Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase (gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30degreesC to 45degreesC). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans-acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

Transposable elements in Coffea (Gentianales : Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK beta-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted.

Vertical inheritance and bursts of transposition have shaped the evolution of the BS non-LTR retrotransposon in Drosophila

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The relationship between obesity, low back pain, and lumbar disc degeneration when genetics and the environment are considered: a systematic review of twin studies

BACKGROUND CONTEXT: The relationships between obesity and low back pain (LBP) and lumbar disc degeneration (LDD) remain unclear. It is possible that familial factors, including genetics and early environment, affect these relationships.PURPOSE: To investigate the relationship between obesity-related measures (eg, weight, body mass index [BMI]) and LBP and LDD using twin studies, where the effect of genetics and early environment can be controlled.STUDY DESIGN: A systematic review with meta-analysis.METHODS: MEDLINE, CINAHL, Scopus, Web of Science, and EMBASE databases were searched from the earliest records to August 2014. All cross-sectional and longitudinal observational twin studies identified by the search strategy were considered for inclusion. Two investigators independently assessed the eligibility, conducted the quality assessment, and extracted the data. Metaanalyses (fixed or random effects, as appropriate) were used to pool studies'estimates of association.RESULTS: In total, 11 articles met the inclusion criteria. Five studies were included in the LBP analysis and seven in the LDD analysis. For the LBP analysis, pooling of the five studies showed that the risk of having LBP for individuals with the highest levels of BMI or weight was almost twice that of people with a lower BMI (odds ratio [OR] 1.8; 95% confidence interval [CI] 1.6-2.0; I-2 = 0%). A dose-response relationship was also identified. When genetics and the effects of a shared early environment were adjusted for using a within-pair twin case-control analysis, pooling of three studies showed a reduced but statistically positive association between obesity and prevalence of LBP (OR 1.5; 95% CI 1.1-2.1; I-2 = 0%). However, the association was further diminished and not significant (OR 1.4; 95% CI 0.8-2.3; I-2 = 0%) when pooling included two studies on monozygotic twin pairs only. Seven studies met the inclusion criteria for LDD. When familial factors were not controlled for, body weight was positively associated with LDD in all five cross-sectional studies. Only two cross-sectional studies investigated the relationship between obesity-related measures and LDD accounting for familial factors, and the results were conflicting. One longitudinal study in LBP and three longitudinal studies in LDD found no increase in risk in obese individuals, whether or not familial factors were controlled for.CONCLUSIONS: Findings from this review suggest that genetics and early environment are possible mechanisms underlying the relationship between obesity and LBP; however, a direct causal link between these conditions appears to be weak. Further longitudinal studies using the twin design are needed to better understand the complex mechanisms underlying the associations between obesity, LBP, and LDD.

High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Selection for Increased Starvation Resistance Using Drosophila melanogaster: Investigating Physiological and Life History Trait Responses to Starvation and Dietary Supplementation in the Context of an Obese Phenotype

Artificial selection for starvation resistance provided insight into the relationships between evolved physiological and life history trait responses following exposure to biologically induced stress. Investigations of alterations to body composition, metabolic rate, movement, and life history traits including development time, female egg production, and longevity in response to brief periods of starvation were conducted on genetically based starvation-resistant and control lines of Drosophila melanogaster. Analysis of the starvation-resistant lines indicated increased energy storage with increased triglyceride deposition and conversion of carbohydrates to lipid, as identified by respiratory quotient values. Correlations between reductions in metabolic rates and movement in the starvation-resistant lines, suggested the presence of an evolved physiological response resulting in energy conservation. Investigations of life history traits in the starvation-resistant lines indicated no significant differences in development time or reproduction between the selected and control lines. Measurements of longevity, however, indicated a significant reduction in starvation-resistant D. melanogaster lifespan. These results suggested that elevated lipid concentrations, similar to that observed with obesity, were correlated with premature mortality. Exposure of the starvation-resistant and control lines to diets supplemented with glucose, palmitic acid, and a 2:1 mixture of casein to albumin were used to investigate alterations in body composition, movement, and life history traits. Results obtained from this study indicated that increased sugar in the diet led to increased carbohydrate, glycogen, total sugar, trehalose, and triglyceride concentrations, while increased fat and protein in the diet resulted in increased soluble protein, carbohydrate, glycogen, total sugar, and trehalose concentrations. Examination of life history trait responses indicated reduced fecundity in females exposed to increased glucose concentrations. Increased supplementations of palmitic acid was consistently correlated with an overall reduction in lifespan in both the starvation-resistant and control Drosophila lines, while measurements of movement indicated increased female activity levels in flies exposed to diets supplemented with fat and protein. Analyses of the physiological and life history trait responses to starvation and dietary supplementation on Drosophila melanogaster used in the present study has implications for investigating the mechanisms underlying the development and persistence of human obesity and associated metabolic disorders.

Mercury-induced autoimmunity : Genetics and immunoregulation

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

Agricultural Genetics and Plant Breeding in Early Twentieth-Century Italy

This thesis is about plant breeding in Early 20th-Century Italy. The stories of the two most prominent Italian plant-breeders of the time, Nazareno Strampelli and Francesco Todaro, are used to explore a fragment of the often-neglected history of Italian agricultural research. While Italy was not at the forefront of agricultural innovation, research programs aimed at varietal innovation did emerge in the country, along with an early diffusion of Mendelism. Using philosophical as well as historical analysis, plant breeding is analysed throughout this thesis as a process: a sequence of steps that lays on practical skills and theoretical assumptions, acting on various elements of production. Systematic plant-breeding programs in Italy started from small individual efforts, attracting more and more resources until they became a crucial part of the fascist regime's infamous agricultural policy. Hybrid varieties developed in the early 20th century survived World War II and are now ancestors of the varieties that are still cultivated today. Despite this relevance, the history of Italian wheat hybrids is today largely forgotten: this thesis is an effort to re-evaluate a part of it. The research did allow previously unknown or neglected facts to emerge, giving a new perspective on the infamous alliance between plant-breeding programs and the fascist regime. This thesis undertakes an analysis of Italian plant-breeding programs as processes. Those processes had a practical as well as a theoretical side, and involved various elements of production. Although a complete history of Italian plant breeding still remains to be written, the Italian case can now be considered along with the other case-studies that other scholars have developed in the history of plant breeding. The hope is that this historical and philosophical analysis will contribute to the on-going effort to understand the history of plants.

Effects of housing, perches, genetics, and 25-hydroxycholecalciferol on keel bone deformities in laying hens

Several studies have shown a high prevalence of keel bone deformities in commercial laying hens. The aim of this project was to assess the effects of perch material, a vitamin D feed additive (25-hydroxyvitamin D(3); HyD, DSM Nutritional Products, Basel, Switzerland), and genetics on keel bone pathology. The study consisted of 2 experiments. In the first experiment, 4,000 Lohmann Selected Leghorn hens were raised in aviary systems until 18 wk of age. Two factors were investigated: perch material (plastic or rubber-coated metal) and feed (with and without HyD). Afterward, the hens were moved to a layer house with 8 pens with 2 aviary systems. Daily feed consumption, egg production, mortality, and feather condition were evaluated. Every 6 wk, the keel bones of 10 randomly selected birds per pen were palpated and scored. In the second experiment, 2,000 Lohmann Brown (LB) hens and 2,000 Lohmann Brown parent stock (LBPS) hens were raised in a manner identical to the first experiment. During the laying period, the hens were kept in 24 identical floor pens but equipped with different perch material (plastic or rubber-coated metal). The same variables were investigated as in the first experiment. No keel bone deformities were found during the rearing period in either experiment. During the laying period, deformities gradually appeared and reached a prevalence of 35% in the first experiment and 43.8% in the second experiment at the age of 65 and 62 wk, respectively. In the first experiment, neither HyD nor the aviary system had any significant effect on the prevalence of keel bone deformities. In the second experiment, LBPS had significantly fewer moderate and severe deformities than LB, and rubber-coated metal perches were associated with a higher prevalence of keel bone deformities compared with plastic perches. The LBPS laid more but smaller eggs than the LB. Again, HyD did not affect the prevalence of keel bone deformities. However, the significant effect of breed affiliation strongly indicates a sizeable genetic component that may provide a basis for targeted selection.

Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized. Many animal and human studies were conducted on copper to provide a rich database from which data suitable for modeling the dose-response relationship for copper may be extracted. Possible dose-response modeling strategies are considered in this review, including those based on the benchmark dose and categorical regression. The usefulness of biologically based dose-response modeling techniques in understanding copper toxicity was difficult to assess at this time since the mechanisms underlying copper-induced toxicity have yet to be fully elucidated. A dose-response modeling strategy for copper toxicity was proposed associated with both deficiency and excess. This modeling strategy was applied to multiple studies of copper-induced toxicity, standardized with respect to severity of adverse health outcomes and selected on the basis of criteria reflecting the quality and relevance of individual studies. The use of a comprehensive database on copper-induced toxicity is essential for dose-response modeling since there is insufficient information in any single study to adequately characterize copper dose-response relationships. The dose-response modeling strategy envisioned here is designed to determine whether the existing toxicity data for copper excess or deficiency may be effectively utilized in defining the limits of the homeostatic range in humans and other species. By considering alternative techniques for determining a point of departure and low-dose extrapolation (including categorical regression, the benchmark dose, and identification of observed no-effect levels) this strategy will identify which techniques are most suitable for this purpose. This analysis also serves to identify areas in which additional data are needed to better define the characteristics of dose-response relationships for copper-induced toxicity in relation to excess or deficiency.

Regulation of woody plant development by auxin and polar auxin transport

Auxin is a key regulator in plant growth and development. This dissertation examines the role of auxin and polar auxin transport in woody growth and development. Strategies of promoter reporter system, microarray expression analysis, transgenic modification, physiological assays, anatomical analysis, and histochemical/biochemical assays were employed to improve our understanding of auxin study in Populus. The results demonstrate various aspects of auxin regulation on shoot growth, root development, wood formation, and gravitropism in woody tissues. We describe the behavior of the DR5 reporter system for measuring auxin concentrations and response in stably transformed Populus trees. Our study shows that DR5 reporter system can be efficiently used in Populus to study auxin biology at a cellular resolution. We investigated the global gene expression in responding to auxin in Populus root. The results revealed groups of IBA up- and down- regulated genes involved in various biological processes including cell wall modification, root growth and lateral root formation, transporter activity and hormone crosstalk. We also verify two of the identified genes' function by transgenic modification in Populus, which encode auxin efflux carrier PtPIN9 and transcription factor PtERF72. We investigated the role of PtPIN9 in woody growth and development, especially in wood formation and gravitropic response in woody stem. We found that overexpressing PtPIN9 enhanced several growth parameters while suppression of PtPIN9 has inhibited tension wood formation. Our results show that PIN9 and other members from PIN family could be possible useful tools for increasing biomass productivity, wood quality, or in modifying plant form.