Bypassing reproductive barriers in hybrid seeds using chemically induced epimutagenesis

The triploid block, which prevents interploidy hybridizations in flowering plants, is characterized by a failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed genes (PEGs), which are upregulated in tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in the CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of “epimutagenized” plants, which may allow epialleles involved in the triploid block response to be identified in future studies. Finally, we demonstrate that chemically induced epimutagenesis facilitates hybridization between different Capsella species, thus potentially emerging as a strategy for producing triploids and interspecific hybrids with high agronomic interest.

Genome-wide loss of DNA methylation induced by 5-Azacytidine allows interploidy and interspecific hybridization barriers to be bypassed in Arabidopsis and Capsella.     

The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state

MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus- intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C- terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.      

Complex interactions on fig trees: ants capturing parasitic wasps as possible indirect mutualists of the fig–fig wasp interaction

Like other mutualisms, pollination mutualisms attract parasites, as well as opportunistic and specialist predators of the pollinators and parasites. These associated species influence the evolutionary dynamics of pairwise mutualisms. Predatory ants are frequent associates of pollination mutualisms, but their effects on the complex interactions between plants, pollinators and parasites have not yet been clearly established, even in the case of the well-described obligate interaction between figs and fig wasps. We attempted to quantify such effects for ants associated with three fig species, two dioecious ( Ficus condensa [Bruneï], F . carica [France]) and one monoecious ( F . racemosa [India]). In all these cases, ant presence on a fig tree strongly reduced the number of parasitic wasps on the figs. Experimental exclusion of ants resulted in an increase in the number of non-pollinating fig wasps on F . condensa and F . racemosa . Experimental ant supplementation led to a decrease in the number of non-pollinating fig wasps on F . carica . Moreover, on F . condensa , the level of reduction of the number of parasitic wasps depended on the number and identity of the ants. On F . carica , non-pollinating fig wasps even avoided trees occupied by the dominant predatory ant. The consistency of the effect of ants in these three cases, representing a geographically, ecologically, and taxonomically broad sample of figs, argues for the generality of the effect we observed. Because reduction of parasitism benefits the pollinator, ants may be considered as indirect mutualists of plants and pollinators in the network of complex interactions supported by fig trees.     

Adrenaline induced changes in the metabolism of glycogen in the teleost retina

A study on the influences of adrenaline upon the metabolism of glycogen in fish retina is presented. The glycogen in the retina of the fish Eugerres plumieri amounted to approximately 7·5 mg glucose/10 g tissue wet weight of which 5·8+0±81 S.D. was present in the lyo-form and 1·7+0·41 S.D. in the desmo-form. The phosphorylase A activity in this tissue was found to be 36·5 μg P/g tissue wet weight/min, which represents 68 % of the total phosphorlyase activity. A definite effect of administered adrenaline upon the retinal glycogen could be observed only when conditions facilitating the penetration of this substance into the retina were given. A dose of 100 μg/kg body weight of adrenaline injected to the animals previously pretreated with 10 mg/kg of isocarboxazid and 20 ml of 20% v/v aqueous solution of ethanol showed a significant increase in phosphorylase activity and a close to 50% drop in the retinal lyo-glycogen. The highest penetration of adrenaline into the retina was observed 20 to 40 min after the injection of adrenaline into a fish pretreated with isocarboxazid 4 h and ethanol 20 min prior to the administration of the amine. Under these conditions the glycemia went up from 70mg% to 90mg% and the MAO-activity dropped from a value of 85 ug 4 HOQ/g tissue wet weight/30 min to zero.     

Some properties of phenylethanolamine-N-methyltransferase of rat brain

Phenylethanolamine-N-methyltransferase (PNMT, EC 2.1.1.28) was partially purified from rat brain. Brain homogenates were subjected to ultracentrifugation, salt fractionation, and gel filtration on Sephadex G-100. To compare the rat brain PNMT with that of adrenals, the same procedure was carried out with rat adrenal homogenates. The brain enzyme was eluted from Sephadex as a single fraction with a molecular weight of 26,900, while the enzyme from adrenals under the same conditions appeared in two fractions with molecular weights of 38,700 and 108,500. The brain fraction separated on Sephadex G-100 was active on phenylethanolamine substrates and inactive on indoleamine and phenylethylamine substrates. Products of the enzyme reaction were identified by bidimensional thin-layer chromatography asN-methyl derivatives of the corresponding amines. Kinetic studies showed that the type of inhibition of PNMT from rat brain and rat adrenals by SK&F 7698 was the same as described for PNMT from rabbit adrenals. Also, when normetanephrine andS-adenosyl-l-methionine were used as substrates, the apparentK _m values found with PNMT from rat adrenals and rat brain were similar.Preliminary reports were presented at XXV Convención Anual AsoVAC, Caracas, Venezuela, October 1975, and at XII Congreso Latinoamericano de Ciencias Fisiológicas, Bogotá, Colombia, November 1975.     

Comparison of the effects of concentration, pH and anion species on astringency and sourness of organic acids

The separate effects of concentration, pH and anion species on intensity of sourness and astringency of organic acids were evaluated. Judges rated sourness and astringency intensity of lactic, malic, tartaric and citric acid solutions at three levels of constant pH varying in normality and at three levels of constant concentration varying in pH. To assess the comparative sourness and astringency of the organic acid anions of study, binary acid solutions matched in pH and titratable acidity were also rated. As pH was decreased in equinormal solutions, both sourness and astringency increased significantly (P < 0.001). By contrast, as the normality of the equi-pH solutions was increased, only sourness demonstrated significant increases (P < 0.001) while astringency remained constant or decreased slightly. At the lowest normality tested, all solutions were more astringent than sour (P < 0.05). Although lactic acid was found to be significantly more sour than citric acid (P < 0.05), no other sourness or astringency differences among the organic acid anions were noted. This study demonstrates for the first time that astringency elicited by acids is a function of pH and not concentration or anion species, and confirms that sourness is independently influenced by concentration, pH and anion species of the acid.      

Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction

A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426-4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain two FRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomal adhE gene in strains of E. coli K-12 and E. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.     

Post-embryonic development of Ophyra aenescens (Wiedemann, 1830) (Diptera: Muscidae) with different diets, under laboratory conditions

The performance of various diets (bovine meat, fish-sardine, shrimp, dog faeces, and banana) in Ophyra aenescens development was evaluated. The biology was studied in an incubator (BOD) at 27 +/- 1 degrees C and 80 +/- 10% of RH. The developmental time from larvae to adult, the developmental time and viability of larvae and pupae, the weight of pupae as well as the sex ratio of the emerging adults were also determined. Beef and shrimp were the more efficient diets for rearing O. aenescens.     

Exposing the confounding in experimental designs to understand and evaluate them,and formulating linear mixed models for analyzing the data from a designed experiment

Comparative experiments involve the allocation of treatments to units, ideally by randomization. This necessarily confounds treatment information with unit information, which we distinguish from the other forms of information blending, in particular aliasing and marginality. We outline a factor-allocation paradigm for describing experimental designs with the aim of (i) exhibiting the confounding in a design, using analysis-of-variance-like tables, so as to understand and evaluate the design and (ii) formulating a linear mixed model based on the factor allocation that the design involves. The approach exhibits the dispersal of treatments information between units sources, allows designers a choice in the strategy that they adopt for including block-treatment interactions, clarifies differences between experiments, accommodates systematic allocation of factors, and provides a consolidated analysis of nonorthogonal designs. It provides insights into the process of designing experiments and issues that commonly arise with designs. The paradigm has pedagogical advantages and is implemented using the R package dae .