Experience-dependent plasticity in the mouse barrel cortex

Abstract : Host-Cell Factor 1 (HCF-1) was first discovered in the study of the herpes simplex virus (HSV) infection. HCF-1 is one of the two cellular proteins that compose the VP16-induced complex, a key activator of HSV lytic infection. lncleed, when HSV infects human cells, it is able to enter two modes of infection: lytic or latent. The V`P16-induced complex promotes the lytic mode and in so doing the virus targets important cellular regulatory proteins, such as HCF-1, to manipulate the status of the infected cell. Indeed, HCF-1 regulates human cell proliferation and the cell cycle at different steps. In human, HCF-1 is unusual in that it undergoes a process of proteolytic maturation that results from cleavages at six centrally located 26 amino acid repeats called HCF-1pro repeats. This generates a heterodimeric complex of stably associated amino- (HCF-1n) and carboxy- (HCF-1c) terminal subunits. The absence of the HCF-1 N or HCF-1; subunit leads predominantly to either G1 or M phase defects, respectively. We have hypothesized that HCF-1 forms a heterodimeric complex to permit communication between the two subunits of HCF-1 involved in regulating different phases of the cell cycle. Indeed, there is evidence for such inter-subunit communication because a point mutation called P134S in the HCF-1N subunit in the temperature-sensitive hamster cell line tsBN67 causes, addition to G1- phase defects associated with the HCF-1n subunit, M-phase defects similar to the defects seen upon loss of HCF-1 function. Furthermore, inhibition of the proteolytic maturation of HCF-1 by deletion of the six HCF-1pro repeats (HCF-1Aimo) also leads to M-phase defects, specifically cytokinesis defects leading to binucleation, indicating that there is loss of HCF-15 function in the absence of HCF-1 maturation. I demonstrate that individual point mutations in each of the six HCF-1pro repeats that prevent HCF-1 proteolytic maturation also lead to binucleation; however, this defect can be latgely rescued by the presence of just one HCF-1pRO sequence in I-ICF»1. These results argue that processing itself is important for the HCF-1g function. In fact, until now, the hypothesis was that the proteolytic processing per se is more important for HCF-1C function than the proteolytic processing region. But I show that processing per se is not sufticient to rescue multinucleation, but that the HCF-lpm sequence itself is crucial. This discovery leads to the conclusion that the I-ICF-1pRO repeats have an additional function important for HCF-le function. From the studies of others, one potential function of the HCF-lrxo tepeats is as a binding site for O-link NAcetyl glycosamine tansferase (OGT) to glycosylate an HCF-1n-sunbunit region called the Basic region. This new function suggests the Basic region of HCF-1n is also implicated in the communication between the two subunits. This inter-subunit communication was analyzed in more detail with the studies of the Pl34S mutation and the residues 382-450 region of HCF-l that when removed prevents HCF-l subunit association. I demonstrate that the point mutation also leads to a binucleation defect in Hela cells as well as in the tsBN67 cells. In addition, the effect of this mutation on the regulation of HCF-1c activity seems to interfere with that of the HCF-lpgg repeats because the sum of the deletion of the proteolytic processing region and the point mutation surprisingly leads to re-establishment of correct cytokinesis. The study of the 382-450 HCF-lN region also yielded surprising results. This region important for the association of the two subunits is also important for both HCF-1c function in M phase and G1 phase progression. Thus, I have discovered two main functions of this region: its role in the regulation of HCF-lc function in M phase and its involvement in the regulation of G1/S phase ?- an HCF-1n function. These results support the importance of inter-subunit communication in HCF-1 functions. My research illuminates the understanding of the interaction of the two subunits by showing that the whole HCF-1n subunit is involved in the inter-subunit communication in order to regulate HCF-1c function. For this work, I was concentrated on the study of cytokinesis; the first phenotype showing the role of HCF-1c in the M phase. Then, I extended the study of the M phase with analysis of steps earlier to cytokinesis. Because some defects in the chromosome segregation was already described in the absence of HCF-1, I decided to continue the study of M phase by checking effects on the chromosome segregation. I showed that the HCF-1n subunit and HCF-1pro repeats are both important for this key step of M phase. I show that the binucleation phenotype resulting from deletion or mutation in HCF-1pro repeats, Pl34S point mutation or the lack of the region 382-450 are correlated with micronuclei, and chromosome segregation and alignment defects. This suggests that HCF«lç already regulates M phase during an early step and could be involved in the complex regulation of chromosome segregation. Because one of the major roles of HCF-1 is to be a transcription regulator, I also checked the capacity of HCF-1 to bind to the chromatin in my different cell lines. All my recombinant proteins can bind the chromatin, except for, as previously described, the HCF-1 with the P134S point mutation, This suggests that the binding of HCF-1 to the chromatin is not dependant to the Basic and proteolytic regions but more to the Kelch domain. Thus, if the function of HCF-ig in M phase is dependant to its chromatin association, the intercommunication and the proteolytic region are not involved in the ability to bind to the chromatin but more to bind to the right place of the chromatin or to be associated with the co-factors. Résumé : L'étude de l'infection par le virus Herpes Simplex (HSV) a permis la découverte de la protéine HCF-1 (Host-Cell Factor). HCF-1 est une des protéines cellulaires qui font partie du complexe induit par VP16 ; ce complexe est la clef pour l'activation de la phase lytique de HSV. Afin de manipuler les cellules infectées, le complexe induit pas le VPIG devrait donc cibler les protéines importantes pour la régulation cellulaire, telles que la protéine HCF-1. Cette dernière s'avère donc être un senseur pour la cellule et devrait également jouer un rôle de régulation lors des différentes phases du cycle cellulaire. Chez l'humain, HCF-1 a la particularité de devoir passer par une phase de maturation pour devenir active. Lors de cette maturation, la protéine subit une coupure protéolytique au niveau de six répétitions composées de 26 acides aminés, appelé HCF-1pro repeats. Cette coupure engendre la formation d'un complexe formé de deux sous-unités, HCF-1n et HCF-1c, associées l'une à l'autre de façon stable. Enlever la sous-unité HCF-IN ou C entraîne respectivement des défauts dans la phase G1 et M. Nous pensons donc que HCF-1 forme un complexe hétérodimérique afin de permettre la communication entre les molécules impliquées dans la régulation des différentes phases du cycle cellulaire. Cette hypothèse est déduite suite à deux études: l'une réalisée sur la lignée cellulaire tsBN67 et l'autre portant sur l'inhibition de la maturation protéolytique. La lignée cellulaire tsBN67, sensible à la température, porte la mutation Pl 345 dans la sous-unité HCF-1n. Cette mutation, en plus d'occasionner des défauts dans la phase G1 (défauts liés à la sous-unité HCF-1N), a aussi pour conséquence d'entrainer des défauts dans la phase M, défauts similaires à ceux dus a la perte de la sous-unité HCF-1c. Quant à la maturation protéolytique, l'absence de la région de la protéolyse provoque la binucléation, défaut lié à la cytokinèse, indiquant la perte de la fonction de la sous-unité HCF-1c. Au cours de ma thèse, j'ai démontré que des mutations dans les HCF-1=no repeats, qui bloquent la protéolyse, engendrent la binucléation ; cependant ce défaut peut être corrigé pas l'ajout d'un HCF-1pro