Gene regulation

A typical character of plants is the production and storage of usually complex mixtures of secondary metabolites (SM). The main function of secondary metabolites is defense against herbivores and microbes; some SM are signal compounds to attract pollinating and seed dispersing animals or play a role in the symbiotic relationships with plants and microbes. The distribution of SM in the plant kingdom shows an interesting pattern. A specific SM is often confined to a particular systematic unit, but isolated occurrences can occur in widely unrelated taxonomic groups. This review tries to explain the patchy occurrence of SM in plants. It could be due to convergent evolution, but evidence is provided that the genes that encode the biosynthesis of SM appear to have a much wider distribution than the actual secondary metabolite. It seems to be rather a matter of differential gene regulation whether a pathway is active and expressed in a given taxonomic unit or not. It is speculated that the...

Members of the abscisic acid-responsive element binding protein (AREB)/abscisic acid-responsive element binding factor (ABF) subfamily of basic leucine zipper (bZIP) transcription factors have been implicated in abscisic acid (ABA) and abiotic stress responses in plants. Here we describe two members identified in cultivated tomato (Solanum lycopersicum), named SlAREB1 and SlAREB2. Expression of SlAREB1 and SlAREB2 is induced by drought and salinity in both leaves and root tissues, although that of SlAREB1 was more affected. In stress assays, SlAREB1-overexpressing transgenic tomato plants showed increased tolerance to salt and water stress compared to wild-type and SlAREB1-down-regulating transgenic plants, as assessed by physiological parameters such as relative water content (RWC), chlorophyll fluorescence and damage by lipoperoxidation. In order to identify SlAREB1 target genes responsible for the enhanced tolerance, microarray and cDNA-amplified fragment length polymorphism (AFLP) analyses were performed. Genes encoding oxidative stress-related proteins, lipid transfer proteins (LTPs), transcription regulators and late embryogenesis abundant proteins were found among the up-regulated genes in SlAREB1-overexpressing lines, especially in aerial tissue. Notably, several genes encoding defence proteins associated with responses to biotic stress (e.g. pathogenesis-related proteins, protease inhibitors, and catabolic enzymes) were also up-regulated by SlAREB1 overexpression, suggesting that this bZIP transcription factor is involved in ABA signals that participate in abiotic stress and possibly in response to pathogens.

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and f lagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Gene regulatory network is a collection of DNA which interact with each other and with other matter in the cell. The lac operon is an example of a relatively simple genetic network and is one of the best-studied structures in the Escherichia coli bacteria. In this work we consider a deterministic model of the lac operon with a noise term, representing the stochastic nature of the regulation. The model is written in terms of a system of simultaneous first order differential equations with delays. We investigate an analytical and numerical solution and analyse the range of values for the parameters corresponding to a stable solution.

The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, L-tryptophan (Trp), at their intracellular concentrations. The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions. TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA. We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR. For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR. The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer. This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression.

In this review, the pathways involving small RNAs are provided followed by a new and updated network that illustrates their interplay with diverse cellular mechanisms in Caenorhabditis elegans. The RNA silencing pathways are now recognized as key factors that connect together the many variations in biological processes, including transcriptional gene regulation, post-transcriptional gene silencing, translational gene silencing, apoptosis, meiosis, and antiviral defense. The utilization of small RNAs represents a specific, energy conserving, and fast mechanism of gene regulation via a core system known as RNA interference.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays containing 1500 cDNAs to profile transcripts regulated by androgens in prostate cancer cells and identified the serine protease TMPRSS2 as a gene exhibiting increased expression upon exposure to androgens. The TMPRSS2 gene is located on chromosome 21 and contains four distinct domains, including a transmembrane region, indicating that it is expressed on the cell surface. Northern analysis demonstrated that TMPRSS2 is highly expressed in prostate epithelium relative to other normal human tissues. In situ hybridization of normal and malignant prostate tissues localizes TMMPRSS2 expression to prostate basal cells and to prostate carcinoma. These results suggest that TMPRSS2 may play a role in prostate carcinogenesis and should be investigated as a diagno...

