Small-scale turbulence affects the division rate and morphology of two red-tide dinoflagellates

The effects of small-scale turbulence on two species of dinoflagellates were examined in cultures where the turbulent forces came randomly from all directions and were intermittent both spatially and temporally; much like small-scale turbulence in the ocean. With Lingulodinium polyedrum (Stein) Dodge (syn. Gonyaulax polyedra), division rate increased linearly (from 0.35 to 0.5 per day) and the mean cross-sectional area (CSA) decreased linearly (from 1100 to 750 μm²) as a function of the logarithmic increase in turbulence energy dissipation rate (). These effects were noted when values increased between 10⁻⁸ and 10⁻⁴ m² s⁻³. However, when increased to 10⁻³ m² s⁻³, division rate sharply decreased and mean CSA increased. Over the same range of , Alexandrium catenella (Wheedon and Kofoid) Balech had its division rate decrease linearly (from 0.6 to 0.45 per day) and its CSA increase linearly (from 560 to 650 μm²) as a function of the logarithmic increase in . Even at the highest examined (10⁻³ m² s⁻³), which may be unrealistically high for their ambits, both L. polyedra and A. catenella still had fairly high division rates, 0.2 and 0.45 per day, respectively. Turbulence strongly affected chain formation in A. catenella. In non-turbulent cultures, the mode was single cells (80–90% of the population), but at of 10⁻⁵ to 10⁻⁴ m² s⁻³, the mode was 8 cells per chain. At the highest (10⁻³ m² s⁻³), the mode decreased to 4 cells per chain. The vertical distributions of A. catenella populations in relation to hydrographic flow fields were studied in the summers of 1997 and 1998 in East Sound, Washington, USA (latitude 48°39′N, 122°53′W). In both summers, high concentrations of A. catenella were found as a subsurface bloom in a narrow depth interval (2 m), where both current shear and turbulence intensity were at a minimum. Other researchers have shown that A. catenella orients its swimming in shear flows, and that swimming speed increases with chain length. These responses, when combined with our observations, support a hypothesis that A. catenella actively concentrates at depths with low turbulence and shear.     

A high-throughput comparison of recombinant gene expression parameters for E. coli-mediated gene transfer to P388D1 macrophage cells

Escherichia coli strain BL21(DE3) was tested as a delivery vector for gene transfer to a murine P388D1 macrophage cell line using a 96-well high-throughput assay. Five recombinant strains of E. coli were compared to identify the effect recombinant listeriolysin O (LLO) and associated gene expression parameters had on final delivery of a luciferase reporter gene. Listeriolysin O, native to Listeria monocytogenes and used here in an effort to improve final gene delivery, was expressed from plasmid and chromosomal locations under the control of constitutive Tet or inducible T7 promoters. The E. coli vectors delivered the luciferase reporter gene to the P388D1 line with success assessed by recording luciferase luminescence activity within the macrophage cells. The assay allowed rapid analysis and evaluation of each E. coli strain tested with strain BL21(DE3) harboring a chromosomal copy of the T7-driven LLO gene showing the greatest relative measure of gene delivery. Strains were separately assayed for LLO activity and exhibited a trend of maximum gene delivery between the lowest and highest recorded LLO activities.     

Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line

Large numbers of maize chromosome 9 can be collected with high purity by flow cytometric sorting of chromosomes isolated from a disomic maize chromosome addition line of oat. Metaphase chromosome suspensions were prepared from highly synchronized seedling root-tips of an oat-maize chromosome-9 addition line (OM9) and its parental oat and maize lines. Chromosomes were stained with propidium iodide for flow cytometric analysis and sorting. Flow-karyotypes of the oat-maize addition line showed an extra peak not present in the parental oat line. This peak is due to the presence of a maize chromosome-9 pair within the genome of OM9. Separation of maize chromosome 9 by flow cytometric sorting of a chromosome preparation from a normal maize line was not possible because of its size similarity (DNA content) to maize chromosomes 6, 7 and 8. However, it is possible to separate maize chromosome 9 from oat chromosomes and chromatids. An average of about 6×10³ chromosomes of maize chromosome 9 can be collected by flow-sorting from chromosomes isolated from 30 root tips (ten seedlings) of the oat-maize addition line. Purity of the maize chromosome 9, sorted from the oat-maize chromosome addition line, was estimated to be more than 90% based on genomic in situ hybridization analysis. Sorting of individual chromosomes provides valuable genomic tools for physical mapping, library construction, and gene isolation. Received: 28 February 2000 / Accepted: 14 July 2000     

