Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions

Background: The trithorax group (trxG) genes absent, small or homeotic discs 1 (ash1) and 2 (ash2) were isolated in a screen for mutants with abnormal imaginal discs. Mutations in either gene cause homeotic transformations but Hox genes are not their only targets. Although analysis of double mutants revealed that ash2 and ash1 mutations enhance each other's phenotypes, suggesting they are functionally related, it was shown that these proteins are subunits of distinct complexes.Results: The analysis of wing imaginal disc transcriptomes from ash2 and ash1 mutants showed that they are highly similar. Functional annotation of regulated genes using Gene Ontology allowed identification of severely affected groups of genes that could be correlated to the wing phenotypes observed. Comparison of the differentially expressed genes with those from other genome-wide analyses revealed similarities between ASH2 and Sin3A, suggesting a putative functional relationship. Coimmunoprecipitation studies and immunolocalization on polytene chromosomes demonstrated that ASH2 and Sin3A interact with HCF (host-cell factor). The results of nucleosome western blots and clonal analysis indicated that ASH2 is necessary for trimethylation of the Lys4 on histone 3 (H3K4).Conclusion: The similarity between the transcriptomes of ash2 and ash1 mutants supports a model in which the two genes act together to maintain stable states of transcription. Like in humans, both ASH2 and Sin3A bind HCF. Finally, the reduction of H3K4 trimethylation in ash2 mutants is the first evidence in Drosophila regarding the molecular function of this trxG gene.

Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes

BACKGROUND: Understanding how alternative phenotypes arise from the same genome is a major challenge in modern biology. Eusociality in insects requires the evolution of two alternative phenotypes - workers, who sacrifice personal reproduction, and queens, who realize that reproduction. Extensive work on honeybees and ants has revealed the molecular basis of derived queen and worker phenotypes in highly eusocial lineages, but we lack equivalent deep-level analyses of wasps and of primitively eusocial species, the latter of which can reveal how phenotypic decoupling first occurs in the early stages of eusocial evolution. RESULTS: We sequenced 20 Gbp of transcriptomes derived from brains of different behavioral castes of the primitively eusocial tropical paper wasp Polistes canadensis. Surprisingly, 75% of the 2,442 genes differentially expressed between phenotypes were novel, having no significant homology with described sequences. Moreover, 90% of these novel genes were significantly upregulated in workers relative to queens. Differential expression of novel genes in the early stages of sociality may be important in facilitating the evolution of worker behavioral complexity in eusocial evolution. We also found surprisingly low correlation in the identity and direction of expression of differentially expressed genes across similar phenotypes in different social lineages, supporting the idea that social evolution in different lineages requires substantial de novo rewiring of molecular pathways. CONCLUSIONS: These genomic resources for aculeate wasps and first transcriptome-wide insights into the origin of castes bring us closer to a more general understanding of eusocial evolution and how phenotypic diversity arises from the same genome.

Muscle transcriptomic profiles in pigs with divergent phenotypes for fatness traits

BACKGROUND: Selection for increasing intramuscular fat content would definitively improve the palatability and juiciness of pig meat as well as the sensorial and organoleptic properties of cured products. However, evidences obtained in human and model organisms suggest that high levels of intramuscular fat might alter muscle lipid and carbohydrate metabolism. We have analysed this issue by determining the transcriptomic profiles of Duroc pigs with divergent phenotypes for 13 fatness traits. The strong aptitude of Duroc pigs to have high levels of intramuscular fat makes them a valuable model to analyse the mechanisms that regulate muscle lipid metabolism, an issue with evident implications in the elucidation of the genetic basis of human metabolic diseases such as obesity and insulin resistance. RESULTS: Muscle gene expression profiles of 68 Duroc pigs belonging to two groups (HIGH and LOW) with extreme phenotypes for lipid deposition and composition traits have been analysed. Microarray and quantitative PCR analysis showed that genes related to fatty acid uptake, lipogenesis and triacylglycerol synthesis were upregulated in the muscle tissue of HIGH pigs, which are fatter and have higher amounts of intramuscular fat than their LOW counterparts. Paradoxically, lipolytic genes also showed increased mRNA levels in the HIGH group suggesting the existence of a cycle where triacylglycerols are continuously synthesized and degraded. Several genes related to the insulin-signalling pathway, that is usually impaired in obese humans, were also upregulated. Finally, genes related to antigen-processing and presentation were downregulated in the HIGH group. CONCLUSION: Our data suggest that selection for increasing intramuscular fat content in pigs would lead to a shift but not a disruption of the metabolic homeostasis of muscle cells. Future studies on the post-translational changes affecting protein activity or expression as well as information about protein location within the cell would be needed to to elucidate the effects of lipid deposition on muscle metabolism in pigs.

White eye phenotypes and their genetic analysis

An interesting case for undergraduate students of general Genetics is to consider that different genes can produce the same or similar phenotypes. We present here an experiment to discover that the same phenotype could be produced by different genes, and then, to carry out the genetic analysis of these genes. For this laboratory study we have used the following Drosophila melanogaster strains: white (white eyes) and scarlet-brown (white eyes).

White eye phenotypes and their genetic analysis

An interesting case for undergraduate students of general Genetics is to consider that different genes can produce the same or similar phenotypes. We present here an experiment to discover that the same phenotype could be produced by different genes, and then, to carry out the genetic analysis of these genes. For this laboratory study we have used the following Drosophila melanogaster strains: white (white eyes) and scarlet-brown (white eyes).

Desarrollo de una colección de Líneas Casi Isogénicas (NIL) para el estudio de caracteres de interés agronómico en especies del género Fragaria

Fragaria vesca posee varias características que la hacen interesante como especie modelo en la familia Rosaceae. Además, su naturaleza diploide permite sortear la complejidad genética de la fresa cultivada. Considerando que los genomas de las especies diploides y octoploides de Fragaria mantienen una relación de sintenia y colinearidad, estamos desarrollando una colección de Líneas Casi Isogénicas (NIL) en Fragaria diploide, usando Fragaria bucharica, una variedad asiática, como donante de introgresiones y Fragaria vesca var. Reine des Vallées, una variedad francesa comúnmente cultivada en España para usos industriales, como parental recurrente. Para obtener introgresiones de F. bucharica en un fondo genético homogéneo de F. vesca, se desarrolló un retrocruzamiento y se obtuvo una población BC1 que fue analizada para la presencia de alelos de F. bucharica a lo largo de los 7 grupos de ligamiento (LG) del mapa de referencia de Fragaria. Los individuos con bajo número de introgresiones de gran tamaño, que en conjunto abarcaron todo el genoma de F. bucharica, fueron seleccionados y retrocruzados con el parental recurrente. La descendencia fue analizada para los loci segregantes y los individuos BC2 con 1 ó 2 introgresiones fueron seleccionados como líneas donadoras de introgresiones de las NIL. Hasta el momento se han descrito tres fenotipos dominantes diferentes bajo condiciones de invernadero en varias NIL heterozigóticas: Las plantas con introgresiones en LG2 presentan estolones. Las plantas con introgresiones en LG1 y LG3 presentan floración temprana. Las plantas con introgresiones en LG6 presentan floración estacional. Actualmente se está trabajando en la selección y caracterización de introgresiones de pequeño tamaño mediante la autofecundación de las líneas seleccionadas. La caracterización fenotípica de los recombinantes seleccionados permitirá localizar y estimar los patrones de herencia de los genes implicados en los caracteres descritos, además de la identificación de nuevos caracteres recesivos que aparecerán en homozigosis.

Estudio de los mecanismos terapéuticos en la inducción de tolerancia immunológica en un modelo animal de esclerosis múltiple

En una serie de estudios previos demostramos que la infusión de células de médula ósea (MO) modificadas genéticamente para la expresión del autoantígeno MOG40-55 en ausencia de mieloablación inducía tolerancia antígenoespecífica en un modelo murino de esclerosis múltiple. También observamos que este efecto terapéutico no requería injerto hematopoyético. Nos propusimos estudiar si el efecto tolerogénico está inducido por una subpoblación de células generadas durante la transducción de la MO y el papel de las células T reguladoras en la inducción de la tolerancia. Las células de MO fueron cultivadas y transducidas usando medio complementado con 20% FCS y medios condicionados como fuente de stem cell factor (SCF) e IL-3 murinos. Las diferentes poblaciones celulares se separaron por citometría de flujo y se analizó la capacidad supresora de las poblaciones candidatas. Por otro lado se analizó la presencia de células T reguladoras en bazo y SNC de los ratones recuperados después de la infusión de células de MO transducidas. A los cinco días de cultivo, la mayoría de células presentaban fenotipo mieloide (Mac-1+Gr-1low/-:31,9+-10,2%; Mac-1+Gr-1high:26,0+-3,3%). Ambos fenotipos se corresponden con dos subpoblaciones de células mieloides supresoras (MDSC, tipo monocí¬tico y granulocítico respectivamente) descritas recientemente. Se estudió la capacidad de ambas poblaciones para suprimir la respuesta proliferativa específica de esplenocitos frente a MOG40-55 in vitro, observando una mayor capacidad de supresión de las MDSC monocíticas, que se correspondí¬a con niveles significativamente superiores de actividad de las enzimas arginasa-1 y sintasa de óxido nítrico (ambos mecanismos supresores característicos de las MDSC). A los 7 días del tratamiento no se observaron diferencias significativas en el porcentaje de células T reguladoras (Treg y Tr1) entre el grupo tratado (liM) y los grupos de control.

Obtenció de jerarquies d'estats cel·lulars via web

Hi ha diversos mètodes d'anàlisi que duen a terme una agrupació global de la sèries de mostres de microarrays, com SelfOrganizing Maps, o que realitzen agrupaments locals tenint en compte només un subconjunt de gens coexpressats, com Biclustering, entre d'altres. En aquest projecte s'ha desenvolupat una aplicació web: el PCOPSamplecl, és una eina que pertany als mètodes d'agrupació (clustering) local, que no busca subconjunts de gens coexpresats (anàlisi de relacions linials), si no parelles de gens que davant canvis fenotípics, la seva relació d'expressió pateix fluctuacions. El resultats del PCOPSamplecl seràn les diferents distribucions finals de clusters i les parelles de gens involucrades en aquests canvis fenotípics. Aquestes parelles de gens podràn ser estudiades per trobar la causa i efecte del canvi fenotípic. A més, l'eina facilita l'estudi de les dependències entre les diferents distribucions de clusters que proporciona l'aplicació per poder estudiar la intersecció entre clusters o l'aparició de subclusters (2 clusters d'una mateixa agrupació de clusters poden ser subclusters d'altres clusters de diferents distribucions de clusters). L'eina és disponible al servidor: http://revolutionresearch.uab.es/

Teleost fish larvae adapt to dietary arachidonic acid supply through modulation of the expression of lipid metabolism and stress response genes

Dietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0·4, 0·8, 1·5 and 3·0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPARα, phospholipase A2, cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0·4 %. Nonetheless, despite practical suppression of phospholipase A2 transcription, higher leukotriene B4 levels were detected in larvae fed 3·0 % ARA, whereas a similar trend was observed regarding PGE2 production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.

Estudi dels factors mol•leculars i cel•lulars implicats en les alteracions corticals de la Síndrome de down

Investigación producida a partir de una estancia en en el Instituto de Neurociencias de la Universidad Miguel Hernández entre enero y mayo del 2007. El SD o trisomía del cromosoma 21 es la aneuploidía cromosómica más frecuente y constituye la principal causa de retraso mental. Las cuestiones que aún son objeto de debate en el SD son: 1) si pueden existir, entre los genes triplicados, algunos que contribuyan de forma más importante a algunos de los fenotipos observables en SD y, 2) hasta qué punto los fenotipos observados derivan de alteraciones del neurodesarrollo o de alteraciones funcionales en el adulto. Con el fin de abordar esta cuestión nos hemos centrado en las alteraciones cognitivas del SD y hemos realizado la caracterización del papel de Dyrk1A en el desarrollo de una estructura clave para esta función: la corteza cerebral. Los resultados obtenidos muestran que la sobrexpresión de Dyrk1A produce un desajuste proliferativo dando lugar a un retraso en la formación de la subplaca, con consecuencias en la laminación de la placa cortical. Las alteraciones en la corticogénesis van a tener consecuencias en el establecimiento de la conectividad tálamo-cortical que se encuentra marcadamente retrasada. En el hipocampo, los ratones transgénicos mostraron una reducción del grosor de las capas. Estos resultados pueden ser relevantes para el SD, puesto que es similar a lo observado en fetos SD.

