Aphaenogaster senilis is a monogynous, monandrous, fission performing ant, in which queen selection occurs during colony fission and when replacement queens are produced to overcome orphaning. First-born queens are usually behaviourally dominant over subsequent queens, and eventually inherit the colony. We investigated the importance of physical dominance in queen selection in orphaned groups by manipulating the fighting ability of first-born queens via mandibular ablation.Many animals face some form of conflict over reproductive opportunities. Queen selection in social insect colonies represents a high-stakes conflict where competition occurs among multiple queens for a few or a single reproductive role(s). The outcome of the contest is critical to the fitness of all colony individuals as most are sterile, and thus represents a conflict at multiple levels. First emerged queens were heavier than second emerged queens, performed almost all aggression, were behaviourally dominant 92% of the time, and prevailed in 76% of groups after co-existing for 16 days on average. Mandibular ablation had no effect on queen behaviour or contest outcome.Aggression is probably ritualised and contests are decided by workers based on relative queen fertility. First-born queens thus have an inherent advantage over second-born queens as they have more time to develop ovaries. Subordinates never retaliated against aggression from dominants and this lack of retaliation can be interpreted as a form of bourgeois strategy as dominants were almost always first-born. However, the lack of alternative reproductive options makes not-fighting effectively a form of suicide. High relatedness between full-sister queens means that subordinates may be better off sacrificing themselves than risking injury to both queens by fighting. Conflict frequently arises in animal societies over reproduction. In many social insects, this conflict is centred on the queen because she is the sole reproductive in a colony of potentially thousands of individuals: workers cannot (or do not) reproduce themselves and only gain fitness indirectly. The identity of the queen is therefore of critical importance as the fitness of all individuals in the colony depends on her. In some species workers may have opportunities to select their queen, and might be expected to do so based on fecundity, relatedness, longevity, and minimising the delay to egg laying . HoweverQueen selection occurs in three main contexts in social insect colonies: i) after worker emergence in pleometrotic (multiple queen) assemblages , ii) assAphaenogaster senilis is a monogynous, monandrous ant that founds colonies by dependent colony foundation (= fission [ fission ). Supern fission ,5. In or fission . As this fission . There s fission  demonstrApis mellifera the ablation of a mandible results in queen avoidance of other queens: mutilation of all queens yields stable polygynous colonies despite this species being strictly monogynous [A. senilis should increase the proportion of second-born queens inheriting the colony if queen choice is based on phenotypic characteristics such as fighting ability, whereas there will be no effect of the treatment if queen choice is based on order of emergence. Mutilation also allows testing whether A. senilis queens show evidence of self-assessment, as has been argued for Apis mellifera [Order of eclosion and any advantages relating to acquired asymmetries are correlated so that it remains unclear which factor is determinant in queen selection. We investigated this by manipulating the ability of first queens to dominate subsequent queens, by ablating a mandible of the first-born queen in experimentally orphaned colonies. In nogynous . We infeellifera . SpecifiA. senilis were collected from Aznalcázar, near Seville, Spain, between the 8th and 12th of November, 2008. One additional colony used was collected in February 2007 and had undergone laboratory hibernation. Entire colonies were placed in open plastic boxes of 16 × 26 cm, the walls of which were treated with fluon to prevent ant escape, and kept in a constant temperature room (~28°C) under a 12/12 hr day/night light cycle. Colonies were housed in nests made of two plastic Petri dishes of 10 × 5.7 cm each (diameter × height) placed on top of one another. The top Petri dish provided the nesting space while the bottom Petri dish contained water, creating a humid environment in the top box via a thin metallic mesh. After one week, queens were removed from all colonies and workers and brood were divided evenly between two identical nest boxes, creating a total of 60 orphaned groups. One half of each colony was assigned to the control group and the other to the treatment group, and thus control and treatment groups were of identical size. Groups were provided with water and sugar cubes ad libitum, and fed freeze-dried crickets and an artificial diet three times a week. Colony size in the initially collected colonies ranged from 550 to 1698 (mean ± SE: 1031 ± 57), with experimental/treatment groups half this size.Twenty-nine colonies of A. senilis workers rear existing brood into replacement queens, and groups were monitored over the following 18 weeks for developing queen pupae. Queens were weighed and paint marked two days after emergence to permit them time to develop a hardened cuticle and be accepted by workers. In experimental groups, the left mandible of the first emerging queen was ablated at this time using small scissors. Queens were given several minutes rest before being returned to their group. Workers seemed to detect the injury to ablated queens, but behaviour soon returned to normal following reintroduction in both groups. Any sexual (queen) pupae reared after the first two queens had emerged were removed to simplify the selection process.In the absence of a queen, Groups were monitored daily for eclosion and survival of queens for the duration of the experiment 18 weeks). In addition, behavioural observations were made for the duration of the association. The scan method  was empl8 weeks. 2. Significance levels are reported for terms when adding them last to this minimal adequate model. Means are quoted with standard errors throughout unless otherwise noted.Statistics were carried out in the 'R' statistical package, version 2.8.1 for Windows . In the 24 = 3.59, p < 0.01, and t22 = 4.27, p < 0.01 respectively).Two-queen associations arose in 54 of 60 orphaned groups (26 treatment and 28 control); four treatment and two control groups (including two treatment-control pairs) did not produce a second queen (hereafter Q2) and were excluded from subsequent analyses. Orphaned groups produced first queens (Q1s) 31.6 ± 2.4 days after being orphaned (range 22-93). In four instances, the first queen died before eclosion of the second queen. This was not a treatment effect as treated queens were no more likely to die than untreated (n = 2 of each). Indeed, treated (ablated) individuals survived the entire observation period (up to 96 days), suggesting no negative effect of the treatment on longevity. In the four cases above monitoring was maintained until further rearing of sexual brood lead to the presence of two queens. Q2s were produced 46.3 ± 3.0 days after orphaning range 27-111), or 14.7 ± 1.9 days after first queens (range 1-65). Third and subsequent sexual brood were detected (and removed) in 28 groups . As pupae were removed before eclosion we cannot assess how this would translate into adults, but these data concur with a previous study in that multiple replacement queens (up to 5) can be produced [-111, or 21 = 13.52, p < 0.001 overall; χ21 = 6.26, p = 0.012 control; χ21 = 7.34, p = 0.007 treatment). GLM analysis with group, queen age, queen weight, date of contest resolution, initial group size, contest duration, treatment and dominance status (see below) as potential explanatory variables indicated that only dominance status had a significant effect (p < 0.001) on the outcome of contests (ie: which queen prevailed).Contests were resolved in favour of one or other queen (i.e. a queen died) in 50 groups, 15.9 ± 1.7 days (range 0-51) after the emergence of Q2s. In two other groups both queens were found dead on the same day, and in the last two groups both queens remained alive when the experiment was terminated (after 19 and 71 days of cohabitation). First eclosed queens were significantly more likely to prevail, and did so in 76% of cases overall (n = 50), 78% (n = 23) of treatment and 74% (n = 27) of control groups  and in treatment and control groups . Aggression did not differ between treatments for Q1s or Q2s . No aggression between queens was observed in 7 groups prior to queen selection. In the remaining 38 groups (where dominance could be ascertained), Q1s were behaviourally dominant in all but 3 cases (92%). Q1s were equally likely to be dominant in control and treatment groups . Q2s emerged victorious in 12 instances overall and behavioural data were available for 10 of these. Q1 was behaviourally dominant in 6 cases, Q2 was dominant in 2 cases, and there was no aggression in the final 2 cases . No behavioural observations were possible in 9 groups as queens were killed before observations. We observed a total of 296 aggressions , as summarised in figure Behavioural profiles for Q1s and Q2s are summarised in figure Subordinates were completely stationary and often assumed a crouched posture or lay flat during biting or stand-overs, and occasionally during antennation. At no time were subordinates observed attempting to retaliate, defend themselves or escape when aggressed by a dominant. Bouts of biting could last many minutes (fights > 35 mins observed), during which time the dominant queen would attack the subordinate repeatedly, often interspersed with short periods of walking away from the subordinate. In contrast, worker aggression occurred only during queen execution. Several executions were observed in which multiple workers (up to around twenty) would immobilise and bite the queen, continuing until the victim was dismembered and eventually devoured. Victims did attempt to flee from workers when under attack, but were never successful. Queens were generally not observed aiding in queen executions and queen aggression did not appear to pre-empt queen execution. The one exception to this was an instance where Q1 was observed immobilising a Q2 with the aid of three workers for ~1 hr before leaving workers, who then executed Q2 (which required a further 3 hrs). Queen aggression never otherwise included immobilisation, except in one further instance in which Q1 was immobilised for several minutes during the execution of Q2.Aphaenogaster senilis indicated that first-born queens were behaviourally dominant over subsequently produced queens and usually survived the queen selection process. Chéron et al [Queen selection is a critical event in the life of a colony. It represents not only a potential conflict of interest between queens, workers, and the colony as a whole, but also a possibility for colony failure. Workers are known to be responsible for selection of queens in several species of ants, and cull queens based on a range of criteria . It is ion et al  proposedOur results are similar with respect to timing of queen replacement and number of queens produced, and concur with Chéron et al  in that A. senilis, the proximate mechanism triggering queen execution remains unclear. Indeed, disentangling worker and queen roles in queen elimination can be problematic: workers attack already injured queens in Solenopsis invicta [Messor (Veromessor)pergandei [A. senilis argues against queens using chemical means to 'mark' subordinates for execution or immobilisation  and did not differ between control and treatment groups. We witnessed multiple bouts of aggression in some groups, some of which were extended (>35 minutes), yet at no time did this clearly result in an injury to either queen or lead to queen culling. In contrast, queen contests in honeybees are resolved fatally and relatively quickly  suggesting they did not consider themselves under direct threat from queen aggression. These data suggest that aggression in A. senilis is ritualised and serves primarily as a dominance display rather than a direct means of resolving contests. This may explain the different response to ablation in honeybees, where fighting between queens is the primary means of contest resolution and bouts are usually fatal [While dominance is important for survival in queens of  invicta ,16,32, aergandei . The lonion eg: ,35) and  and A. s-15 mins ). Second-15 mins , the behLeptothorax sp. A dominance was correlated with ovarian status and attractiveness to workers, and lead to preferential feeding and eventual expulsion or killing of subordinates [Dinoponera quadriceps [Aphaenogaster cockerelli and workers kill "cheating" (laying) workers [Dominance is important in reproductive success in many species, and can be correlated with productivity or fitness . In Leptrdinates ,38. We drdinates ,40. The rdinates ,8, this rdinates ). Develoadriceps , which i workers ,44. Anot workers  and indiA. senilis have few options other than inheriting the colony, as dispersal is only possible through colony fission which can only occur in large colonies and under certain conditions [Trissolcus basalis, the first arriving female at a patch of host eggs is more likely to win contests than intruders, who back down before escalated conflict [A. senilis are effectively prior residents, and in most cases this translated to behavioural dominance and eventual victory. Queens are thus basically following a 'bourgeois' convention. However, game theory models also predict that when the value of the contested resources represents the majority of an individual's possible lifetime fitness, contestants should fight regardless of ability, kinship or asymmetries [Apis mellifera with one mandible ablated avoid contests, but will fight if challenged [A. senilis that may be important is the high relatedness between queens, which are full sisters (r = 0.75; [indirect fitness even if it is killed, and this indirect fitness may be greater than the benefits of fighting for inheritance if fighting entails risk to both queens. By obeying a bourgeois strategy and ceding to first-born queens, second-born queens can minimise the chance of both queens being injured while also facilitating rapid commencement of reproductive activity in the colony.Aggression is, however, important in the establishment of dominance, and a problem that remains is explaining the lack of aggression from subordinate queens. Although some subordinate queens were able to prevail, dominant queens succeeded in 73% of cases for which dominance could be ascertained. Queens produced during emergency queen production in nditions . Thus, nnditions . Prior rnditions , though nditions . The prinditions ,48. For conflict . Similarmmetries -52. Hencallenged . Our datAphaenogaster senilis queens were behaviourally dominant over second-born queens, and inherited the colony in most cases. Ablating one mandible of the first-born queen did not affect contest outcomes, suggesting aggression is probably a form of ritualised dominance. Queens are likely selected by workers based on reproductive quality, with first-born queens gaining a significant time-related advantage over subsequently-born queens. Queens may follow a bourgeois strategy in contests, with second-born queens effectively committing suicide by ceding to first-born queens. This minimises the chance of colony failure and reproductive delay through injury or death of both queens, and is possible because queens are full sisters and thus highly related.Queen selection is an important process in the lifecycle of a colony as it influences the fitness of all colony members. First-born The authors declare that they have no competing interests.Both authors conceived of the study, designed and executed the experiment, and contributed to the final manuscript. ALC performed analyses and drafted the manuscript.


Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown.Urease is a virulence factor that plays a role in the resistance of ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium.Re-examination of the ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.Based on these results, we concluded that the operon  These equilibrate in water causing a rise of the pH of the medium. Accordingly, bacterial ureases serve two main purposes: to neutralize acidic conditions, and to provide a source of assimilable nitrogen.Urease catalyzes the chemical hydrolysis of the urea molecule into COBrucella spp, as well as in Helicobacter pylori, Klebsiella and Yersinia, urease allows bacteria to survive the acidic conditions encountered in the stomach during the gastrointestinal infection [Pathogenic bacteria exploit urease activity in different ways along the infectious process. In nfection -5. The rnfection .Brucella contains two urease operons, both located in chromosome I. The Brucella ure1 operon contains the genes ureDABCEFG, and the Brucella ure2 locus shows the structure ureABCEFGDT [ure2, ureT, encodes a putative urea transporter homologous to Yut from Yersinia pseudotuberculosis [Brucella species show a strong urease activity, derived from ure1 but not from ure2, and this activity is responsible for the ability of Brucella to survive stomachal transit and to establish a systemic infection [B. ovis is not able to infect the host by the gastrointestinal route, a fact that has been linked to its lack of urease activity [B. suis showed the presence of urease subunits from ure1 but not from ure2 [ure2 genes are transcribed in vivo [Brucella.Ureases are complex enzymes generally composed of three structural subunits (UreABC). To assemble a functional urease, the cooperation of several accessory proteins is required (UreEFGD) and, as a consequence, large gene clusters are needed to encode for functional ureases. rculosis . Most Bractivity . Furtherrom ure2 . StrikinureDEFG that form part of both Brucella urease operons. High affinity nickel transport systems of bacteria fall into several categories: the ATP-binding cassette (ABC) systems represented by NikABCDE of E. coli [Brucella suis, a nickel ABC transporter coded by the nikABCDE gene cluster has been identified. This gene cluster has been shown to contribute towards the urease activity of the bacteria when Ni ions are chelated with EDTA in the growth medium, but not in control media without EDTA. This implies, as noted by the authors, that there is at least another functional nickel transport system in this bacteria [Urease is one of the few enzymes known to contain nickel atoms in their active centers . Because E. coli , the new E. coli  and seco E. coli , UreH [1 E. coli , and Hup E. coli ,15. The  E. coli ,16. In Bbacteria .Desulfovibrio vulgaris urea transporter [Helicobacter UreI, ten for Yersinia Yut), that in the case of Yut and UreT form two repeated domains linked by a large periplasmic loop. However, the most important difference between UreI and Yut is their response to acidic pH. While Yut shows similar activity at a range of different pH [Urease activity is also dependent on the supply of urea. There are at least three urea uptake systems in bacteria. The ABC-type urea transporter is energy-dependent and requires ATP to transport urea across the cytoplasmic membrane. The other two urea transporters, Yut and UreI, are energy-independent and appear to be channel-like structures that allow urea to enter the cytoplasm through a pore powered by a favorable concentration gradient that is maintained by rapid hydrolysis of the incoming urea by intrabacterial ureases. The recent determination of the crystal structure of the nsporter  confirmserent pH , UreI sherent pH . The preerent pH . Both niure2 includes genes for the transport of urea and nickel, and that these genes are expressed and active, contributing to urease activity and to resistance to the acidic conditions present in the oral route of infection.The evidence presented here shows that the urease operon Brucella urease operon ure2 did not contribute to the urease activity of the bacteria [ure2 operon of Brucella abortus 2308 was considered to be composed of eight genes ureABCEFGDT (BAB1_1376-1383). A re-evaluation of the chromosomal region suggested that some genes immediately downstream of ureT could be part of the same operon, because: 1) the distance between ureT and the contiguous gene nikM was only 26 bp, 2) there was a good ribosome binding site upstream the putative start codon of nikM, and 3) there was no obvious transcriptional terminator between the two genes. PCR amplification of reverse transcribed Brucella RNA using the pairs of primers indicated in Table nikKMLQO) was to code for a nickel transport system belonging to the novel ECF class of modular transporters [ure2 operon is thus composed of thirteen genes putatively involved in three different functions, namely urease production, urea transport, and nickel transport.We have previously reported that the bacteria . The uresporters . Accordiure2 genes on urease activity, we constructed three mutants as described in the Methods section: i) a polar mutant created by replacing part of ureT with a kanamycin resistance gene that has a transcriptional termination signal (ΔureTp), ii) a non-polar mutant lacking the aph transcriptional terminator, which only affects ureT function (ΔureT), and iii) a ΔnikO mutant, affecting the ATP binding protein of the putative nickel transport system encoded by nikO, the last gene of the operon, and predicted to have the biggest impact on the correct function of the transporter while still maintaining basal activity [In order to analyze the impact of the activity .ΔureTp and ΔnikO mutants had their urease activity reduced to about 50% of the activity observed in the wild type strain 2308, while the urease activity was rather unaffected in the ΔureT mutant. To confirm that the observed effects were direct consequence of the introduced mutations, mutants were complemented with plasmids carrying either ureT or nikO genes. Mutants ΔureTp and ΔnikO were complemented by the nikO containing plasmid pFJS245  was used to complement the ΔureTp, ΔureT, or ΔnikO mutants (data not shown).Urease activity was measured in crude protein extracts from the mutants and the wild type strain. The results in Figure 5 Figure , but no nikKMLQO, currently annotated in the Brucella genomes as components of a cobalt transport system, are found downstream of the ure2 genes, and form part of the same operon, so we tested whether they were involved in the transport of nickel, which is essential for urease activity. The addition of an excess of nickel in the form of NiCl2 would supply the metal needed for urease assembly in spite of the inactivation of the nik transport system. We tested the urease activity of the different strains grown in the presence or absence of 0.5 mM NiCl2 in the culture medium. The results in Figure nik genes of the ure2 operon in nickel transport. These results are also a further evidence for the extension of the operon until the nikO gene; that is a polar ureT mutation has a lower urease activity than the corresponding non-polar mutant, and identical activity to that of the nikO mutant, suggesting that the observed phenotype is the result of a polar effect on the genes downstream of ureT.Nickel and cobalt are transition metals that can share the same bacterial import systems . The genBrucella urease assayed in vitro shows a pH-dependent activity that is maximal at pH 7.3 [B. abortus 2308 cells, the activity was higher at low pH values and dropped to near one third as the pH of the medium reached a value of 6  were exposed for one hour to increasing concentrations of urea (pH 4.2). The urease activity of both the wild type and the complemented strains increased steadily with the available urea. However, the ΔureT mutant showed significantly lower activities at all the urea concentrations tested, except for 75 and 100 mM, where urease activity reached wild type levels . At 50 mM urea the ΔureT mutant was as resistant as the parental strain, while the ΔureTp and nikO mutants remained significantly susceptible  exposures to highly acidic environments pH 2.0), urease activity in the presence of urea in the medium enables 5 min exp.0, ureasnikO was completely reversed by complementing it with pFJS245 in trans. The mutant ΔureTp could not be complemented in this assay with either pFJS243 or pFJS245 (data not shown). However the acid sensitivity of both mutants could be compensated by the addition of NiCl2 to the growth medium (data not shown).The susceptibility to low pH of the mutant Brucella had already been reported. Evidence from our laboratory and elsewhere [ure1 contributed towards the urease activity of Brucella. Explanations based on a degeneration in the sequence of ure2 were ruled out and early evidence suggested that ure2 genes were transcribed [ure2 should have some function that ensures its conservation in the genome of Brucella [The presence of two operons encoding urease in the genome of lsewhere ,2,9 showure2 operon by RT-PCR, confirming that the ure2 genes are transcribed and that transcription goes beyond ureT, up to the gene nikO (BAB1_1388)  was pH-dependent. The activity observed at pH 5.8 or higher would be the result of urea diffusing through the inner membrane. That UreT is an acid-activated urea transporter is somewhat surprising, given that its closest homolog, Yut of Y. enterocolitica, is not pH-regulated [H. pylori [D. vulgaris shows that all the residues that form the pore are conserved (data not shown). The only one minor difference is that in one of the two urea slots present in UreT, one of the phenylalanines forming the slot is changed to leucine (L201F), and the corresponding leucine in the slot is changed to phenylalanine (F304L) (data not shown).By compairing the mutant strains to the wild type progenitor we observed that there was no significant difference in urease activity between protein extracts from egulated , while t. pylori , shares . pylori  revealedYersinia spp, the unrestricted entry of urea would alkalinize the cytoplasm to lethal levels. Yersinia has solved this problem by expressing a urease with an acidic pH-optimum, that has little or no activity at ~pH 8.0 [Brucella urease has a pH optimum of 7.3, and although its activity is much lower at pH 8.0, it is still significant. In this case, the problem of lethal alkalinization is prevented by the existence of a pH-regulated urea transporter that reduces urea uptake to just the amount that diffuses through the inner membrane.Since urea uptake is not pH regulated in  ~pH 8.0 . BrucellΔureT mutant, mutants ΔureTp and ΔnikO showed around a 40% decrease in urease activity in cell extracts. Both phenotypes were reversed by complementation of the mutant strains with a nikO-containing plasmid or, alternatively, with high concentrations of nickel in the culture medium suggesting that the amount of active urease in these mutants was limited by nickel availability. Complementation of the urease activity of the ΔureTp mutant with the nikO plasmid was rather surprising if we consider that the mutant should be defective not only in nikO but also in the other nik genes. Furthermore, the susceptibity to low pH of the ΔureTp mutant was not complemented by the nikO gene in trans, suggesting that other factors may be implicated in the acid resistance phenotype of Brucella.In contrast to the B. suis, NikABCDE (10). nikA mutants were not affected in urease activity unless a chelating agent was added to the medium. As both the ΔureTp and ΔnikO mutants show lower urease activity than the wild type when grown