PCR bias toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work factor IX suggests that this assumption is invalid for one case of near-sequence identity To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify, wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For kras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods ape described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R, solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74-97, 455-475, 1454-1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division TI (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii, or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum, 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.

Detection of lead(II) and silver(I) in aqueous solution using a piezoelectric device

The gold surface of a quartz crystal microbalance was modified by the attachment of silica particles derivatised with N-[(3-trimethoxysilyl)propyl] ethylenediaminetriacetic acid. The device was employed to study the kinetics of the interaction of aqueous solutions of lead(II) nitrate and silver(I) nitrate with the surface and for the selective separation of the metal ions.

Engineered detoxification confers resistance against a pathogenic bacterium

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.

Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug

A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia, The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794(T) (= DSM 12717(T)).

A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: Phylogenetic, physiological, and preliminary biochemical studies

A previously unknown chemolithoautotrophic arsenite-oxidizing bacterium has been isolated from a gold mine in the Northern Territory of Australia. The organism, designated NT-26, was found to be a gram-negative motile rod with two subterminal flagella. In a minimal medium containing only arsenite as the electron donor (5 mM), oxygen as the electron acceptor, and carbon dioxide-bicarbonate as the carbon source, the doubling time for chemolithoautotrophic growth was 7.6 h. Arsenite oxidation was found to be catalyzed by a periplasmic arsenite oxidase (optimum pH, 5.5). Based upon 16S rDNA phylogenetic sequence analysis, NT-26 belongs to the Agrobacterium/Rhizbium branch of the alpha-Proteobacteria and may represent a new species. This recently discovered organism is the most rapidly growing chemolithoautotrophic arsenite oxidizer known.

Detection of dimethyl sulfone in the human brain by in vivo proton magnetic resonance spectroscopy

We wish to report the detection of dimethyl sulfone (methylsulfonylmethane, C2H6O2S) in the brain of a normal 62-year-old male using in vivo proton magnetic resonance spectroscopy. The presence of this exogenous metabolite resulted from ingestion of a dietary supplement containing dimethyl sulfone. The concentration of this compound in the brain was measured to be 2.4 mmol, with a washout half life of approximately 7.5 days. The in vivo T-1 and T-2 relaxation times of dimethyl sulfone were measured to be 2180 ms and 385 ms, respectively. The concentration of major brain metabolites, namely N-acetylaspartate, total Creatine and Choline, and myo-Inositol were within normal limits. (C) 2000 Elsevier Science Inc. All rights reserved.

Weak-force detection with superposed coherent states

We investigate the utility of nonclassical states of simple harmonic oscillators, particularly a superposition of coherent states, for sensitive force detection. We find that like squeezed states, a superposition of coherent states allows displacement measurements at the Heisenberg limit. Entangling many superpositions of coherent states offers a significant advantage over a single-mode superposition state with the same mean photon number.

Spatial and temporal variation in distribution of mangroves in Moreton Bay, subtropical Australia: a comparison of pattern metrics and change detection analyses based on aerial photographs

An assessment of the changes in the distribution and extent of mangroves within Moreton Bay, southeast Queensland, Australia, was carried out. Two assessment methods were evaluated: spatial and temporal pattern metrics analysis, and change detection analysis. Currently, about 15,000 ha of mangroves are present in Moreton Bay. These mangroves are important ecosystems, but are subject to disturbance from a number of sources. Over the past 25 years, there has been a loss of more than 3800 ha, as a result of natural losses and mangrove clearing (e.g. for urban and industrial development, agriculture and aquaculture). However, areas of new mangroves have become established over the same time period, offsetting these losses to create a net loss of about 200 ha. These new mangroves have mainly appeared in the southern bay region and the bay islands, particularly on the landward edge of existing mangroves. In addition, spatial patterns and species composition of mangrove patches have changed. The pattern metrics analysis provided an overview of mangrove distribution and change in the form of single metric values, while the change detection analysis gave a more detailed and spatially explicit description of change. An analysis of the effects of spatial scales on the pattern metrics indicated that they were relatively insensitive to scale at spatial resolutions less than 50 m, but that most metrics became sensitive at coarser resolutions, a finding which has implications for mapping of mangroves based on remotely sensed data. (C) 2003 Elsevier Science B.V. All rights reserved.