Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.     

Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Influences of habitat features and human disturbance on use of breeding sites by a declining population of southern fur seals (Arctocephalus australis)

Southern fur seals Arctocephalus australis in Peru have declined gradually over the past decade, and declined dramatically (72%) as a result of low food availability during the severe El Niño in 1997–98. In 1999, seals abandoned some historically important breeding sites. This is particularly alarming because new sites were not colonized. Our objective was to examine how habitat features and human disturbance influenced whether sites were currently used, abandoned or apparently not used in the past by fur seals for breeding. Data were collected on 14 variables at 70 potential breeding sites at three guano reserves in Peru. Discriminant analysis revealed significant multivariate differences among sites currently used for breeding, abandoned sites and unused sites ( F =5.97, P <0.00001), and the model classified 74% of sites correctly. Currently used sites were less likely to have human disturbance and more likely to have offshore islands, stacked rocks, tide pools and abundant shade. Separate discriminant analyses for each reserve produced similar results. Habitat associated with thermoregulation (e.g. shade or pools) may be more important to fur seals in Peru, which breed at lower latitudes and are at greater risk of overheating on land than other populations. Habitat with minimized human access may be especially important to seals in small populations in which individuals may perceive themselves as more vulnerable because of decreased vigilance and dilution effects. Seals in our study selected breeding habitat with stacked rocks, which create shade and tide pools for thermoregulation and make human access difficult; but pups might suffer higher mortality in this habitat. We hypothesize that fur seals in Peru may exhibit an Allee effect, whereby suitability of habitat varies with population abundance.     

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION1

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A₀, A₁ and Fe-S_X. Fe-S_X is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-S_X is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-S_A/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.Dedicated to the memory of Daniel I. Arnon.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones

Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×10⁸; 1.2×0⁹ bacteria ml^-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical -glucuronidase assays, and Southern blot hybridization analyses.Abbreviations BA benzyladenine - GUS -glucuronidase - IBA indolebutyric acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - nptII neomycin phosphotransferase II gene - uidA -glucuronidase gene - WPM Woody Plant Medium     

Sequence analysis of an actin isoform in the flatworm Diphyllobothrium dendriticum (Cestoda)

We have examined actin cDNA of the flatworm Diphyllobothrium dendriticum (Cestoda). Actin is a contractile protein that has been implicated in a variety of developmental and cellular processes. It is highly conserved and present in all eukaryotic cells. It is of particular interest to analyze evolutionary preserved genes in flatworms, because ancestral flatworms are regarded to play a central role in the evolution of the metazoans (Barnes et al., 1998). Screening a cDNA library of D. dendriticum (UniZap XR, Stratagene) with a human -actin probe resulted in several positive clones. One of the cDNA inserts, Didactl, consisting of 1392 bp was completely sequenced. The established nucleotide sequence revealed a 5 untranslated region of 33 bp, the entire open reading frame of 1128 bp and a 3 untranslated region of 231 bp which ends in a stretch of 21 A residues. The potential polyadenylation signal (AATAAA) is located 14 bp upstream of the poly (A) tail. The deduced amino acid sequence of Didactl is 376 amino acids long. It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins. Didactl is for example 96% homologous to human cytoplasmic -actin but only 92.6% identical with human smooth muscle -actin. The actin proteins are generally encoded by a multigene family which differs in size from species to species. Most organisms have four to eight genes coding for actin in their genome, but the number of actin genes can also be over 20 (Hamelin et al., 1988). Sequence comparisons of Didactl and the partly sequenced cDNA clones indicate that D. dendriticum has at least four different genes coding for actin in its genome.