Giving SBML support to ByoDyn

Report for the scientific stay at the California Institute of Technology during the summer of 2005. ByoDyn is a tool for simulating the dynamical expression of gene regulatory networks (GRNs) and for parameter estimation in uni- and multicellular models. A software support was carried out describing GRNs in the Systems Biology Markup Language (SBML). This one is a computer format for representing and storing computational models of biochemical pathways in software tools and databases. Supporting this format gives ByoDyn a wide range of possibilities to study the dynamical properties of multiple regulatory pathways.

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.

Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line

Background: The understanding of whole genome sequences in higher eukaryotes depends to a large degree on the reliable definition of transcription units including exon/intron structures, translated open reading frames (ORFs) and flanking untranslated regions. The best currently available chicken transcript catalog is the Ensembl build based on the mappings of a relatively small number of full length cDNAs and ESTs to the genome as well as genome sequence derived in silico gene predictions.Results: We use Long Serial Analysis of Gene Expression (LongSAGE) in bursal lymphocytes and the DT40 cell line to verify the quality and completeness of the annotated transcripts. 53.6% of the more than 38,000 unique SAGE tags (unitags) match to full length bursal cDNAs, the Ensembl transcript build or the genome sequence. The majority of all matching unitags show single matches to the genome, but no matches to the genome derived Ensembl transcript build. Nevertheless, most of these tags map close to the 3' boundaries of annotated Ensembl transcripts.Conclusions: These results suggests that rather few genes are missing in the current Ensembl chicken transcript build, but that the 3' ends of many transcripts may not have been accurately predicted. The tags with no match in the transcript sequences can now be used to improve gene predictions, pinpoint the genomic location of entirely missed transcripts and optimize the accuracy of gene finder software.

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

DnaSP, DNA polymorphism analyses by the coalescent and other methods

DnaSP is a software package for the analysis of DNA polymorphism data. Present version introduces several new modules and features which, among other options allow: (1) handling big data sets (~5 Mb per sequence); (2) conducting a large number of coalescent-based tests by Monte Carlo computer simulations; (3) extensive analyses of the genetic differentiation and gene flow among populations; (4) analysing the evolutionary pattern of preferred and unpreferred codons; (5) generating graphical outputs for an easy visualization of results. Availability: The software package, including complete documentation and examples, is freely available to academic users from: http://www.ub.es/dnasp

Thermal evolution of gene expression profiles in Drosophila subobscura

Background: Despite its pervasiveness, the genetic basis of adaptation resulting in variation directly or indirectly related to temperature (climatic) gradients is poorly understood. By using 3-fold replicated laboratory thermal stocks covering much of the physiologically tolerable temperature range for the temperate (i.e., cold tolerant) species Drosophila subobscura we have assessed whole-genome transcriptional responses after three years of thermal adaptation, when the populations had already diverged for inversion frequencies, pre-adult life history components, and morphological traits. Total mRNA from each population was compared to a reference pool mRNA in a standard, highly replicated two-colour competitive hybridization experiment using cDNA microarrays.Results: A total of 306 (6.6%) cDNA clones were identified as 'differentially expressed' (following a false discovery rate correction) after contrasting the two furthest apart thermal selection regimes (i.e., 13°C vs . 22°C), also including four previously reported candidate genes for thermotolerance in Drosophila (Hsp26, Hsp68, Fst, and Treh). On the other hand, correlated patterns of gene expression were similar in cold- and warm-adapted populations. Analysis of functional categories defined by the Gene Ontology project point to an overrepresentation of genes involved in carbohydrate metabolism, nucleic acids metabolism and regulation of transcription among other categories. Although the location of differently expressed genes was approximately at random with respect to chromosomes, a physical mapping of 88 probes to the polytene chromosomes of D. subobscura has shown that a larger than expected number mapped inside inverted chromosomal segments.Conclusion: Our data suggest that a sizeable number of genes appear to be involved in thermal adaptation in Drosophila, with a substantial fraction implicated in metabolism. This apparently illustrates the formidable challenge to understanding the adaptive evolution of complex trait variation. Furthermore, some clustering of genes within inverted chromosomal sections was detected. Disentangling the effects of inversions will be obviously required in any future approach if we want to identify the relevant candidate genes.

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.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines.

Minimization of biosynthetic costs in adaptive gene expression responses of yeast to environmental changes

Yeast successfully adapts to an environmental stress by altering physiology and fine-tuning metabolism. This fine-tuning is achieved through regulation of both gene expression and protein activity, and it is shaped by various physiological requirements. Such requirements impose a sustained evolutionary pressure that ultimately selects a specific gene expression profile, generating a suitable adaptive response to each environmental change. Although some of the requirements are stress specific, it is likely that others are common to various situations. We hypothesize that an evolutionary pressure for minimizing biosynthetic costs might have left signatures in the physicochemical properties of proteins whose gene expression is fine-tuned during adaptive responses. To test this hypothesis we analyze existing yeast transcriptomic data for such responses and investigate how several properties of proteins correlate to changes in gene expression. Our results reveal signatures that are consistent with a selective pressure for economy in protein synthesis during adaptive response of yeast to various types of stress. These signatures differentiate two groups of adaptive responses with respect to how cells manage expenditure in protein biosynthesis. In one group, significant trends towards downregulation of large proteins and upregulation of small ones are observed. In the other group we find no such trends. These results are consistent with resource limitation being important in the evolution of the first group of stress responses.

Use of physiological constraints to identify quantitative design principles for gene expression in yeast adaptation to heat shock

Background: Understanding the relationship between gene expression changes, enzyme activity shifts, and the corresponding physiological adaptive response of organisms to environmental cues is crucial in explaining how cells cope with stress. For example, adaptation of yeast to heat shock involves a characteristic profile of changes to the expression levels of genes coding for enzymes of the glycolytic pathway and some of its branches. The experimental determination of changes in gene expression profiles provides a descriptive picture of the adaptive response to stress. However, it does not explain why a particular profile is selected for any given response. Results: We used mathematical models and analysis of in silico gene expression profiles (GEPs) to understand how changes in gene expression correlate to an efficient response of yeast cells to heat shock. An exhaustive set of GEPs, matched with the corresponding set of enzyme activities, was simulated and analyzed. The effectiveness of each profile in the response to heat shock was evaluated according to relevant physiological and functional criteria. The small subset of GEPs that lead to effective physiological responses after heat shock was identified as the result of the tuning of several evolutionary criteria. The experimentally observed transcriptional changes in response to heat shock belong to this set and can be explained by quantitative design principles at the physiological level that ultimately constrain changes in gene expression. Conclusion: Our theoretical approach suggests a method for understanding the combined effect of changes in the expression of multiple genes on the activity of metabolic pathways, and consequently on the adaptation of cellular metabolism to heat shock. This method identifies quantitative design principles that facilitate understating the response of the cell to stress.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

Background: The zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians. Results: In this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern. Conclusions: The absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

Background: The zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians. Results: In this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern. Conclusions: The absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations.