Conference Review REGIA, an EU project on functional genomics of transcription factors from Arabidopsis thaliana

Abstract Transcription factors (TFs) are regulatory proteins that have played a pivotal role in the evolution of eukaryotes and that also have great biotechnological potential. REGIA (REgulatory Gene Initiative in Arabidopsis) is an EU-funded project involving 29 European laboratories with the objective of determining the function of virtually all transcription factors from the model plant, Arabidopsis thaliana. REGIA involves: 1. the definition of TF gene expression patterns in Arabidopsis; 2. the identification of mutations at TF loci; 3. the ectopic expression of TFs (or derivatives) in Arabidopsis and in crop plants; 4. phenotypic analysis of the mutants and mis-expression lines, including both RNA and metabolic profiling; 5. the systematic analysis of interactions between TFs; and 6. the generation of a bioinformatics infrastructure to access and integrate all this information. We expect that this programme will establish the full biotechnological potential of plant TFs, and provide insights into hierarchies, redundancies, and interdependencies, and their evolution. The project involves the preparation of both a TF gene array for expression analysis and a normalised full length open reading frame (ORF) library of TFs in a yeast two hybrid vector; the applications of these resources should extend beyond the scope of this programme

The NER-related gene GTF2H5 predicts survival in high-grade serous ovarian cancer patients

Gayarre, Javier et al[Objective] We aimed to evaluate the prognostic and predictive value of the nucleotide excision repair-related gene GTF2H5, which is localized at the 6q24.2-26 deletion previously reported by our group to predict longer survival of high-grade serous ovarian cancer patients.[Methods] In order to test if protein levels of GTF2H5 are associated with patients' outcome, we performed GTF2H5 immunohistochemical staining in 139 high-grade serous ovarian carcinomas included in tissue microarrays. Upon stratification of cases into high- and low-GTF2H5 staining categories (> and ≤ median staining, respectively) Kaplan-Meier and logrank test were used to estimate patients’ survival and assess statistical differences. We also evaluated the association of GTF2H5 with survival at the transcriptional level by using the on-line Kaplan-Meier plotter tool, which includes gene expression and survival data of 855 high-grade serous ovarian cancer patients from 13 different datasets. Finally, we determined whether stable short hairpin RNA-mediated GTF2H5 downregulation modulates cisplatin sensitivity in the SKOV3 and COV504 cell lines by using cytotoxicity assays.[Results] Low expression of GTF2H5 was associated with longer 5-year survival of patients at the protein (hazard ratio [HR], 0.52; 95% CI, 0.29 to 0.93; p=0.024) and transcriptional level (HR, 0.80; 95% CI, 0.65 to 0.97; p=0.023) in high-grade serous ovarian cancer patients. We confirmed the association with 5-year overall survival (HR, 0.55; 95% CI, 0.38 to 0.78; p=0.0007) and also found an association with progression-free survival (HR, 0.72; 95% CI, 0.54 to 0.96; p=0.026) in a homogenous group of 388 high-stage (stages III-IV using the International Federation of Gynecology and Obstetrics staging system), optimally debulked high-grade serous ovarian cancer patients. GTF2H5- silencing induced a decrease of the half maximal inhibitory concentration upon cisplatin treatment in GTF2H5-silenced ovarian cancer cells.[Conclusion] Low levels of GTF2H5 are associated with enhanced prognosis in high-grade serous ovarian cancer patients and may contribute to cisplatin sensitization.This study was financially supported by the Fondo de Investigación Sanitaria (FIS), Instituto de Salud Carlos III (grant PI12/01319) and by FEDER funds (2014-2020 Program). MJG is recipient of a research contract from the Instituto de Salud Carlos III of the Ministerio Español de Sanidad y Consumo (Miguel Servet tipo II Program, CPII 13/00047). JG has a contract from CIBERER and MMK was a holder of a La Caixa international PhD fellowship. LPA and JP are recipients of financial support from Red Temática de Investigación Cooperativa en Cáncer (RTICC) (grants RD12/0036/0028 and RD12/0036/0064, respectively). Gayarre, Javier et al.Peer Reviewe

Ubiquitinación de proteínas nucleares en la señalización del ayuno de fosfato en plantas

Comunicaciones a congreso

Cloning and nucleotide sequence of a cDNA encoding the precursor of the barley toxin a-hordothionin

A cDNA library, prepared from developing barley endosperm, was screened for thionin recombinants. Clone pTH1 was that with the largest insert out of three identified. The longest reading frame in the 610-base-pair insert codes for a protein of 127 amino acids that includes an internal sequence of 45 amino acids, which is identical to that obtained for the α-hordothionin by direct protein sequencing. The deduced thionin sequence is preceded by a leader sequence of 18 residues and followed by a sequence that corresponds to an acidic protein of 64 amino acids. This structure supports previous evidence indicating that thionin is synthesized as a much larger precursor, which undergoes two processing steps: the cotranslational cleavage of a leader sequence and the post-translational one of a larger peptide. The size of the mRNA was estimated to be about 950 bases by Northern analysis. Thionin concentration in mature endosperm of barley cv. Bomi was about twice that of its high-lysine mutant Risç 1508. The same difference was observed in thionin mRNA in the corresponding developing endosperms, indicating that gene expression is partially blocked in the mutant at a pretranslational leve

Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare)

Thionin is a lysine-rich polypeptide (mol. wt. 5000) which is synthesized in developing barley endosperm from - 8 days to - 30 days after anthesis. Two thionin precursors (THPl and THP2) have been identified using monospecific antibodies (A-TH) prepared against the mature protein. THPl, which is the only polypeptide recognized in vitro by A-TH, is encoded by a 7.5S mRNA obtained from membrane-bound polysomes, and its alkylated derivative has an apparent mol. wt. of 17 800. THP2, which is selected together with mature thionin by A-TH among labelled proteins in vivo, differs from THPl in apparent mol. wt. (17 400 alkylated) and in electrophoretic mobility at pH 3.2. Both THPl and THP2 are competed out of the antigen-antibody complex by purified thionin. The conversión of THP2 into thionin, which has been demonstrated in a pulse-chase experiment in vivo, is a post-translational process. As it has not been possible to detect THPl in vivo it is assumed that it is converted cotranslationally into THP2. Final deposition of thionin as an extrínsic membrane protein, possibly associated with the endoplasmic reticulum, has been tentatively established on the basis of subcellular fractionation experíments

A dimeric inhibitor of insect a-amylase from barley. Cloning of the cDNA and identification of the protein

A cDNA clone, designated pUP-44, whose longest open reading frame codes for a protein that is homologous to the wheat α-amylase inhibitors, has been isolated from a library obtained from developing barley endosperm. The deduced sequence for the mature protein, which is 122 residues long, is preceded by a sequence of 30 residues which has the typical features of a signal peptide. A closely corresponding protein, designated BDAI-1, has been isolated from mature endosperm. BDAI-1 behaves as a dimer and inhibits the α-amylase from the insect Tenebrio molitor at concentrations that have no effect on salivary or pancreatic α-amylases

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts

The CM-proteins from cereal endosperm: Immunochemical relationships

The CM-proteins, which are salt-soluble proteins that can be extracted with chloroform: methanol (2: 1, v/v), seem to be present in the endosperm of all the cereal species investigated. Antibodies raised against a mixture of the barley CM-proteins (A-H) cross-reacted with wheat and rye proteins in Ouchterlony tests and a detailed study was carried out for purified wheat (CM1, CM2. CM3. CM3') and barley (CMa, CMb, CMc, CMd) CM-proteins. [35Sl-Cysteine-labelled endosperm proteins from wheat and barley were investigated by immuno-precipitation, electrophoresis and fluorography using the antibodies (A-H) and also those to a mixture of wheat CM-proteins and to CMd. There was complete antigenic identity for all the wheat proteins and CMd, some of the other proteins showed partial antigenic identity. Previously proposed genetic and biochemical relationships among these proteins were confirmed in the present study

Actualización de las recomendaciones para la determinación de biomarcadores en el carcinoma de pulmón avanzado de célula no pequeña. Consenso Nacional de la Sociedad Española de Anatomía Patológica y de la Sociedad Española de Oncología Médica

En el año 2011 se inició un proyecto conjunto entre la Sociedad Española de Oncología Médica (SEOM) y la Sociedad Española de Anatomía Patológica (SEAP) para establecer unas recomendaciones basadas en la evidencia actual con respecto a la determinación de biomarcadores en pacientes con carcinoma de pulmón de célula no pequeña (CPCNP) avanzado. La mayoría de estas recomendaciones siguen siendo válidas; sin embargo, existen nuevas evidencias que hacen necesaria la actualización de algunos aspectos. En concreto, se modifica la recomendación de qué biomarcadores hay que analizar y en qué pacientes, y se define el manejo óptimo de la muestra tumoral así como las características del material mínimo necesario para la determinación de biomarcadores. Además, se revisan las técnicas adecuadas para la determinación de las mutaciones de EGFR y el reordenamiento de ALK, y se consensúa en qué situaciones se debe llevar a cabo una re-biopsi

CM-proteins and thionins in cereals: characterization and cloning of cDNA

The study of cereal albumins and globulins has lagged somewhat behind that of the prolamins, which nave been considered as typical reserve proteins. However, these protein fractions merit closer attention for a variety of reasons. The main individual albumins and globulins are at least as abundant as many prolamin components, and it can be speculated that in a tissue, such as the cereal endosperm, which is completely consumed during germination, al 1 abundant proteins may play a reserve role. They have also a higher proportion of essential amino acids, as compared with the prolamins, and thus may be releyant in connec^ tion with the genetic alteration of overall grain composition. Finally, a high proportion of the main components of these protein fractions have inhibitory and even toxic properties, which may be related to the protection of this tissue during development and germination, and might influence the nutritional valué of the cereal products. We report here the characterization in barley of cDNA clones encoding two major groups of proteins: the CM-proteins, a family that inciudes inhibitors of trypsin and a-amylase, and the thionins, a group of high-lysine toxic polypeptides