutive Ste7 activates Kss1, but not Fus3, in vivo and promotes filamentation gene expression while suppressing mating gene expression. Differences in the ability of constitutive Ste7 variants to bind the MAPKs and Ste5 account for the selective activation of Kss1. These findings support the model that Fus3 activation in vivo requires binding to both Ste7 and the scaffold-Ste5 but that Kss1 activation is independent of Ste5. This scaffold-independent activation of Kss1 by constitutive Ste7 and the existence of mechanisms for pathway-specific promoter discrimination impose a unique developmental fate independently of any distinguishing external stimuli.

"Death was a blessing"--should it ever be pharmaceutically hastened? British pharmacists' views.

OBJECTIVES: To investigate the views and experiences of British pharmacists in physician-assisted suicide (PAS) and voluntary active euthanasia (VAE) and to examine whether differences in views were associated with personal characteristics. METHOD: A postal questionnaire was mailed to a random sample of 500 registered pharmacists, with addresses in England or Wales, stratified according to sex and community/hospital working environments. MAIN OUTCOME MEASURE: Pharmacists' reports of their views and experiences of PAS and VAE. RESULTS: Completed questionnaires were received from 295 respondents (response rate: 59%). The majority of respondents (83%) reported that at times, a person had the right to choose their own manner of death; 61% and 53% thought that there should be changes in the Law to legalise VAE and PAS, respectively. If it were legal to do so, 63% stated that they would be willing to dispense medication for VAE and 64% for PAS. However, only 45% agreed that the pharmacist should have a role in advising the prescriber on the choice and dose of drugs used. Sex, age, and years registered as a pharmacist were not found to be significantly associated with views on the practices of PAS and VAE. However, pharmacists who reported having a religion were significantly less likely to favour such practices. On comparing the views of community and hospital pharmacists, community pharmacists were significantly less likely than expected to want to be informed about the intended purpose of lethal drugs. CONCLUSION: In this survey, while over half of British pharmacists did not disagree in principle to the legalisation of VAE and/or PAS, they were less supportive of direct involvement in such procedures. Religion was a discriminatory factor associated with negative views of VAE and PAS. The significant minority of pharmacists not wanting to know the purpose of drugs they suspected might be for PAS or VAE is not in accordance with professional accountability, reflecting the complex and sometimes conflicting legal and moral aspects of such practices when deciding upon a course of action.