Socioeconomic per-case costs of stroke, myocardial infarction, and preterm birth attributable to air pollution in Sweden.

BACKGROUND: Incident cases of stroke, myocardial infarction, and preterm birth have established exposure-response functions associated with air pollution. However, there are no studies reporting detailed costs per case for these health outcomes that are adapted to the cost-benefit tools that guide the regulation of air pollution. OBJECTIVES: The primary objective was to establish non-fatal per-case monetary estimates for stroke, myocardial infarction, and preterm birth attributable to air pollution in Sweden, and the secondary objective was to ease the economic evaluation process of air pollution morbidity effects and their inclusion in cost-benefit assessments. METHODS: Based on recommendations from the literature, the case-cost analysis considered direct and indirect medical costs, as well as production losses and informal costs relevant for the calculation of the net present value. A literature search was conducted to estimate the costs of each category for each incident case in Sweden. Informal costs were estimated using the quality-adjusted life-years approach and the corresponding willingness-to-pay in the Swedish population. The total average per-case cost was estimated based on specific health outcome durations and severity and was discounted by 3.5% per year. Sensitivity analysis included varying discount rates, severity of health outcome, and the range of societal willingness to pay for quality-adjusted life years. RESULTS: The average net present value cost estimate was €2016 460k (185k-1M) for non-fatal stroke, €2016 24k (16k-38k) for myocardial infarction, and €2016 34k (19k-57k) for late preterm birth. The main drivers of the per-case total cost estimates were health outcome severity and societal willingness to pay for risk reduction. Varying the discount rate had the largest effect on preterm birth, with costs changing by ±30% for the discount rates analysed. RECOMMENDATION: Because stroke, myocardial infarction, and preterm birth have established exposure-response functions linking these to air pollution, cost-benefit analyses should include the costs for these health outcomes in order to adequately guide future air pollution and climate change policies.

Sir2 and Reb1 antagonistically regulate nucleosome occupancy in subtelomeric X-elements and repress TERRAs by distinct mechanisms.

Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibiting the DNA damage response. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins bind to terminal repeats and to subtelomeric X-elements, resulting in transcriptional silencing. Herein, we show that sir2 mutant strains display a specific loss of a nucleosome residing in the X-elements and that this deficiency is remarkably consistent between different telomeres. The X-elements contain several binding sites for the transcription factor Reb1 and we found that Sir2 and Reb1 compete for stabilizing/destabilizing this nucleosome, i.e. inactivation of Reb1 in a sir2 background reinstated the lost nucleosome. The telomeric-repeat-containing RNAs (TERRAs) originate from subtelomeric regions and extend into the terminal repeats. Both Sir2 and Reb1 repress TERRAs and in a sir2 reb1 double mutant, TERRA levels increased synergistically, showing that Sir2 and Reb1 act in different pathways for repressing TERRAs. We present evidence that Reb1 restricts TERRAs by terminating transcription. Mapping the 5'-ends of TERRAs from several telomeres revealed that the Sir2-stabilized nucleosome is the first nucleosome downstream from the transcriptional start site for TERRAs. Finally, moving an X-element to a euchromatic locus changed nucleosome occupancy and positioning, demonstrating that X-element nucleosome structure is dependent on the local telomere environment.

Valuating environmental impacts from ship emissions - The marine perspective.

