Rapid evolution of cancer/testis genes on the X chromosome.

BACKGROUND: Cancer/testis (CT) genes are normally expressed only in germ cells, but can be activated in the cancer state. This unusual property, together with the finding that many CT proteins elicit an antigenic response in cancer patients, has established a role for this class of genes as targets in immunotherapy regimes. Many families of CT genes have been identified in the human genome, but their biological function for the most part remains unclear. While it has been shown that some CT genes are under diversifying selection, this question has not been addressed before for the class as a whole. RESULTS: To shed more light on this interesting group of genes, we exploited the generation of a draft chimpanzee (Pan troglodytes) genomic sequence to examine CT genes in an organism that is closely related to human, and generated a high-quality, manually curated set of human:chimpanzee CT gene alignments. We find that the chimpanzee genome contains homologues to most of the human CT families, and that the genes are located on the same chromosome and at a similar copy number to those in human. Comparison of putative human:chimpanzee orthologues indicates that CT genes located on chromosome X are diverging faster and are undergoing stronger diversifying selection than those on the autosomes or than a set of control genes on either chromosome X or autosomes. CONCLUSION: Given their high level of diversifying selection, we suggest that CT genes are primarily responsible for the observed rapid evolution of protein-coding genes on the X chromosome.

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders.

Predator-prey chemical warfare determines the expression of biocontrol genes by rhizosphere-associated Pseudomonas fluorescens.

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.

Evaluation of virulence factors in environmental isolates of Vibrio species

Strains of Vibrio parahaemolyticus, Vibrio fluvialis and Vibrio mimicus isolated from seafood and seawater were examined for characteristics related to infectivity, such as enzymatic activity and animal assays. All strains hydrolysed DNA, starch, gelatin and chitin. Variable results were obtained with the haemolysin, chondroitin, collagen, elastin and lecithin tests. Production of thermostable direct haemolysin by V. parahaemolyticus was detected in 7.1% strains derived from seafood and 2%from seawater. In the animal assays, strains of V. fluvialis showed positive results at skin PF (75%), mouse lethality (100%), but no fluid accumulation in the suckling mice model was noted. Concerning V. mimicus, results showed skin PF (100%), mouse lethality (100%) and fluid accumulation in suckling mice (66.6%).

Ribonucleotide reductase genes of Bacillus prophages: a refuge to introns and intein coding sequences.

The ribonucleotide reductase gene tandem bnrdE/bnrdF in SPbeta-related prophages of different Bacillus spp. isolates presents different configurations of intervening sequences, comprising one to three of six non-homologous splicing elements. Insertion sites of group I introns and intein DNA are clustered in three relatively short segments encoding functionally important domains of the ribonucleotide reductase. Comparison of the bnrdE homologs reveals mutual exclusion of a group I intron and an intein coding sequence flanking the codon that specifies a conserved cysteine. In vivo splicing was demonstrated for all introns. However, for two of them a part of the mRNA precursor molecules remains unspliced. Intergenic bnrdE-bnrdF regions are unexpectedly long, comprising between 238 and 541 nt. The longest encodes a putative polypeptide related to HNH homing endonucleases.

Study of Staphylococcus aureus pathogenic genes by transfer and expression in the less virulent organism Streptococcus gordonii.

Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.

Bioinformática: interfaz web para estudiar el efecto de diferentes condiciones sobre la expresión de los genes

El trabajo realizado se divide en dos bloques bien diferenciados, ambos relacionados con el análisis de microarrays. El primer bloque consiste en agrupar las condiciones muestrales de todos los genes en grupos o clústers. Estas agrupaciones se obtienen al aplicar directamente sobre la microarray los siguientes algoritmos de agrupación: SOM,PAM,SOTA,HC y al aplicar sobre la microarray escalada con PC y MDS los siguientes algoritmos: SOM,PAM,SOTA,HC y K-MEANS. El segundo bloque consiste en realizar una búsqueda de genes basada en los intervalos de confianza de cada clúster de la agrupación activa. Las condiciones de búsqueda ajustadas por el usuario se validan para cada clúster respecto el valor basal 0 y respecto el resto de clústers, para estas validaciones se usan los intervalos de confianza. Estos dos bloques se integran en una aplicación web ya existente, el applet PCOPGene, alojada en el servidor: http://revolutionresearch.uab.es.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

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.

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.

Courtship song genes and speciation in sand flies

Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) is a vector of visceral leishmaniasis in the Americas and it might represent a complex of sibling species. Reproductive isolation between closely related species often involves differences in courtship behaviour. cacophony (cac) and period (per) are two Drosophila genes that control features of the "lovesong" males produce during courtship that has been implicated in the sexual isolation between closely related species. We are using gene fragments from L. longipalpis' homologues of these two genes to study the speciation process in this putative species complex.

Genes as leaders and followers in evolution.

A major question for the study of phenotypic evolution is whether intra- and interspecific diversity originates directly from genetic variation, or instead, as plastic responses to environmental influences initially, followed later by genetic change. In species with discrete alternative phenotypes, evolutionary sequences can be inferred from transitions between environmental and genetic phenotype control, and from losses of phenotypic alternatives. From the available evidence, sequences appear equally probable to start with genetic polymorphism as with polyphenism, with a possible dominance of one or the other for specific trait types. We argue in this review that to evaluate the prevalence of each route, an investigation of both genetic and environmental cues for phenotype determination in several related rather than in isolated species is required.

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Promoter and transcription analysis of penicillin-binding protein genes in Streptococcus gordonii.

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.

The development of targeted treatment strategies adapted to individual patients requires identification of the different tumor classes according to their biology and prognosis. We focus here on the molecular aspects underlying these differences, in terms of sets of genes that control pathogenesis of the different subtypes of astrocytic glioma. By performing cDNA-array analysis of 53 patient biopsies, comprising low-grade astrocytoma, secondary glioblastoma (respective recurrent high-grade tumors), and newly diagnosed primary glioblastoma, we demonstrate that human gliomas can be differentiated according to their gene expression. We found that low-grade astrocytoma have the most specific and similar expression profiles, whereas primary glioblastoma exhibit much larger variation between tumors. Secondary glioblastoma display features of both other groups. We identified several sets of genes with relatively highly correlated expression within groups that: (a). can be associated with specific biological functions; and (b). effectively differentiate tumor class. One prominent gene cluster discriminating primary versus nonprimary glioblastoma comprises mostly genes involved in angiogenesis, including VEGF fms-related tyrosine kinase 1 but also IGFBP2, that has not yet been directly linked to angiogenesis. In situ hybridization demonstrating coexpression of IGFBP2 and VEGF in pseudopalisading cells surrounding tumor necrosis provided further evidence for a possible involvement of IGFBP2 in angiogenesis. The separating groups of genes were found by the unsupervised coupled two-way clustering method, and their classification power was validated by a supervised construction of a nearly perfect glioma classifier.