Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

A new clinical and molecular form of Unverricht-Lundborg disease localized by homozygosity mapping

Progressive myoclonus epilepsy (PME) has a number of causes, of which Unverricht-Lundborg disease (ULD) is the most common. ULD has previously been mapped to a locus on chromosome 21 (EPM1). Subsequently, mutations in the cystatin B gene have been found in most cases. In the present work we identified an inbred Arab family with a clinical pattern compatible with ULD, but mutations in the cystatin B gene were absent. We sought to characterize the clinical and molecular features of the disorder. The family was studied by multiple field trips to their town to clarify details of the complex consanguineous relationships and to personally examine the family. DNA was collected for subsequent molecular analyses from 21 individuals. A genome-wide screen was performed using 811 microsatellite markers. Homozygosity mapping was used to identify loci of interest. There were eight affected individuals. Clinical onset was at 7.3 +/- 1.5 years with myoclonic or tonic-clonic seizures. All had myoclonus that progressed in severity over time and seven had tonic-clonic seizures. Ataxia, in addition to myoclonus, occurred in all. Detailed cognitive assessment was not possible, but there was no significant progressive dementia. There was intrafamily variation in severity; three required wheelchairs in adult life; the others could walk unaided. MRI, muscle and skin biopsies on one individual were unremarkable. We mapped the family to a 15-megabase region at the pericentromeric region of chromosome 12 with a maximum lod score of 6.32. Although the phenotype of individual subjects was typical of ULD, the mean age of onset (7.3 years versus 11 years for ULD) was younger. The locus on chromosome 12 does not contain genes for any other form of PME, nor does it have genes known to be related to cystatin B. This represents a new form of PME and we have designated the locus as EPM1B.

CleanEx: a database of heterogeneous gene expression data based on a consistent gene nomenclature.

The main goal of CleanEx is to provide access to public gene expression data via unique gene names. A second objective is to represent heterogeneous expression data produced by different technologies in a way that facilitates joint analysis and cross-data set comparisons. A consistent and up-to-date gene nomenclature is achieved by associating each single experiment with a permanent target identifier consisting of a physical description of the targeted RNA population or the hybridization reagent used. These targets are then mapped at regular intervals to the growing and evolving catalogues of human genes and genes from model organisms. The completely automatic mapping procedure relies partly on external genome information resources such as UniGene and RefSeq. The central part of CleanEx is a weekly built gene index containing cross-references to all public expression data already incorporated into the system. In addition, the expression target database of CleanEx provides gene mapping and quality control information for various types of experimental resource, such as cDNA clones or Affymetrix probe sets. The web-based query interfaces offer access to individual entries via text string searches or quantitative expression criteria. CleanEx is accessible at: http://www.cleanex.isb-sib.ch/.

CleanEx: a database of heterogeneous gene expression data based on a consistent gene nomenclature and linked to an improved annotation system