repeat dans un HCF-1 ne contenant pas la région protéolytique. Ces résultats soutiennent l'idée que la région protéolytique est importante pour le bon fonctionnement de HCF-1c. En réalité jusqu'a maintenant on supposait que le mécanisme de coupure était plus important que la région impliquée pour la régulation de la fonction de HCF-1;. Mais mon étude montre que la protéolyse n'est pas suffisante pour éviter la binucléation ; en effet, les HCF-1pro repeats semblent jouer le rôle essentiel dans le cycle cellulaire. Cette découverte conduit à la conclusion que les HCF-1pro repeats ont sûrement une fonction autre qui serait cruciale pour la foncton de HCF-1c. Une des fonctions possibles est d'être le site de liaison de l'O-linked N-acetylglucosamine transférase (OGT) qui glycosylerait la région Basique de HCF-1n. Cette nouvelle fonction suggère que la région Basique est aussi impliquée dans la communication entre les deux sous- unités. L'intercommunication entre les deux sous-unités ai été d'ailleurs analysée plus en détail dans mon travail à travers l'étude de la mutation Pl34S et de la région 382-450, essentielle pour l'association des deux sous»unités. J'ai ainsi démontré que la mutation P134S entraînait aussi des défauts dans la cytokinése dans la lignée cellulaire Hela, de plus, son influence sur HCF-1c semble interférer avec celle de la région protéolytique. En effet, la superposition de ces deux modifications dans HCF-1 conduit au rétablissement d'une cytokinése correcte. Concernant la région 382 à 450, les résultats ont été assez surprenants, la perte de cette région provoque l'arrêt du cycle en G1 et la binucléation, ce qui tend à prouver son importance pour le bon fonctionnement de HCF-1n et de HCF-1c. Cette découverte appuie par conséquent l'hypotl1èse d'une intercommunicatzion entre les deux sous-unités mettant en jeu les différentes régions de HCF-1n. Grâce à mes recherches, j'ai pu améliorer la compréhension de l'interaction des deux sous-unités de HCF-1 en montrant que toutes les régions de HCF-1n sont engagées dans un processus d'intercommunication, dont le but est de réguler l'action de HCF-1c. J'ai également mis en évidence une nouvelle étape de la maturation de HCF-1 qui représente une phase importante pour l'activation de la fonction de HCF-1c. Afin de mettre à jour cette découverte, je me suis concentrée sur l'étude de l'impact de ces régions au niveau de la cytokinése qui fut le premier phénotype démontrant le rôle de HCF-1c dans la phase M. A ce jour, nous savons que HCF-1c joue un rôle dans la cytokinèse, nous ne connaissons pas encore sa fonction précise. Dans le but de cerner plus précisément cette fonction, j'ai investigué des étapes ultérieures ai la cytokinèse. Des défauts dans la ségrégation des chromosomes avaient déjà été observés, ai donc continué l'étude en prouvant que HCF-1n et les HCF-1pro repeats sont aussi importants pour le bon fonctionnement de cette étape clef également régulée par HCF-1c. J' ai aussi montré que la région 382-450 et la mutation P134S sont associées à un taux élevé de micronoyaux, de défauts dans la ségrégation des chromosomes. L'une des fonctions principales de HCF-1 étant la régulation de la transcription, j'ai aussi contrôlé la capacité de HCF-1 à se lier à la chromatine après insertion de mutations ou délétions dans HCF-1n et dans la région protéolytique. Or, à l'exception des HCF-1 contenant la mutation P134S, la sous-unité HCF-1c des HCF-1 tronquées se lie correctement à la chromatine. Cette constatation suggère que la liaison entre HCF-1c et chromatine n'est pas dépendante de la région Basique ou Protéolytique mais peut-être vraisemblablement de la région Kelch. Donc si le rôle de HCF-1c est dépendant de sa capacité â activer la transcription, l'intercommunication entre les deux sous-unités et la région protéolytique joueraient un rôle important non pas dans son habileté à se lier à la chromatine, mais dans la capacité de HCF-1 à s'associer aux co-facteurs ou à se placer sur les bonnes régions du génome.

