Systematic Dissection of the Sequence Determinants of Gene 3’ End Mediated Expression Control

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Disentangling host,pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research

The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co‐evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen “hot spots,” we need to identify pathogen “cold spots” so that we can better understand what limits the pathogen''s distribution. Finally, we introduce the concept of “the Ghost of Epizootics Past” to discuss expected patterns in postepizootic host communities.     

Ophiostomatoid fungi including two new fungal species associated with pine root-feeding beetles in northern Spain

Many bark beetles live in a symbiosis with ophiostomatoid fungi but very little is known regarding these fungi in Spain. In this study, we considered the fungi associated with nine bark beetle species and one weevil infesting two native tree species (Pinus sylvestris and Pinus nigra) and one non-native (Pinus radiata) in Cantabria (Northern Spain). This included examination of 239 bark beetles or their galleries. Isolations yielded a total of 110 cultures that included 11 fungal species (five species of Leptographium sensu lato including Leptographium absconditum sp. nov., five species of Ophiostoma sensu lato including Ophiostoma cantabriense sp. nov, and one species of Graphilbum). The most commonly encountered fungal associates of the bark beetles were Grosmannia olivacea, Leptographium procerum, and Ophiostoma canum. The aggressiveness of the collected fungal species was evaluated using inoculations on two-year-old P. radiata seedlings. Leptographium wingfieldii, Leptographium guttulatum, and Ophiostoma ips were the only species capable of causing significant lesions.     

Is caseinomacropeptide from milk proteins,an inhibitor of gastric secretion?

The aim of this work was to study, in vivo, the effect of the ingestion of not glycosylated caseinomacropeptide (CMP) on gastric secretion. In Experiments #1 and #2, 7 calves fitted with a gastric pouch received either a diet without CMP (C diet) or C diet in which CMP was introduced (equal to and 5 folds that of CMP quantity contained in cow milk, diets CMP1 and CMP5, respectively). In Experiment #3, 2 calves (with gastric pouch) were fed C diet followed by an “iv perfusion” of CMP. In Experiment #4, 25 calves fed either C, CMP1 or CMP5 diets were fitted with a blood catheter for sample collections. The quantities of daily gastric secretions seemed few modified by CMP ingestion but the profile of these secretions was changed along the day. The most important result is that CMP can inhibit gastric secretions (mainly hydrochloric acid) stimulated by the meal, but there was no dose-dependent response. No similar observations were obtained after perfusion of CMP in jugular vein. CMP was not detected in blood. Results obtained in our experiments are not in favor of its significant intestinal absorption. Gastrin, somatostatin and VIP could be implicated in the mechanisms of regulation.     

'Genome order index' should not be used for defining compositional constraints in nucleotide sequences - a case study of the Z-curve

Background  

The Z-curve is a three dimensional representation of DNA sequences proposed over a decade ago and has been extensively applied to sequence segmentation, horizontal gene transfer detection, and sequence analysis. Based on the Z-curve, a "genome order index," was proposed, which is defined as S = a ²+ c ²+t ²+g ², where a, c, t, and g are the nucleotide frequencies of A, C, T, and G, respectively. This index was found to be smaller than 1/3 for almost all tested genomes, which was taken as support for the existence of a constraint on genome composition. A geometric explanation for this constraint has been suggested. Each genome was represented by a point P whose distance from the four faces of a regular tetrahedron was given by the frequencies a, c, t, and g. They claimed that an inscribed sphere of radius r = 1/ contains almost all points corresponding to various genomes, implying that S <r ². The distribution of the points P obtained by S was studied using the Z-curve.     

Validation of QTLs for Orobanche crenata resistance in faba bean (Vicia faba L.) across environments and generations

Broomrape (Orobanche crenata Forsk.) is a major root–parasite of faba bean (Vicia faba L.), that seriously limits crop cultivation in the whole Mediterranean area. This parasitic weed is difficult to control, difficult to evaluate and the resistance identified so far is of polygenic nature. This study was conducted to identify genetic regions associated with broomrape resistance in recombinant inbred lines (RILs) and to validate their previous location in the original F₂ population derived from the cross between lines Vf6 and Vf136. A progeny consisting of 165 F₆ RILs was evaluated in three environments across two locations in 2003 and 2004. Two hundred seventy seven molecular markers were assigned to 21 linkage groups (9 of them assigned to specific chromosomes) that covered 2,856.7 cM of the V. faba genome. The composite interval mapping on the F₆ map detected more quantitative trait loci (QTL) than in the F₂ analysis. In this sense, four QTLs controlling O. crenata resistance (Oc2–Oc5) were identified in the RI segregant population in three different environments. Only Oc1, previously reported in the F₂ population, was not significant in the advanced lines. Oc2 and Oc3 were found to be associated with O. crenata resistance in at least two of the three environments, while the remaining two, Oc4 and Oc5, were only detected in Córdoba-04 and Mengíbar-04 and seemed to be environment dependent.