in standard medium, we concluded that NikKMLQO is the main nickel transport system in Brucella. B. suis nikA mutants have an intact NikKMLQO nickel transporter, whose function can override the nikA mutation. In B. abortus 2308 by contrast, the single nikO mutation produced a significant decrease in urease activity. Sequence analysis reveals that the three B. abortus strains sequenced so far are natural nikA mutants, explaining why the nikO mutation caused such a decrease in urease activity. When supplemented with 500 mM NiCl2, B. abortus 2308 showed an increased urease activity, which probably reflects that the nickel content is not optimal in B. abortus and that this could be one of the factors that determines a lower urease activity in B. abortus when compared to B. suis.NikO is predicted to be the ATPase component of an ECF-type nickel transporter, and its mutation should abolish most of the activity of the transporter. There is another nickel transport system already described in Brucella possesses several genetic resources to cope with its needs of urease. At least three loci, nik, ure1 and ure2 play a role in this function. There are also some additional genes, like cobT, that contribute in a yet unknown way to the overall urease activity [Brucella spp. not only has at least one active urease, but also a specific, proton-gated urea transporter, and two nickel transport systems that contribute to the overall urease activity. While the urease structural genes and nickel transport systems affect the intrinsic urease activity, UreT would not affect it, but would be important for physiological processes such as the resistance to low acid conditions by increasing the efflux of urea into the bacteria, affecting in this way the overall urease activity, specially at low urea concentrations. These are the conditions faced by the bacteria in the gastrointestinal route, that it is been again recognized in the last years as an important route of infection in Brucella [Brucella ,24, reinB. abortus strains were grown in Brucella broth (BB) or Brucella agar (BA) plates . Escherichia coli strains were grown in Luria-Bertani broth (LB) or plates (LA). When required, media were supplemented with the following antibiotics: kanamycin (Km) 50 μg/ml, ampicillin (Ap) 100 μg/ml, or chloramphenicol (Cm) 25 μg/ml, or with 500 μM of NiCl2. Mating mixtures were plated in BA plates made selective with Brucella Selectavial, (BAF) . All experiments with live Brucella were performed in a Biosafety Level 3 facility at the Department of Molecular Biology of the University of Cantabria.The bacterial strains and plasmids used in this study are listed in Table B. abortus 2308 genomic DNA extracted as previously described [Vent polymerase (New England Biolabs), and standard PCR was performed using Taq (Qiagen). PCR products were purified using GenElute™ PCR Clean-Up (Sigma). Amplified products were cloned in pGEM®-T Easy (Promega) or pJET1.2 (Fermentas) depending on the polymerase used. The DNA sequence of the final plasmids was determined to rule out mutations introduced by PCR. Gateway cloning was made according to the manufacturer instructions (Invitrogen). The oligonucleotides used are listed in Table DNA manipulations were performed according to standard techniques . Restricescribed . High-fiaphT) was constructed as follows. Primer aphT.F, derived from pUC4K [aph gene, and primer aphT.R, derived from the aph sequence [PstI and cloned into pUC4K/PstI, yielding plasmid pFJS235. The aphT gene can be retrieved from pFJS235 by using PstI, HincII, SalI, or EcoRI.Plasmid pFJS235 carrying the aminoglycoside 3'-phosphotransferase gene (which encodes for kanamycin resistance) devoid of its transcription terminator  from B. abortus strain 2308, ureT was replaced by aph. DNA fragments both upstream and downstream of ureT were amplified with the following set of primers: U_BMEI0642_XbaI.F and U_BMEI0642_BamHI.R were used to amplify a region of 578 bp upstream of ureT (U_ureT) and D_BMEI0642_BglII.F and D_BMEI0642_PstI.R were used to amplify a region of 589 downstream of ureT (D_ureT). PCR fragments of the expected size were gel-purified and cloned into pGEM®-T Easy resulting in plasmids pFJS225 and pFJS226 respectively. pFJS225 was linearized with BamHI and pFJS226 with BglII, and ligated to a 1.2 kb BamHI fragment from pUC4K, containing aph with its transcription terminator. An XbaI &PstI fragment of 1.4 kb was obtained directly from the partially digested ligation mixture, and cloned into pDS132 digested with PstI and partially with XbaI, to obtain pFJS227b, that was used to construct the corresponding ΔureTp mutants in Brucella, as described below.To construct a polar ΔureT mutant from B. abortus 2308, two PCR fragments of 578 bp and 619 bp were generated immediately upstream and downstream of the ureT gene with oligonucleotides U_BMEI0642_XbaI.F and U_BMEI0642_BamHI.R and oligonucleotides D_BMEI0642.F and D_BMEI0642_PstI.R respectively. The reaction conditions for both PCRs were 30 cycles at 55°C, and 45 seconds at 72°C, using Vent polymerase. Both fragments (containing complementary regions) were ligated by overlapping PCR using oligonucleotides U_BMEI0642_XbaI.F and D_BMEI0642_PstI.R and Taq polymerase from Qiagen, for 25 cycles at 55°C and extension time of 1 minute at 72°C. The resulting fragment containing the ureT deletion allele was gel-purified and cloned into pGEM®-T Easy to obtain pFJS236. A BamHI fragment from pFJS235 containing aphT was introduced into the BamHI site of pFJS236, resulting in plasmid pFJS238. An XbaI & PstI fragment from this plasmid containing the replaced ureT gene was cloned into pDS132 digested with PstI and partially with XbaI, resulting in plasmid pFJS241b, that was used to create the corresponding Brucella mutant as described below.For the construction of a non-polar ΔnikO non-polar mutant, two PCR fragments of 501 bp and 499 bp were generated immediately upstream and downstream of the nikO gene with oligonucleotides BAB1_1388_XbaI.F and RT_BAB1_1388.R, and oligonucleotides BAB1_1388_BglII.F and BAB1_1388_PstI.R respectively, using Vent polymerase. Both fragments (containing complementary regions) were ligated by overlapping PCR using oligonucleotides BAB1_1388_XbaI.F and BAB1_1388_PstI.R and Taq polymerase, and the resulting fragment containing the deleted nikO allele was cloned into pGEM®-T Easy (pFJS237). A BamHI fragment from pFJS235 containing aphT was introduced into the BglII site of pFJS237, resulting in plasmid pFJS239. An XbaI &PstI fragment from this plasmid containing the replaced nikO gene was cloned into pDS132 digested with PstI and partially with XbaI, resulting in plasmid pFJS242b, that was used to create the corresponding Brucella mutant as described below.For the construction of a ΔE. coli S17-1 λ pir, and mobilized to the corresponding Brucella recipient strain, by mixing equal volumes (100 μl) of liquid cultures of both donor and recipient cells on a 0.22-μm-pore-size filter. The filter was left for 4 h on a BA plate without antibiotics, soaked in PBS, and then different dilutions were plated onto BAF plates containing Cm and Km. Colonies growing in this medium represented single-crossover events. Five colonies of each construct were pooled and grown in BB, and 108 CFU were plated on BA containing 5% sucrose to select for the double crossover. Sucrose-resistant colonies were replicated in BA Cm plates, and CmS colonies were selected and analyzed by PCR and southern blot to ensure that the right mutant had been constructed.To construct the different mutants, replacement plasmids were transformed into ureT was cloned by using the Gateway recombination cloning technology (Invitrogen) [Brucella ORFeome generated previously [attR1 and attR2 recombinational sites. The recombinational cloning procedure was performed as recommended by the manufacturer, to produce pFJS243. nikO was amplified by PCR with oligonucleotides nikO_SalI.F and nikO_PstI.R, cloned into pGEM®-T Easy to obtain pFJS244, and then subcloned into pBBR1 MCS/SalI &PstI to give pFJS245. Both pFJS243 and pFJS245 were transformed into E. coli S17-1 λ pir to be mobilized to Brucella. Complemented strains were selected in BAF Cm.To complement the different mutants complementation plasmids were constructed as follows: itrogen) . The enteviously . The deseviously , carriesB. abortus strains were grown in BB until the end of the exponential phase, washed in sterile water and resuspended at a concentration of 108 CFU/ml in citrate buffer pH 2.0 for 30 min in the presence or absence of different concentrations of urea. Bacteria were washed three times in phosphate-buffered saline (PBS), and survivors counted after dilution and plating.2 as indicated, were recovered by centrifugation, washed, and resuspended in PBS to a concentration of 108 CFU/ml. The preparations were then lysed using three 10-s cycles with a FastPrep system  at the maximum setting, cooled on ice, and centrifuged for 5 min at 25,000 × g at 4°C to remove the cell debris. Crude extracts were stored at -80°C until they were used. For standard urease reactions, 5 to 10 μl of extract were added to a tube containing 200 μl of 50 mM urea in PBS and incubated for 5 min at 37°C. Urease activitiy was also measured in intact cells, in this case the pelleted bacteria were resuspended in 200 μl of either PBS (pH 7.7) or citrate buffer at different pH , supplemented or not with urea at different concentrations , and incubated at 37°C for 1 hour. The amount of ammonia released from urea hydrolysis was determined colorimetrically by the modified Berthelot reaction [-1mg-1 protein (for crude extracts) and pmol of NH3 min-1 log10 cfu-1 (for intact cells).Urease activity was determined by measuring the amount of ammonia released from urea. Exponential cultures of bacteria grown in BB, supplemented or not with 500 μM of NiClreaction , and thereaction . Urease 600 = 0.6-0.7) were stabilized with RNAprotect Bacteria Reagent (Qiagen). After harvesting the cells, they were resuspended in 300 μl of TE containing lysozyme 1 mg/ml, and incubated for 15 min at room temperature. They were treated with 15 μl of 10% Zwittergent 3-16  and 6 μl of 20 mg/ml of proteinase K for 1 h at 37°C, and then total RNA was extracted with the RNeasy Mini System (Qiagen) in combination with the RNase-Free DNase Set (Qiagen). cDNA was generated by using Superscript III RT (Invitrogen) according to the manufacturer's protocol. 1 μl of the resulting cDNA was used for each PCR. As a negative control, reactions were also run on RNA templates without RT treatment, and as a positive control, each reaction was also made with purified genomic DNA as template. The cycling parameters were 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min. The resulting amplicons were analyzed in 0.8% agarose gels. Primers were designed with Primer3 software [3 ml of a bacterial culture in mid-log phase (ODsoftware .B. abortus 2308 strain was obtained fron GenBank (Accession numbers AM040264 and AM040265 for chromosomes I and II respectively).The complete genome sequence and annotation of the Blast comparisons against the microbial genome database were performed via web at the NCBI Blast server .t test. A P value of < 0.05 was considered statistically significant.A statistical analysis was performed using Prism3, version 3.0. Statistical significance wascalculated using either a nonparametric Mann-Whitney test or an unpaired FJS designed and supervised the work and wrote the paper. AC performed all the microbiological work and the different urease activity assays. AS did the transcriptional analysis of the urease operon. JMGL performed the genomic analysis and bioinformatic work and also wrote the paper.


