Genomics of wine yeast: functional analysis of new and variable genes

Projecte de recerca elaborat a partir d’una estada a l’Institut National de la Recherche Agronomique, França, entre 2007 i 2009. Saccharomyces cerevisiae ha estat el llevat utilitzat durant mil.lenis en l'elaboració de vins. Tot i així, es té poc coneixement sobre les pressions de selecció que han actuat en la modelització del genoma dels llevats vínics. S’ha seqüenciat el genoma d'una soca vínica comercial, EC1118, obtenint 31 supercontigs que cobreixen el 97% del genoma de la soca de referència, S288c. S’ha trobat que el genoma de la soca vínica es diferencia bàsicament en la possessió de 3 regions úniques que contenen 34 gens implicats en funcions claus per al procés fermentatiu. A banda, s’han dut a terme estudis de filogènia i synteny (ordre dels gens) que mostren que una d'aquestes tres regions és pròxima a una espècie relacionada amb el gènere Saccharomyces, mentre que les altres dos regions tenen un origen no-Saccharomyces. S’ha identificat mitjançant PCR i seqüenciació a Zygosaccharomyces bailii, una espècie contaminant de les fermentacions víniques, com a espècie donadora d'una de les dues regions. Les hibridacions naturals entre soques de diferents espècies dins del grup Saccharomyces sensu stricto ja han estat descrites. El treball és el primer que presenta hibridacions entre espècies Saccharomyces i no-Saccharomyces (Z. bailii, en aquest cas). També s’assenyala que les noves regions es troben freqüent i diferencialment presents entre els clades de S. cerevisiae, trobant-se de manera gairebé exclusiva en el grup de les soques víniques, suggerint que es tracta d'una adquisició recent de transferència gènica. En general, les dades demostren que el genoma de les soques víniques pateix una constant remodelació mitjançant l'adquisició de gens exògens. Els resultats suggereixen que aquests processos estan afavorits per la proximitat ecològica i estan implicats en l'adaptació molecular de les soques víniques a les condicions d'elevada concentració en sucres, poc nitrogen i elevades concentracions en etanol.

ALK rearrangement in a selected population of advanced non small cell lung cancer patients. FISH and inmunohistochemistry diagnostic methods, prevalence and clinical outcomes

Anaplastic lymphoma kinase (ALK) rearrangements represents a new driver oncogenic event in non-small cell lung cancer (NSCLC). ALK positive patients account for a 1-7% of NSCLC patients. The objective of this study is to know the prevalence and clinical characteristics of ALK positive patients in a cohort of NSCLC patients and to compare inmunohistochemistry with D5F3 monoclonal antibody with gold standard method fluorescence in situ hybridation

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Polytene chromosome map and inversion polymorphism in Drosophila mediopunctata

Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.

