Sequential and counter-selectable cassettes for fission yeast

BACKGROUND: Fission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. However, the currently available cassettes all share the same tef promoter and terminator sequences. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes. RESULTS: Here we have generated new selection cassettes that do not recombine with those traditionally used. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2'-deoxyuridine (FdU, Floxuridin or FUDR). By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method. CONCLUSIONS: The plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study

Telomere protein Rap1 is a charge resistant scaffolding protein in chromosomal bouquet formation

© 2015 Amelina et al. Background: Chromosomes reorganize in early meiotic prophase to form the so-called telomere bouquet. In fission yeast, telomeres localize to the nuclear periphery via interaction of the telomeric protein Rap1 with the membrane protein Bqt4. During meiotic prophase, the meiotic proteins Bqt1-2 bind Rap1 and tether to the spindle pole body to form the bouquet. Although it is known that this polarized chromosomal arrangement plays a crucial role in meiotic progression, the molecular mechanisms of telomere bouquet regulation are poorly understood. Results: Here, we detected high levels of Rap1 phospho-modification throughout meiotic prophase, and identified a maximum of 35 phosphorylation sites. Concomitant phosphomimetic mutation of the modification sites suggests that Rap1 hyper-phosphorylation does not directly regulate telomere bouquet formation or dissociation. Despite the negative charge conferred by its highly phosphorylated state, Rap1 maintains interactions with its binding partners. Interestingly, mutations that change the charge of negatively charged residues within the Bqt1-2 binding site of Rap1 abolished the affinity to the Bqt1-2 complex, suggesting that the intrinsic negative charge of Rap1 is crucial for telomere bouquet formation. Conclusions: Whereas Rap1 hyper-phosphorylation observed in meiotic prophase does not have an apparent role in bouquet formation, the intrinsic negative charge of Rap1 is important for forming interactions with its binding partners. Thus, Rap1 is able to retain bouquet formation under heavily phosphorylated status.This work is supported mainly by the European Research Council (281722-HRMCB) and partly by Cancer Research UK (C36439/A12097) and the Cancer Research UK - UCL Centre

LARP7 family proteins have conserved function in telomerase assembly

Understanding the intricacies of telomerase regulation is crucial due to the potential health benefits of modifying its activity. Telomerase is composed of an RNA component and reverse transcriptase. However, additional factors required during biogenesis vary between species. Here we have identified fission yeast Lar7 as a member of the conserved LARP7 family, which includes the Tetrahymena telomerase-binding protein p65 and human LARP7. We show that Lar7 has conserved RNA-recognition motifs, which bind telomerase RNA to protect it from exosomal degradation. In addition, Lar7 is required to stabilise the association of telomerase RNA with the protective complex LSm2-8, and telomerase reverse transcriptase. Lar7 remains a component of the mature telomerase complex and is required for telomerase localisation to the telomere. Collectively, we demonstrate that Lar7 is a crucial player in fission yeast telomerase biogenesis, similarly to p65 in Tetrahymena, and highlight the LARP7 family as a conserved factor in telomere maintenance

LARP7 family proteins have conserved function in telomerase assembly

Understanding the intricacies of telomerase regulation is crucial due to the potential health benefits of modifying its activity. Telomerase is composed of an RNA component and reverse transcriptase. However, additional factors required during biogenesis vary between species. Here we have identified fission yeast Lar7 as a member of the conserved LARP7 family, which includes the Tetrahymena telomerase-binding protein p65 and human LARP7. We show that Lar7 has conserved RNA-recognition motifs, which bind telomerase RNA to protect it from exosomal degradation. In addition, Lar7 is required to stabilise the association of telomerase RNA with the protective complex LSm2–8, and telomerase reverse transcriptase. Lar7 remains a component of the mature telomerase complex and is required for telomerase localisation to the telomere. Collectively, we demonstrate that Lar7 is a crucial player in fission yeast telomerase biogenesis, similarly to p65 in Tetrahymena, and highlight the LARP7 family as a conserved factor in telomere maintenance

