The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

The Function and Regulation of Photobodies in Phytochrome Signaling

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

On the Regulation of Small Actors: Three Experimental Essays about Policies based on Voluntary Compliance and Decentralized Monitoring

Monitoring and enforcement are perhaps the biggest challenges in the design and implementation of environmental policies in developing countries where the actions of many small informal actors cause significant impacts on the ecosystem services and where the transaction costs for the state to regulate them could be enormous. This dissertation studies the potential of innovative institutions based on decentralized coordination and enforcement to induce better environmental outcomes. Such policies have in common that the state plays the role of providing the incentives for organization but the process of compliance happens through decentralized agreements, trust building, signaling and monitoring. I draw from the literatures in collective action, common-pool resources, game-theory and non-point source pollution to develop the instruments proposed here. To test the different conditions in which such policies could be implemented I designed two field-experiments that I conducted with small-scale gold miners in the Colombian Pacific and with users and providers of ecosystem services in the states of Veracruz, Quintana Roo and Yucatan in Mexico. This dissertation is organized in three essays.

The first essay, “Collective Incentives for Cleaner Small-Scale Gold Mining on the Frontier: Experimental Tests of Compliance with Group Incentives given Limited State Monitoring”, examines whether collective incentives, i.e. incentives provided to a group conditional on collective compliance, could “outsource” the required local monitoring, i.e. induce group interactions that extend the reach of the state that can observe only aggregate consequences in the context of small-scale gold mining. I employed a framed field-lab experiment in which the miners make decisions regarding mining intensity. The state sets a collective target for an environmental outcome, verifies compliance and provides a group reward for compliance which is split equally among members. Since the target set by the state transforms the situation into a coordination game, outcomes depend on expectations of what others will do. I conducted this experiment with 640 participants in a mining region of the Colombian Pacific and I examine different levels of policy severity and their ordering. The findings of the experiment suggest that such instruments can induce compliance but this regulation involves tradeoffs. For most severe targets – with rewards just above costs – raise gains if successful but can collapse rapidly and completely. In terms of group interactions, better outcomes are found when severity initially is lower suggesting learning.

The second essay, “Collective Compliance can be Efficient and Inequitable: Impacts of Leaders among Small-Scale Gold Miners in Colombia”, explores the channels through which communication help groups to coordinate in presence of collective incentives and whether the reached solutions are equitable or not. Also in the context of small-scale gold mining in the Colombian Pacific, I test the effect of communication in compliance with a collective environmental target. The results suggest that communication, as expected, helps to solve coordination challenges but still some groups reach agreements involving unequal outcomes. By examining the agreements that took place in each group, I observe that the main coordination mechanism was the presence of leaders that help other group members to clarify the situation. Interestingly, leaders not only helped groups to reach efficiency but also played a key role in equity by defining how the costs of compliance would be distributed among group members.

The third essay, “Creating Local PES Institutions and Increasing Impacts of PES in Mexico: A real-Time Watershed-Level Framed Field Experiment on Coordination and Conditionality”, considers the creation of a local payments for ecosystem services (PES) mechanism as an assurance game that requires the coordination between two groups of participants: upstream and downstream. Based on this assurance interaction, I explore the effect of allowing peer-sanctions on upstream behavior in the functioning of the mechanism. This field-lab experiment was implemented in three real cases of the Mexican Fondos Concurrentes (matching funds) program in the states of Veracruz, Quintana Roo and Yucatan, where 240 real users and 240 real providers of hydrological services were recruited and interacted with each other in real time. The experimental results suggest that initial trust-game behaviors align with participants’ perceptions and predicts baseline giving in assurance game. For upstream providers, i.e. those who get sanctioned, the threat and the use of sanctions increase contributions. Downstream users contribute less when offered the option to sanction – as if that option signal an uncooperative upstream – then the contributions rise in line with the complementarity in payments of the assurance game.

Factors influencing malaria control policy-making in Kenya, Uganda and Tanzania.

BACKGROUND: Policy decisions for malaria control are often difficult to make as decision-makers have to carefully consider an array of options and respond to the needs of a large number of stakeholders. This study assessed the factors and specific objectives that influence malaria control policy decisions, as a crucial first step towards developing an inclusive malaria decision analysis support tool (MDAST). METHODS: Country-specific stakeholder engagement activities using structured questionnaires were carried out in Kenya, Uganda and Tanzania. The survey respondents were drawn from a non-random purposeful sample of stakeholders, targeting individuals in ministries and non-governmental organizations whose policy decisions and actions are likely to have an impact on the status of malaria. Summary statistics across the three countries are presented in aggregate. RESULTS: Important findings aggregated across countries included a belief that donor preferences and agendas were exerting too much influence on malaria policies in the countries. Respondents on average also thought that some relevant objectives such as engaging members of parliament by the agency responsible for malaria control in a particular country were not being given enough consideration in malaria decision-making. Factors found to influence decisions regarding specific malaria control strategies included donor agendas, costs, effectiveness of interventions, health and environmental impacts, compliance and/acceptance, financial sustainability, and vector resistance to insecticides. CONCLUSION: Malaria control decision-makers in Kenya, Uganda and Tanzania take into account health and environmental impacts as well as cost implications of different intervention strategies. Further engagement of government legislators and other policy makers is needed in order to increase funding from domestic sources, reduce donor dependence, sustain interventions and consolidate current gains in malaria.

A tale of two market failures: Technology and environmental policy

Market failures associated with environmental pollution interact with market failures associated with the innovation and diffusion of new technologies. These combined market failures provide a strong rationale for a portfolio of public policies that foster emissions reduction as well as the development and adoption of environmentally beneficial technology. Both theory and empirical evidence suggest that the rate and direction of technological advance is influenced by market and regulatory incentives, and can be cost-effectively harnessed through the use of economic-incentive based policy. In the presence of weak or nonexistent environmental policies, investments in the development and diffusion of new environmentally beneficial technologies are very likely to be less than would be socially desirable. Positive knowledge and adoption spillovers and information problems can further weaken innovation incentives. While environmental technology policy is fraught with difficulties, a long-term view suggests a strategy of experimenting with policy approaches and systematically evaluating their success. © 2005 Elsevier B.V. All rights reserved.