Development of a set of PCR-based anchor markers encompassing the tomato genome and evaluation of their usefulness for genetics and breeding experiments

Tomato and potato expressed sequence tag (EST) sequences contained in the solanaceae genomics network (SGN) database were screened for simple sequence repeat (SSR) motifs. A total of 609 SSRs were identified and assayed on Solanum lycopersicum LA925 (formerly Lycopersicon esculentum) and S. pennellii LA716 (formerly L. pennellii). The SSRs that did not amplify, gave multiple band products, or did not exhibit a polymorphism that could be readily detected on standard agarose gels in either of these species were eliminated. A set of 76 SSRs meeting these criteria was then placed on the S. lycopersicum (LA925) x S. pennellii (LA716) high-density map. A set of 76 selected cleaved amplified polymorphism (CAP) markers was also developed and mapped onto the same population. These 152 PCR-based anchor markers are uniformly distributed and encompass 95% of the genome with an average spacing of 10.0 cM. These PCR-based markers were further used to characterize S. pennellii introgression lines (Eshed and Zamir, Genetics 141:1147-1162, 1995) and should prove helpful in utilizing these stocks for high-resolution mapping experiments. The majority of these anchor markers also exhibit polymorphism between S. lycopersicum and two wild species commonly used as parents for mapping experiments, S. pimpinellifolium (formerly L. pimpinellifolium) and S. habrochaites (formerly L. hirsutum), indicating that they will be useful for mapping in other interspecific populations. Sixty of the mapped SSRs plus another 49 microsatellites were tested for polymorphism in seven tomato cultivars, four S. lycopersicum var. cerasiforme accessions and eight accessions of five different wild tomato species. Polymorphism information content values were highest among the wild accessions, with as many as 13 alleles detected per locus over all accessions. Most of the SSRs (90%) had accession-specific alleles, with the most unique alleles and heterozygotes usually found in accessions of self-incompatible species. The markers should be a useful resource for qualitative and quantitative trait mapping, marker-assisted selection, germplasm identification, and genetic diversity studies in tomato. The genetic map and marker information can be found on SGN (http://www.sgn.cornell.edu).     

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of S?o Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.     

Bis(3-hydroxy-4-pyridinone)-EDTA derivative as a potential therapeutic Al-chelating agent. Synthesis, solution studies and biological assays

The 3-hydroxy-4-pyridinones are chelating agents of current interest due to their high affinity for hard metal ions and potential clinical applications as metal-decorporation agents. A new bis-(3-hydroxy-4-pyridinone) derivative of EDTA have been developed, and herein we describe the results of solution studies to determine the protonation constants and the partition coefficient. Biodistribution studies, performed with 67Ga-overload mice, showed rapid clearance of the radiotracer from the body, thus indicating that the new ligand should be a quite effective agent for the in vivo aluminium removal.     

A phytochemical study of the quinolizidine alkaloids from Genista tenera by gas chromatography-mass spectrometry

Gas chromatography coupled with mass spectrometry has been used to analyse the alkaloids present in the aerial parts of Genista tenera. Anagyrine, cytisine, N-formylcytisine, N-methylcytisine and lupanine were the major compounds, the last two alkaloids being known for their hypoglycaemic activity. Dehydrocytisine, 5,6-dehydrolupanine, rhombifoline, aphylline and thermopsine were the minor alkaloids. The characterisation of the constituents was based on comparison of their Kovats retention indexes and electron impact-mass spectrometric data recorded on-line with those of reference compounds and literature data.     

Proteolytic modification of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B-512F dextransucrase

Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209–Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.Martha Argüello-Morales and Mónica Sánchez-González equally contributed to this work.     

Role of histone tails in the conformation and interactions of nucleosome core particles

The goal of this work was to test the role of the histone tails in the emergence of attractive interactions between nucleosomes above a critical salt concentration that corresponds to the complete tail extension outside the nucleosome [Mangenot, S., et al (2002) Biophys. J. 82, 345-356; Mangenot, S., et al (2002) Eur. Phys. J. E 7, 221-231]. Small angle X-ray scattering experiments were performed in parallel with intact and trypsin tail-deleted nucleosomes with 146 +/- 3 bp DNA. We varied the monovalent salt concentration from 10 to 300 monovalent salt concentration and followed the evolution of (i) the second virial coefficient that characterizes the interactions between particles and (ii) the conformation of the particle. The attractive interactions do not emerge in the absence of the tails, which validates the proposed hypothesis.     

Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

A functional analysis of the crown architecture of tropical forest<Emphasis Type="Italic"> Psychotria</Emphasis> species: do species vary in light capture efficiency and consequently in carbon gain and growth?

The crown architectures of 11 Psychotria species native to Barro Colorado Island, Panama were reconstructed from field measurements of leaf and branch geometry with the three-dimensional simulation model Y-plant. The objective was to assess the role of species differences in architecture in light capture and carbon gain in their natural understory environment. When species were grouped according to their putative light environment preference, the shade tolerant species were found to have a small but significantly higher efficiency of light capture for both diffuse and direct light as compared to the light demanding species. Within each grouping, however, there were few significant differences in light capture efficiency among species. The lower efficiencies of light demanding species was due to slightly higher self-shading and slightly lower angular efficiencies. Simulations of whole plant assimilation showed that light demanding species had greater daily assimilation in both direct and diffuse light due to the significantly greater light availability in the sites where light demanding species were found, as compared to those where shade tolerant species occurred. Among light demanding species, the above ground relative growth rate measured over a 1-year period by applying allometric equations for mass versus linear dimensions, was positively correlated with diffuse PFD and with mean daily assimilation estimated from Y-plant. For the shade tolerant plants, there was no significant correlation between RGR and mean daily assimilation or with any measure of light availability, probably because they occurred over a much narrower range of light environments. Overall, the results reveal a strong convergence in light capture efficiencies among the Psychotria species at lower values than previously observed in understory plants using similar approaches. Constraints imposed by other crown functions such as hydraulics and biomechanical support may place upper limits on light capture efficiency.Abbreviations E_a Efficiency of light absorption (dimensionless) - E_adir Efficiency of direct light absorption (dimensionless) - E_adif Efficiency of diffuse light absorption (dimensionless) - D_E Display efficiency (dimensionless) - P_E Projection efficiency (dimensionless) - CosI Mean cosine of incidence (dimensionless) - aLAR_e Effective leaf area ratio (m² g^–1) - A_tot Daily assimilation (mmol m^–2 day^–1) - A_dir Daily assimilation in direct PFD (mmol m^–2 day^–1) - A_dif Daily assimilation in diffuse PFD (mmol m^–2 day^–1)