A new locus for congenital cataract, microcornea, microphthalmia, and atypical iris coloboma maps to chromosome 2.

OBJECTIVE: To report a novel phenotype of autosomal dominant atypical congenital cataract associated with variable expression of microcornea, microphthalmia, and iris coloboma linked to chromosome 2. Molecular analysis of this phenotype may improve our understanding of anterior segment development. DESIGN: Observational case study, genome linkage analysis, and gene mutation screening. PARTICIPANTS: Three families, 1 Egyptian and 2 Belgians, with a total of 31 affected were studied. METHODS: Twenty-one affected subjects and 9 first-degree relatives underwent complete ophthalmic examination. In the Egyptian family, exclusion of PAX6, CRYAA, and MAF genes was demonstrated by haplotype analysis using microsatellite markers on chromosomes 11, 16, and 21. Genome-wide linkage analysis was then performed using 385 microsatellite markers on this family. In the 2 Belgian families, the PAX6 gene was screened for mutations by direct sequencing of all exons. MAIN OUTCOME MEASURES: Phenotype description, genome-wide linkage of the phenotype, linkage to the PAX6, CRYAA, and MAF genes, and mutation detection in the PAX6 gene. RESULTS: Affected members of the 3 families had bilateral congenital cataracts inherited in an autosomal dominant pattern. A novel form of hexagonal nuclear cataract with cortical riders was expressed. Among affected subjects with available data, 95% had microcornea, 39% had microphthalmia, and 38% had iris coloboma. Seventy-five percent of the colobomata were atypical, showing a nasal superior location in 56%. A positive lod score of 4.86 was obtained at theta = 0 for D2S2309 on chromosome 2, a 4.9-Mb common haplotype flanked by D2S2309 and D2S2358 was obtained in the Egyptian family, and linkage to the PAX6, CRYAA, or MAF gene was excluded. In the 2 Belgian families, sequencing of the junctions and all coding exons of PAX6 did not reveal any molecular change. CONCLUSIONS: We describe a novel phenotype that includes the combination of a novel form of congenital hexagonal cataract, with variably expressed microcornea, microphthalmia, and atypical iris coloboma, not caused by PAX6 and mapping to chromosome 2. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Non-classical karyotypic features in relapsed childhood B-cell precursor acute lymphoblastic leukemia.

Karyotype analysis of acute lymphoblastic leukemia (ALL) at diagnosis has provided valuable prognostic markers for treatment stratification. However, reports of cytogenetic studies of relapsed ALL samples are limited. We compared the karyotypes from 436 nonselected B-cell precursor ALL patients at initial diagnosis and of 76 patients at first relapse. We noticed a relative increase of karyotypes that did not fall into the classic ALL cytogenetic subgroups (high hyperdiploidy, t(12;21), t(9;22), 11q23, t(1;19), <45 chromosomes) in a group of 29 patients at relapse (38%) compared to 130 patients at presentation (30%). Non-classical cytogenetic aberrations in these 29 patients were mostly found on chromosomes 1, 2, 7, 9, 13, 14, and 17. We also describe six rare reciprocal translocations, three of which involved 14q32. The most frequent abnormalities were found in 9p (12/29 cases) and were associated with a marked decrease in the duration of the second remission, but not of the probability of 10-year event-free survival after relapse treatment. From 29 patients with non-classical cytogenetic aberrations, only 8 (28%) had been stratified to a high risk-arm on the first treatment protocol, suggesting that this subgroup might benefit from the identification of new prognostic markers in future studies.

Short- and long-term changes in chromosomal inversion polymorphism and global warming: Drosophila subobscura from the Balkans

The chromosomal inversion polymorphism of Drosophila subobscura is adaptive to environmental changes. The population of Petnica, Serbia, was chosen to analyze short- and long-term changes in this polymorphism. Short-term changes were studied in the samples collected in May, June, and August of 1995. The inversion polymorphism varied over these months, although various interpretations are possible. To analyze long-term changes, samples obtained in May 1995 and May 2010 were compared. The frequency of the 'cold' adapted inversions (Ast, Jst, Ust, Est, and Ost) decreased and that of the 'warm' adapted inversions (A2, J1, U1+2, and O3+4) increased, from 1995 to 2010. These changes are consistent with the general increase in temperature recorded in Petnica for the same period. Finally, the possible response of chromosomal polymorphism to global warming was analyzed at the regional level (Balkan peninsula). This polymorphism depends on the ecological conditions of the populations, and the changes observed appear to be consistent with global warming expectations. Natural selection seems to be the main mechanism responsible for the evolution of this chromosomal polymorphism.

