Structure and evolution of gene regulatory networks in microbial genomes

With the availability of genome sequences for hundreds of microbial genomes, it has become possible to address several questions from a comparative perspective to understand the structure and function of regulatory systems, at least in model organisms. Recent studies have focused on topological properties and the evolution of regulatory networks and their components. Our understanding of natural networks is paving the way to embedding synthetic regulatory systems into organisms, allowing us to expand the natural diversity of living systems to an extent we had never before anticipated.     

Positive and negative regulatory elements contribute to CpG oligonucleotide-mediated regulation of human IL-6 gene expression

Oligonucleotides (ODN) expressing immunostimulatory "CpG motifs" activate human RPMI 8226 myeloma cells to secrete IL-6. Using deletion and site-directed mutagenesis of the human (h)IL-6 promoter region, two positive regulatory elements (the binding sites for the 5'-CCAAT / enhancer binding protein-beta and NF-kappaB) were identified. Two negative regulatory elements, the 3'-retinoblastoma control element (RCE) and the binding site for Epstein-Barr virus C-promoter binding factor 1 (CBF1), also contributed to CpG ODN induction of hIL-6 gene expression. Of interest, CpG ODN treatment induced the dissociation of a repressor protein from its 3'-RCE binding site. Thus, CpG ODN regulation of hIL-6 gene expression involves both enhancer and derepression mechanisms.     

Epidemiology of insect venom sensitivity in children and its correlation to clinical and atopic features

The aim of this study was to obtain more accurate figures of the prevalence of cutaneous sensitivity to Hymenoptera venoms (HV) and its correlation with other parameters of atopy in a population of primary schoolchildren. Parents filled out a structured questionnaire and children were tested with a panel of inhalant and food allergens as well as standardized freeze-dried extracts of HV. Among the 1175 children who completed the study there was a personal history of rhinoconjunctivitis in 242 (20.8%) and a current wheezing in 114 (9.78%). Two-hundred twenty-eight (19.40%) children had a history of Hymenoptera sting (HS) reactions (224 or 19.06% of local reactions and 4 or 0.34% of local and systemic reactions). Positive skin-prick tests (SPT) to any given HV extract were present in 43 children (3.66%). Most subjects had positive SPT to honey bee venom (35/1175; 2.98%); 17/1175 (1.45%) had positive SPT to wasp and only 12 subjects (1.02%) had positive SPT to polistes venom. There was a correlation between a positive SPT to HV and the history of clinical reactions to HS (P = 0.0026). Positive SPT to at least one of the inhalant and food allergens tested were found in 353 subjects (30.04%). Factors such as age, sex, reactions to HV, positive SPT to mite, cat dander, grass, Alternaria, Parietaria, cow's milk, egg white and wheat were significantly associated with a positive SPT to HV using a univariate regression analysis. Only age, reactions to HV, a positive SPT to grass, Parietaria, cow's milk, and egg white were significantly associated with a positive SPT to HV using a multiple regression analysis. In this study, the frequency of immunological sensitization to HV in a population of unselected children is not so high as in adults. There is an association between the presence of positive SPT to HV and an atopy linked humoral IgE response. The presence of a significant and independent association between positive SPT to food of animal origin and positive SPT to HV is surprising and needs further study.     

An 80-bp cis-acting regulatory region controls cAMP and development regulation of a prestalk gene in Dictyostelium

