Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.     

Diterpenes from the leaves of Croton zambesicus

Two new trachylobane- and one isopimarane-type diterpenoids: ent-18-hydroxy-trachyloban-3-one; ent-trachyloban-3-one; isopimara-7,15-dien-3beta-ol, were isolated from the leaves of Croton zambesicus, together with trans-phytol, beta-sitosterol, alpha-amyrin and stigmasterol. The structures were determined by extensive NMR techniques and X-ray analysis. The cytotoxicity of these compounds has been evaluated on cancer and non-cancer cell-lines.     

A two-dimensional proteome map of maize endosperm

We have established a proteome reference map for maize (Zea mays L.) endosperm by means of two-dimensional gel electrophoresis and protein identification with LC-MS/MS analysis. This investigation focussed on proteins in major spots in a 4-7 pI range and 10-100 kDa M(r) range. Among the 632 protein spots processed, 496 were identified by matching against the NCBInr and ZMtuc-tus databases (using the SEQUEST software). Forty-two per cent of the proteins were identified against maize sequences, 23% against rice sequences and 21% against Arabidopsis sequences. Identified proteins were not only cytoplasmic but also nuclear, mitochondrial or amyloplastic. Metabolic processes, protein destination, protein synthesis, cell rescue, defense, cell death and ageing are the most abundant functional categories, comprising almost half of the 632 proteins analyzed in our study. This proteome map constitutes a powerful tool for physiological studies and is the first step for investigating the maize endosperm development.     

Self-incompatibility genotypes in almond re-evaluated by PCR,stylar ribonucleases,sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S₅ and S₁₀ alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S₅ allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S₂₅ This new S allele, previously reported to be S₁₀, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S₂S₂₅), Ardechoise (S₁S₂₅), Desmayo Largueta (S₁S₂₅), Ferrastar (S₂S₂₅) and Gabaix (S₁₀S₂₅). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Description of two new species of Glypthelmins Stafford, 1905 (Digenea: Macroderoididae) in Rana spp. from Mexico, based on morphology and mtDNA and rDNA sequences

Glypthelmins Stafford, 1905 includes 29 putative species commonly found in the intestine and liver of anurans from all over the world but mainly in the Americas. Partial sequences of the cytochrome c oxidase subunit 1 ( cox 1), ribosomal internal transcribed spacer region 2 (ITS2) and the large subunit 28S rDNA gene were obtained and analysed using pairwise distance matrices and parsimony methods in order to characterise the interrelationships between 14 isolates of four nominal species of Glypthelmins recognised on morphological grounds. The highest intra-specific sequence divergence occurred in the cox 1 (18.53%) sequence, followed by that of the ITS2 (5.44%) and 28S (4.63%). Genetic variability was detected between the three isolates originally identified as G. facioi Brenes et al., 1959 from two localities in Mexico and one locality in Costa Rica. Sequence divergence exhibited among these isolates ranged from 10.70 to 11.22%, from 0.48 to 0.97% and from 1.33 to 1.88% for cox 1, ITS2 and 28S, respectively. Phylogenetic analysis combining all three data-sets generated a single most parsimonious tree. The three isolates of G. facioi form a clade, with an isolate collected from frogs in Veracruz State as the sister group to an isolate from Tabasco State + G. facioi from Costa Rica. The information derived from pairwise distance of independent data-sets plus the phylogenetic information indicate that each of the two isolates from Mexico, identified a priori as G. facioi, represent separate species. A re-examination of specimens was carried out and a re-evaluation made of the morphological characters to find reliable differences that had been overlooked. As a consequence, G. brownorumae n. sp. from Tabasco and G. tuxtlasensis n. sp. from Veracruz are described based on molecular and morphological differences.