Short- and long-term changes in chromosomal inversion polymorphism and global warming: Drosophila subobscura from the Balkans

The chromosomal inversion polymorphism of Drosophila subobscura is adaptive to environmental changes. The population of Petnica, Serbia, was chosen to analyze short- and long-term changes in this polymorphism. Short-term changes were studied in the samples collected in May, June, and August of 1995. The inversion polymorphism varied over these months, although various interpretations are possible. To analyze long-term changes, samples obtained in May 1995 and May 2010 were compared. The frequency of the 'cold' adapted inversions (Ast, Jst, Ust, Est, and Ost) decreased and that of the 'warm' adapted inversions (A2, J1, U1+2, and O3+4) increased, from 1995 to 2010. These changes are consistent with the general increase in temperature recorded in Petnica for the same period. Finally, the possible response of chromosomal polymorphism to global warming was analyzed at the regional level (Balkan peninsula). This polymorphism depends on the ecological conditions of the populations, and the changes observed appear to be consistent with global warming expectations. Natural selection seems to be the main mechanism responsible for the evolution of this chromosomal polymorphism.

Numerous potentially functional but non-genic conserved sequences on human chromosome 21.

The use of comparative genomics to infer genome function relies on the understanding of how different components of the genome change over evolutionary time. The aim of such comparative analysis is to identify conserved, functionally transcribed sequences such as protein-coding genes and non-coding RNA genes, and other functional sequences such as regulatory regions, as well as other genomic features. Here, we have compared the entire human chromosome 21 with syntenic regions of the mouse genome, and have identified a large number of conserved blocks of unknown function. Although previous studies have made similar observations, it is unknown whether these conserved sequences are genes or not. Here we present an extensive experimental and computational analysis of human chromosome 21 in an effort to assign function to sequences conserved between human chromosome 21 (ref. 8) and the syntenic mouse regions. Our data support the presence of a large number of potentially functional non-genic sequences, probably regulatory and structural. The integration of the properties of the conserved components of human chromosome 21 to the rapidly accumulating functional data for this chromosome will improve considerably our understanding of the role of sequence conservation in mammalian genomes.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination.

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27-amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 pmol/l). GIP binding was displaced by &lt; 20% by 1 mumol/l glucagon, glucagon-like peptide (GLP-I)(7-36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9-39) at 1 mumol/l displaced binding by approximately 70 and approximately 100% at 10 mumol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6-0.8 nmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9-39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 mumol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

Structural variation and its effect on expression.

Structural variation, whether it is caused by copy number variants or present in a balanced form, such as reciprocal translocations and inversions, can have a profound and dramatic effect on the expression of genes mapping within and close to the rearrangement, as well as affecting others genome wide. These effects can be caused by altering the copy number of one or more genes or regulatory elements (dosage effect) or from physical disruption of links between regulatory elements and their associated gene or genes, resulting in perturbation of expression. Similarly, large-scale structural variants can result in genome-wide expression changes by altering the positions that chromosomes occupy within the nucleus, potentially disrupting not only local cis interactions, but also trans interactions that occur throughout the genome. Structural variation is, therefore, a significant factor in the study of gene expression and is discussed here in more detail.

Reciprocal Translocations in Cattle: frequency estimation

Chromosomal anomalies, like Robertsonian and reciprocal translocations represent a big problem in cattle breeding as their presence induces, in the carrier subjects, a well documented fertility reduction. In cattle reciprocal translocations (RCPs, a chromosome abnormality caused by an exchange of material between nonhomologous chromosomes) are considered rare as to date only 19 reciprocal translocations have been described. In cattle it is common knowledge that the Robertsonian translocations represent the most common cytogenetic anomalies, and this is probably due to the existence of the endemic 1;29 Robertsonian translocation. However, these considerations are based on data obtained using techniques that are unable to identify all reciprocal translocations and thus their frequency is clearly underestimated. The purpose of this work is to provide a first realistic estimate of the impact of RCPs in the cattle population studied, trying to eliminate the factors which have caused an underestimation of their frequency so far. We performed this work using a mathematical as well as a simulation approach and, as biological data, we considered the cytogenetic results obtained in the last 15 years. The results obtained show that only 16% of reciprocal translocations can be detected using simple Giemsa techniques and consequently they could be present in no less than 0,14% of cattle subjects, a frequency five times higher than that shown by de novo Robertsonian translocations. This data is useful to open a debate about the need to introduce a more efficient method to identify RCP in cattle.