State work and the testing concours of citizenship

Anyone trying to be a citizen has to pass through a set of practices trying to be a state. This paper investigates some of the ways testing practices calibrate citizens, and in doing so, perform “the state.” The paper focuses on three forms of citizenship testing, which it considers exemplary forms of “state work,” and which all, in various ways, concern “migration.” First, the constitution of a “border crossing,” which requires an identity test configured by deceptibility. Second, the Dutch asylum process, in which “being gay” can, in certain cases, be reason for being granted asylum, but where “being gay” is also the outcome of an examination organized by suspicion. And third, the Dutch measurement of immigrants’ “integration,” which is comprised of a testing process in which such factishes as “being a member of society” and “being modern” surface. Citizenship is analyzed in this paper as accrued and (re)configured along a migration trajectory that takes shape as a testing concours, meaning that subjects become citizens along a trajectory of testing practices. In contributing both to work on states and citizenship, and to work on testing, this paper thus puts forward the concept of citizenship testing as state work, where “state work is the term for that kind of labor that most knows itself as comparison, equivalency, and exchange in the social realm” (Harney, 2002, pp. 10–11). Throughout the testing practices discussed here, comparison, equivalency, and exchange figure prominently as the practical achievements of crafting states and citizens

The telomere bouquet facilitates meiotic prophase progression and exit in fission yeast

During meiotic prophase, chromosome arrangement and oscillation promote the pairing of homologous chromosomes for meiotic recombination. This dramatic movement involves clustering of telomeres at the nuclear membrane to form the so-called telomere bouquet. In fission yeast, the telomere bouquet is formed near the spindle pole body (SPB), which is the microtubule organising centre, functionally equivalent to the metazoan centrosome. Disruption of bouquet configuration impedes homologous chromosome pairing, meiotic recombination and spindle formation. Here, we demonstrate that the bouquet is maintained throughout meiotic prophase and promotes timely prophase exit in fission yeast. Persistent DNA damages, induced during meiotic recombination, activate the Rad3 and Chk1 DNA damage checkpoint kinases and extend the bouquet stage beyond the chromosome oscillation period. The auxin-inducible degron system demonstrated that premature termination of the bouquet stage leads to severe extension of prophase and consequently spindle formation defects. However, this delayed exit from meiotic prophase was not caused by residual DNA damage. Rather, loss of chromosome contact with the SPB caused delayed accumulation of CDK1-cyclin B at the SPB, which correlated with impaired SPB separation. In the absence of the bouquet, CDK1-cyclin B localised near the telomeres but not at the SPB at the later stage of meiotic prophase. Thus, bouquet configuration is maintained throughout meiotic prophase, by which this spatial organisation may facilitate local and timely activation of CDK1 near the SPB. Our findings illustrate that chromosome contact with the nuclear membrane synchronises meiotic progression of the nucleoplasmic chromosomes with that of the cytoplasmic SPB.the European Research Council and Cancer Research U

Towards framing the global in Global Development: prospects for development geography

This paper examines data in the public sphere on the global scope of geography’s UKRI Global Challenges Research Fund (GCRF) projects. Building on decolonial critiques of development research, I argue that geography should frame ‘the global’ of global research as a sphere of ethical choices in research design and practice. The distribution of funded projects in the UKRI Gateway data suggests geographers succeed where they extend on the more worthy aspects of the discipline’s Area Studies legacy. The discipline’s engagements with Early Career Researchers, international colleagues, and the development sector, however, have potentially been reshaped by GCRF and thus need closer examination. While the UK government has brought the GCRF programme to a close, further work on these themes should inform the next iteration of global research. The ethical choices which make research global will remain fundamental to equitable design and impact in Global Development projects, thus scholars in development geography should prepare to make their projects more transparent and accountable

an association between diversity and exoticism

Focusing on contemporary Iranian artists and intellectuals, I examine the creation of collective identifications from an internal perspective. Drawing on research on migrant associations and ethnic and racial boundaries in Germany, the ethnographic account alternates between internal relations, member's participation in the transnational field of Iranian artists, and representative activities in the German public sphere. It explains how the members' unequal resources and varying politics of value caused a shift in the association's system of value. From a critique of assimilationism and the promotion of the value of diversity, the group came to largely comply with the system of value prevailing in the German public sphere, sustained by its intersection with the one that shapes the transnational social field of Iranian artists