We have analyzed an 80-bp region containing the cis-acting sequences necessary for regulation of the Dictyostelium discoideum prestalk gene pst-cathepsin at the appropriate stage during multicellular development and in response to cAMP in single-cell culture. The region lies between approximately -280 and -200 bp upstream from the Cap site and contains two intertwined G/C-rich sequences, including a palindromic sequence and a direct repeat of the 3' portion of the palindrome. In a previous set of experiments, we showed that the direct repeat, or G-box, is important in the regulation of pst-cath expression. In this paper, we use a series of nested internal deletions to define other regions within the promoter required for cAMP and developmental expression, to further examine the importance of the two G-boxes, and to examine the functional relationship of the G-boxes with respect to the other regulatory sequences. Analysis of the expression of these constructs in transformed cells showed that both the 5' portion of the palindrome and the two G-boxes are required for promoter function and are capable of developmentally regulating pst-cath expression. An A/T-rich sequence located 5' to the G/C-rich sequence is also essential for maximal expression, whereas insertion of a linker 5' to this region suggests the presence of a negative element functional during multicellular development. The spacing between the G-box sequences proved to be important for the full induction of gene expression. Constructs containing the G-boxes at wild-type spacing or closer show wild-type or near wild-type levels of expression, whereas expansion of the region between the G-boxes leads to a substantial drop in the level of gene expression in response to cAMP. Insertion of an oligonucleotide containing one of the G-boxes and surrounding sequences can partially complement deletions in which this region has been removed. Analysis of the expression of the cassette constructs, in some cases, revealed significant differences in the quantitative level of expression under the two developmental conditions. This suggests that there are either qualitative or quantitative differences in the factors controlling the expression of this gene under these two conditions.     

Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress

A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Tharappel C. James and Jane Usher contributed equally to this work.     

HLA linked immune response to S and pre-S2 gene products in hepatitis B vaccination

In order to detect a possible HLA linked genetic control of human immune responses to hepatitis B virus, forty healthy adult persons of the same age typed for HLA-A, -B and -DR antigens, were vaccinated against virus hepatitis B and sequentially tested for anti-HBs and anti-pre-S2 antibodies. They received three injections of Hevac-B Pasteur vaccine, the second 1 month and the third 3 months after the first. Following the third immunization, 38 individuals (95%) had a protective level of anti-HBs antibodies and 17 (42.3%) had a positive level of anti-pre-S2 antibodies. HLA-A11 antigen was significantly more frequent (pc = 0.007) among anti-HBs high responders than low responders. In addition, anti-HBs high responders were more frequently HLA-DR1, and less frequently HLA-DR4 and DR7 positive; corrected values, however, were not significant. Anti-pre-S2 high responders showed an apparent increase of HLA-B7, B14 or DR3 antigens, when compared to low responders (pc not significant).     

Mice deficient in the X-linked lymphoproliferative disease gene sap exhibit increased susceptibility to murine gammaherpesvirus-68 and hypo-gammaglobulinemia

X-linked lymphoproliferative disease is characterized by immune dysregulation and uncontrolled lymphoproliferation on exposure to Epstein-Barr virus (EBV). This disease has been attributed to mutations in the SAP gene (also denominated as SH2D1A or DSHP). To delineate the role of SAP in the pathophysiology of X-linked lymphoproliferative disease, a strain of sap-deficient mice has been generated by deleting exon 2 of the gene. After infection with murine gammaherpesvirus-68, which is homologous to EBV, the mutant mice exhibit more vigorous CD8+ T cell proliferation and more disseminated lymphocyte infiltration compared to their wild-type littermates. Chronic tissue damage and hemophagocytosis were evident in sap-deficient mice but not in their wild-type littermates. Concordantly, murine gammaherpesvirus-68 reactivation was observed in sap-deficient mice, indicating an impaired control of the virus. Notably, IgE deficiency and decreased serum IgG level were observed in mutant mice prior to and after murine gammaherpesvirus-68 infection, which reproduces hypo-gammaglobulinemia in X-linked lymphoproliferative disease patients. This mouse model will therefore be a useful tool for dissecting the various phenotypes of X-linked lymphoproliferative disease.     

Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera)

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of two large protein families, G protein-coupled olfactory receptors (ORs) and odorant binding proteins (OBPs). In olfactory sensilla, OBPs deliver hydrophobic airborne molecules to ORs, but their expression in nonolfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. Here we used bioinformatic and experimental approaches to characterize the OBP-like gene family in a highly social insect, the Western honey bee. Comparison with other insects shows that the honey bee has the smallest set of these genes, consisting of only 21 OBPs. This number stands in stark contrast to the more than 70 OBPs in Anopheles gambiae and 51 in Drosophila melanogaster. In the honey bee as in the two dipterans, these genes are organized in clusters. We show that the evolution of their structure involved frequent intron losses. We describe a monophyletic subfamily of OBPs where the diversification of some amino acids appears to have been accelerated by positive selection. Expression profiling under a wide range of conditions shows that in the honey bee only nine OBPs are antenna-specific. The remaining genes are expressed either ubiquitously or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs are not restricted to olfaction and are likely to be involved in broader physiological functions.     