Shipping is an activity responsible for a range of different pressures affecting the marine environment, air quality and human welfare. The methodology on how ship emissions impact air quality and human health are comparatively well established and used in cost-benefit analysis of policy proposals. However, the knowledge base is not the same for impacts on the marine environment and a coherent environmental and socio-economic impact assessment of shipping has not yet been made. This risk policies to be biased towards air pollution whilst trading off impacts on the marine environment. The aim of the current study was to develop a comprehensive framework on how different pressures from shipping degrade marine ecosystems, air quality and human welfare. A secondary aim was to quantify the societal damage costs of shipping due to the degradation of human welfare in a Baltic Sea case study. By adding knowledge from marine ecotoxicology and life-cycle analysis to the existing knowledge from climate, air pollution and environmental economics we were able to establish a more comprehensive conceptual framework that allows for valuation of environmental impacts from shipping, but it still omits economic values for biological pollution, littering and underwater noise. The results for the Baltic Sea case showed the total annual damage costs of Baltic Sea shipping to be 2.9 billion €2010 (95% CI 2.0-3.9 billion €2010). The damage costs due to impacts on marine eutrophication (768 million €2010) and marine ecotoxicity (582 million €2010) were in the same range as the total damage costs associated with reduced air quality (816 million €2010) and climate change (737 million €2010). The framework and the results from the current study can be used in future socio-economic assessments of ship emissions to prioritize cost efficient measures. The framework can be used globally but the damage costs presented on the marine environment are restricted to emissions on the Baltic Sea and Kattegat region as they are based on willingness to pay studies conducted on citizens around the Baltic Sea where eutrophication and emissions of chemicals are particularly threats to the state of the Baltic Sea.

MutSα deficiency increases tolerance to DNA damage in yeast lacking postreplication repair.

By combining mutations in DNA repair genes, important and unexpected interactions between different repair pathways can be discovered. In this study, we identified a novel link between mismatch repair (MMR) genes and postreplication repair (PRR) in Saccharomyces cerevisiae. Strains lacking Rad5 (HLTF in mammals), a protein important for restarting stalled replication forks in the error-free PRR pathway, were supersensitive to the DNA methylating agent methyl methanesulfonate (MMS). Deletion of the mismatch repair genes, MSH2 or MSH6, which together constitutes the MutSα complex, partially suppressed the MMS super-sensitivity of the rad5Δ strain. Deletion of MSH2 also suppressed the MMS sensitivity of mms2Δ, which acts together with Rad5 in error-free PRR. However, inactivating the mismatch repair genes MSH3 and MLH1 did not suppress rad5Δ, showing that the suppression was specific for disabling MutSα. The partial suppression did not require translesion DNA synthesis (REV1, REV3 or RAD30), base excision repair (MAG1) or homologous recombination (RAD51). Instead, the underlying mechanism was dependent on RAD52 while independent of established pathways involving RAD52, like single-strand annealing and break-induced replication. We propose a Rad5- and Rad51-independent template switch pathway, capable of compensating for the loss of the error-free template-switch subpathway of postreplication repair, triggered by the loss of MutSα.

Helicase/SUMO-targeted ubiquitin ligase Uls1 interacts with the Holliday junction resolvase Yen1.

Resolution of branched DNA structures is pivotal for repair of stalled replication forks and meiotic recombination intermediates. The Yen1 nuclease cleaves both Holliday junctions and replication forks. We show that Yen1 interacts physically with Uls1, a suggested SUMO-targeted ubiquitin ligase that also contains a SWI/SNF-family ATPase-domain. Yen1 is SUMO-modified in its noncatalytic carboxyl terminus and DNA damage induces SUMOylation. SUMO-modification of Yen1 strengthens the interaction to Uls1, and mutations in SUMO interaction motifs in Uls1 weakens the interaction. However, Uls1 does not regulate the steady-state level of SUMO-modified Yen1 or chromatin-associated Yen1. In addition, SUMO-modification of Yen1 does not affect the catalytic activity in vitro. Consistent with a shared function for Uls1 and Yen1, mutations in both genes display similar phenotypes. Both uls1 and yen1 display negative genetic interactions with the alternative HJ-cleaving nuclease Mus81, manifested both in hypersensitivity to DNA damaging agents and in meiotic defects. Point mutations in ULS1 (uls1K975R and uls1C1330S, C1333S) predicted to inactivate the ATPase and ubiquitin ligase activities, respectively, are as defective as the null allele, indicating that both functions of Uls1 are essential. A micrococcal nuclease sequencing experiment showed that Uls1 had minimal effects on global nucleosome positioning/occupancy. Moreover, increased gene dosage of YEN1 partially alleviates the mus81 uls1 sensitivity to DNA damage. We suggest a preliminary model in which Uls1 acts in the same pathway as Yen1 to resolve branched DNA structures.

Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching.

Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast.

Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATα in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1-MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HMLα) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MATa locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed -1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATα genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase-derived endonuclease vital for sexual differentiation.

A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis.

Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.

Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival.

The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.

Ixr1 is required for the expression of the ribonucleotide reductase Rnr1 and maintenance of dNTP pools.

The Saccharomyces cerevisiae Dun1 protein kinase is a downstream target of the conserved Mec1-Rad53 checkpoint pathway. Dun1 regulates dNTP pools during an unperturbed cell cycle and after DNA damage by modulating the activity of ribonucleotide reductase (RNR) by multiple mechanisms, including phosphorylation of RNR inhibitors Sml1 and Dif1. Dun1 also activates DNA-damage-inducible genes by inhibiting the Crt1 transcriptional repressor. Among the genes repressed by Crt1 are three out of four RNR genes: RNR2, RNR3, and RNR4. The fourth RNR gene, RNR1, is also DNA damage-inducible, but is not controlled by Crt1. It has been shown that the deletion of DUN1 is synthetic lethal with the deletion of IXR1, encoding an HMG-box-containing DNA binding protein, but the reason for this lethality is not known. Here we demonstrate that the dun1 ixr1 synthetic lethality is caused by an inadequate RNR activity. The deletion of IXR1 results in decreased dNTP levels due to a reduced RNR1 expression. The ixr1 single mutants compensate for the reduced Rnr1 levels by the Mec1-Rad53-Dun1-Crt1-dependent elevation of Rnr3 and Rnr4 levels and downregulation of Sml1 levels, explaining why DUN1 is indispensible in ixr1 mutants. The dun1 ixr1 synthetic lethality is rescued by an artificial elevation of the dNTP pools. We show that Ixr1 is phosphorylated at several residues and that Ser366, a residue important for the interaction of HMG boxes with DNA, is required for Ixr1 phosphorylation. Ixr1 interacts with DNA at multiple loci, including the RNR1 promoter. Ixr1 levels are decreased in Rad53-deficient cells, which are known to have excessive histone levels. A reduction of the histone gene dosage in the rad53 mutant restores Ixr1 levels. Our results demonstrate that Ixr1, but not Dun1, is required for the proper RNR1 expression both during an unperturbed cell cycle and after DNA damage.

An assessment of costs and benefits associated with mercury emission reductions from major anthropogenic sources.

Several measures are available for reducing mercury emissions; however, these measures differ with regard to emission control efficiency, cost, and environmental benefits obtained through their implementation. Measures that include the application of technology, such as technology to remove mercury from flue gases in electric power plants, waste incinerators, and smelters, are rather expensive compared with nontechnological measures. In general, dedicated mercury removal is considerably more expensive than a co-benefit strategy, using air pollution control equipment originally designed to limit emissions of criterion pollutants, such as particulate matter, sulfur dioxide, or oxides of nitrogen. Substantial benefits can be achieved globally by introducing mercury emission reduction measures because they reduce human and wildlife exposure to methyl mercury. Although the reduction potential is greatest with the technological measures, technological and nontechnological solutions for mercury emissions and exposure reductions can be carried out in parallel.

Alpha3, a transposable element that promotes host sexual reproduction.

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism.

Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair.

The role of nonhomologous end-joining components in telomere metabolism in Kluyveromyces lactis.

The relationship between telomeres and nonhomologous end-joining (NHEJ) is paradoxical, as NHEJ proteins are part of the telomere cap, which serves to differentiate telomeres from DNA double-strand breaks. We explored these contradictory functions for NHEJ proteins by investigating their role in Kluyveromyces lactis telomere metabolism. The ter1-4LBsr allele of the TER1 gene resulted in the introduction of sequence altered telomeric repeats and subsequent telomere-telomere fusions (T-TFs). In this background, Lig4 and Ku80 were necessary for T-TFs to form. Nej1, essential for NHEJ at internal positions, was not. Hence, T-TF formation was mediated by an unusual NHEJ mechanism. Rad50 and mre11 strains exhibited stable short telomeres, suggesting that Rad50 and Mre11 were required for telomerase recruitment. Introduction of the ter1-4LBsr allele into these strains failed to result in telomere elongation as normally observed with the ter1-4LBsr allele. Thus, the role of Rad50 and Mre11 in the formation of T-TFs was unclear. Furthermore, rad50 and mre11 mutants had highly increased subtelomeric recombination rates, while ku80 and lig4 mutants displayed moderate increases. Ku80 mutant strains also contained extended single-stranded 3' telomeric overhangs. We concluded that NHEJ proteins have multiple roles at telomeres, mediating fusions of mutant telomeres and ensuring end protection of normal telomeres.

Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis.

Illegitimate recombination (IR) is the process by which two DNA molecules not sharing homology to each other are joined. In Kluyveromyces lactis, integration of heterologous DNA occurred very frequently therefore constituting an excellent model organism to study IR. IR was completely dependent on the nonhomologous end-joining (NHEJ) pathway for DNA double strand break (DSB) repair and we detected no other pathways capable of mediating IR. NHEJ was very versatile, capable of repairing both blunt and non-complementary ends efficiently. Mapping the locations of genomic IR-events revealed target site preferences, in which intergenic regions (IGRs) and ribosomal DNA were overrepresented six-fold compared to open reading frames (ORFs). The IGR-events occurred predominantly within transcriptional regulatory regions. In a rad52 mutant strain IR still preferentially occurred at IGRs, indicating that DSBs in ORFs were not primarily repaired by homologous recombination (HR). Introduction of ectopic DSBs resulted in the efficient targeting of IR to these sites, strongly suggesting that IR occurred at spontaneous mitotic DSBs. The targeting efficiency was equal when ectopic breaks were introduced in an ORF or an IGR. We propose that spontaneous DSBs arise more frequently in transcriptional regulatory regions and in rDNA and such DSBs can be mapped by analyzing IR target sites.

The Drosophila melanogaster sir2+ gene is nonessential and has only minor effects on position-effect variegation.

Five Drosophila melanogaster genes belong to the highly conserved sir2 family, which encodes NAD(+)-dependent protein deacetylases. Of these five, dsir2(+) (CG5216) is most similar to the Saccharomyces cerevisiae SIR2 gene, which has profound effects on chromatin structure and life span. Four independent Drosophila strains were found with P-element insertions near the dsir2 transcriptional start site as well as extraneous linked recessive lethal mutations. Imprecise excision of one of these P elements (PlacW07223) from a chromosome freed of extraneous lethal mutations produced dsir2(17), a null intragenic deletion allele that generates no DSIR2 protein. Contrary to expectations from the report by Rosenberg and Parkhurst on their P-mobilization allele dSir2(ex10), homozygosity for dsir2(17) had no apparent deleterious effects on viability, developmental rate, or sex ratio, and it fully complemented sir2(ex10). Moreover, through a genetic test, we ruled out the reported effect of dSir2(ex10) on Sex-lethal expression. We did observe a modest, strictly recessive suppression of white(m4) position-effect variegation and a shortening of life span in dsir2 homozygous mutants, suggesting that dsir2 has some functions in common with yeast SIR2.

Functional diversity of silencers in budding yeasts.

We studied the silencing of the cryptic mating-type loci HMLa and HMRa in the budding yeast Kluyveromyces lactis. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLalpha. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REBI gene derepressed the transcription of the HMLalpha1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces.