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Maintaining a symbiotic oral microbiota is essential for oral and dental health, and host genetic factors may affect the composition or function of the oral microbiota through a range of possible mechanisms, including immune pathways. The study included 836 Swedish twins divided into separate groups of adolescents (n= 418) and unrelated adults (n= 418). Oral microbiota composition and functions of non-enzymatically lysed oral bacteria samples were evaluated using 16S rRNA gene sequencing and functional bioinformatics tools in the adolescents. Adaptive immune responses were assessed by testing for serum IgG antibodies against a panel of common oral bacteria in adults. In the adolescents, host genetic factors were associated with both the detection and abundance of microbial species, but with considerable variation between species. Host genetic factors were associated with predicted microbiota functions, including several functions related to bacterial sucrose, fructose, and carbohydrate metabolism. In adults, genetic factors were associated with serum antibodies against oral bacteria. In conclusion, host genetic factors affect the composition of the oral microbiota at a species level, and host-governed adaptive immune responses, and also affect the concerted functions of the oral microbiota as a whole. This may help explain why some people are genetically predisposed to the major dental diseases of caries and periodontitis.

Corynebacterium matruchotii may be key in tooth biofilm formation, but information about demographics, bacterial partners, and binding ligands is limited. The aims of this study were to explore C. matruchotii's demography by age and colonization site (plaque and saliva), in vitro bacterial-bacterial interactions in coaggregation and coadhesion assays, and glycolipids as potential binding ligands in thin-layer chromatogram binding assays. C. matruchotii prevalence increased from 3 months to 18 years old, with 90% and 100% prevalence in saliva and tooth biofilm, respectively. C. matruchotii aggregated in saliva in a dose-dependent manner but lacked the ability to bind to saliva-coated hydroxyapatite. In vivo, C. matruchotii abundance paralleled that of Actinomyces naeslundii, Capnocytophaga sp. HMT 326, Fusobacterium nucleatum subsp. polymorphum, and Tannerella sp. HMT 286. In vitro, C. matruchotii bound both planktonic and surface-bound A. naeslundii, Actinomyces odontolyticus, and F. nucleatum. In addition, C. matruchotii exhibited the ability to bind glycolipids isolated from human erythrocytes (blood group O), human granulocytes, rabbit intestine, human meconium, and rat intestine. Binding assays identified candidate carbohydrate ligands as isoglobotriaosylceramide, Gal alpha 3-isoglobotriaosylceramide, lactotriaosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and neolactohexaosylceramide. Thus, C. matruchotii likely uses specific plaque bacteria to adhere to the biofilm and may interact with human tissues through carbohydrate interactions.

Purpose: Advanced glycation end products (AGEs) can be formed in foods by the reaction of reducing sugars with proteins, and have been shown to induce insulin resistance and obesity in experimental studies. We examined the association between dietary AGEs intake and changes in body weight in adults over an average of 5 years of follow-up. Methods: A total of 255,170 participants aged 25–70 years were recruited in ten European countries (1992–2000) in the PANACEA study (Physical Activity, Nutrition, Alcohol, Cessation of smoking, Eating out of home in relation to Anthropometry), a sub-cohort of the EPIC (European Prospective Investigation into Cancer and Nutrition). Body weight was measured at recruitment and self-reported between 2 and 11 years later depending on the study center. A reference database for AGEs was used containing UPLC–MS/MS-measured Nε-(carboxymethyl)-lysine (CML), Nε-(1-carboxyethyl)-lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) in 200 common European foods. This reference database was matched to foods and decomposed recipes obtained from country-specific validated dietary questionnaires in EPIC and intake levels of CEL, CML, and MG-H1 were estimated. Associations between dietary AGEs intake and body weight change were estimated separately for each of the three AGEs using multilevel mixed linear regression models with center as random effect and dietary AGEs intake and relevant confounders as fixed effects. Results: A one-SD increment in CEL intake was associated with 0.111 kg (95% CI 0.087–0.135) additional weight gain over 5 years. The corresponding additional weight gain for CML and MG-H1 was 0.065 kg (0.041–0.089) and 0.034 kg (0.012, 0.057), respectively. The top six food groups contributing to AGEs intake, with varying proportions across the AGEs, were cereals/cereal products, meat/processed meat, cakes/biscuits, dairy, sugar and confectionary, and fish/shellfish. Conclusion: In this study of European adults, hig

Bacteria colonizing the mouth induce an adaptive immune response with the systemic and local presence of species or strain-specific immunoglobulins. Few studies have addressed global antibody patterns for oral bacteria or potential population time trends. We assessed these aspects in relation to a panel of oral bacteria. Using multiplex immunoblotting, IgG levels for 26 oral bacterial species (54 strains) were determined in 888 plasma samples from 30-year-old early pregnant women (n = 516) and 50-year-old men and women (n = 372) collected between 1976 and 2018. Inter-species correlations were found and age-dependent profiles and levels of immune responses to oral bacteria confirmed. We found temporal trends in the global and single-species antibody responses, but this was age-specific with both inclining and declining shifts. Prominent shifts in the younger group increased IgG towards health-associated Streptococcus salivarius and Streptococcus sanguinis, and in the older group towards disease-associated Aggregatibacter actinomycetemcomitans, Filifactor alocis, and Streptococcus mutans, among others. We concluded that temporal shifts occurred from 1976 to 2018, which may reflect improved oral health (more remaining teeth) and altered lifestyle habits, but this needs to be evaluated in observational studies considering more aspects.

The role of dairy products in cancer is unclear. We assessed consumption of fermented milk, non-fermented milk, cheese, and butter, estimated from semi-quantitative food frequency questionnaires, in relation to prospective risk of breast, prostate, colorectal, smoking-, and obesity-related cancers in 101,235 subjects, including 12,552 cancer cases, in the population-based Northern Sweden Health and Disease Study. Most analyses (n = 20) rendered null results. In men, we observed an increased prostate cancer risk among high-consumers of cheese (hazard ratio (HR) for highest vs. lowest quintile (Q5-Q1), 1.11; 95% CI, 0.97-1.27; Ptrend = 0.013). In women, high-consumers of cheese had a decreased risk of overall cancer (HR Q5-Q1, 0.95; 95% CI, 0.88-1.04; Ptrend = 0.039), smoking-related (HR Q5-Q1, 0.84; 95% CI, 0.72-0.97; Ptrend ≤ 0.001), and colorectal cancers (HR Q5-Q1, 0.82; 95% CI, 0.63-1.07; Ptrend = 0.048). Butter yielded a weak decreased obesity-related cancer risk in women (HR Q5-Q1, 0.91; 95% CI, 0.81-1.02; Ptrend = 0.049). Fermented milk yielded HRs below zero in women, but with no clear linear associations. In conclusion, this study does not support any major adverse or beneficial effects of fermented milk, non-fermented milk, cheese, and butter in the diet from a cancer risk perspective.

Supplement: 1Meeting Abstract: THU0296

Previous studies report that dental caries is partially heritable, but there is uncertainty in the magnitude of genetic effects and little understanding of how genetic factors might influence caries progression or caries subtypes. This study aimed to estimate the relative importance of genetic and environmental factors in the etiology of different caries outcomes using a twin-based design. Analysis included up to 41,678 twins in the Swedish Twin Register aged 7 to 97 y, and dental data were obtained from preexisting dental records. The outcome measures were 1) summary indices of caries experience, 2) parameters representing trajectory in caries progression derived from longitudinal modeling, and 3) caries scores in groups of biologically similar tooth surfaces derived from hierarchical clustering of tooth surfaces (termed caries clusters). Additive genetic factors explained between 49.1% and 62.7% of variation in caries scores and between 50.0% and 60.5% of variation in caries trajectories. Seven caries clusters were identified, which had estimates of heritability lying between 41.9% and 54.3%. Shared environmental factors were important for only some of these clusters and explained 16% of variation in fissure caries in molar teeth but little variation in other clusters of caries presentation. The genetic factors influencing these clusters were only partially overlapping, suggesting that different biological processes are important in different groups of tooth surfaces and that innate liability to some patterns of caries presentation may partially explain why groups of tooth surfaces form clusters within the mouth. These results provide 1) improved quantification of genetic factors in the etiology of caries and 2) new data about the role of genetics in terms of longitudinal changes in caries status and specific patterns of disease presentation, and they may help lay the foundations for personalized interventions in the future.

Oral microbiota ecology is influenced by environmental and host conditions, but few studies have evaluated associations between untargeted measures of the entire oral microbiome and potentially relevant environmental and host factors. This study aimed to identify salivary microbiota cluster groups using hierarchical cluster analyses (Wards method) based on 16S rRNA gene amplicon sequencing, and identify lifestyle and host factors which were associated with these groups. Group members (n = 175) were distinctly separated by microbiota profiles and differed in reported sucrose intake and allelic variation in the taste-preference-associated genes TAS1R1 (rs731024) and GNAT3 (rs2074673). Groups with higher sucrose intake were either characterized by a wide panel of species or phylotypes with fewer aciduric species, or by a narrower profile that included documented aciduric- and caries-associated species. The inferred functional profiles of the latter type were dominated by metabolic pathways associated with the carbohydrate metabolism with enrichment of glycosidase functions. In conclusion, this study supported in vivo associations between sugar intake and oral microbiota ecology, but it also found evidence for a variable microbiota response to sugar, highlighting the importance of modifying host factors and microbes beyond the commonly targeted acidogenic and acid-tolerant species. The results should be confirmed under controlled settings with comprehensive phenotypic and genotypic data.

Aim The aim of this study was to explore which peri-implant crevicular fluid (PICF) protein pattern is associated with the active peri-implantitis process. Materials and methods Peri-implant crevicular fluid from 25 peri-implantitis sites were subjected to proteomic analysis using liquid chromatography-tandem mass spectrometry before and at 3, 6 and 12 months after treatment, to identify associations between PICF protein pattern and implant loss, bleeding on probing, pocket depth and enamel matrix derivative (EMD) treatment. Results Clustering of subjects based on their 3-12 months PICF proteomic profiles by principal component analysis defined two major clusters. Cluster 2 differentiated from cluster 3 by 52 proteins (R-2 = 90%, Q(2) = 80%) and belonging to cluster 2 was associated with implant loss (p = 0.009) and bleeding on probing (p = 0.001). Cluster 3 was associated with implant survival and EMD treatment (p = 0.044). Conclusion Here, we demonstrate that a specific PICF proteomic profile associates with active peri-implantitis process and implant loss.

Dairy products are important constituents of most diets, and their association with adverse health outcomes remains a focus. We characterized dairy food intake and examined associations with the incidence of type 2 diabetes (T2D), myocardial infarction (MI) or stroke among 108,065 Swedish men and women. Hazard ratios (HRs) and 95% CIs were estimated using the multivariable Cox proportional hazards models in a population characterized by high milk tolerance. During a mean follow-up of 14.2 years, 11,641 first-time events occurred. Non-fermented milk intake decreased, whereas butter intake increased over the period. For high intake of non-fermented milk, the HR (95% CI) for developing T2D and MI was 1.17 (1.03, 1.34) and 1.23 (1.10, 1.37), respectively, in men. A greater intake of butter, fermented milk, and cheese tended to be associated with a reduced risk of T2D and/or MI. Non-consumers and those who chose low-fat variants of the targeted dairy products had increased risk for T2D, MI, or stroke compared to those in the non-case group. Generally, effect-sizes were small. This prospective study found that non-fermented milk was associated with an increased risk for developing T2D and MI and that subjects abstaining from dairy products or choosing low-fat variants were at greater risk. However, the overall cardiometabolic risk of non-fermented milk intake was judged as low, since the effect sizes were small.

Taste and diet preferences are complex and influenced by both environmental and host traits while affecting both food selection and associated health outcomes. The present study genotyped 94 single nucleotide polymorphisms (SNPs) in previously reported taste and food intake related genes and assessed associations with taste threshold (TT) and preferred intensity (PT) of sweet, sour and bitter, food preferences, habitual diet intake, and caries status in healthy young Swedish men and women (n = 127). Polymorphisms in the GNAT3, SLC2A4, TAS1R1 and TAS1R2 genes were associated with variation in TT and PT for sweet taste as well as sweet food intake. Increasing PT for sweet was associated with increasing preference and intake of sugary foods. Similarly, increasing TT for sour was associated with increasing intake of sour foods, whereas the associations between food preference/intake and TT/PT for bitter was weak in this study group. Finally, allelic variation in the GNAT3, SLC2A2, SLC2A4, TAS1R1 and TAS1R2 genes was associated with caries status, whereas TT, PT and food preferences were not. It was concluded that variations in taste receptor, glucose transporter and gustducin encoding genes are related to taste perception, food preference and intake as well as the sugar-dependent caries disease.

Carbonic anhydrase VI (CA6) catalyses the reversible hydration of carbon dioxide in saliva with possible pH regulation, taste perception, and tooth formation effects. This study assessed effects of variation in the CA6 gene on oral microbiota and specifically the acidophilic and caries-associated Streptococcus mutans in 17-year old Swedish adolescents (n = 154). Associations with caries status and secreted CA6 protein were also evaluated. Single Nucleotide Polymorphisms (27 SNPs in 5 haploblocks) and saliva and tooth biofilm microbiota from Illumina MiSeq 16S rDNA (V3-V4) sequencing and culturing were analysed. Haploblock 4 (rs10864376, rs3737665, rs12138897) CCC associated with low prevalence of S. mutans (OR (95% CI): 0.5 (0.3, 0.8)), and caries (OR 0.6 (0.3, 0.9)), whereas haploblock 4 TTG associated with high prevalence of S. mutans (OR: 2.7 (1.2, 5.9)) and caries (OR: 2.3 (1.2, 4.4)). The TTG-haploblock 4 (represented by rs12138897(G)) was characterized by S. mutans, Scardovia wiggsiae, Treponema sp. HOT268, Tannerella sp. HOT286, Veillonella gp.1 compared with the CCC-haploblock 4 (represented by rs12138897(C)). Secreted CA6 in saliva was weakly linked to CA6 gene variation. In conclusion, the results indicate that CA6 gene polymorphisms influence S. mutans colonization, tooth biofilm microbiota composition and risk of dental caries in Swedish adolescents.

Streptococcus mutans is a key bacterial species in the caries process, which affects >90% of the population worldwide. However, other acidogenic and aciduric/acidophilic species may contribute to disease development. In Sweden, a country with low prevalences of caries and S. mutans, a significant portion of caries-affected adolescents lack detectable levels of S. mutans. The objectives of the present study were 1) to characterize the tooth biofilm and saliva microbiota of adolescents with caries disease, with or without detectable S. mutans, from tooth biofilm and saliva samples and 2) to assess taxa clustering in the tooth biofilm and saliva samples and relate this information to caries status. For 17-y-old participants ( N = 154), enamel and dentin caries (the total number of present carious surfaces in the enamel and dentin) and caries experience (the number of decayed and filled tooth surfaces) were recorded, dental biofilm and saliva samples obtained, and information on medical and lifestyle habits collected. Multiplex 16S rDNA (V3-V4) sequencing of bacterial DNA was performed with the Illumina MiSeq platform. The Human Oral Microbiome Database and the ProbeSeq pipeline were used in the HOMI NGS procedure. In subjects with caries experience, high levels of S. mutans were associated with a few species and low levels with a panel of saccharolytic species. Present caries was similarly associated with a panel of saccharolytic species in subjects without S. mutans. Furthermore, tooth biofilm microbiota could be used to establish 4 clusters of subjects with different caries experiences. In particular, high levels of S. mutans were associated with the presence of a few influential species in multivariate modeling, including Scardovia wiggsiae. By contrast, a panel of less avid lactic acid-producing species was influential in patients with undetectable or low S. mutans levels in such modeling. These findings support a prominent role of S. mutans in infected adolesce

Bovine milk intake has been associated with various disease outcomes, with modulation of the gastro-intestinal microbiome being suggested as one potential mechanism. The aim of the present study was to explore the oral microbiota in relation to variation in self-reported milk intake. Saliva and tooth biofilm microbiota was characterized by 16S rDNA sequencing, PCR and cultivation in 154 Swedish adolescents, and information on diet and other lifestyle markers were obtained from a questionnaire, and dental caries from clinical examination. A replication cohort of 31,571 adults with similar information on diet intake, other lifestyle markers and caries was also studied. Multivariate partial least squares (PLS) modelling separated adolescents with low milk intake (lowest tertile with <0.4 servings/day) apart from those with high intake of milk (≥3.7 servings/day) based on saliva and tooth biofilm, respectively. Taxa in several genera contributed to this separation, and milk intake was inversely associated with the caries causing Streptococcus mutans in saliva and tooth biofilm samples by sequencing, PCR and cultivation. Despite the difference in S. mutans colonization, caries prevalence did not differ between milk consumption groups in the adolescents or the adults in the replication cohort, which may reflect that a significant positive association between intake of milk and sweet products was present in both the study and replication group. It was concluded that high milk intake correlates with different oral microbiota and it is hypothesized that milk may confer similar effects in the gut. The study also illustrated that reduction of one single disease associated bacterial species, such as S. mutans by milk intake, may modulate but not prevent development of complex diseases, such as caries, due to adverse effects from other causal factors, such as sugar intake in the present study.

Dental caries is a chronic infectious disease that affects billions of people with large individual differences in activity. We investigated whether PRH1 and PRH2 polymorphisms in saliva acidic proline-rich protein (PRP) receptors for indigenous bacteria match and predict individual differences in the development of caries. PRH1 and PRH2 variation and adhesion of indigenous and cariogenic (Streptococcus mutans) model bacteria were measured in 452 12-year-old Swedish children along with traditional risk factors and related to caries at baseline and after 5-years. The children grouped into low-to-moderate and high susceptibility phenotypes for caries based on allelic PRH1, PRH2 variation. The low-to-moderate susceptibility children (P1 and P4a−) experienced caries from eating sugar or bad oral hygiene or infection by S. mutans. The high susceptibility P4a (Db, PIF, PRP12) children had more caries despite receiving extra prevention and irrespective of eating sugar or bad oral hygiene or S. mutans-infection. They instead developed 3.9-fold more caries than P1 children from plaque accumulation in general when treated with orthodontic multibrackets; and had basic PRP polymorphisms and low DMBT1-mediated S. mutans adhesion as additional susceptibility traits. The present findings thus suggest genetic autoimmune-like (P4a) and traditional life style (P1) caries, providing a rationale for individualized oral care., Wos title: eGenetic- and Lifestyle-dependent Dental Caries Defined by the Acidic Proline-rich Protein Genes PRH1 and PRH2

Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um) nucleosides in the anticodon at the wobble position (U34). Earlier we showed that mutants unable to form the side chain at position 5 (ncm5 or mcm5) or lacking sulphur at position 2 (s2) of U34 result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm5 and s2 side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.

The commensal pathogen Streptococcus mutans uses AgI/II adhesins to adhere to gp340 adsorbed on teeth. Here we analyzed isolates of S. mutans (n = 70 isolates) from caries and caries-free human extremes (n = 19 subjects) by multilocus sequence typing (MLST), AgI/II full-length gene sequencing, and adhesion to parotid saliva matched from the strain donors (nested from a case-control sample of defined gp340 and acidic proline-rich protein [PRP] profiles). The concatenated MLST as well as AgI/II gene sequences showed unique sequence types between, and identical types within, the subjects. The matched adhesion levels ranged widely (40% adhesion range), from low to moderate to high, between subjects but were similar within subjects (or sequence types). In contrast, the adhesion avidity of the strains was narrow, normally distributed for high, moderate, or low adhesion reference saliva or pure gp340 regardless of the sequence type. The adhesion of S. mutans Ingbritt and matched isolates and saliva samples correlated (r = 0.929), suggesting that the host specify about four-fifths (r(2) = 0.86) of the variation in matched adhesion. Half of the variation in S. mutans Ingbritt adhesion to saliva from the caries cases-controls (n = 218) was explained by the primary gp340 receptor and PRP coreceptor composition. The isolates also varied, although less so, in adhesion to standardized saliva (18% adhesion range) and clustered into three major AgI/II groups (groups A, B-1, and B-2) due to two variable V-region segments and diverse AgI/II sequence types due to a set of single-amino-acid substitutions. Isolates with AgI/II type A versus types B-1 and B-2 tended to differ in gp340 binding avidity and qualitative adhesion profiles for saliva gp340 phenotypes. In conclusion, the host saliva phenotype plays a more prominent role in S. mutans adhesion than anticipated previously.

BACKGROUND: Iwr1, a protein conserved throughout eukaryotes, was originally identified by its physical interaction with RNA polymerase (Pol) II. PRINCIPAL FINDINGS: Here, we identify Iwr1 in a genetic screen designed to uncover proteins involved in Pol III transcription in S. cerevisiae. Iwr1 is important for Pol III transcription, because an iwr1 mutant strain shows reduced association of TBP and Pol III at Pol III promoters, a decreased rate of Pol III transcription, and lower steady-state levels of Pol III transcripts. Interestingly, an iwr1 mutant strain also displays reduced association of TBP to Pol I-transcribed genes and of both TBP and Pol II to Pol II-transcribed promoters. Despite this, rRNA and mRNA levels are virtually unaffected, suggesting a post-transcriptional mechanism compensating for the occupancy defect. CONCLUSIONS: Thus, Iwr1 plays an important role in preinitiation complex formation by all three nuclear RNA polymerases.

The diploid isolate EM93 is the main ancestor to the widely used Saccharomyces cerevisiae haploid laboratory strain, S288C. In this study, we generate a high-resolution overview of the genetic differences between EM93 and S288C. We show that EM93 is heterozygous for >45,000 polymorphisms, including large sequence polymorphisms, such as deletions and a Saccharomyces paradoxus introgression. We also find that many large sequence polymorphisms (LSPs) are associated with Ty-elements and sub-telomeric regions. We identified 2,965 genetic markers, which we then used to genotype 120 EM93 tetrads. In addition to deducing the structures of all EM93 chromosomes, we estimate that the average EM93 meiosis produces 144 detectable recombination events, consisting of 87 crossover and 31 non-crossover gene conversion events. Of the 50 polymorphisms showing the highest levels of non-crossover gene conversions, only three deviated from parity, all of which were near heterozygous LSPs. We find that non-telomeric heterozygous LSPs significantly reduce meiotic recombination in adjacent intervals, while sub-telomeric LSPs have no discernable effect on recombination. We identified 203 recombination hotspots, relatively few of which are hot for both non-crossover gene conversions and crossovers. Strikingly, we find that recombination hotspots show limited conservation. Some novel hotspots are found adjacent to heterozygous LSPs that eliminate other hotspots, suggesting that hotspots may appear and disappear relatively rapidly.

Kluyveromyces lactis gamma-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae [see text] between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) residue at position 34 (wobble position) of which the mcm(5) group is required for efficient cleavage. However, the different cleavage efficiencies of mcm(5)s(2)U(34)-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by gamma-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate [see text] is cleaved at the same position as the natural substrate. In [see text], the nucleotides U(34)U(35)C(36)A(37)C(38) are required for optimal gamma-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast [see text] reinforced the strong stimulatory effects of the mcm(5) group, revealed a weak positive effect of the s(2) group and a negative effect of the bacterial 5-methylaminomethyl (mnm(5)) group. The data underscore the high specificity of this yeast tRNA toxin.