Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.

A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanτ) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, β-galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.     

On the integrated interpretation of indirect site ordinations: a case study using semi-arid vegetation in southeastern Spain

The need for rigorous methods of interpreting indirect site ordinations is discussed and the problem of oblique habitat and phytosociological trends is emphasized. A sequential approach, termed integrated interpretation, is introduced to overcome this problem. It first involves a technique termed rotational correlation a linear form of trend surface analysis, which locates the best fit between a habitat variable and the coordinates of a pluridimensional ordination. The coordinates at positions of best fit are then used to produce two-way site-species tables (seriation arrays) which summarize floristic variation in relation to each major habitat trend. The seriation arrays can also be used to identify groups of differential species. Integrated interpretation is demonstrated using semi-arid vegetation from Murcia Province, SE Spain. A two-axis site ordination of vegetation data by non-metric multidimensional scaling is shown to have two oblique trends related to aspect-induced topoclimates and types of past anthropogenic disturbance. Rotation of the configuration to maximum linear correlation achieves very high levels of explanation for each factor. Rotated ordination coordinates from the correlation analysis are used to obtain two seriation arrays and floristic noda which relate directly to the two underlying habitat trends. A floristic discontinuity is revealed on the topoclimate continuum and it is hypothesized that temperature conditions are a major determinant of floristic composition, but that moisture partly determines vegetational cover and phytomass. Several patterns of past cultivation are detected and associated with five main kinds of geomorphological unit, together with some evidence that the rate of succession is partly determined by topoclimate. The integrated approach to interpretation is compared with visual methods and other multivariate techniques. A case is made for increased interpretational rigour in site coordinate studies, especially the discontinuation of trend-seeking using individual unrotated ordination axes.Nomenclature of vascular plants follows Tutin et al. (1964–1980) Flora Europaea 5 Vols. Cambridge University Press, Cambridge. Nomenclature of syntaxa follows Rivas-Goday & Rivas-Martinez (1967).I am indebted to J. A. Michel, Dr M. A. Abdelrahman and Monica M. Dargie for field assistance. Field discussion with Dr R. L. Wright, Dr M. P. Austin and Professor F. Esteve Chueca was most helpful; the latter assisted with confirmation of vascular plant voucher specimens. Non-vascular taxonomy was determined by Dr C. Humphries (mosses and one alga) and Dr O. L. Gilbert (lichens). An anonymous referee suggested an approximation of the analytical solution to rotational correlation, and Dr N. R. J. Fieller assisted with its confirmation. The project was assisted by a grant from the University of Sheffield Research Fund.     

Evidence that the crystalline arrays in the outer membrane of Neurospora mitochondria are composed of the voltage-dependent channel protein

Antibodies were raised in rabbits against the outer membrane of Neurospora mitochondria. Antibodies were obtained that were specific for this membrane's major polypeptide ($\text{M, 31 000}$) and its slower-migrating derivatives on SDS-polyacrylamide gels. These antibodies inhibited the insertion into phospholipid bilayers of voltage-dependent ion channels from detergent extracts of the mitochondrial outer membranes. The same antibodies bound preferentially to membranes containing crystalline surface arrays in outer mitochondrial membrane fractions. These results indicate that the 31 kDa polypeptide is a component both of the ion channels and of the membrane arrays, suggesting identity between the functional and structural entities.     

Simulation of the Viking biology experiments: An overview

Summary Several ground-based investigations have been carried out since the Viking biology results were received from Mars. Many of these have resulted in reasonable simulations of the Martian data, using as analogues of Mars either strong oxidants, UV-treated materials, iron-containing clays, or iron salts. The ambiguity between the GCMS experiment, in which no organic compounds were found on Mars, and the Labeled Release experiment, in which added organics were decomposed, may well be accounted for by these simulations.     

An enzymatic method for microdetermination of aphidicolin: a promising anticancer drug

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.     