Four hundred specimens were collected from pediatric patients hospitalized in Singapore; 21 of these specimens tested positive for human metapneumovirus (HMPV), with the A2 genotype predominating. A 5% infection rate was estimated, suggesting that HMPV is a significant cause of morbidity among the pediatric population of Singapore. Human metapneumovirus (HMPV) is a new member of the family Paramyxoviridae. It was first identified in children with respiratory diseases in the Netherlands and is now recognized as a substantial cause of acute respiratory infection in pediatric patients . Viral RNA (vRNA) was extracted from each of the thawed nasopharyngeal swabs with the QIAamp viral RNA minikit  according to the manufacturer’s instructions. Of the total RNA extracted from the clinical specimens, 5 μL was subjected to real-time reverse transcription–PCR (RT-PCR) testing by using the N gene specific primer set NL–N . Specimens that tested positive by real-time RT-PCR analysis were confirmed by conventional RT–PCR by using the NL–N primer set. The amplified products (163 bp) were detected by using agarose gel electrophoresis, and their identity was confirmed by DNA sequencing.Kandang Kerbau Women’s and Children’s hospital is one of the major centers in Singapore for the admission of sick children, including those showing respiratory illness. After obtaining prior approval from the hospital’s ethics committee , we collected nasopharyngeal swabs from 400 pediatric patients between October 2005 and January 2007. When admitted to the hospital, these patients exhibited symptoms of acute lower respiratory tract infections (LRTI)  and URTI (pharnygitis). Specimens were sent to the hospital’s microbiologic laboratory for routine testing for influenza A and B viruses, RSV, adenovirus, and parainfluenza virus (serotypes 1–3) by immunofluorescence assay . The clinical specimens were stored at –80Of the 400 samples collected, 21 tested positive for HMPV infection, which suggests an incidence rate of ≈5.3%, compared with an 11.5% incidence rate for RSV . PreviouoC for 5 min followed by 30 cycles of 94oC for 15 s, 62oC (reducing by 0.5oC/cycle) for 30 s, 72oC for 1 min, and a final extension step of 72oC for 7 min. The sizes of the respective PCR-amplified products were examined by using agarose gel electrophoresis, gel-purified, and confirmed by DNA sequencing. The sequences were submitted to GenBank under accession nos. EF409351–EF409371. The genetic relationship between the Singapore HMPV isolates and those HMPV isolates described previously was analyzed by comparing the P gene sequences . PCR cycling was performed on a conventional thermal cycler by using a “touch-down” procedure; conditions were 94equences . This an<1 year of age compared with 63% of RSV-infected children. Of the HMPV-infected patients, 52% exhibited LRTI; of these, 82% were infected with the HMPV sublineage A2. In contrast, ≈43% of the patients exhibited URTI caused by the sublineages A2 and B2. In comparison, 61% and 20% of the RSV patients had a clinical diagnosis of LRTI or URTI, respectively. Our data suggested an increased association of sublineage A2 with LRTI in the HMPV-infected patients. The implications of this are unclear, but several reports note a correlation between severity of infection and the presence of the A genotype (,,The age and clinical characteristics of the HMPV patients were next compared with the different HMPV lineages . ChildreOur study is the first, to our knowledge, that has attempted to assess the importance of HMPV among the pediatric population in Singapore. We analyzed 400 samples that were collected from pediatric patients who were admitted to a hospital over a 16-month period. An infection rate of 5.3% was observed, which is consistent with the reported infection rates of several other industrialized countries. We also noted that of the viruses detected, ≈67% were of the A subtype and 33% were of the B subtype, which suggests that the former was the predominant HMPV subtype causing illness in these patients. Furthermore, a significant proportion of the HMPV-infected patients had LRTI. Our findings suggest that HMPV is a substantial cause of illness among the pediatric population of Singapore.


SLIT3 gene (P = 2×10−3). Extending the controls with 30,000 subjects typed on the Illumina 550 k array, we found the CNV to remain exclusive to MDD cases (P = 3.2×10−9). Duplication was observed in 5 unrelated MDD cases encompassing 646 kb with highly similar breakpoints. SLIT3 is integral to repulsive axon guidance based on binding to Roundabout receptors. Duplication of 5q35.1 is a highly penetrant variation accounting for 0.7% of the subset of 647 cases harboring large CNVs, using a threshold of a minimum of 10 SNPs and 100 kb. This study leverages a large dataset of MDD cases and controls for the analysis of CNVs with matched platform and ethnicity. SLIT3 duplication is a novel association which explains a definitive proportion of the largely unknown etiology of MDD.Major depressive disorder (MDD) is a common psychiatric and behavioral disorder. To discover novel variants conferring risk to MDD, we conducted a whole-genome scan of copy number variation (CNV), including 1,693 MDD cases and 4,506 controls genotyped on the Perlegen 600K platform. The most significant locus was observed on 5q35.1, harboring the  Major depressive disorder (MDD) is characterized by prolonged sadness and is often observed in conjunction with poor perception of self, frequent thoughts of suicide, lack of energy, and abnormal sleep PCLOATP6V1B2SP4GRM7GLYATL1RYR3Genome-wide association studies of SNP genotype frequencies in large MDD populations have revealed significance in We processed genotype and intensity data for all 599,164 probes of the Perlegen 600 K dataset for 3,761 MDD cases and controls, 1,682 Psoriasis cases and controls, and 2,788 ADHD trio samples available through dbGaP. We first converted the Cartesian coordinate values X and Y into Polar coordinates Theta and R which correlate clearly to genotype and intensity values. The observed values were then clustered to establish null expected values for Theta and R for AA, AB, and BB states. Clustering was successful on 586,730 probes. Values for B allele frequency (BAF) and log R ratio (LRR) were then calculated for each SNP in each sample based on this cluster. A HMM model was trained specifically for the resulting data using PennCNV The Perlegen data has proven to have higher noise content than comparable genome-wide SNP arrays from Illumina and Affymetrix. The essential chemistry problem which dilutes CNV signal in Perlegen 600 k data is that PCR is run to the point of saturation. Long-range PCR products are pooled together to facilitate probe binding and boost signal for each SNP. However, as a result the relative differences in intensity for different regions are diminished. The problem is that many CNV calling algorithms use intensity alone, rather than intensity and genotype in tandem as PennCNV does. While there is a higher noise content to the data, we trained a HMM specifically for this data and we have visually validated all CNVs called by the Perlegen 600 K platform We used PennCNV-Affy which evaluates intensity in terms of Log R Ratio rather than Log 2 Ratio. The Log 2 Ratio is based on quantile normalization, which derived from the sum of signal intensity for A allele and B allele for each sample, the median across all samples, and for a given sample, the A+B allele intensity is divided by the median value and logarithm base 2 applied. In contrast, the Log R Ratio is based on defined signal intensity clusters of AA, AB and BB genotypes across a large group of samples. Given this expected intensity value, the observed A+B signal intensity data is divided by this expected value, and the logarithm taken.We evaluated the SNP-based frequency of deletions and duplications between cases and controls to identify CNV regions (CNVRs) common to cases and not observed in controls . We evalSLIT3, CCDC99, and DOCK2 (P = 2.0×10−3). We ran permutation analysis through 10,000 simulations of case and controls to determine the probability of such an observation by chance (P = 2.7×10−3). Depression cases 05D01518, 06D02197, 06D06042, 06D06073, and 06D11362 were clearly scored with large duplication CNVs. The calls were near identical with coordinates on chr5 ranging from 168,423,758–169,070,607 shared by all 5 samples (−9).The CNV with the highest frequency in cases, exclusivity with respect to controls, and with clear duplication signals was a locus on 5q35.1 which impacts exons of  samples . There w samples . To evalTo gain further confidence in the duplication calls on 5q35.1, we extracted and viewed BAF and LRR values derived from the Perlegen 600 k data for the CNV region and flanking diploid regions . The BAFSLIT3, CCDC99, and DOCK2 on 5q35.1. Duplication was observed in 5 unrelated MDD cases encompassing 646 kb with highly similar breakpoints. While the Perlegen 600 k array has been deemed to be an array with data that is difficult to interpret CNV calling from, we have established an approach, using the PennCNV-Affy methodology, to robustly define the reference diploid state and extract not only informative intensity data but also relative genotype content, optimized HMM, and careful review of CNV calls, loci recurrently impacted by CNVs, raw BAF and LRR values, and raw Cartesian coordinates all support that these CNVs are real biological phenomenon. We have experimentally validated a large number of Perlegen-generated CNV calls with the independent method qPCR and demonstrated their inheritance through families as a further validation for the CNV calling Our report represents the first genome wide association analysis of CNVs in MDD. The study cohort consisted of 1,693 MDD cases compared to 4,506 controls typed on the Perlegen 600 K platform. The most significant locus included SLIT3 (slit homolog 3), the entire CCDC99 gene and the first 15 exons of DOCK2 were impacted by the duplication in all cases. Based on functional classifications of these genes, SLIT3 is the strongest functional candidate due to its role in axon development. SLIT3 is integral to repulsive axon guidance based on binding to Roundabout receptors. Slits are large, secreted repulsive axon guidance molecules which function as ligands for Roundabout (Robo) receptors. Slit3 has been characterized as an important player in the genesis of the diaphragm and kidney. Slit3 promotes angiogenesis, a process essential for proper organogenesis during embryonic development CCDC99 (coiled-coil domain containing 99) was predicted to be a mitotic spindle protein, suggested to be a human homologue of Drosophila Spindly, and proven to localize at the spindle pole by immunofluorescence microscopy DOCK2 (dedicator of cytokinesis 2) encodes a protein specific to hematopoietic cells and is indispensable for lymphocyte migration by triggering cytoskeleton reorganization via Rac activation SLIT3, an axon guidance gene.We found that the first 4 exons of The controls collected within the MDD study  were screened based on never scoring high (>0.65) on a general factor score for anxious depression and never reported a history of MDD in any survey, whereas the psoriasis  and ADHD  control supplement lacked MDD screening in the controls. Therefore, MDD cases could exist within the controls where they could mask true association signals. Assuming the population frequency of MDD at 16% SLIT3 locus. Although 77 clinical parameters were available for these individuals, no clear bias for specific co-morbidities was evident. The age of onset for depression symptoms was 8, 16, 33, 30, and 30. Two were males and three were females. Interestingly, the total scores for Neuroticism Extraversion Openness (NEO) five-factor inventory short form neuroticism were notably elevated at an average of 47 whereas the MDD population average was 34. Additional characteristics including items from NEO, Inventory of Depression Severity (IDS) depression, and Beck Anxiety Inventory (BAI) anxiety are included in We sought to evaluate the specific phenotypes of the 5 MDD cases with the duplication of the SLIT3 locus duplication with an independent technology such as qPCR, FISH, or MLPA. Without this data, the possibility of false positives exists, albeit minimal from our experience. 83 SNPs showed AAB or ABB genotypes which are only possible in duplications throughout 198 SNP probe signals in agreement of the duplication signal with physical size 646 kb. We similarly used conservative inclusion thresholds to mitigate the false positives from this array. Parent and sibling samples were not collected or available to determine if these duplications were inherited but family history of MDD was reported by all 5 individuals.We were unable to acquire the 5 samples to validate the presence of the SLIT3, CCDC99, and DOCK2 on 5q35.1 shared by 5 MDD cases. These results offer a highly penetrant variation which underlies MDD involving axon structure and guidance, thereby extending previous observations involving neurotransmitter actions as the underlying cause of MDD.We conducted a large scale CNV study on 1,693 MDD cases and 4,506 controls typed on the Perlegen 600 K platform. This report represents the first successful CNV analysis using the Perlegen 600 K platform and the first scan for CNVs contributing to MDD susceptibility. CNVs were associated to MDD based on the frequency in cases and exclusivity when compared to controls. The most significant locus included a 646 kb duplication of This research was approved by the institutional review board or the Children's Hospital of Philadelphia. Informed consent from the participants was obtained and data were analyzed with anonymous encrypted labels. Written consent was given by the patients for their information to be stored in the hospital database and used for research.Raw genotyping data from three Genetic Association Information Network (GAIN) MDD cases were recruited through mental health care organizations, general practices and in the community setting as previously described Control subjects matched for age and gender were mainly derived from the Netherlands Twin Register, for which data collection in twins, their parents, spouses and siblings occurred in 1991, 1993, 1995, 1997, 2000, 2002/3 and 2004/5. A total of 1860  controls were selected (only one member from each family) with the following inclusion criteria: 1) age 18 through 65 years, 2) never scoring high (>0.65) on a general factor score for anxious depression . The psoriasis cases were diagnosed by dermatologists and their matched controls had no history of psoriasis, no family history of psoriasis or other auto-immune disorders. All subjects were 18 years of age or older. The second control cohort included parents from the ADHD parent-offspring trios study who were also genotyped on the Perlegen platform and included as controls .Additional controls on the Illumina platform were typed on the We performed high-throughput genome-wide SNP genotyping using the InfiniumII HumanHap550 BeadChip technology (Illumina San Diego CA), at the Center for Applied Genomics at CHOP. Subjects were primarily recruited from the Philadelphia region through the Hospital's Health Care Network, including four primary care clinics and several group practices and outpatient practices that performed well child visits. Eligibility criteria for this study included all of the following: (1) disease-free children and parents of these children in the age range of 0–18 yr of age who had high quality, genome-wide genotyping data from blood samples ; (2) self-reported ethnic background; and (3) no serious underlying medical disorder, including but not limited to neurodevelopmental disorders, cancer, chromosomal abnormalities, and known metabolic or genetic disorders.http://www.openbioinformatics.org/penncnv/penncnv_tutorial_affy_gw6.html.The CNV calling on the Perlegen platform used a highly similar algorithm to those used on the Illumina arrays, but the signal pre-processing steps differ. Unlike the Illumina platform, where normalized signal intensities (Log R Ratio and B Allele Frequency) can be exported directly from the BeadStudio software, these signal intensity measures in the Perlegen 600 K platform need to be calculated from the collection of genotyped samples based on raw X and Y values. To perform data normalization and signal extraction from raw final report files generated in genotyping experiments, we first reformatted data from dbGaP into the format produced by Affymetrix Power Tools: birdseed.calls.txt, birdseed.confidences.txt, and quant-norm.pm-only.med-polish.expr.summary.txt. The X and Y values provided in the sample based report files from dbGaP were reduced to a smaller range by taking the logarithm base 10. For each SNP marker, we then relied on the allele-specific signal intensity for the AA, AB and BB genotypes on all genotyped samples to construct three canonical genotype clusters in polar coordinates theta and R, similar to the Illumina clustering generation approach. The “-conf 2” option was included in running generate_affy_geno_cluster.pl since 1 was coded as the best score. Once the canonical genotype clusters were constructed, we then transformed the signal intensity values for each SNP to Log R Ratio (LRR) and B Allele Frequency (BAF) values using normalize_affy_geno_cluster.pl. For more technical details, see 10 in three successive batches of thirty. The first training used the samples with the lowest standard deviation of LRR while the other two runs, using the file created as a new reference, included more random representative samples. We also created definition files providing inter-SNP distance and population b-allele frequency to further inform CNV calling specifically adapted to the observed Perlegen data. This allowed for CNV calls to be made with good quality metric scores in 6,199 out of 8,231 Perlegen 600 K samples available. Although the global standard deviation of LRR was below 0.2 for the majority (84%) of samples, the intensity data was notably noisier and often showed a subpopulation of SNPs unable to differentiate a deletion signal, perhaps due to PCR saturation during the lab processing. Nevertheless, the deletion and duplication features were still detected with confirmation of homozygote and AAB/ABB genotypes respectively shown for the same SNPs. The SNP level data underlying each CNV call was reviewed to ensure clean signal quality (To optimize the Hidden Markov Model (HMM), we used the baseline reference file HH550.hmm and ran “-train” in PennCNV quality . To ensu quality .We calculated Quality Control (QC) measures on our Perlegen 600 K GWAS data based on statistical distributions to exclude poor quality DNA samples and false positive CNVs. The first threshold is the percentage of attempted SNPs which were successfully genotyped. Only samples with call rate >98% were included. The genome wide intensity signal must have as little noise as possible. Only samples with the standard deviation (SD) of normalized intensity (LRR) <0.25 were included. All samples must have Caucasian ethnicity based on Eigenstrat principle components analysis of genotypes and all other samples were excluded. Wave artifacts roughly correlating with GC content resulting from hybridization bias of low full length DNA quantity are known to interfere with accurate inference of copy number variations CNV frequency between cases and controls was evaluated at each SNP using Fisher's exact test. We only considered loci where cases in the MDD cohort had the same variation and were not observed in any of the control subjects. We report statistical local minimums to narrow the association in reference to a region of nominal significance including SNPs residing within 1 Mb of each other. Resulting nominally significant CNVRs were excluded if they met any of the following criteria: i) residing on telomere or centromere proximal cytobands; ii) arising in a “peninsula” of common CNV arising from variation in boundary truncation of CNV calling; iii) genomic regions with extremes in GC content which produces hybridization bias; or iv) samples contributing to multiple CNVRs.Table S1SLIT3 locus to evaluate co-morbiditiesSpecific phenotypes of the 5 MDD cases with the duplication of the (DOC)Click here for additional data file.


Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in  E. coli cells, when the stringent response is in effect, complete chromosomal replication but do not initiate new rounds and arrest with an integer number of chromosomes. The number of chromosomes corresponds to the growth rate prior to arrest. In polyploid arrested cells, the chromosomal regions at which replication initiates are segregated, whereas the termini regions remain colocalized. The E. coli chromosome remains decondensed and unsegregated during arrest and rapidly resumes replication and segregation, concomitant with chromosome condensation, upon release. The protein SeqA, a DNA binding protein and negative regulator of replication, is necessary for enforcing this arrest.Management of cell growth and division in response to environmental conditions is important for all cells. In bacteria, nutritional downturns are signaled by accumulation of the nucleotide ppGpp. Amino acid starvation causes a programmed change in transcription, known as the “stringent response”; ppGpp also causes an arrest of cell cycle in bacteria, whose mechanism has not been thoroughly investigated. Here, we show that  Bacterial cells encounter varied environmental stresses and make appropriate adjustments to ensure survival. One such stress is amino acid starvation, which triggers physiological reprogramming known as the “stringent response”. Signaling of the stringent response is achieved by accumulation of effector nucleotides, guanosine tetra- and pentaphosphate ppGpp and pppGpp , a period with ongoing DNA synthesis  and a period after completion of replication but before cell division . In medium rich in nutrients, E. coli cell cycle is accelerated such that it becomes faster than the time necessary to replicate the entire chromosome; under this circumstance, replication cycles overlap and cells are born with partially replicated chromosomes, whose replication was initiated in its mother or even grandmother The cell cycle of E. coli, replication initiates at the oriC locus and proceeds bidirectionally to completion within the terminator region, teroriC followed by loading of the DnaB helicase depends on levels of DnaA-ATP oriC locus.In oriC, SeqA binding prolongs its hemimethylated status and blocks DnaA-origin interaction until the region becomes fully methylated by Dam methyltransferase “Sequestration”, the binding of SeqA to a hemimethylated origin is a component of initiation control E. coli, in a manner independent of sequestration at oriCIn addition, SeqA is implicated in chromosome segregation and nucleoid organization E. coli promotes replication arrest via inhibition of initiation oriC after stringent onset It has been reported that stringent response in relA mutants that fail to arrest, nucleoid compaction is apparent, similar to that seen upon treatment of cells with the translational inhibitor, chloramphenicol. Although marker frequency analysis shows that the ratio of oriC to ter is 1 after stringent arrest, visualization of oriC and ter by ParB-GFP binding shows that sister loci at the termini remain colocalized, whereas the oriC loci have separated. Stringent cell cycle arrest is dependent on SeqA and Dam, in a manner that is only partly dependent on GATC sites near the origin.Our studies reveal that under the stringent response, cells appear to complete replication and arrest at an integer DNA content corresponding to their pre-arrest growth conditions. This arrest appears to be dependent on RelA-dependent ppGpp synthesis. Stringent cells always contain one decondensed nucleoid; in Escherichia coli K12 cells growing in defined media, we used flow cytometry for DNA content using the stain PicoGreen (Invitrogen). We examined DNA content in cells at different growth rates, with and without treatment with serine hydroxamate, a derivative of serine that acts as a competitive inhibitor of seryl-tRNA synthetase, eliciting the stringent response E. coli. These antibiotics block initiation of replication and cell division, respectively; under this regimen, ongoing replication forks are completed and cells arrest in the D period. Under run-out conditions, the DNA content reflects the number of oriC loci in the cell at the time of treatment.To determine the extent of DNA replication in populations of With no treatment, there is a broad distribution of cellular DNA content, indicative of DNA replication, asynchronous in the population . After tIn each growth medium, treatment with serine hydroxamate to induce the stringent response for 1.5 hr caused the population to assume an integer DNA content, similar to treatment with rifampicin and cephalexin, indicating an inhibition of cell cycle progression and DNA replication. In each case, the integer DNA content at stringent arrest was somewhat lower than that seen with rifampicin and cephalexin treatment. This indicates that some stringent arrested cells undergo cell division after cessation of replication. Although some stringent cells may arrest in D period  as do rifampicin and cephalexin-treated cells, there is a preference for stringent arrest in B period .relA mutants, defective in ppGpp production elicited by serine hydroxamate. Mutants in relA were noticeably impaired for arrest after serine hydroxamate treatment and the cellular DNA content of the population was broadly distributed, indicative of ongoing replication  over unmethylated sites  strains . As seqAphalexin , althougseqA and dam mutants. To test this, we engineered otherwise wild-type cells to overexpress Dam from an arabinose-inducible promoter. This strain exhibited cell cycle arrest after treatment with serine hydroxamate when grown in the presence of glucose but failed to shift DNA content fully to an integer amount when Dam was induced by arabinose  allele lowers the frequency of initiation at cold-temperatures and suppresses the overinitiation property of ΔseqA mutants at its permissive temperature dnaA(Sx) itself does not influence stringent cell cycle arrest. dnaA(Sx) cells displayed a stringent arrest after treatment with serine hydroxamate as indicated by a shift to an integer number of chromosomes, similar to run-out conditions (dnaA(Sx) allele, ΔseqA cells maintain a DNA content similar to wild-type cells in the absence of any treatment (dnaA(Sx) ΔseqA mutant did not successfully arrest after treatment with serine hydroxamate, although DNA content remained within the range that was comparable to arrested wild-type cells. This suggests that failed stringent arrest in ΔseqA cells is due to a lack of SeqA function and is not an indirect consequence of hyper-initiation or excessive DNA content.DNA content is notably higher in nditions . When coreatment . The dnaoriCm3 strain, which is specifically defective in oriC sequestration. oriCm3 strains have eight of the GATC sequences within oriC converted to GTTC, causing a decreased affinity for SeqA, asynchronous replication, shortened eclipse periods and asymmetric cell division, equivalent to strains lacking SeqA altogether oriCm3 cells shows a modest increase in DNA content, presumably due to hyperinitiation caused by defective origin sequestration. After treatment with serine hydroxamate, the DNA content indicates that stringent arrest is partially intact in the oriCm3 strain, with a prominent 4N arrest peak. This suggests that there is a critical role of SeqA in enforcing the stringent response by interactions to DNA sites outside the origin. Mutants in oriCm3, like those in dam and seqA, showed poor run-out to integer chromosomal content after treatment with rifampicin and cephalexin.SeqA appears to have two distinct functions: it binds to hemimethylated DNA sites at the origin and, somewhat more transiently, to newly replicated DNA throughout the chromosome. To distinguish between the origin and other sites of SeqA binding, we examined DNA content after stringent arrest in an parS sequence inserted adjacent to the origin or to the terminus. The parS sequence provides a binding site for the plasmid-expressed fusion protein, GFP-ParB, to allow visualization of the locus of interest To examine segregation patterns of specific locations of the chromosome, we used strains that contain a oriC and ter foci via ParB-GFP showed that stringently-arrested wild-type cells have two or four distinct oriC foci  seqA mutant cells was 2.8 before treatment and 2.7 after serine hydroxamate, confirming that replication is ongoing after induction of the stringent response in these strains.For growth in minimal medium plus casamino acids at 34°, examination of riC foci . However was 2.5 , indicat was 1.0 , indicatreatment . In simioriC to ter foci after serine hydroxamate treatment of wild-type cells, despite the fact that the chromosome appears to have completely duplicated from the marker frequency analysis, suggests that ter loci remain colocalized after replication. In contrast, colocalization of ter was not observed in wild-type cells arrested in the cell cycle by rifampicin and cephalexin: under these conditions the number of ter foci are equivalent to those seen for oriC  seqA strains that have more normal DNA content per cell. This may indicate that seqA affects only a subset of the responses to ppGpp (see below). Mutants in oriCm3 also exhibited nucleoid decondensation similar to wild-type cells after serine hydroxamate treatment.We also examined nucleoid morphology in Δ strains . UntreatWe wondered how cell cycle patterns would be reset after release from arrest. Would cells resume replication in a pattern similar to that prior to arrest? Or would cells be obligated to segregate each chromosome, divide and initiate replication from 1N progeny? To examine this, after 90 minutes of stringent arrest induced by serine hydroxamate, wild-type cells in minimal M9 Glucose CAA medium were washed and allowed to resume growth. DNA content was followed over time by flow cytometry and nucleoid appearance was monitored by DAPI staining . As earlEscherichia coli cells with elevated ppGpp levels and they are unable to form colonies dam and ΔseqA would permit colony formation during chronic ppGpp production. Wild type, ΔseqA, Δdam, and oriCm3 cells containing the IPTG-inducible pALS13 plasmid producing truncated RelA′, were grown on M9 minimal CAA ampicillin plates containing various concentrations of IPTG. A control plasmid, pALS14, contains a truncated 1.2 kb fragment of RelA that has no enzymatic activity + (data not shown). ΔseqA and Δdam cells, however, maintained the ability to form some colonies in the presence of elevated ppGpp concentrations, with plating efficiencies dramatically elevated, over 1000-fold, in the presence of IPTG , indicating that no heritable change arising during growth, which is a special concern for dam mutator strains, had allowed them to escape preferentially.Growth is inhibited in wild-type seqA and dam is not 100%, indicating that transcriptional reprogramming, translational inhibition or other aspects of the stringent response, which remain intact in seqA and dam mutants (see below), may also be inhibitory to growth and division.The inability of cells to form colonies under constitutive ppGpp accumulation appears to be, in part, a consequence of cell cycle arrest. We do note that the rescue of plating by dam and ΔseqA strains to arrest cell cycle during the stringent response is that production of ppGpp, or its ability to interact with RNA polymerase, for some reason, is defective. To investigate this, we examined ppGpp production directly by 32P-phosphate labeling and separation of nucleotides by thin-layer chromatography , appeared to be intact in seqA mutants. This implies that SeqA and Dam specifically control cell cycle and do not have global effects on the stringent response, including levels of ppGpp.We have identified mutants, ΔIn bacteria, as well as other organisms, control of the cell cycle is likely to be an important component of stress responses. Our previous work suggested that SeqA may limit replication in the face of chronic DNA damage dam or seqA are dramatically relieved of growth inhibition by overexpression of