Copy number variation and chromatin structure

The functional consequences of structural variation in the human genome range from adaptation, to phenotypic variation, to predisposition to diseases. Copy number variation (CNV) was shown to influence the phenotype by modifying, in a somewhat dose-dependent manner, the expression of genes that map within them, as well as that of genes located on their flanks. To assess the possible mechanism(s) behind this neighboring effect, we compared histone modification status of cell lines from patients affected by Williams-Beuren, Williams-Beuren region duplication, Smith-Magenis or DiGeorge Syndrome and control individuals using a high-throughput version of chromatin immuno-precipitation method (ChIP), called ChlP-seq. We monitored monomethylation of lysine K20 on histone H4 and trimethylation of lysine K27 on histone H3, as proxies for open and condensed chromatin, respectively. Consistent with the changes in expression levels observed for multiple genes mapping on the entire length of chromosomes affected by structural variants, we also detected regions with modified histone status between samples, up- and downstream from the critical regions, up to the end of the rearranged chromosome. We also gauged the intrachromosomal interactions of these cell lines utilizing chromosome conformation capture (4C-seq) technique. We observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together, possibly forming an interacting cluster with each other and the WBSCR. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We conclude, that large genomic rearrangements can lead to changes in the state of the chromatin spreading far away from the critical region, thus possibly affecting expression globally and as a result modifying the phenotype of the patients. - Les conséquences fonctionnelles des variations structurelles dans le génome humain sont vastes, allant de l'adaptation, en passant par les variations phénotypiques, aux prédispositions à certaines maladies. Il a été démontré que les variations du nombre de copies (CNV) influencent le phénotype en modifiant, d'une manière plus ou moins dose-dépendante, l'expression des gènes se situant à l'intérieur de ces régions, mais également celle des gènes se trouvant dans les régions flanquantes. Afin d'étudier les mécanismes possibles sous-jacents à cet effet de voisinage, nous avons comparé les états de modification des histones dans des lignées cellulaires dérivées de patients atteints du syndrome de Williams-Beuren, de la duplication de la région Williams-Beuren, du syndrome de Smith-Magenis ou du syndrome de Di- George et d'individus contrôles en utilisant une version haut-débit de la méthode d'immunoprécipitation de la chromatine (ChIP), appelée ChIP-seq. Nous avons suivi la mono-méthylation de la lysine K20 sur l'histone H4 et la tri-méthylation de la lysine K27 sur l'histone H3, marqueurs respectifs de la chromatine ouverte et fermée. En accord avec les changements de niveaux d'expression observés pour de multiples gènes tout le long des chromosomes affectés par les CNVs, nous avons aussi détecté des régions présentant des modifications d'histones entre les échantillons, situées de part et d'autre des régions critiques, jusqu'aux extrémités du chromosome réarrangé. Nous avons aussi évalué les interactions intra-chromosomiques ayant lieu dans ces cellules par l'utilisation de la technique de capture de conformation des chromosomes (4C-seq). Nous avons observé qu'un groupe de gènes flanquants la région critique du syndrome de Williams-Beuren (WBSCR) forment souvent une boucle, constituant un groupe d'interactions privilégiées entre ces gènes et la WBSCR. La délétion de la WBSCR perturbe l'expression de ce groupe de gènes flanquants, mais également les interactions à grande échelle entre eux et la région réarrangée. Nous en concluons que les larges réarrangements génomiques peuvent aboutir à des changements de l'état de la chromatine pouvant s'étendre bien plus loin que la région critique, affectant donc potentiellement l'expression de manière globale et ainsi modifiant le phénotype des patients.

Molecular characterization of enterococci harboring genotype and phenotype incongruence related to glycopeptide resistance isolated in Brazilian hospitals

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.

The effect of translocation-induced nuclear reorganization on gene expression.

Translocations are known to affect the expression of genes at the breakpoints and, in the case of unbalanced translocations, alter the gene copy number. However, a comprehensive understanding of the functional impact of this class of variation is lacking. Here, we have studied the effect of balanced chromosomal rearrangements on gene expression by comparing the transcriptomes of cell lines from controls and individuals with the t(11;22)(q23;q11) translocation. The number of differentially expressed transcripts between translocation-carrying and control cohorts is significantly higher than that observed between control samples alone, suggesting that balanced rearrangements have a greater effect on gene expression than normal variation. Many of the affected genes are located along the length of the derived chromosome 11. We show that this chromosome is concomitantly altered in its spatial organization, occupying a more central position in the nucleus than its nonrearranged counterpart. Derivative 22-mapping chromosome 22 genes, on the other hand, remain in their usual environment. Our results are consistent with recent studies that experimentally altered nuclear organization, and indicated that nuclear position plays a functional role in regulating the expression of some genes in mammalian cells. Our study suggests that chromosomal translocations can result in hitherto unforeseen, large-scale changes in gene expression that are the consequence of alterations in normal chromosome territory positioning. This has consequences for the patterns of gene expression change seen during tumorigenesis-associated genome instability and during the karyotype changes that lead to speciation.

Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis.

The S. pombe cdc15 gene is essential for cell division. cdc15ts mutants do not form a septum, but growth and nuclear division continue, leading to formation of multinucleate cells. The earliest step in septum formation and cytokinesis, rearrangement of actin to the center of the cell, is associated with appearance of hypophosphorylated cdc15p and formation of a cdc15p ring, which colocalizes with actin. Loss of cdc15p function impairs formation of the actin ring. The abundance of cdc15 mRNA varies through the cell division cycle, peaking in early mitosis before septation. Expression of cdc15 in G2-arrested cells induces actin rearrangement to the center of the cell. These data implicate cdc15p as a key element in mediating the cytoskeletal rearrangements required for cytokinesis.

Trial of recombinant follicle-stimulating hormone pretreatment for gnrh-induced fertility in patients with congenital hypogonadotropic hypogonadism.

CONTEXT AND OBJECTIVE: The optimal strategy for inducing fertility in men with congenital hypogonadotropic hypogonadism (CHH) is equivocal. Albeit a biologically plausible approach, pretreatment with recombinant FSH (rFSH) before GnRH/human chorionic gonadotropin administration has not been sufficiently assessed. The objective of the study was to test this method. DESIGN AND SETTING: This was a randomized, open-label treatment protocol at an academic medical center. PATIENTS AND INTERVENTIONS: GnRH-deficient men (CHH) with prepubertal testes (<4 mL), no cryptorchidism, and no prior gonadotropin therapy were randomly assigned to either 24 months of pulsatile GnRH therapy alone (inducing endogenous LH and FSH release) or 4 months of rFSH pretreatment followed by 24 months of GnRH therapy. Patients underwent serial testicular biopsies, ultrasound assessments of testicular volume, serum hormone measurements, and seminal fluid analyses. RESULTS: rFSH treatment increased inhibin B levels into the normal range (from 29 ± 9 to 107 ± 41 pg/mL, P < .05) and doubled testicular volume (from 1.1 ± 0.2 to 2.2 ± 0.3 mL, P < .005). Histological analysis showed proliferation of both Sertoli cells (SCs) and spermatogonia, a decreased SC to germ cell ratio (from 0.74 to 0.35), and SC cytoskeletal rearrangements. With pulsatile GnRH, the groups had similar hormonal responses and exhibited significant testicular growth. All men receiving rFSH pretreatment developed sperm in their ejaculate (7 of 7 vs 4 of 6 in the GnRH-only group) and showed trends toward higher maximal sperm counts. CONCLUSIONS: rFSH pretreatment followed by GnRH is successful in inducing testicular growth and fertility in men with CHH with prepubertal testes. rFSH not only appears to maximize the SC population but also induces morphologic changes, suggesting broader developmental roles.

Prenatal detection of rare chromosomal autosomal abnormalities in Europe.

The aim of the present study was to evaluate the prenatal detection of rare chromosomal autosomal abnormalities by ultrasound (US) examination. Data were obtained from 19 congenital malformation registries from 11 European countries, between 01/07/96 and 31/12/98. A total of 664,340 births were covered and 7,758 cases with congenital malformations were recorded. Rare autosomal abnormalities were diagnosed in 114 cases (6.6%) from a total of 1,738 chromosome abnormalities. There were a wide variety of autosomal abnormalities: the most common were deletions (33 cases), duplications (32 cases), trisomies of chromosomes 8, 9, 10, 14, 15, and 16 (23 cases), and unbalanced rearrangements (19 cases). Out of these cases, 45.6% were detected prenatally by US examination due to the presence of congenital anomaly. As for the types of chromosomal anomaly, unbalanced rearrangements and deletions were the most frequently detected by US. A high percentage of cases with balanced rearrangements were associated with severe congenital anomalies. The most frequent congenital anomalies detected by US were cystic hygroma (20.6%), central nervous system defects (17.6%), cardiac defects (13.2%), and diaphragm defects (10.3%). This large series offers useful information about prenatal diagnosis by US of congenital defects associated with rare autosomal abnormalities and it provides a valuable knowledge about outcome. Fetal anomalies detected by US that were associated with rare autosomal abnormalities were significantly more frequent than those associated with common chromosomal abnormalities (45.6 vs. 34.7%). This study indicates the need to increase the detection of congenital anomalies by US.

Current cytogenetic map of the common shrew, Sorex araneus L.: localization of 7 genes and 4 microsatellites

Here we present information on the assignment of 7 genes, ACADVL, ADORA3, ATP7A, MTMR4, MYH2, HBB, TSPAN-3, and 4 common shrew microsatellites to chromosomes of the common shrew (Sorex araneus) and on the current status of its cytogenetic map. Comparative mapping data were used for the analysis of evolutionary chromosomal rearrangements in the common shrew genome.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodium vivaxmalaria cases in China that are imported across its Southeast Asia border, especially in the China-Myanmar border area (CMB). To date, little is known about the genetic diversity of P. vivaxin this region. In this paper, we report the first genome sequencing of a P. vivaxisolate (CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto 96.43% of the P. vivaxSalvador I reference strain (Sal I) genome with 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Using the de novoassembly approach, we generated 8,541 scaffolds and assembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, we identified all 295 known virgenes, which is the largest subtelomeric multigene family in malaria parasites. These results provide an important foundation for further research onP. vivaxpopulation genetics.