Implicación del fenotipo de cáncer de mama en pacientes con carcinomatosis leptomeníngea.

Aquest estudi permet conèixer les implicacions dels diferents subtipus de cáncer de mama: luminal A, luminal B, HER2+, triple negatiu en el desenvolupament y pronostic de la carcinomatosi leptomeníngea (CL). Es van identificar 38 pacients, major proporció: luminal B y HER2+, 53% va rebre quimioteràpia sistémica (QTS). La mitjana de supervivencia post CL: 2,6 messos. A l’anàlisis univariat: ECOG de 0-2 y tractament amb QT van ser variables pronòstiques i al multivariant nomès QTS. En conclussió el subtipus de cáncer de mama influiex en el temps d’aparició de la CL, no afectant la supervivencia desprès del diagnóstic.

Evolució clínica després de la retirada d’infliximab en pacients amb MII en remissió clínica i endoscòpica sota tractament combinat amb tiopurines

Objectius: Avaluar la retirada d’infliximab en pacients amb MII, en remissió mantinguda i curació mucosa sota tractament combinat. Mètodes: Pacients amb MC luminal o CU sota tractament combinat (infliximab i azatioprina) durant mínim un any, als quals se’ls retira l’infliximab per remissió clínica, biològica i endoscòpica. Resultats: S’inclogueren 26 pacients. Un 73% es van mantenir en remissió. La recidiva no s’associà al fracàs previ d’immunomoduladors. A les recidives se’ls va reiniciar infliximab amb bona resposta. Cap factor va associar-se a la recidiva. Conclusions: La retirada de l’infliximab en aquests pacients, s’acompanya d’una baixa probabilitat de recidiva a mig termini.

GDAP1-related autosomal dominant Charcot-Marie-Tooth disease: phenotypical and MRI features

El present estudi es basa en la descripció de quatre famílies portadores d’una mateixa mutació puntual (p.R120W) en el gen GDAP1 que segreguen d’una manera autosòmica dominant. Les trobades més rellevants foren:  Els individus afectats varen tindre un començament més tardà i un fenotipus més lleu que les mutacions recessives del gen GDAP-1, però amb una gran variabilitat clínica.  La Resonància Magnètica Muscular va demostrar una afectació selectiva de la musculatura intrínseca del peu i la part distal del panxell. El compartiment posterior superficial de la musculatura del panxell estava més afectat que el compartiment anterolateral.

Characterization and evolution of the novel gene family FAM90A in primates originated by multiple duplication and rearrangement events

Genomic plasticity of human chromosome 8p23.1 region is highly influenced by two groups of complex segmental duplications (SDs), termed REPD and REPP, that mediate different kinds of rearrangements. Part of the difficulty to explain the wide range of phenotypes associated with 8p23.1 rearrangements is that REPP and REPD are not yet well characterized, probably due to their polymorphic status. Here, we describe a novel primate-specific gene family, named FAM90A (family with sequence similarity 90), found within these SDs. According to the current human reference sequence assembly, the FAM90A family includes 24 members along 8p23.1 region plus a single member on chromosome 12p13.31, showing copy number variation (CNV) between individuals. These genes can be classified into subfamilies I and II, which differ in their upstream and 5′-untranslated region sequences, but both share the same open reading frame and are ubiquitously expressed. Sequence analysis and comparative fluorescence in situ hybridization studies showed that FAM90A subfamily II suffered a big expansion in the hominoid lineage, whereas subfamily I members were likely generated sometime around the divergence of orangutan and African great apes by a fusion process. In addition, the analysis of the Ka/Ks ratios provides evidence of functional constraint of some FAM90A genes in all species. The characterization of the FAM90A gene family contributes to a better understanding of the structural polymorphism of the human 8p23.1 region and constitutes a good example of how SDs, CNVs and rearrangements within themselves can promote the formation of new gene sequences with potential functional consequences.

Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.