Lampreys are a jawless vertebrate species belonging to an ancient vertebrate lineage that diverged from a common ancestor with humans ~500 million years ago. The sea lamprey (Petromyzon marinus) has a filter feeding ammocoete larval stage that metamorphoses into a parasitic adult, feeding both on teleost and elasmobranch fish. Lampreys are a valuable comparative model species for vertebrate immunity and physiology due to their unique phylogenetic position, unusual adaptive immune system, and physiological adaptions such as tolerance to salinity changes and urea. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which catalyses posttranslational deimination/citrullination in target proteins, enabling proteins to gain new functions (moonlighting). The identification of deiminated protein targets in species across phylogeny may provide novel insights into post-translational regulation of physiological and pathophysiological processes. Extracellular vesicles (EVs) are membrane vesicles released from cells that carry cargos of small molecules and proteins for cellular communication, involved in both normal and pathological processes. The current study identified deimination signatures in proteins of both total plasma and plasma-EVs in sea lamprey and furthermore reports the first characterisation of plasma-EVs in lamprey. EVs were poly-dispersed in the size range of 40-500 nm, similar to what is observed in other taxa, positive for CD63 and Flotillin-1. Plasma-EV morphology was confirmed by transmission electron microscopy. Assessment of deimination/citrullination signatures in lamprey plasma and plasma-EVs, revealed 72 deimination target proteins involved in immunity, metabolism and gene regulation in whole plasma, and 37 target proteins in EVs, whereof 24 were shared targets. Furthermore, the presence of deiminated histone H3, indicative of gene-regulatory mechanisms and also a marker of neutrophil extracellular trap formation (NETosis), was confirmed in lamprey plasma. Functional protein network analysis revealed some differences in KEGG and GO pathways of deiminated proteins in whole plasma compared with plasma-EVs. For example, while common STRING network clusters in plasma and plasma-EVs included Peptide chain elongation, Viral mRNA translation, Glycolysis and gluconeogenesis, STRING network clusters specific for EVs only included: Cellular response to heat stress, Muscle protein and striated muscle thin filament, Nucleosome, Protein processing in endoplasmic reticulum, Nucleosome and histone deacetylase complex. STRING network clusters specific for plasma were: Adipokinetic hormone receptor activity, Fibrinogen alpha/beta chain family, peptidase S1A, Glutathione synthesis and recycling-arginine, Fructose 1,6-bisphosphate metabolic process, Carbon metabolism and lactate dehydrogenase activity, Post-translational protein phosphorylation, Regulation of insulin-like growth factor transport and clotting cascade. Overall, for the EV citrullinome, five STRING network clusters, 10 KEGG pathways, 15 molecular GO pathways and 29 Reactome pathways were identified, compared with nine STRING network clusters, six KEGG pathways, two Molecular GO pathways and one Reactome pathway specific for whole plasma; while further pathways were shared. The reported findings indicate that major pathways relevant for immunity and metabolism are targets of deimination in lamprey plasma and plasma-EVs, with some differences, and may help elucidating roles for the conserved PAD enzyme family in regulation of immune and metabolic function throughout phylogeny.

There are several methods to introduce purified DNA into mammalian cells. These include microinjection into the nuclei of the recipient cells and complexing the DNA with facilitating agents such as calcium-phosphate. After it enters the nucleus, the DNA is expressed in a large proportion of the cells. This expression is dependent upon cis-acting sequences that are recognized by the mammalian transcriptional and translational machinery. In a smaller proportion of cells, the exogenous DNA becomes covalently integrated into the host cell DNA at random sites. Non-selectable genes can be introduced into mammalian cells by ligating them to a selectable marker or mixing the DNA with carrier DNA containing a selectable marker. The DNA that is introduced into mammalian cells can be rescued for examination and analysis. These gene transfer methods have already proven to be useful in identification of sequences which are necessary for normal gene expression as well as gene regulation. In addition a number of genes have been isolated using gene transfer methods. DNA mediated gene transfer holds much promise to target genes to specific sites in the chromosomes, to understand mechanisms of mammalian development and cell differentiation and is expected to provide a method to produce important and novel gene products that may be used for diagnostic and therapeutic purposes.

KEYWORDS Recurrent pregnancy loss; immunity; pro-inflammatory cytokines; homogenous samples ... ABSTRACT It is a well-known fact that pro-inflammatory cytokines exert an adverse effect on conceptus and result in pregnancy failure and there are mutations reported in ...