RelA and are able to form colonies, albeit with some loss of plating efficiency, likely the result of down-regulation of translational capacity Bacterial cells fail to proliferate when ppGpp levels rise, such as caused by overexpression of RelA or loss of the ppGpp hydolase SpoT (reviewed in Previous results suggested that initiation of replication is blocked when ppGpp levels are high and that elongation of replication continues to completion after ppGpp accumulation Escherichia coli and Bacillus subtilis. B. subtilis arrests replication elongation by ppGpp inhibition of primase activity E. coli does not perform this C-period (“intra-S phase”-like) arrest, although it remains possible that elongation is slower in stringent cells or punctuated with stalling events. We also do not know the threshold levels of ppGpp or starvation that elicit replication arrest in the two organisms. It remains possible that elongation arrest in E. coli could occur under more stringent conditions or occurs in a subpopulation of cells, obscured by arrest in B and D periods. During the stringent response in E. coli, GTP levels do not fall more than 50% Bacillus subtilis experiences a drop in GTP concomitant with ppGpp accumulation and its stringent response may be an indirect consequence of depletion of GTP pools E. coli may respond more sensitively to ppGpp, enabling it to complete replication before collapse of nucleotide pools that would stall replication. E. coli and its relatives are unique in their acquisition of adenine methylation and SeqA function, perhaps allowing an extra level of control of replication and chromosome segregation, lacking in other bacteria.Stringent cell cycle arrest points differ between oriCm3oriC during the stringent response. Our data indicates that in this mutant stringent arrest is partially intact. Some arrest, as judged by flow cytometry for DNA content, was seen in this mutant upon serine hydroxamate treatment. During chronic exposure to ppGpp, this mutation relieved inhibition of colony formation only modestly, approximately 10-fold, relative to wild-type strains. This was in contrast to the ΔseqA strain, in which no evidence of replication arrest was apparent and which was 1,000-fold more efficient in colony formation during chronic exposure to ppGpp. It is possible that the oriCm3 mutation only produces a partial loss of SeqA binding to the origin, although the first characterization of this mutant suggests that loss of sequestration at oriC is complete oriCm3 but dependent on the property of SeqA for self-aggregation. It may be this mode of SeqA binding that is required for stringent arrest.The origin region is particularly rich in GATC sites and SeqA binding to these sites is relatively long-lived, up to 1/3 of the cell cycle, when the sites remain hemimethylated oriC regions marked by GFP-ParB but only one half the predicted number of ter regions. In contrast, after cell cycle arrest by rifampicin and cephalexin, the number of observed ter foci is equivalent to those of oriC. Loss of ter cohesion may be a prerequisite for chromosome segregation and a point of regulation by the stringent response.This study provides new evidence that late stages of chromosome replication or chromosome segregation can be regulated in response to environmental conditions. The nature of this arrest is intriguing and suggests possible mechanisms. We observed segregated Bacillus subtilis Obg and E. coli ObgE bind ppGpp  interacts with ppGpp synthetase/hydrolase SpoT Vibrio choleraeInterestingly, the nature of stringent arrest of chromosome segregation is very similar to the arrest seen in cells depleted of the GTPase ObgE, which is required for chromosome segregation and survival after treatment with replication inhibitors E. coli cells and loci segregate as they are duplicated. In some studies ter relative to other regions of the chromosome, suggesting that there could be special factors that control its segregation. The 2∶1 ratio of oriC to ter foci may suggest a post-replication cohesion, either because regions near ter have not fully duplicated or because fully-replicated sister chromosomes are held together by DNA or protein linkages. Although the marker frequency analysis showing a 1∶1 oriC/ter ratio appears to support the latter explanation, we cannot rule out the possibility that short or heterogeneous unreplicated sequences in the ter region after ppGpp accumulation escape our detection. In any case, this “cohesion” may assist in the organization of chromosome segregation that will occur when cells are released from arrest with multiple chromosomes. We observed a rapid segregation of 4 nucleoids upon release, concomitant with nucleoid compaction, with segregation occurring first at midcell and later at the quarter position. Cohesion at the termini , the mechanism of this control is still obscure, since promoter activity at seqA defects in stringent arrest by dnaA mutants with reduced initiation efficiency. Moreover, DnaA is unlikely to directly influence chromosome segregation arrest during the stringent response. Nonetheless, it is possible that multiple mechanisms of cell cycle control, such as the DnaA levels and the regulatory inactivation of DnaA (RIDA), cooperate to control cell cycle in response to ppGpp.Previous reports have shown that DnaA expression decreases during the stringent response Escherichia coli K-12 strains  with 0.2% glucose, 0.4% glucose, or 0.4% arabinose, and 0.2% casamino acids with 1.5% agar for plates. For P1 transductions and phage lysates, cultures were grown in LCG, LB medium supplemented with 1% glucose with an additional 2 mM calcium chloride; for plates, 1% agar was added. Antibiotics were used in the following concentrations: ampicillin (Ap), 100 µg/ml; kanamycin (Km), 60 µg/ml; tetracycline (Tc) and chloramphenicol (Cm), 15 µg/ml. Isogenic strains in MG1655 were constructed by P1 vira transduction. Cultures employed M9 minimal media described above with the addition of 1 mg/ml DL-Serine hydroxamate (Sigma) or, in the case of strains harboring pALS13 (pRelA′), 1 mM isopropyl β-D-1-thiogalactopyranoside IPTG for 1.5 hrs. Addition was made in early logarithmic growth of the culture, at OD600 of ∼0.2. Experiments examining chloramphenicol-induced translational inhibition were performed with the addition of 300 µg/ml chloramphenicol for 1.5 hrs. Release from DL-serine hydroxamate was accomplished by centrifugation of the treated culture and resuspension of the cell pellet in an equal volume of fresh medium without the drug. For experiments employing radioactive inorganic phosphate labeling, MOPS-buffered minimal medium was used . strains  were grodnaA46 strain, temperature sensitive for replication initiation, was analyzed similarly after 3 hr of growth at 42°C.DNA content per cell was determined as described Overnight cultures were inoculated into fresh medium at a dilution of 1∶100 and grown with aeration for 3 hours. Nucleoid staining was performed as previously described EcoRI, HindIII, and EcoRV and the fragments were separated on a 1.0% agarose gel. The DNA was vacuum-transferred to a nylon membrane (Amersham). The membrane was prehybridized with BSA for more than 1 h at 65°C and hybridized overnight at 65°C with 32P a-dATP. The probe consisted of two DNA fragments that anneal to the chromosomal regions gidA (84.3 min), and relB (34.8 min). The DNA fragments were labeled using Random Primer Labeling (Molecular Cloning). After hybridization, the membrane was washed with 0.21 M Na2HPO4 (pH 7.3) / 6% SDS / 0.85 mM EDTA 3× for 10 minutes at 25°C followed by 2× for 5 minutes at 65°C. The membrane was exposed on a Phophoimaging screen (Molecular Dynamics) and scanned on a Bio–Rad Molecular Imager FX (Bio-Rad). Analysis of bands was carried out using Quantity One imaging software (Bio-Rad). Normalization of the bands was accomplished using genomic DNA from dnaA46 that was grown at the non-permissive temperature for 2 h.Cells were grown to exponential growth phase in M9 0.4% glucose CAA medium. Samples were taken and chromosomal DNA was extracted using the MasterPure DNA Purification Kit (Epicentre). Restriction digest and preparation of the probe were done as described previously 600∼0.3. 10-fold serially diluted cultures were plated on M9 minimal media plates containing ampicillin and 100 µM IPTG. Total colony numbers were determined by plating on M9 ampicillin medium without IPTG. Colonies were counted after 48 hours of growth at 37°C. The number of independent cultures, as indicated in figure legends, was determined on at least three different days.Overnight cultures grown in M9 CAA media were diluted 1∶100 in fresh media and grown to an OD600∼0.2. Where indicated, cells were treated with serine hydroxamate. Pulse labeling was initiated by the addition of 20 µCi of [3H] uridine per 2 ml of culture. Uracil was added to 0.9 mg/ml after a ten minute pulse. Total RNA was extracted from 0.5 ml by the RNAeasy kit (Qiagen). Purified RNA was resuspended in RNase free water and stored at −20°C. Approximately 1 µg of RNA sample was analyzed by electrophoresis in a 1% non-denaturing agarose gel. After electrophoresis, the gel was photographed under UV light. Amount of rRNA was determined by band intensity of the 16S, 23S, and unprocessed rRNA bands in the image using Quantity Oneâ imaging software (Bio-Rad). To determine the amount of [3H] labeled rRNA, corresponding rRNA bands were cut out from the gel, dissolved, and analyzed in a scintillation counter. Normalization was achieved by dividing the obtained cpm by the volume of rRNA band intensity.Overnight cultures were diluted 1/50 in M9 CAA media and grown to an O.D.2PO4) and 0.2% casamino acids were diluted 1∶100 into MOPS medium containing 0.4 mM phosphate and 0.2% casamino acids. Treated samples received 1 mg/ml SHX before the addition of [32P]H3PO4 to a final concentration of 200 µCi/ml when the culture OD600 reached ∼0.05. Total RNA was isolated after 15 minutes of incubation with [32P]H3PO4. RNA was isolated using the RiboPure Bacteria Kit from Ambion according to the manufacturer's instructions. Total RNA was electrophoresed on a native agarose gel and stained with ethidium bromide. rRNA bands were visualized and quantified by UV- light induced fluorescence using Quantity One software (Bio-Rad). RNA was then transferred to a nylon membrane (Amersham) using the Vacuum Blotter 785 (Bio-Rad). The amount of radiolabeled rRNA was determined by autoradiography and quantified using MolecularImager FX PhosphorImager and Quantity One software (Bio-Rad). Amount of radiolabeled rRNA was normalized to the amount of RNA observed by UV-light induced fluorescence.Overnight cultures grown in MOPS pH 7.2 medium containing 2 mM phosphate (KH2PO4) and 0.2% casamino acids were diluted 1∶100 into the same MOPS medium except containing 0.4 mM phosphate. [32P]H3PO4 was added to a final concentration of 100 µCi/ml when the OD600 reached ∼0.05 and cultures grew for an additional 3 hours before the first sample was taken. SHX was added at time 0, and samples were isolated every ten minutes by mixing 100 µl of culture with an equal volume of 13 M formic acid and chilling on dry ice. The samples were subjected to two rounds of freezing and thawing before microcentrifugation at 14, 000 rpm for 2 minutes to remove cellular debris. 6 µl of supernatant were spotted onto 20×20 cm polyethyleneimine cellulose on polyester TLC plates (Sigma) in 1.5 KH2PO4 (pH 3.4) for 2 h. After chromatography, nucleotides were visualized by autoradiography and quantified with a MolecularImager FX PhosphorImager and Quantity One software (Bio-Rad). Unlabeled GDP and GTP were spotted on the plates as markers and visualized after chromatography by UV light-induced fluorescence. The identities of the labeled (p)ppGpp were inferred from their positions in the chromatograph relative to the origin and GTP. (p)ppGpp levels are normalized to levels of GTP observed in the same sample.(p)ppGpp measurements were performed as previously described


