A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

Production of trace levels of carbon monoxide during methanogenesis on acetate and methanol

Summary Production of trace levels of carbon monoxide was consistently observed in the off-gas of a laboratory anaerobic digester fed Waste Activated Sludge. Inocula from this digester was enriched for acetate and methanol utilizing methanogenic populations. These enriched inocula were then monitored in batch assays for carbon monoxide and hydrogen production. Results demonstrated that carbon monoxide is produced during methanogenesis on both substrates. Subsequent utilization of CO was observed to occur after methane production was essentially complete for the assays conducted with methanol. Carbon monoxide evolution during methanogenesis on acetate displayed a markedly different trend from that observed from methanol.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Development and characterization of continuous avian cell lines depleted of mitochondrial DNA

Summary Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy. This work was supported by a grant from the Medical Research Council of Canada.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

M orais , P.V. & D a C osta , M.S. 1990. Alterations in the major heterotrophic bacterial populations isolated from a. still bottled mineral water. Journal of Applied Bacteriology 69 , 750–757.
The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9–12 months. The plate counts in R₂A medium incubated at 22 and 37C were low initially, increasing to 10⁴-10⁵ cfu/ml within a few days of bottling. The number of bacteria recovered at 22C from PVC bottles was fairly constant during the storage period, but the population isolated at 37C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis . Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Computer-aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS-PAGE of wholecell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating that populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.     

Yeast communities as descriptors of habitat use by the Drosophila fasciola subgroup (repleta group) in Atlantic rain forests

Yeast communities associated with four species of the Drosophila fasciola subgroup (repleta group) in tropical rain forests were surveyed in an abandoned orchard, and rain forest sites of Rio de Janeiro and Ilha Grande, State of Rio de Janeiro, Brazil. Adult flies of Drosophila carolinae, Drosophila coroica, Drosophila fascioloides and Drosophila onca frequently carried Candida colliculosa, Geotrichum sp, Kloeckera apiculata and a Pichia membranaefaciens-like species. The most frequent yeasts in the crop of flies included Candida collicullosa, C. krusei, Pichia kluyveri and a P. membranaefaciens-like species. The physiological abilities and species composition of these yeast communities differed from those of other forest-inhabiting Drosophila. The narrow feeding niches of the fasciola subgroup suggested the use of only part of the substrates available to the flies as food in the forest environment, as noted previously for cactophilic Drosophila serido (mulleri subgroup of the repleta group) in a sand dune ecosystem. The cactophilic yeasts that were isolated have not been previously found in forests. The fasciola subgroup probably used epiphytic cactus substrates as breeding and feeding sites in the forest. The physiological profile of yeasts associated with the fasciola flies was broader than that of yeasts associated with the cactophilic Drosophila serido, suggesting that the fasciola subgroup represents an older lineage from which the South American repleta species evolved.