Age-related features of cardiac contractility and its adrenergic regulation in catecholamine damage to the myocardium

Studies on isolated rat hearts showed that in contrast to 8-month-old animals, in 26-month-old rats the contractile activity of the myocardium increases by the 3rd day after administration of toxic adrenalin doses (1 mg/kg i.m.). The maximum functional response of catecholamine injured hearts to stimulation of alpha- and beta-adrenoceptors reduced in both age groups, while the sensitivity of the myocardium to beta-adrenoceptor agonists in mature rats increased.     

Mutations to constitutivity and derepression are separate and separable in a regulatory gene of Aspergillus nidulans

Summary Previous work has shown that expression of the structural genes for the enzymes of nitrate and nitrite assimilation in Aspergillus nidulans requires the products of two positively acting regulatory genes — nirA, mediating induction, and areA, mediating nitrogen metabolite repression. Here we show that, in addition to previously described mutations in nirA leading to constitutivity, other mutations can be selected in nirA leading to nitrogen metabolite derepression. These constitutivity and depression mutations in nirA are additive and separable by intragenic recombination. This suggests that the nirA gene product contains two separate domains, a co-inducer binding region, defined by constitutivity mutations, and a region interacting with the areA gene product or with initiator sites adjacent to structural genes under areA and nirA control, defined by derepression mutations. These findings might indicate a striking similarity of action between the eukaryotic regulatory gene nirA and a comparable prokaryotic regulatory gene.     

The fat mass and obesity gene is linked to reduced verbal fluency in overweight and obese elderly men

Humans carrying the prevalent rs9939609 A allele of the fat mass and obesity-associated (FTO) gene are more susceptible to developing obesity than noncarries. Recently, polymorphisms in the FTO gene of elderly subjects have also been linked to a reduced volume in the frontal lobe as well as increased risk for incident Alzheimer disease. However, so far there is no evidence directly linking the FTO gene to functional cognitive processes. Here we examined whether the FTO rs9939609 A allele is associated with verbal fluency performance in 355 elderly men at the age of 82 years who have no clinically apparent cognitive impairment. Retrieval of verbal memory is a good surrogate measure reflecting frontal lobe functioning. Here we found that obese and overweight but not normal weight FTO A allele carriers showed a lower performance on verbal fluency than non-carriers (homozygous for rs9939609 T allele). This effect was not observed for a measure of general cognitive performance (i.e., Mini-Mental State Examination score), thereby indicating that the FTO gene primarily affects frontal lobe-dependent cognitive processes in elderly men.     

Down-regulation of the oncogene cyclin D1 increases migratory capacity in breast cancer and is linked to unfavorable prognostic features

The oncogene cyclin D1 is highly expressed in many breast cancers and, despite its proliferation-activating properties, it has been linked to a less malignant phenotype. To clarify this observation, we focused on two key components of malignant behavior, migration and proliferation, and observed that quiescent G(0)/G(1) cells display an increased migratory capacity compared to cycling cells. We also found that the down-regulation of cyclin D1 in actively cycling cells significantly increased migration while also decreasing proliferation. When analyzing a large set of premenopausal breast cancers, we observed an inverse proliferation-independent link between cyclin D1 and tumor size and recurrence, suggesting that this protein might abrogate infiltrative malignant behavior in vivo. Finally, gene expression analysis after cyclin D1 down-regulation by siRNA confirmed changes in processes associated with migration and enrichment of our gene set in a metastatic poor prognosis signature. This novel function of cyclin D1 illustrates the interplay between tumor proliferation and migration and may explain the attenuation of malignant behavior in breast cancers with high cyclin D1 levels.     

A gene for cytochrome c oxidase subunit III (COXIII) in broad bean mitochondrial DNA: structural features and sequence evolution

Summary A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains the coxIII gene is presented, and compared to corresponding sequences of Oenothera and corn mtDNAs. Upstream from the broad bean coxIII gene are three potential secondary structures: a single stem and loop (hairpin) that is conserved in the Oenothera and corn sequences; a second single stem and loop; and a double stem and loop. The rate of evolution of the coxIII gene has been slower in plants than in mammals. Constraints on the fixation of at least some kinds of mutations in silent (synonymous) third position nucleotides, as well as of mutations that cause amino acid replacements, seem to have contributed to this slower rate.     

Organization and sequence of five tRNA genes and of an unidentified reading frame in the wheat chloroplast genome: evidence for gene rearrangements during the evolution of chloroplast genomes

Summary The genes for the initiator tRNA_CAU ^Met. tRNA_UCC ^Gly, tRNA_GGU ^Thr, tRNA_UUC ^Glu and tRNA_GUA ^Tyr and an open reading frame of 62 codons have been identified by sequencing a 2,358 by BamHl and a 1,378 by BamHI-Sst2 DNA fragments from wheat chloroplasts. A comparison of the organization of these five tRNA genes and of the open reading frame on the wheat, tobacco and spinach chloroplast genomes suggests that at least three genomic inversions must have occurred during the evolution of the wheat chloroplast genome from a spinach-like ancestor genome. Furthermore, it seems that in wheat the 91 by intergenic region between the genes for the initiator tRNA^Met and the gene for tRNA_UCC ^Gly is one end-point of the 20 kbp genomic inversion proposed by Palmer and Thompson in the case of maize (Palmer and Thompson 1982). A 119 bp duplication is located at this junction: the first copy comprises the 91 by of the intergenic region and the first 28 by of the tRNA^Met gene, the second copy is found downstream of the tRNA^Met gene.     

Revisit of multiple epiphyseal dysplasia: ethnic difference in genotypes and comparison of radiographic features linked to the COMP and MATN3 genes

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous group of diseases characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. The purpose of this study was to investigate the frequency of mutations in individuals with a clinical and radiographic diagnosis of MED and to test the hypothesis that characteristic radiological findings may be helpful in predicting the gene responsible. The radiographs of 74 Korean patients were evaluated by a panel of skeletal dysplasia experts. Six genes known to be associated with MED (COMP, MATN3, COL9A1, COL9A2, COL9A3, and DTDST) were screened by sequencing. Mutations were found in 55 of the 63 patients (87%). MATN3 mutations were found in 30 patients (55%), followed by COMP mutations in 23 (41%), and COL9A2 and DTDST mutations in one patient (2%) each. Comparisons of radiographic findings in patients with COMP and MATN3 mutations showed that albeit marked abnormalities in hip and knee joints were observed in both groups, the degree of involvement and the morphology of dysplastic epiphyses differed markedly. The contour of the pelvic acetabulum, the presence of metaphyseal vertical striations, and/or the brachydactyly of the hand were also found to be highly correlated with the genotypes. The study confirms that MATN3 and COMP are the genes most frequently responsible for MED and that subtle radiographic signs may give precious indications on which gene(s) should be prioritized for mutational screening in a given individual.     

On the mechanisms of radiobiological events in relation to the regulatory role of p53 gene and protein

The paper analyzes the topical problems of radiobiology in the light of the present-day data on the molecular biology and biochemistry of p53 protein that is an integrator of stress signals from various damaging exposures and that fulfills the function of genome guard by regulating the checking points of a cellular cycle, DNA reparation, and apoptosis. It also considers the mechanisms of radiation cell death and radiosensitivity/radioresistance in the light of data on p53 protein, as well as the problems of searching for antiradiation agents, the radiation-induced instability of genome, the biological aftereffects of small-dose radiation, as well as radiation-induced carcinogenesis due to the important regulatory role of p53 protein. The lines of further studies of the above problems are outlined to refine the understanding of pathogenetic processes in radiation damages, to extend the therapeutic, diagnostic, predictive capacities of clinical radiobiology and radiation medicine.