Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

<p>Abstract</p> <p>Background</p> <p>The sequencing of the <it>D.melanogaster </it>genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of <it>Drosophila </it>protein-coding genes contain one or more alternative exons. A recent transcription map of the <it>Drosophila </it>embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of <it>Drosophila </it>transcriptome.</p> <p>Results</p> <p>Bioinformatic analysis of 1,303 <it>Drosophila </it>ORESTES clusters identified 68 sequences derived from unannotated regions in the current <it>Drosophila </it>genome version (4.3). Of these, a set of 38 was analysed by polyA⁺northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The <it>SP212 </it>gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this <it>locus </it>is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury.</p> <p>Conclusion</p> <p>Using the ORESTES methodology we identified 17 novel exons from low abundance <it>Drosophila </it>transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.</p

Influence of Milk-Feeding Type and Genetic Risk of Developing Coeliac Disease on Intestinal Microbiota of Infants: The PROFICEL Study

Interactions between environmental factors and predisposing genes could be involved in the development of coeliac disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated measures. In the whole population, breast-feeding promoted colonization of C. leptum group, B. longum and B. breve, while formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover, increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B. fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants' gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could partly explain the protective role attributed to breast milk in this disorder

Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE

The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function.

The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding

Investigación educativa y práctica escolar : programas de acción en el aula

Se trata de un documento que aporta diferentes perspectivas de la investigación educativa y presenta modelos y programas de práctica educativa. Los temas son: integración entre la praxia educativa y l ateoría pedagógica, la reflexibidad como objetivo educativo, hiperactividad y atención en el aula y la prevención del fracaso escolar.MadridBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín 5 -3 Planta; 28014 Madrid; Tel. +34917748000; [email protected]

Tras las huellas de los asentamientos asturienses. Intervenciones arqueológicas en El Alloru, la Sierra Plana de la Borbolla y otros sitios mesolíticos del oriente de Asturias

A pesar de la gran abundancia de yacimientos atribuibles a este período (unos 130 sólo en el oriente de Asturias) (Fano 1998), la información disponible para el estudio de las áreas de hábitat de las sociedades relacionadas con el complejo arqueológico costero conocido como asturiense (viii-vi milenios cal BC) es muy escasa. Es éste un problema que ya preocupó al propio conde de la Vega del Sella, quien abogó por la existencia de espacios habitacionales al aire libre próximos a las cavidades (Vega del Sella 1923), pues éstas no habrían podido ser utilizadas para esta finalidad, dado que en algunos casos los concheros las habrían colmatado completamente. Años más tarde, G. A. Clark trató de contrastar esta hipótesis excavando frente a la boca de la cueva de La Riera (Clark, 1974), donde creyó haber documentado indicios de ocupaciones asturienses al aire libre. No obstante, estos resultados fueron cuestionados por revisiones posteriores (González Morales, 1982, Arias, 1991). Por el contrario, en la base del conchero de Mazaculos II se encontraron indicios de un asentamiento en la entrada de la cueva (González Morales, 1982), lo que volvió a poner sobre la mesa la posibilidad de la existencia de asentamientos hipogeos en el Mesolítico Cantábrico, planteamiento que fue considerado en trabajos posteriores, al evaluar la habitabilidad de las cavidades con conchero (Arias, 1991). Observaciones efectuadas en los años 1990, como la relativa a la topografía del terreno inmediato a las cuevas, no siempre favorable a la existencia de ocupaciones exteriores, corroboraron esa posibilidad, pero sin negar la probable presencia de asentamientos al aire libre (Fano, 1998).info:eu-repo/semantics/publishedVersio

Plan of the intended New Port, Adelaide, South Australia as designed by his Excellency Col. G. Gawler, K. H. &c. &c. &c. divided into sections of 80 and 134 acres [cartographic material]

Cadastral map of Port Adelaide when it was first settled as a port with notes on vegetation and landform. Subdivided land has been referenced to major landholders as well as annotations on soundings and shoreline, and includes refences on sailing directions. An inset of Encounter Bay shows numbered land blocks. Relief shown by hachures and bathymetric soundings.; Also available in an electronic version via the Internet at: http://nla.gov.au/nla.map-rm1131. Insets: Plan of sections at Encounter Bay. Scale [1:63,360] -- Chart of the outlet of Lake Alexandrina / by J.W.L. Pullen, Esqr. Comr. R.N., 1839. Scale not given