Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography

Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a C0t (moles of nucleotides times seconds per liter), or standard reassociation value. From this C0t value, C0t values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated C0t values, the Mr values of 1.96 X 10(9) for E. coli, 2.02 X 10(9) for B. alba, and 3.28 X 10(9) for S. coelicolor were estimated. Plasmid concentration was determined from cleared lysates by comparing the integrated area under the phosphate buffer-eluted plasmid peak to values obtained with known amounts of plasmid. The plasmid copy number was estimated by multiplying the ratio between the amounts of plasmid and chromosomal DNA by the ratio between the Mr values of the chromosome and the plasmid. A copy number of 29 was obtained from a culture of E. coli HB101 harboring pBR322 grown to a culture density of 1.6 X 10(9) CFU . ml-1.     

Cytokeratin provides a specific marker for human arachnoid cells grown in vitro

Cells from cranial and spinal arachnoid membranes of humans were grown in culture. Their growth characteristics, morphology and details of their cytoskeletal composition are described. Arachnoid membranes, obtained at autopsy, were finely minced and incubated in tissue culture medium. Monolayers of cells of homogeneous morphology grew from these tissue fragments. The cells were flat and polygonal. They divided slowly to form non-overlapping monolayers of low cell density. Electron microscopic examination of cultured arachnoid cells revealed numerous desmosome-like tight junctions and abundant intermediate filaments (tonofilaments). Both morphological features are characteristic of arachnoid cells in situ, but not of cells in the fibroblast-rich dura mater. Immunofluorescence microscopy with monoclonal antibodies demonstrated cytokeratin in the cytoplasm of primary cultures of arachnoid cells. Thus we demonstrated that these cultured cells retained certain of the specific differentiated properties of arachnoid cells in situ and that they are not fibroblasts (which lack tight junctions and cytokeratins). To our knowledge, there have been no previous reports of in vitro growth of arachnoid cells. This in vitro model should be useful in studying the response of arachnoid cells to a variety of substances thought to be involved in the chronic inflammatory condition of the meninges known as arachnoiditis.     

Effect of Selected Herbicides on Bacterial Growth Rates

Specific growth rate constants were used to evaluate the effects of selected herbicides on Erwinia carotovora, Pseudomonas fluorescens, and Bacillus sp. Comparison of growth rate constants permitted the identification of either stimulatory or inhibitory effects of these substances. E. carotovora was inhibited by 6,7-dihydrodipyrido(1,2-a:2'-c)pyrazinediium (diquat) and 4-hydroxy-3,5-diiodobenzonitrile (ioxynil) at 25 mug/ml; 1,1'-dimethyl-4,4'-bipyridinium (paraquat) at 50 mug/ml; and pentachlorophenol (PCP) at 10 mug/ml. P. fluorescens was inhibited by paraquat and PCP at 25 mug/ml and by 4-amino-3,5,6-trichloropicolinic acid (picloram) at 50 mug/ml. Stimulation of P. fluorescens was observed with 4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline (nitralin) at 25 mug/ml. The Bacillus species was inhibited by diquat (25 mug/ml), ioxynil (10 mug/ml), and paraquat and PCP (5 mug/ml). No significant effect of 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin), or 1,1-dimethyl-3-(alpha,alpha,alpha-trifluoro-m-tolyl)urea (fluometuron) on growth rates of the bacteria was observed at 25 and 50 mug/ml.     

Host tree resistance against the polyphagous wood-boring beetle Anoplophora glabripennis

Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae: Lamiini) is an invasive wood‐boring beetle with an unusually broad host range and a proven ability to increase its host range as it colonizes new areas and encounters new tree species. The beetle is native to eastern Asia and has become an invasive pest in North America and Europe, stimulating interest in delineating host and non‐host tree species more clearly. When offered a choice among four species of living trees in a greenhouse, adult A. glabripennis fed more on golden‐rain tree (Koelreuteria paniculata Laxmann) and river birch (Betula nigra L.) than on London planetree (Platanus × acerifolia (Aiton) Willdenow) or callery pear (Pyrus calleryana Decaisne). Oviposition rate was highest in golden‐rain tree, but larval mortality was also high and larval growth was slowest in this tree species. Oviposition rate was lowest in callery pear, and larvae failed to survive in this tree species, whether they eclosed from eggs laid in the trees or were manually inserted into the trees. Adult beetles feeding on callery pear had a reduced longevity and females feeding only on callery pear failed to develop any eggs. The resistance of golden‐rain tree against the larvae appears to operate primarily through the physical mechanism of abundant sap flow. The resistance of callery pear against both larvae and adults appears to operate through the chemical composition of the tree, which may include compounds that are toxic or which otherwise interfere with normal growth and development of the beetle. Unlike river birch or London planetree, both golden‐rain tree and callery pear are present in the native range of A. glabripennis and may therefore have developed resistance to the beetle by virtue of exposure to attack during their evolutionary history.