Background: Multi-genome comparative analysis has yielded important insights into the molecular details of gene regulation. We have developed EvoPrinter, a web-accessed genomics tool that provides a single uninterrupted view of conserved sequences as they appear in a species of interest. An EvoPrint reveals with near base-pair resolution those sequences that are essential for gene function.

We have developed phiSITE, database of gene regulation in bacteriophages. To date it contains detailed information about more than 700 experimentally confirmed or predicted regulatory elements (promoters, operators, terminators and attachment sites) from 32 bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. The database is manually curated, the data are collected mainly form scientific papers, cross-referenced with other database resources (EMBL, UniProt, NCBI taxonomy database, NCBI Genome, ICTVdb, PubMed Central) and stored in SQL based database system. The system provides full text search for regulatory elements, graphical visualization of phage genomes and several export options. In addition, visualizations of gene regulatory networks for five phages (Bacillus phage GA-1, Enterobacteria phage lambda, Enterobacteria phage Mu, Enterobacteria phage P2 and Mycoplasma phage P1) have been defined and made available. The phiSITE is accessible at http://www.phisite.org/.

Office of the Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (PtaC) and the trpA terminator and then cloned between Not1 sites to construct the CAS-Nm (Ptac-npt//-TtpA) and CAS-Gm ( Pt , CP, , , c/ -aacC/ -T, )

Plant non-speciWc lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an eYcient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel Triticum aestivum nsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1-7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the -Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no -Glucuronidase (GUS) activity was detected in stems. In Xowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther Wlaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta.

We present MEDUSA, an integrative method for learning motif models of transcription factor binding sites by incorporating promoter sequence and gene expression data. We use a modern large-margin machine learning approach, based on boosting, to enable feature selection from the high-dimensional search space of candidate binding sequences while avoiding overfitting. At each iteration of the algorithm, MEDUSA builds a motif model whose presence in the promoter region of a gene, coupled with activity of a regulator in an experiment, is predictive of differential expression. In this way, we learn motifs that are functional and predictive of regulatory response rather than motifs that are simply overrepresented in promoter sequences. Moreover, MEDUSA produces a model of the transcriptional control logic that can predict the expression of any gene in the organism, given the sequence of the promoter region of the target gene and the expression state of a set of known or putative transcription factors and signaling molecules. Each motif model is either a k-length sequence, a dimer, or a PSSM that is built by agglomerative probabilistic clustering of sequences with similar boosting loss. By applying MEDUSA to a set of environmental stress response expression data in yeast, we learn motifs whose ability to predict differential expression of target genes outperforms motifs from the TRANSFAC dataset and from a previously published candidate set of PSSMs. We also show that MEDUSA retrieves many experimentally confirmed binding sites associated with environmental stress response from the literature.

Simplified mathematical representation using explicitly computed time delays to maintain instabilities permits the analysis of negative feedback loops like those observed in biological pathways. ABSTRACT | Negative feedback loops are a common cellular motif underlying many important gene regulation pathways, and therefore a clear understanding of their dynamics is important for the study of gene regulation. Here we analyze the linear stability of negative feedback loops with and without explicit time delays. When the degradation rates of each loop component are identical, we derive the analytical solution of Manuscript

Many microorganisms from extreme environments have been well characterized, and increasing access to genomic sequence data has recently allowed the analysis of the protein families related to stress responses. Heat shock proteins appear to be ubiquitous in extremophiles. In this review, we focus on the family of small heat shock proteins (sHSPs) from extremophiles, which are a-crystallin homologues. Like the a-crystallin eye lens proteins, sHSPs act as molecular chaperones and prevent aggregation of denatured proteins under heat and desiccation stress. Many putative sHSP homologues have been identified in the genomic sequences of all classes of extremophiles. Current studies of shsp gene expression have revealed mechanisms of regulation and activity distinct from other known hsp gene regulation systems. Biochemical studies on sHSPs are limited to thermophilic and hyperthermophilic organisms, and the only two available crystal structures of sHSPs from Methanocaldococcus jannaschii, a hyperthermophilic archaeon and a mesophilic eukaryote, have contributed significantly to an understanding of the mechanisms of action of sHSPs, although many aspects remain unclear. Keywords a-Crystallins AE Extremophiles AE Molecular chaperones AE Small heat shock proteins AE Subunit assembly

The heterogeneity of neuronal subpopulations within particular brain regions plays an important role in the information processing of those regions. Although it is of critical importance, the issue of heterogeneity has been largely ignored for aminergic nuclei; these neurons have been long considered as rather functionally homogenous populations. This is in spite of the fact that neurons within these nuclei often exhibit different connectivity profiles. In this review we will describe electro-

The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial -glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells.

Many methanotrophs contain both a soluble and a particulate methane monooxygenase. A unique metabolic switch, mediated by copper ions, regulates the expression of these enzymes. When the copper-to-biomass ratio of the cell is low, the soluble enzyme is expressed, and when the copper-to-biomass ratio is high, the particulate enzyme is expressed. A model for the mechanism of this switch is proposed.

In Streptomyces coelicolor, AtrA is an activator of transcription of the actinorhodin cluster-situated regulator gene actII-ORF4. In previous work, we showed that S. coelicolor AtrA binds in vitro to the promoter of S. griseus strR, the streptomycin cluster-situated regulator. We show here that S. griseus carries a single close homologue of atrA and that expression of S. coelicolor AtrA in S. griseus causes a DNA binding-dependent reduction in streptomycin production and in the mRNA levels of strR and genes of streptomycin biosynthesis. However, there is no effect on the level of the mRNA of adpA, which is the only transcription factor that has so far been characterised for strR. The adpA gene is directly regulated by ArpA, the receptor protein for the c-butyrolactone signalling molecule A-factor. Therefore, to our knowledge, our results provide the first in vivo evidence that A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. The potential biotechnological application of our findings is discussed.

Conserved non-coding sequences (CNS) are islands of non-coding sequence that, like protein coding exons, show less divergence in sequence between related species than functionless DNA. Several CNSs have been demonstrated experimentally to function as cis-regulatory regions. However, the specific functions of most CNSs remain unknown. Previous searches for CNS in plants have either anchored on exons and only identified nearby sequences or required years of painstaking manual annotation. Here we present an open source tool that can accurately identify CNSs between any two related species with sequenced genomes, including both those immediately adjacent to exons and distal sequences separated by >12 kb of non-coding sequence. We have used this tool to characterize new motifs, associate CNSs with additional functions, and identify previously undetected genes encoding RNA and protein in the genomes of five grass species. We provide a list of 15,363 orthologous CNSs conserved across al...

RNA processing is a tightly regulated and highly complex pathway which includes transcription, splicing, editing, transportation, translation and degradation. It has been well-documented that splicing of RNA polymerase II medicated nascent transcripts occurs co-transcriptionally and is functionally coupled to other RNA processing. Recently, increasing experimental evidence indicated that pre-mRNA splicing influences RNA degradation and vice versa. In this review, we summarized the recent findings demonstrating the coupling of these two processes. In addition, we highlighted the importance of splicing in the production of intronic miRNA and circular RNAs, and hence the discovery of the novel mechanisms in the regulation of gene expression.

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml−1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.

Insulin resistance has been shown to be the major contributing factor to the metabolic syndrome, which comprises a cluster of risk factors for metabolic aberrations such as obesity, dyslipidemia, hypertension, and hyperglycemia. Additionally, insulin resistance has been associated with the occurrence of cardiovascular disease and type 2 diabetes. Epidemiological studies indicate that obesity and diabetes have become alarmingly prevalent in recent years. Substantial evidence suggests that dietary interventions and regular exercise greatly improve body mass index and lipid profile as well as alleviate insulin resistance. Therefore, dietary supplements such as insulinsensitizing agents may be beneficial in the prevention and treatment of obesity and type 2 diabetes. Numerous in vitro and in vivo studies suggest that chromium supplements, particularly niacin-bound chromium or chromium-nicotinate, may be effective in attenuating insulin resistance and lowering plasma cholesterol levels. Utilizing the powerful technology of nutrigenomics to identify the genes regulated by chromium supplementation may shed some light on the underlying mechanisms of chromium-gene interactions, and thus provide strategies to mitigate and prevent insulinresistance-related disorders.

Chronic renal failure (CRF) is frequently associated with increased plasma levels of homocysteine (Hcy), an amino acid that can be considered a new uremic toxin according to recent evidence. Studies on Hcy described first homocystinuria, an inherited disease characterized by high plasma Hcy levels and premature cardiovascular disease, resulting in high mortality rates. Hyperhomocysteinemia was then shown to be associated with cardiovascular events both in the general population and in CRF patients.

Orange fruit quality is highly dependent on time-specific expression of developmental genes in peel tissues, especially those controlling physical structure and synthesis of pigments and aromatic secondary metabolites. Using a custom microarray platform, we examined ...

Background: The importance of in silico predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of cis regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/β-catenin pathway target genes potentially involved in brain diseases.

Post-traumatic stress disorder (PTSD) is a severely debilitating psychiatric condition. Although a lifetime trauma incidence of 40e90% has been reported in the general population, the overall lifetime prevalence for PTSD ranges between 7e12%, suggesting individual-specific differences towards the susceptibility to PTSD. While studies investigating main genetic effects associated with PTSD have yielded inconsistent findings, there is growing evidence supporting the role of geneeenvironment (G Â E) interactions in PTSD. The hypothalamus pituitary adrenal (HPA) axis is one of the main systems activated after exposure to a trauma and perturbations in this system are one of the more consistent neurobiological abnormalities observed in PTSD. Genes regulating the HPA-axis are therefore interesting candidates for G Â E studies in PTSD.

The study of inherited monogenic diseases has contributed greatly to our mechanistic understanding of pathogenic mutations and gene regulation, and to the development of effective diagnostic tools. But interest has gradually shifted away from monogenic diseases, which collectively affect only a small fraction of the world's population, towards multifactorial, common diseases. The quest for the genetic variability associated with common traits should not be done at the expense of Mendelian disorders, because the latter could still contribute greatly to understanding the aetiology of complex traits.

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize, such as wheat or barley among others, in particular in certain regions (Southern Europe). The phylogenetic study performed in this work using a wide sample of isolates from diverse hosts and origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCR assay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1, which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significant correlation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosis of metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influence on environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widely distributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides. Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact of fumonisin risk in a number of commodities might need a revision.

In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli [1]. This proposal was followed by an explication of allosteric behavior by Monod and colleagues [2]. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function.

Recent years have seen great advances in the understanding of epigenetic gene regulation. Many of the molecular players involved have recently been identified and are rapidly being characterized in detail. Genome scale studies, using chromatin immunoprecipitation followed by expression arrays ('ChIP-Chip') or next generation sequencing ('ChIP-Seq'), have been applied to the study of transcription factor binding, DNA methylation, alternative histone use, and covalent histone modifications such as acetylation, ubiquitination and methylation. Initial studies focused on yeast, and embryonic stem cells. Genome-wide studies are now also being employed to characterize cancer and specifically leukemia genomes, with the prospect of improved diagnostic accuracy and discovery of novel therapeutic strategies. Here, we review some of the epigenetic modifications and their relevance for leukemia.

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modificationspecific antibodies.

We have investigated the regulation by N-acetyl-glucosamine of the nag1 gene of the mycoparasitic biocontrol fungus Trichoderma atroviride (=T. harzianum P1), which encodes a 73-kDa N-acetyl-β-D-glucosaminidase. The use of translational fusions revealed that a 290-bp fragment of the 5′ regulatory region of nag1 is sufficient to confer inducibility on the Aspergillus niger goxA gene. The region between positions –150 and –290, upstream of the nag1 coding region, was investigated using in vivo methylation protection analysis and electrophoretic mobility shift assays (EMSAs). Two neighbouring regions that interacted with regulatory proteins were identified, and bases essential for these interactions were determined in vitro. These data reveal protein binding to a CCCCT element at –240, a CCAGN13CTGG motif at –284, and a CCAAT-box which is present in the spacer of the latter motif. Evidence for the binding of a Hap2/3/5 complex to this CCAAT motif is presented. Protein binding to all three motifs was constitutive, and no differences were observed between induced and non-induced cultures. Mutation of either the CCAGN13CTGG or the AGGGG motif resulted in loss of inducibility of nag1 expression by N-acetyl-D-glucosamine in vivo.