草鱼出血病病毒对其它鱼的感染性研究

用草鱼出血病病鱼分离出的草鱼出血病病毒(Grass carp hemorrhage virus,GCHV)感染其它常见鱼并用ELISA方法检查感染鱼组织提取液,结果表明:青鱼、鲢鱼、布氏鳌条对GCHV抗体呈阳性反应;鲤鱼、鳙鱼、鲫鱼、团头鲂、泥鳅则呈阴性反应。综合感染鱼发病症状及死亡特征,初步认为:青鱼对GCHV是易感的,GCHV能在鲢鱼、布氏蟹条体内增值,但毒力较低,鳙鱼、鲫鱼、团头鲂、鲤鱼、泥鳅能抗GCHV感染。     

Silver Enhancement of Nickel-Diaminobenzidine as Applied to Single and Double Immunoperoxidase Staining

A reliable and practical method is proposed for increasing sensitivity and detection efficiency of immunocytochemical techniques, based on silver enhancement of the nickel-diaminobenzidine product of the peroxidase reaction. The procedure produces a strong signal at the site of the end product of the peroxidase reaction which is visible as black grains at the light microscopic level. The method has been used to detect peroxidase labeled probes in immunocytochemical tissue preparations and blotting assays and is ideal for the purposes of double staining and photographic documentation.     

Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare)

Summary In an attempt to transfer traits of agronomic importance from H. bulbosum into H. vulgare we carried out crosses between four diploid barley cultivars and a tetraploid H. bulbosum. Eleven viable triploid F₁ plants were produced by means of embryo rescue techniques. Meiotic pairing between H. vulgare and H. bulbosum chromosomes was evidenced by the formation of trivalents at a mean frequency of 1.3 with a maximum of five per cell. The resulting triploid hybrids were backcrossed to diploid barley, and nine DC₁ plants were obtained. Three of the BC₁ plants exhibited H. bulbosum DNA or disease resistance. A species specific 611-bp DNA probe, pSc119.2, located in telomeres of the H. bulbosum genome, clearly detected five H. bulbosum DNA fragments of about 2.1, 2.4, 3.4, 4.0 and 4.8 kb in size present in one of the BC₁ plants (BC₁-5) in BamHI-digésted genomic Southern blots. Plant BC₁-5 also contained a heterozygous chromosomal interchange involving chromosomes 3 and 4 as identified by N-banding. One of the two translocated chromosomes had the H. bulbosum sequence in the telomeric region as detected using in situ hybridization with pSc119.2. Two other BC₁ plants (BC₁-1 and BC₁-2) were resistant to the powdery mildew isolates to which the barley cultivars were susceptible. Seventy-nine BC₂ plants from plant BC₁-2 segregated 32 mildew resistant to 47 susceptible, which fits a ratio of 11, indicating that the transferred resistance was conditioned by a single dominant gene. Reciprocal crosses showed a tendency towards gametoselection that was relative to the resistance. Mildew resistant plant BC₁-2 also had a 1-kb H. bulbosum DNA fragment identified with a ten-base random primer using polymerase chain reaction (PCR). Forty-three BC₁ plants, randomly sampled from the 79 BC₁ plants, also segregated 2320 for the presence versus absence of this 1-kb H. bulbosum DNA fragment, thereby fitting a 11 ratio and indicating that the PCR product originated from a single locus. The 1-kb DNA fragment and disease resistance were independently inherited as detected by PCR analysis of bulked DNA from 17 resistant and 17 susceptible plants as well as by trait segregation in the 43 individual plants. The progenies produced could serve as an important resistant source in plant breeding. This is the first conclusive report of the stable transfer of disease resistance and DNA from H. bulbosum to H. vulgare.     

Rapid determination of plasmid-carrying yeast cells by using an imaging sensor system

A novel imaging sensor system for the determination of plasmid carrying yeast cells was developed. The sensor system consisted of an Silicon Intensifier Target (SIT) video camera, a fluorescent microscope, and a personal computer system equipped with an image memory board. This system was based on the fact that the membrane integrity of only plasmid-carrying cells is lost following cell growth in 5-fluoro-orotic acid (5-FOA) containing medium, and consequently these target cell can be stained with fluorescent probes and detected. In this study, plasmid-carrying cells were detected and their fraction determined in a mixture of both plasmid-carring and plasmid-free cells. A good correlation was observed between the values determined by this sensor system and the conventional method in the 30%-80% range, and one assay was possible within 4 h. This sensor system could be used for the monitoring of plasmid-carrying fraction in recombinant yeast cells during cultivation.     

Circular dichroism detection in HPLC

A circular dichroism-based detection system presents several advantages in the HPLC analysis of chiral compounds because of the selective monitoring of optically active molecules. Its use allows reliable determination of enantiomeric excesses and elution order. To this end, the application of empirical, semiempirical, and nonempirical methods to get stereochemical information from the CD signal is reported. Furthermore, recording the CD spectra on line and evaluation of the dissymetry factor make the CD detection very powerful in characterizing the stereochemistry of chiral eluates.