Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. (*) 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. (*) 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.     

HERITABILITY AND GENETIC CORRELATION OF COROLLA SHAPE AND SIZE IN ERYSIMUM MEDIOHISPANICUM

Flower shape has evolved in most plants as a consequence of pollinator-mediated selection. Unfortunately, no study has explored the genetic variation of flower shape, despite that this information is crucial to understand its adaptive evolution. Our main goal here is to determine heritability of corolla shape in Erysimum mediohispanicum (Brassicaceae). Also, we explore heritability of other pollinator-selected traits in this plant species, such as plant size, flower display, and corolla size. In addition, we investigate genetic correlations between all these traits. We found significant heritability for one plant-size trait (stalk height), for number of flowers, for all corolla-size traits (corolla diameter, corolla tube length and corolla tube width), and for corolla shape. Consequently, this species retains a high ability to respond to the selection exerted by its pollinators. Genetic correlation was strong between all functionally related traits and between flower number and plant size, weak between corolla size and plant size and no correlation between corolla shape and any other trait. Thus, selection affecting some E. mediohispanicum traits would also indirectly affect other functionally related and unrelated traits. More importantly, the observed genetic correlation seems to be at least partially adaptive because positive correlational selection currently acts on the covariance between some of these traits ( Gómez 2003 ; Gómez et al. 2006 ).     

Ameliorative property of Sesbania grandiflora on carbohydrate metabolic enzymes in the liver and kidney of streptozotocin-induced diabetic rats

In diabetic condition, endogenous glucose synthesis will be elevated due to defect in the action of vital enzymes involved in carbohydrate metabolism, which is the main cause for hyperglycemia. The current study was designed to explore the anti-hyperglycemic efficacy of Sesbania grandiflora flower (SGF) extract by evaluating the concentration of C-peptide, insulin, glucose, glycosylated hemoglobin (HbA_1C), hemoglobin (Hb), glycogen and carbohydrate metabolic enzymes activities in diabetic rats. The study found to lower the level of glucose, HbA_1C and simultaneously ameliorated concentrations of C-peptide, insulin, hemoglobin (Hb), glycogen and carbohydrate metabolic enzymes activities in SGF treated (250 mg/kg body weight for 45 days) diabetic rats. Moreover, SGF administered diabetic rats showed diminished consumption of food and water at the same time improved body weight. The results obtained from the present study were compared with glibenclamide treated (600 µg/kg body weight) diabetic rats. SGF were supplemented to normal rats to rule out toxic effect of SGF, to explore any significant alteration in the above parameters. Hence, the results depict that SGF modulated the carbohydrate metabolic enzymes activities through ameliorating the secretion of insulin and diminishing the level of glucose concentration in STZ-induced diabetic rats by its bioactive compounds.     

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.     

What can a weevil teach a fly,and reciprocally? Interaction of host immune systems with endosymbionts in Glossina and Sitophilus

The tsetse fly (Glossina genus) is the main vector of African trypanosomes, which are protozoan parasites that cause human and animal African trypanosomiases in Sub-Saharan Africa. In the frame of the IAEA/FAO program ‘Enhancing Vector Refractoriness to Trypanosome Infection’, in addition to the tsetse, the cereal weevil Sitophilus has been introduced as a comparative system with regards to immune interactions with endosymbionts. The cereal weevil is an agricultural pest that destroys a significant proportion of cereal stocks worldwide. Tsetse flies are associated with three symbiotic bacteria, the multifunctional obligate Wigglesworthia glossinidia, the facultative commensal Sodalis glossinidius and the parasitic Wolbachia. Cereal weevils house an obligatory nutritional symbiosis with the bacterium Sodalis pierantonius, and occasionally Wolbachia. Studying insect host-symbiont interactions is highly relevant both for understanding the evolution of symbiosis and for envisioning novel pest control strategies. In both insects, the long co-evolution between host and endosymbiont has led to a stringent integration of the host-bacteria partnership. These associations were facilitated by the development of specialized host traits, including symbiont-housing cells called bacteriocytes and specific immune features that enable both tolerance and control of the bacteria. In this review, we compare the tsetse and weevil model systems and compile the latest research findings regarding their biological and ecological similarities, how the immune system controls endosymbiont load and location, and how host-symbiont interactions impact developmental features including cuticle synthesis and immune system maturation. We focus mainly on the interactions between the obligate symbionts and their host’s immune systems, a central theme in both model systems. Finally, we highlight how parallel studies on cereal weevils and tsetse flies led to mutual discoveries and stimulated research on each model, creating a pivotal example of scientific improvement through comparison between relatively distant models.

     

Molecular confirmation of founder mutation c.-167A>G in Tunisian patients with PMLD disease

Pelizaeus Merzbacher disease and Pelizaeus Merzbacher like disease (PMLD) are hypomyelinating leucodystrophies of the central nervous system (CNS) with a very similar phenotype. PMD is an X-linked recessive condition caused by mutations, deletion duplication or triplication of the proteolipid protein 1 gene (PLP1). However, PMLD is a recessive autosomal hypomyelinating leukodystrophy caused by mutations of the GJC2 gene. In this study, we analyzed 5 patients belonging to 4 Tunisian families. Direct sequencing of GJC2 gene in all probands showed the same homozygous founder mutation c.-167A>G localized in the promoter region. We also generated two microsatellite markers GJC2 195GT and GJC2 76AC closed to the GJC2 gene to confirm the presence of a founder effect for this mutation. Haplotype study showed that the c.-167A>G promoter mutation occurred in a specific founder haplotype in Tunisian population. The identification of this founder mutation has important implications towards genetic counseling in relatives of these families and the antenatal diagnosis.     

Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Neisseria meningitidis strain Z2491

Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram-negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of the N. meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter. The IPTG-induced GAPN was purified ca. 48-fold from E. coli cells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibiting absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic polypeptide with an isoelectric point of 6.3. The optimum pH of the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64 °C. Molecular phylogenetic data suggest that N. meningitidis GAPN has a closer relationship with archaeal GAPNs and glyceraldehyde dehydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukaryotes.