Synthesis of novel tyrosinyl FRET cassettes, terminators, and their potential use in DNA sequencing

Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.     

Culture-based and non-growth-dependent detection of the Burkholderia cepacia complex in soil environments

Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens. A total of 91 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22 degrees C. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc. The four soil samples from which these clones were generated did not yield isolates identified as Bcc. Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bcc may underestimate environmental populations.     

Myosin and Ca2+-sensitive streaming in the alga Chara: detection of two polypeptides reacting with a monoclonal anti-myosin and their localization in the streaming endoplasm

A monoclonal antibody to the heavy chain of myosin from mouse 3T3 cells was used to detect and localize related proteins in the green alga Chara. Proteins of 200,000 and 110,000 Mr reacted on immunoblots of proteins precipitated rapidly with trichloroacetic acid to minimize proteolysis. Immunofluorescence of whole cells localized these proteins to organelles of the streaming endoplasm, to a system of endoplasmic strands and to the subcortical actin bundles. Except that fewer endoplasmic strands and organelles were found and the strands were tangled, the localization pattern was similar in cells rapidly perfused to remove the bulk of the streaming endoplasm. Actin was confined almost entirely to the system of subcortical actin bundles in both whole and perfused cells. Myosin that was associated with the tangled endoplasmic strands but not that associated with the organelles or actin bundles was removed by concentrations of Ca2+ inhibiting ATP-dependent streaming in perfused cells. ATP extracted both organelles and endoplasmic strands but left a continuous pattern of myosin immunostaining along the actin bundles. The findings are discussed in relation to the possible existence of two forms of myosin and of separate mechanisms moving the bulk endoplasm and individual organelles.     

The protective effects of L-ascorbic acid and DL-alpha-tocopherol on cultured rat embryos treated with xanthine/xanthine oxidase

Abnormalities of the neural suture were observed in cultured rat embryos exposed to oxygen radicals generated by xanthine and xanthine oxidase. The distribution of the severity of these abnormalities was altered by the addition of L-ascorbic acid (AA) or DL-alpha-tocopherol (AT). The antioxidant effect of AA and AT were probably responsible for the protection of the embryos from the damaging effects of oxygen radicals.     

Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii

Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii. Slow-growing B. japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M. The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well. Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response. Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate. In contrast, fast-growing R. trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B. japonicum. The R. trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B. japonicum strain.     

Assay of lactogenic hormones using receptors isolated from rabbit liver.

Using 125-I-labelled ovine prolactin and receptors isolated from the livers of rabbits, a sensitive method has been developed suitable for the assay of ovine, bovine, porcine, human and rat prolactins. These hormones showed competitive displacement of 125-I-ovine prolactin which was in general agreement with their respective activities in the pigeon crop sac bioassay. Human and monkey growth hormones and human placental lactogen, which have marked prolactin-like actions on mammary tissue were also effective competitors. Non-primate growth hormones (ovine, bovine, equine and canine) which do not have prolactin-like activity gave little if any displacement as did human FSH, LH, TSH, ACTH and bovine insulin. Preparations of equine and canine prolactin of varying purity gave dose-response curves although their activity as competitors relative to ovine prolactin was poor and not related to their pigeon crop stimulating activity. This indicates species differences between prolactins in hormone-receptor interaction. Experiments with antiserum to human growth hormone have suggested an effective method of making the assay specific in species such as man in which prolactin is not the sole hormone with lactogenic activity.     

Construction of mobilizable vectors derived from plasmids RP4, pUC18 and pUC19

Mobilizable narrow-host-range plasmids were constructed from pUC18 and pUC19 by addition of a segment of pSUP2021 bearing the basis of mobilization (bom) site and origin of transfer (oriT) of RP4. One pair of expression vectors, pARO180 and pARO190, retains the beta-lactamase (bla) gene and twelve of the 13 restriction enzyme multiple cloning sites (MCS) of pUC18/19. Another pair was created by replacing the bla gene with the gene encoding kanamycin resistance (kan) from Tn5. The molecules replicate to high copy number in Escherichia coli and Enterobacter aerogenes. They can be transferred efficiently to other Gram- bacteria from the mobilizing strain, E. coli S17-1. In non-enteric strains, the new plasmids can be used as suicide vectors in site-specific insertional mutagenesis.     

The metabolism of shikimate in the rat.

In the rat, shikimate was metabolized and excreted as hippurate, hexahydrohippurate, 3,4,5,6-tetrahydrohippurate, t-3,t-4-dihydroxycyclohexane-r-1-carboxylate and c-3,t-4-dihydroxycyclophexane-r-1-carboxylate, conjugates of catechol and CO2. The metabolism was entirely dependent on various initial microbial transformations in the gut, metabolite formation being suppressed in animals pretreated with antibiotics. Shikimate was not metabolized by mammalian tissues, and products of microbial metabolism were excreted either unchanged or after further biotransformation in the animal tissues.     

Use of cluster and discriminant analyses to compare rhizosphere bacterial communities following biological perturbation

We present an approach to comparing the diversity and composition of bacterial communities from different habitats and for identifying which members of a community are most affected by an introduced bacterium. We use this method to explore both previously published and new data from field and growth chamber experiments in which we isolated heterotrophic bacteria from samples of root-free soil, roots of nontreated soybean seedlings, and from the roots of soybean seedlings grown from Bacillus cereus UW85nl—treated seeds. We characterize bacterial isolates for 40 physiological attributes, and grouped the isolates hierarchically using two-stage density-linkage cluster analysis. Multivariate analysis of variance and discriminant analysis of the relative frequencies of the clusters in the soil and rhizosphere habitats were then used to determine whether there were differences among the bacterial communities from the various habitats, and which of the clusters were most useful in discriminating among the communities. We used rarefied estimates of richness as a measure of community diversity in the various habitats. Introduction of UW85n 1 affected the composition and/or diversity of rhizosphere communities in three of four experiments. Present address: Department of Environmental Science, Policy and Management, 108 Hilgard Hall, University of California, Berkeley, CA 94720 Correspondence to: G.S. Gilbert.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.