Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy

Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.     

Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.     

Powdery mildew of tobacco (Erysiphe cichoracearum DC.)

Percentage germination, and growth of hyphae from single conidia of Erysiphe cichoracearum DC., were measured on leaf discs from topped and intact tobacco plants, grown in aerated nutrient solutions consisting of basal medium plus large or small amounts of potassium. The effect of supplying sodium was also studied. Discs were incubated on water and on 10% sucrose solution. Changes in free amino nitrogen and carbohydrate in comparable uninfected leaf discs, before and after incubation, were also measured. Potassium deficiency resulted in more free amino nitrogen and soluble carbohydrate and less insoluble carbohydrate, per cm.² of leaf. Spore germination was not greatly affected by treatments, though it was usually less on discs from potassium-deficient leaves. The pathogen grew slower on potassium-deficient leaf discs, whether they were incubated on water or on sucrose. Incubating discs from some leaves on sucrose, compared with water, gave greatly increased sugar content and less fungal growth; discs from other leaves had a much smaller increase in sugar, and hyphal length was similar to that on discs incubated on water. Sodium, when potassium was scarce, increased potassium deficiency symptoms, free amino nitrogen and sugar content, and resistance to powdery mildew.     

The effects on a population of potato-root eelworm (Heterodera rostochiensis) of growing potatoes resistant to pathotype B

The effects on a potato-root eelworm (Heterodera rostochiensis Woll.) population of growing potatoes resistant to pathotype B of the eelworm were investigated. The eelworm population, which was originally nearly entirely pathotype A, had been changed into a mixture of pathotypes A, B and C before 1961, when the experiments described in this paper began, by growing potatoes resistant only to pathotype A. The resistant potatoes grown in the years 1961 and 1962 were certain clones of the hybrid triploid species Solarium xjuzepczukii Buk., clones bred from S. multidissectum Hawkes with the gene H₂ and clones bred from S. vernei Bitt. et Wittm. In the years 1963-65 the clone used was D 40 which has both the gene H₂ from Andigena and the gene H₂ from 5. multidissectum and is resistant to both pathotypes A and B of the eelworm. The only potatoes which caused a reduction in the egg population were those bred from S. vernei. The clone D 40, although it had little effect on the egg population, appeared to have more resistance than could be accounted for from possessing genes H₁ and H₂.     

Pectic Enzymes and Phenolic Substances in Apples Rotted by Fungi

The activities of pectic enzymes in extracts from sound applesand from apples rotted by different fungi are described. Sclerotiniafructigenaand Botrytis cinerea rots had little or no polygalacturonaseor macerating enzyme activity, but Penicillium expansum rotswere very active in these respects. Extracts from each of therots had very high pectinesterase activity, and contained galacturonicacid. None of the rots had any cellulase activity. Each of thefungi produced polygalacturonase, macerating enzymes, and pectinesterasein liquid media. The effects of adding extracts of apples tothese media are described. Filtrates from cultures of S. fructigenaand P. expansum liberated galacturonic acid from apple fruitfibre which had been thoroughly extracted with cold water. The phenolic jsubstances present in healthy and rotted tisueswere estimated. B. cinerea and S. fructigena rots containedvery little, but P. expansum rots contained as much as healthytissue which had been allowed to brown. An extract of healthyapple tissue reduced the activity of the polygalacturonase ina culture filtrate of S. fructigena. The substances responsiblefor this were tentatively identified as leuco-anthocyanins whichhad been changed to other compunds following the action of polyphenoloxidase.Thej significance of these results is discussed.     

Root Aeration and Respiration in Young Mangrove Plants (Avicennia marina (Forsk.) Vierh.)

The roots of young plants of Avicennia marina (Forsk.) Vierh.grown under simulated tidal conditions were harvested so asto obtain the entire root system. The roots were subdividedand weighed and subsamples taken for manometric determinationof respiration rates at different temperatures. The supply capacityof the above-ground portion of the root system was determinedand the results compared in terms of supply and demand. Theoxygen consumption rate of the roots at 15°C was found tobe 1·69±0·07 µmol kg^–1 s^–1for cable roots and 3·27±0·12 µmolkg^–1 s^–1 for fine roots. The Q₁₀ for respirationwas 2·55 for oxygen consumption in both fine and cableroots, and for carbon dioxide production was 2·66 forfine roots and 3·04 for cable roots. The respiratoryquotient varied with temperature but was less than unity. Concentrationdifferences of between 1·8 mol m^–3 and 3·4mol m^–3 between the inside of root and the air were sufficientto permit aeration of the root system by diffusion alone, andthe aerenchyma contained sufficient oxygen to maintain aerobicconditions while the roots were covered with water. The effectof tide and seasonal temperature change on gas exchange, togetherwith the possibility of some form of carbon dioxide fixationwithin the root, are examined and the implications of theseeffects on growth and development are discussed. Key words: Mangrove, root aeration, respiration, aerenchyma     

Contributions of Electron Microscopy to Fungal Classification

Such characters as surface ultrastructure of asexual and sexualpropagules, wall and septal ultrastructure, the fine structureof ascal tips, and ultrastructural aspects of nuclear divisionhave taxonomic significance for major groups of fungi. Informationderived from fine-structural analyses can be correlated withthat obtained from light-microscopic, chemical, and developmentalinvestigations. The versatility of electron-microscopic facilitiesmakes them powerful research tools in the hands of the innovativetaxonomist.     

Microevolutionary analysis of Clostridium difficile genomes to investigate transmission

Background

The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing.

Results

Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions.

Conclusions

Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.