Résumé: L'automatisation du séquençage et de l'annotation des génomes, ainsi que l'application à large échelle de méthodes de mesure de l'expression génique, génèrent une quantité phénoménale de données pour des organismes modèles tels que l'homme ou la souris. Dans ce déluge de données, il devient très difficile d'obtenir des informations spécifiques à un organisme ou à un gène, et une telle recherche aboutit fréquemment à des réponses fragmentées, voir incomplètes. La création d'une base de données capable de gérer et d'intégrer aussi bien les données génomiques que les données transcriptomiques peut grandement améliorer la vitesse de recherche ainsi que la qualité des résultats obtenus, en permettant une comparaison directe de mesures d'expression des gènes provenant d'expériences réalisées grâce à des techniques différentes. L'objectif principal de ce projet, appelé CleanEx, est de fournir un accès direct aux données d'expression publiques par le biais de noms de gènes officiels, et de représenter des données d'expression produites selon des protocoles différents de manière à faciliter une analyse générale et une comparaison entre plusieurs jeux de données. Une mise à jour cohérente et régulière de la nomenclature des gènes est assurée en associant chaque expérience d'expression de gène à un identificateur permanent de la séquence-cible, donnant une description physique de la population d'ARN visée par l'expérience. Ces identificateurs sont ensuite associés à intervalles réguliers aux catalogues, en constante évolution, des gènes d'organismes modèles. Cette procédure automatique de traçage se fonde en partie sur des ressources externes d'information génomique, telles que UniGene et RefSeq. La partie centrale de CleanEx consiste en un index de gènes établi de manière hebdomadaire et qui contient les liens à toutes les données publiques d'expression déjà incorporées au système. En outre, la base de données des séquences-cible fournit un lien sur le gène correspondant ainsi qu'un contrôle de qualité de ce lien pour différents types de ressources expérimentales, telles que des clones ou des sondes Affymetrix. Le système de recherche en ligne de CleanEx offre un accès aux entrées individuelles ainsi qu'à des outils d'analyse croisée de jeux de donnnées. Ces outils se sont avérés très efficaces dans le cadre de la comparaison de l'expression de gènes, ainsi que, dans une certaine mesure, dans la détection d'une variation de cette expression liée au phénomène d'épissage alternatif. Les fichiers et les outils de CleanEx sont accessibles en ligne (http://www.cleanex.isb-sib.ch/). Abstract: The automatic genome sequencing and annotation, as well as the large-scale gene expression measurements methods, generate a massive amount of data for model organisms. Searching for genespecific or organism-specific information througout all the different databases has become a very difficult task, and often results in fragmented and unrelated answers. The generation of a database which will federate and integrate genomic and transcriptomic data together will greatly improve the search speed as well as the quality of the results by allowing a direct comparison of expression results obtained by different techniques. The main goal of this project, called the CleanEx database, is thus to provide access to public gene expression data via unique gene names and to represent heterogeneous expression data produced by different technologies in a way that facilitates joint analysis and crossdataset comparisons. A consistent and uptodate gene nomenclature is achieved by associating each single gene expression experiment with a permanent target identifier consisting of a physical description of the targeted RNA population or the hybridization reagent used. These targets are then mapped at regular intervals to the growing and evolving catalogues of genes from model organisms, such as human and mouse. The completely automatic mapping procedure relies partly on external genome information resources such as UniGene and RefSeq. The central part of CleanEx is a weekly built gene index containing crossreferences to all public expression data already incorporated into the system. In addition, the expression target database of CleanEx provides gene mapping and quality control information for various types of experimental resources, such as cDNA clones or Affymetrix probe sets. The Affymetrix mapping files are accessible as text files, for further use in external applications, and as individual entries, via the webbased interfaces . The CleanEx webbased query interfaces offer access to individual entries via text string searches or quantitative expression criteria, as well as crossdataset analysis tools, and crosschip gene comparison. These tools have proven to be very efficient in expression data comparison and even, to a certain extent, in detection of differentially expressed splice variants. The CleanEx flat files and tools are available online at: http://www.cleanex.isbsib. ch/.

Molecular Characterization and Physical Mapping of Two Classes of 5S rDNA in the Genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) not only is a useful source of milk, it also provides meat and works as a natural source of labor and biogas. To establish a project for buffalo genome mapping a 5,000-rad whole genome radiation hybrid panel was constructed for river buffalo and used to build preliminary RH maps from two chromosomes (BBU 3 and BBU10). The preliminary maps contain 66 markers, including coding genes, cattle ESTs and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci, in particular, genes and expressed sequence tags that will allow detailed comparative maps between buffalo, cattle and other species to be constructed. A large quantity of DNA has been prepared from the cell lines forming the RH panel reported here and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.

Mapping eIF5A binding sites for Dys1 and Lia1: In vivo evidence for regulation of eIF5A hypusination

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal α-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Molecular Characterization and Physical Mapping of Two Classes of 5S rDNA in the Genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I. Copyright (C) 2012 S. Karger AG, Basel

Homozygous deletion mapping in myeloma samples identifies genes and an expression signature relevant to pathogenesis and outcome.

PURPOSE: Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes. EXPERIMENTAL DESIGN: Using single nucleotide polymorphism-based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome. RESULTS: We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the "cell death" network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets. CONCLUSIONS: We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma.

AFLP-based genetic mapping of the " bud-flowering" trait in heather (Calluna vulgaris)

Background: Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait " flower type" .Results: The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the " two-way pseudo-testcross" and the " integrated" mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris.Conclusions: All maps confirmed the independent inheritance of the most important horticultural traits " flower type" , " flower colour" , and " leaf colour". An AFLP marker for the most important breeding target " flower type" was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers. © 2013 Behrend et al.; licensee BioMed Central Ltd.

Genetic Analysis of Age-at-Onset for Cardiovascular Risk Factors in a Brazilian Family Study

Background/Aims: Statistical analysis of age-at-onset involving family data is particularly complicated because there is a correlation pattern that needs to be modeled and also because there are measurements that are censored. In this paper, our main purpose was to evaluate the effect of genetic and shared family environmental factors on age-at-onset of three cardiovascular risk factors: hypertension, diabetes and high cholesterol. Methods: The mixed-effects Cox model proposed by Pankratz et al. [2005] was used to analyze the data from 81 families, involving 1,675 individuals from the village of Baependi, in the state of Minas Gerais, Brazil. Results: The analyses performed showed that the polygenic effect plays a greater role than the shared family environmental effect in explaining the variability of the age-at-onset of hypertension, diabetes and high cholesterol. The model which simultaneously evaluated both effects indicated that there are individuals which may have risk of hypertension due to polygenic effects 130% higher than the overall average risk for the entire sample. For diabetes and high cholesterol the risks of some individuals were 115 and 45%, respectively, higher than the overall average risk for the entire population. Conclusions: Results showed evidence of significant polygenic effects indicating that age-at-onset is a useful trait for gene mapping of the common complex diseases analyzed. In addition, we found that the polygenic random component might absorb the effects of some covariates usually considered in the risk evaluation, such as gender, age and BMI. Copyright (C) 2008 S. Karger AG, Basel

An integrative approach for studying the etiology of alcoholism and other addictions

Studies of alcoholism etiology often focus on genetic or psy-chosocial approaches, but not both. Greater understanding of the etiology of alcohol, tobacco and other addictions will come from integration of these research traditions. A research approach is outlined to test three models for the etiology of addictions — behavioral undercontrol, pharmacologic vulnerability, negative affect regulation — addressing key questions including (i) mediators of genetic effects, (ii) genotype-environment correlation effects, (iii) genotype x environment interaction effects, (iv) the developmental unfolding of genetic and environmental effects, (v) subtyping including identification of distinct trajectories of substance involvement, (vi) identification of individual genes that contribute to risk, and (vii) the consequences of excessive use. By using coordinated research designs, including prospective assessment of adolescent twins and their siblings and parents; of adult substance dependent and control twins and their MZ and DZ cotwins, the spouses of these pairs, and their adolescent offspring; and of regular families; by selecting for gene-mapping approaches sibships screened for extreme concordance or discordance on quantitative indices of substance use; and by using experimental (drug challenge) as well as survey approaches, a number of key questions concerning addiction etiology can be addressed. We discuss complementary strengths and weaknesses of different sampling strategies, as well as methods to implement such an integrated approach illustrated for the study of alcoholism etiology. A coordinated program of twin and family studies will allow a comprehensive dissection of the interplay of genetic and environmental risk-factors in the etiology of alcoholism and other addictions.

Predictors of non-response to a questionnaire survey of a volunteer twin panel: Findings from the Australian 1989 twin cohort

Questionnaire surveys, while more economical, typically achieve poorer response rates than interview surveys. We used data from a national volunteer cohort of young adult twins, who were scheduled for assessment by questionnaire in 1989 and by interview in 1996-2000, to identify predictors of questionnaire non-response. Out of a total of 8536 twins, 5058 completed the questionnaire survey (59% response rate), and 6255 completed a telephone interview survey conducted a decade later (73% response rate). Multinomial logit models were fitted to the interview data to identify socioeconomic, psychiatric and health behavior correlates of non-response in the earlier questionnaire survey. Male gender, education below University level, and being a dizygotic rather than monozygotic twin, all predicted reduced likelihood of participating in the questionnaire survey. Associations between questionnaire response status and psychiatric history and health behavior variables were modest, with history of alcohol dependence and childhood conduct disorder predicting decreased probability of returning a questionnaire, and history of smoking and heavy drinking more weakly associated with non-response. Body-mass index showed no association with questionnaire non-response. Despite a poor response rate to the self-report questionnaire survey, we found only limited sampling biases for most variables. While not appropriate for studies where socioeconomic variables are critical, it appears that survey by questionnaire, with questionnaire administration by telephone to non-responders, will represent a viable strategy for gene-mapping studies requiring that large numbers of relatives be screened.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.