COMT and ESR1 are associated with muscle properties and assessed their interaction and their combined effects with physical activity.Muscle strength declines on average by one percent annually from midlife on. In postmenopausal women this decrement coincides with a rapid decline in estrogen production. The genetics underlying the effects of estrogen on skeletal muscle remains unclear. In the present study, we examined whether polymorphisms within A cross-sectional data analysis was conducted with 434 63-76-year-old women from the population-based Finnish Twin Study on Aging. Body anthropometry, muscle cross-sectional area (mCSA), isometric hand grip and knee extension strengths, and leg extension power were measured. COMT Val158Met and ESR1 PvuII genotypes were determined by the RFLP method. mCSA differed by COMT genotypes (p = 0.014) being significantly larger in LL than HL individuals in unadjusted (p = 0.001) and age- and height-adjusted model (p = 0.004). When physical activity and age were entered into GEE model, COMT genotype had a significant main effect (p = 0.038) on mCSA. Furthermore, sedentary individuals with the HH genotype had lower muscle mass, strength and power, but they also appeared to benefit the most from physical activity. No association of ESR1 PvuII polymorphism with any of the muscle outcomes was observed.The present study suggests that the COMT polymorphism, affecting the activity of the enzyme, is associated with muscle mass. Furthermore, sedentary individuals with potential high enzyme activity were the weakest group, but they may potentially benefit the most from physical activity. This observation elucidates the importance of both environmental and genetic factors in muscle properties. One of the most important functions of skeletal muscle is voluntary movement. Muscle also serves as an amino acid reservoir and a site for various metabolic activities participating e.g. to the glucose metabolism of the whole body In women, menopause is characterized by rapid decline in the production of estrogen, an anabolic female sex hormone, and coincides with an accelerated deterioration in muscle performance O-methyltransferase (COMT) into more inactive methoxyestrogens. These O-methylated metabolites no longer bind to estrogen receptors (ESRs) COMT gene results in a valine to methionine amino acid transition at codon 158  leading to thermolability The synthesis and degradation of estrogens are mediated by several enzymes involved in multiple and complex metabolic pathways. After initial hydroxylation of estrogens by isoenzymes belonging to the cytochrome P450 family, they are further metabolized by catechol-ESR1 gene (ESR1 PvuII polymorphism) identifies a nucleotide T to C transition resulting in the loss of PvuII restriction site ESR1 was recently shown to be expressed in human skeletal muscle COMT and ESR1, may have shared effects on estradiol signaling in target tissues. More precisely, a polymorphism affecting the activity of COMT may directly or indirectly modulate the amount of estradiol available to be bound by membrane-bound or intracellular estrogen receptors, whereas a polymorphism potentially modulating the amount of ESR1 transcript may further affect the availability of these receptors. On the other hand, the two estrogen-related polymorphisms under investigation may act in conjunction with physical activity resulting in a specific muscle phenotype. Our aim was to determine whether the two estrogenic polymorphisms, COMT Val158Met and ESR1 PvuII, are associated with serum estradiol levels and skeletal muscle phenotypes or not. Furthermore, we studied the interaction of these polymorphisms and their interaction with physical activity.Theoretically, polymorphisms residing in genes related to estradiol metabolism and action, in this case This study is a part of the Finnish Twin Study on Aging (FITSA), which investigates the genetic and environmental effects on the disablement process in older female twins. The detailed study design, including selection procedures, determination of zygosity and description of the participants, has been reported elsewhere In the whole study sample the prevalence of various diseases potentially effecting muscle properties were: 12 % for coronary heart disease, 8 % for asthma, 7 % for cerebrovascular disease, 6 % for type 2 diabetes, 4 % for rheumatoid arthritis, 29 % for knee, 14 % for hip and 12 % for foot and ankle osteoarthritis. 67 % of all the subjects had never used HRT, whereas 14 % were former users and 19 % current users. Moreover, 3 % of the subjects in the entire population were current cortisol users and 12 % former users, whereas 85 % had never been under long-term cortisol treatment. Subjects with the above-mentioned diseases and different HRT or cortisol status were equally distributed between genotypes.Nonfasting blood samples were collected between 0830 and 0930 hours and sera stored at −70°C for later analysis. Total serum 17β-estradiol was determined by competitive immunoenzymatic colorimetric assay  and serum sex hormone binding globulin (SHBG) levels using solid-phase, chemiluminescent immunometric assay . Free estradiol levels were calculated from 17β-estradiol and SHBG levels according to a previously presented method Body mass and body height were measured using standard procedures. Lean body mass and total body fat were assessed by bioelectrical impedance . The CV in our laboratory has been less than 2 % for lean body mass and less than 3 % for body fat Maximal isometric strength, defined as the maximum voluntary contraction performed at a specific joint ankle against unyielding resistance, was measured for knee extension and hand grip. Maximal isometric hand grip and knee extension strengths were measured on the dominant side in a sitting position using an adjustable dynamometer chair . After familiarization with the test, three to five maximal efforts separated by a one-minute interval were conducted. Mid-life maximal isometric hand grip strength, strength of a non-bearing limb, has been reported to correlate with strength of other muscle groups thus being a good indicator of overall strength Information concerning physical activity was collected using the scale of Grimby Genomic DNA was extracted from EDTA-anticoagulated whole blood according to standard procedures . Genotyping for Val158Met and PvuII polymorphisms was performed using PCR  followed by restriction fragment length polymorphism (RFLP) analysis th codon in the COMT gene was determined by copying a 109-bp fragment with a primer pair  including forward (5′-CTCATC ACCATC GAGATC AA) and reverse primers (5′-CCAGGT CTGACA ACGGGT CA) The G to A transition at the 1585′-GATATC CAGGGT TATGTG GCA) and reverse primers (5′-TTACCT CTTGCC GTCTGT TGC) in a 10 µl reaction mixture containing 20 ng of DNA, 0.1 µM of both primers, 0.4 mM of dNTP mix  and 0.35 U of HotMaster Taq polymerase . PCR reaction was carried out with the following steps: preheating at 95°C for 2 min followed by 36 cycles, in which denaturation was performed at 94°C for 45 s, annealing for 45 s with gradually declining temperature (36 cycles with declining temperature from 67°C to 60°C) and extension at 72°C for 45 s. The PCR product was digested by adding 5 U of PvuII restriction endonuclease  and incubated for two hours at 37°C. The digested products were further separated by electrophoresis in a 3 % agarose gel. Genotypes were determined due to resulting fragments and coded as PP, Pp and pp. Uppercase letters indicate the absence and lowercase letters the presence of a restriction site.In ESR1 PvuII genotyping a 373-bp PCR fragment was produced using a primer pair  consisting of forward , which allows taking into account the correlation between sisters within a twin pair. All outcome variables were normally distributed except for estradiol concentrations, which were skewed towards low concentrations and were considered to follow the gamma-distribution. Two types of single genotype models were constructed, one including the unadjusted main effects of the genotypes, and the other adjusted for age and height. To assess genotype-genotype and genotype-physical activity interactions a reference category was selected for the categorical predictor variables of physical activity (sedentary level), COMT (the HH genotype) and ESR1 Pvull (the pp genotype). Planned contrasts were used in comparing mean levels of each outcome variable between the predictor variable levels and their interactions against the reference category. Test-wise type I error rate was set at 0.05 in all analyses and partial correlation coefficients from the GEE model contrasts We hypothesized that subjects with assumed lower amount of circulating estradiol (HH genotype) and potential lower levels of ESR1 transcript (pp genotype) would be less responsive to estradiol and thus have worse muscle properties in comparison with other combinations. In the models including physical activity, subjects with potential low amount of circulating estradiol (HH genotype) or suggested low amount of ESR1 transcript (pp genotype) combined with sedentary life-style, were assumed to be weaker and have smaller muscles than other combinations. In our approach, reference groups were chosen according to these initial hypothesis and the mean values of other groups compared to that of the reference groups. The reference groups for the interaction effect were formed based on the combination of the main effect reference categories. The main effects of the two components of interest are always presented in contrast to the reference group.2 = 0.004, p = 0.95) suggesting that the subjects represented a homogeneous genetic background. Subject characteristics according to Val158Met genotypes are presented in In our study population 79 subjects (18.0 %) were homozygous for the high activity allele (HH), 208 (47.9 %) heterozygotes (HL) and 137 (31.6 %) homozygous for the low active allele (LL). The allele frequencies were 0.43 for the H and 0.57 for the L allele. The genotype distribution of the entire cohort was in Hardy-Weinberg equilibrium  suggesting that our study sample may not be representative of the target population. Physical characteristics, including hormone levels, were similar in all PvuII genotypes. Furthermore, PvuII polymorphism was not associated with any of the measured muscle variables , whereas pp genotype was more frequent  than PP . The allele frequencies were 0.44 and 0.56 for the P and p alleles, respectively. The genotypes were slightly out of Hardy-Weinberg equilibrium  and dynamic (leg extension power) muscle performance as well as measures from both lower and upper limbs. Furthermore, during isometric testing, the speed of muscle contraction is not as essential as in muscle power measurements. On the other hand, these data provide a multifaceted estimate of the effects of the chosen genotypes on whole body musculature. The results provided by our cross-sectional data set should be further confirmed in a follow-up study and, if possible, with a larger sample and an intervention trial to see, if the response to training is actually genotype-dependent.COMT gene and muscle mass. Furthermore, the interaction effect of this polymorphism and physical activity on muscle mass, strength and power elucidates the interplay of environmental and genetic factors in muscle properties and represents an example of how an unfavorable genetic background may perhaps be compensated for by healthy living habits, in this case physical activity. Overall, our results imply that COMT gene, related to the metabolism of estrogens, may be connected with muscle properties, albeit the exact mechanisms remain unknown.In conclusion, the identification of the genetic susceptibility factors predisposing the elderly to impaired muscle performance could provide novel insights into the etiology of sarcopenia and would enable the recognition of those people at high risk of disability. Analysis of genetic variants, such as SNPs, represents a powerful approach to examine the role of candidate genes in the progression of this obviously multifactorial state. In the present study, we found an association between a polymorphism in the