Physical mapping of a pollen modifier locus controlling self-incompatibility in apricot and synteny analysis within the Rosaceae

S-locus products (S-RNase and F-box proteins) are essential for the gametophytic self-incompatibility (GSI) specific recognition in Prunus. However, accumulated genetic evidence suggests that other S-locus unlinked factors are also required for GSI. For instance, GSI breakdown was associated with a pollen-part mutation unlinked to the S-locus in the apricot (Prunus armeniaca L.) cv. 'Canino'. Fine-mapping of this mutated modifier gene (M-locus) and the synteny analysis of the M-locus within the Rosaceae are here reported. A segregation distortion loci mapping strategy, based on a selectively genotyped population, was used to map the M-locus. In addition, a bacterial artificial chromosome (BAC) contig was constructed for this region using overlapping oligonucleotides probes, and BAC-end sequences (BES) were blasted against Rosaceae genomes to perform micro-synteny analysis. The M-locus was mapped to the distal part of chr.3 flanked by two SSR markers within an interval of 1.8?cM corresponding to ~364?Kb in the peach (Prunus persica L. Batsch) genome. In the integrated genetic-physical map of this region, BES were mapped against the peach scaffold_3 and BACs were anchored to the apricot map. Micro-syntenic blocks were detected in apple (Malus?×?domestica Borkh.) LG17/9 and strawberry (Fragaria vesca L.) FG6 chromosomes. The M-locus fine-scale mapping provides a solid basis for self-compatibility marker-assisted selection and for positional cloning of the underlying gene, a necessary goal to elucidate the pollen rejection mechanism in Prunus. In a wider context, the syntenic regions identified in peach, apple and strawberry might be useful to interpret GSI evolution in Rosaceae.     

Exohedral complexes of large fullerenes,a theoretical approach

The possibility of existence of exohedral organometallic complexes of fullerenes larger than C₆₀ in which their coordination can have η⁶ hapticity was studied from a theoretical point of view. Complexes containing C₇₀, C₇₄ or C₆₀ cages, as well as cyclopentadienyl (Cp), pentamethyl-cyclopentadienyl (Me₅Cp), benzene rings and hexamethyl-phenyl (Me₆Ph) fragments as ligands, were designed and studied. The results show that many of these molecules can be thermodynamically stable and can have electronic interesting behavior.     

Plasmid Transfer between Spatially Separated Donor and Recipient Bacteria in Earthworm-Containing Soil Microcosms

Most gene transfer studies have been performed with relatively homogeneous soil systems in the absence of soil macrobiota, including invertebrates. In this study we examined the influence of earthworm activity (burrowing, casting, and feeding) on transfer of plasmid pJP4 between spatially separated donor (Alcaligenes eutrophus) and recipient (Pseudomonas fluorescens) bacteria in nonsterile soil columns. A model system was designed such that the activity of earthworms would act to mediate cell contact and gene transfer. Three different earthworm species (Aporrectodea trapezoides, Lumbricus rubellus, and Lumbricus terrestris), representing each of the major ecological categories (endogeic, epigeic, and anecic), were evaluated. Inoculated soil microcosms, with and without added earthworms, were analyzed for donor, recipient, and transconjugant bacteria at 5-cm-depth intervals by using selective plating techniques. Transconjugants were confirmed by colony hybridization with a mer gene probe. The presence of earthworms significantly increased dispersal of the donor and recipient strains. In situ gene transfer of plasmid pJP4 from A. eutrophus to P. fluorescens was detected only in earthworm-containing microcosms, at a frequency of (symbl)10(sup2) transconjugants per g of soil. The depth of recovery was dependent on the burrowing behavior of each earthworm species; however, there was no significant difference in the total number of transconjugants among the earthworm species. Donor and recipient bacteria were recovered from earthworm feces (casts) of all three earthworm species, with numbers up to 10(sup6) and 10(sup4) bacteria per g of cast, respectively. A. trapezoides egg capsules (cocoons) formed in the inoculated soil microcosms contained up to 10(sup7) donor and 10(sup6) recipient bacteria per g of cocoon. No transconjugant bacteria, however, were recovered from these microhabitats. To our knowledge, this is the first report of gene transfer between physically isolated bacteria in nonsterile soil, using burrowing earthworms as a biological factor to facilitate cell-to-cell contact.     

Soil factors determining the change in forests between dry and wet Chacos

This work consists of an extensive study of soil properties and floristic composition of the Paraguayan Chacoan forests. The role of the soil factors was determined conditioning the classical landscape differentiation between the dry western Chaco and the wet eastern Chaco. Soil horizon A best explains the previous classification of the forests. It highlights the soil differences between the eastern and western Chaco. CCA ordination including all forest, soil and geographical factors primarily arranged the plant associations along a W-E gradient. A significant relationship was found between the composition of the northern Paraguayan Chaco forest and geographic longitude, drainage and altitude. CCA ordination focusing on alluvial forests and soil features identified clay content as the primary soil factor discriminating forest types; it also underlined the importance of cation exchange and the content of exchange bases in the soil for differentiating dry and wet Chacoan forests.     

Influence of power supply in the feasibility of Phaeodactylum tricornutum cultures

The influence of fluid-dynamic conditions on the yield of Phaeodactylum tricornutum microalgal cultures was analyzed in two stages: first, the influence of air flow rate; second, the influence of using fluid-moving pumps for recirculating the culture. With respect to the air flow rate, the yield of the cultures increased with the aeration rate up to values of 2.0 v/v/min, then stress was observed and the yield of the cultures decreased. With respect to the influence of mechanical power supply for liquid impulsion, three different types of pumps--centrifugal, pulse, and peristaltic--were essayed at different power supplies. The cultures were stressed for the three types of pumps essayed. For each pump, the higher the power supply the lower was the Fv/Fm value and the higher was the stress at which cells were exposed. The highest measured stress was when the culture was moved with the centrifugal pump. Despite measured stress, for all the experiments stable steady states were reached, thus indicating that cells reduced their yield but did not die, as was verified by cell viability measurements. It was observed that the increase of the power supply improved the frequency of light exposition thus enhancing the yield of the cultures. However, the higher the power supply, the lower the microeddy length scale; therefore, stress could appear. Data demonstrated that the microeddy length scale was always much higher than cell size and therefore the turbulence was not responsible for stress. Also, the mass transfer was discarded as responsible for yield reduction. It was concluded that the shear rate was the factor determining the existence of stress phenomena. The evaluation of these shear rates demonstrated that values above 30-80 s(-1) damaged the cells strongly. These data were verified in an outdoor pilot-scale tubular photobioreactor that was implemented with the same type of pumps, thus demonstrating the necessity to take into account this factor in the design and scale-up of microalgal photobioreactors.     

Hydrogen bonding is the prime determinant of carboxyl pKa values at the N-termini of alpha-helices

Experimentally determined mean pK(a) values of carboxyl residues located at the N-termini of alpha-helices are lower than their overall mean values. Here, we perform three types of analyses to account for this phenomenon. We estimate the magnitude of the helix macrodipole to determine its potential role in lowering carboxyl pK(a) values at the N-termini. No correlation between the magnitude of the macrodipole and the pK(a) values is observed. Using the pK(a) program propKa we compare the molecular surroundings of 18 N-termini carboxyl residues versus 233 protein carboxyl groups from a previously studied database. Although pK(a) lowering interactions at the N-termini are similar in nature to those encountered in other protein regions, pK(a) lowering backbone and side-chain hydrogen bonds appear in greater number at the N-termini. For both Asp and Glu, there are about 0.5 more hydrogen bonds per residue at the N-termini than in other protein regions, which can be used to explain their lower than average pK(a) values. Using a QM-based pK(a) prediction model, we investigate the chemical environment of the two lowest Asp and the two lowest Glu pK(a) values at the N-termini so as to quantify the effect of various pK(a) determinants. We show that local interactions suffice to account for the acidity of carboxyl residues at the N-termini. The effect of the helix dipole on carboxyl pK(a) values, if any, is marginal. Backbone amide hydrogen bonds constitute the single biggest contributor to the lowest carboxyl pK(a) values at the N-termini. Their estimated pK(a) lowering effects range from about 1.0 to 1.9 pK(a) units.     

Macrophages and T cells do not express Mlsa determinants

In order to test the tissue distribution of Mlsa determinants, we have generated highly purified stimulator cell populations. First, Mlsa expression in bone marrow derived macrophages (M phi) of Mlsa genotype was tested in primary MLR and on Mlsa-specific T cell hybridomas (THy). Second, a similar experimental approach was used to analyze thioglycolate, peptone or Con A elicited peritoneal M phi. In all cases, these M phi cell populations were able to generate an excellent alloresponse, whereas no functional Mlsa determinants could be detected. Third, to further investigate whether the expression of Mlsa is lymphocyte specific, but dependent on expression of class II molecules, we have transfected I-Ek alpha and beta cDNA into a panel of thymomas of Mlsa genotype. Although we achieved a high level of surface I-Ek expression in all of these T cell tumors, none of them was able to trigger the Mlsa-specific THy. These results strongly suggest that Mlsa expression is limited to B cells. It is likely that Mlsa is a tissue-specific self-peptide that associates with class II molecules.     

T-lymphocyte behaviour modification in vitro by cancer patient sera

Cell mediated immune response blocking factor(s) in the serum of colon adenocarcinoma patients inhibit blast transformation of lymphocytes from normal individuals. The blocking capacity of the sera has been shown to correlate with the infiltration of the tumor. This correlation suggests that these phenomena may be mediated by identical serum factors and through a common cell receptor present in the lymphocytes of normal individuals.