Relationship between grain colour and preharvest sprouting-resistance in wheat

Since red alleles (R) of the genes that control grain colour are important for the improvement of preharvest sprouting resistance in wheat and there are three independently inherited loci, on chromosomes 3A, 3B and 3D of hexaploid wheat, it is possible to vary the dosage of dominant alleles in a breeding program. The objective of this work was to evaluate the dosage effect of R genes on preharvest sprouting, in a single seed descent population, named TRL, derived from the cross between Timgalen, white-grained wheat, and RL 4137, red-grained wheat. The study was carried out using sprouting data in ripe ears obtained under artificial conditions in a rainfall simulator over three years. According to the results there is a significant effect on preharvest sprouting provided by colour and a weaker effect of increasing R dosage. However, the significant residual genotypic variation between red lines and all lines (reds and whites) at 0.1% level showed that preharvest sprouting was also controlled by other genes. There are no significant correlations between sprouting and date of ripeness or between ripeness, R dosage and colour intensity.

Mapping of quantitative trait loci for thermosensitive genic male sterility in indica rice

The objective of this work was to select and use microsatellite markers, to map genomic regions associated with the genetic control of thermosensitive genic male sterility (TGMS) in rice. An F2 population, derived from the cross between fertile and TGMS indica lines, was used to construct a microsatellite-based genetic map of rice. The TGMS phenotype showed a continuous variation in the segregant population. A low level of segregation distortion was detected in the F2 (14.65%), whose cause was found to be zygotic selection. There was no evidence suggesting a cause-effect relationship between zygotic selection and the control of TGMS in this cross. A linkage map comprising 1,213.3 cM was constructed based on the segregation data of the F2 population. Ninety-five out of 116 microsatellite polymorphic markers were assembled into 11 linkage groups, with an average of 12.77 cM between two adjacent marker loci. The phenotypic and genotypic data allowed for the identification of three new quantitative trait loci (QTL) for thermosensitive genic male sterility in indica rice. Two of the QTL were mapped on chromosomes that, so far, have not been associated with the genetic control of the TGMS trait (chromosomes 1 and 12). The third QTL was mapped on chromosome 7, where a TGMS locus (tms2) has recently been mapped. Allelic tests will have to be developed, in order to clarify if the two regions are the same or not.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.

Case-control analysis of paternal age and trisomic anomalies.

OBJECTIVES: To determine whether older paternal age increases the risk of fathering a pregnancy with Patau (trisomy 13), Edwards (trisomy 18), Klinefelter (XXY) or XYY syndrome. DESIGN: Case-control: cases with each of these syndromes were matched to four controls with Down syndrome from within the same congenital anomaly register and with maternal age within 6 months. SETTING: Data from 22 EUROCAT congenital anomaly registers in 12 European countries. PARTICIPANTS: Diagnoses with observed or (for terminations) predicted year of birth from 1980 to 2005, comprising live births, fetal deaths with gestational age ≥ 20 weeks and terminations after prenatal diagnosis of the anomaly. Data include 374 cases of Patau syndrome, 929 of Edwards syndrome, 295 of Klinefelter syndrome, 28 of XYY syndrome and 5627 controls with Down syndrome. MAIN OUTCOME MEASURES: Odds ratio (OR) associated with a 10-year increase in paternal age for each anomaly was estimated using conditional logistic regression. Results were adjusted to take account of the estimated association of paternal age with Down syndrome (1.11; 95% CI 1.01 to 1.23). RESULTS: The OR for Patau syndrome was 1.10 (95% CI 0.83 to 1.45); for Edwards syndrome, 1.15 (0.96 to 1.38); for Klinefelter syndrome, 1.35 (1.02 to 1.79); and for XYY syndrome, 1.99 (0.75 to 5.26). CONCLUSIONS: There was a statistically significant increase in the odds of Klinefelter syndrome with increasing paternal age. The larger positive associations of Klinefelter and XYY syndromes with paternal age compared with Patau and Edwards syndromes are consistent with the greater percentage of these sex chromosome anomalies being of paternal origin.