Mapping maize sequences to chromosomes using oat-maize chromosome addition materials

Oat- (Avena sativa) maize (Zea mays) chromosome additions are produced by crossing maize and oat. During early embryo development maize chromosomes are preferentially eliminated, and oat plants are often recovered that retain a single maize chromosome. Each of the 10 maize chromosomes recently has been isolated as a separate oat-maize addition. We describe here the mapping of 400 maize sequences to chromosomes using polymerase chain reaction and DNA from the oat-maize addition material. Fifty of the sequences were from cloned markers that had been previously mapped by linkage analysis, and our results were consistent with those obtained using Southern-blot analysis. Previously unmapped expressed sequence tags and sequence tagged sites (350) were mapped to chromosomes. Maize gene sequences and expression data are rapidly being accumulated. Coupling this information with positional information from high throughput mapping programs provides plant biologists powerful tools for identifying candidate genes of interest.     

Use of paromomycin as a selective agent for oat transformation

Summary Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with two different plasmids containing the E. coli tn5 neomycin phosphotransferase II gene (npt II). Selection was accomplished using the antibiotic paromomycin sulfate following microprojectile bombardment. From two independent experiments, 88 paromomycin-resistant tissue cultures were shown to be transgenic based on Southern blot analysis and detection of the neomycin phosphotransferase (NPT II) protein using ELISA. Copy numbers of the npt II gene ranged from one to eight copies per haploid oat genome integrated into high molecular weight DNA of the paromomycin-resistant cultures. Plants were regenerated from 32 of the 88 transgenic tissue cultures. Plants from 17 of the 32 regenerable cultures exhibited fertility. Stable transformation was shown by segregation patterns of the NPT II protein in R₁ seedlings produced from 16 fertile culture lines that were tested. The overall results demonstrate that the combination of the npt II gene and paromomycin provides efficient selection of transgenic oat tissue cultures. Oat plants transformed with the npt II gene present reduced ecological risk compared to the previously used herbicide-resistance selection system.Abbreviations GUS beta-glucuronidase - uid A E. coli gene coding for GUS - NPT II neomycin phosphotransferase II of Tn 5 - npt II gene for NPT II - 2,4-D 2,4-dichlorophenoxy acid - X-gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexyl-ammonium salt - NOS nopaline synthase - NAA naphthalene acetic acid - BAP benzylaminopurine - ELISA enzyme-linked immunosorbant assay     

Maize individualized chromosome and derived radiation hybrid lines and their use in functional genomics

The duplicated and rearranged nature of plant genomes frequently complicates identification, chromosomal assignment and eventual manipulation of DNA segments. Separating an individual chromosome from its native complement by adding it to an alien genetic background together with the generation of radiation hybrids from such an addition line can enable or simplify structural and functional analyses of complex duplicated genomes. We have established fertile disomic addition lines for each of the individual maize chromosomes, except chromosome 10, with oat as the host species; DNA is available for chromosome 10 in a haploid oat background. We report on instability and transmission in disomic additions of maize chromosomes 1, 5, and 8; the chromosome 2, 3, 4, 6, 7, and 9 additions appear stable. The photoperiodic response of the two recovered maize chromosome 1 addition lines contrasts to the long-day flowering response of the oat parents and the other addition lines. Only when grown under short days did maize chromosome 1 addition lines set seed, and only one line transmitted the maize chromosome 1 to offspring. Low resolution radiation hybrid maps are presented for maize chromosomes 2 and 9 to illustrate the use of radiation hybrids for rapid physical mapping of large numbers of DNA sequences, such as ESTs. The potential of addition and radiation hybrid lines for mapping duplicated sequences or gene families to chromosome segments is presented and also the use of the lines to test interactions between genes located on different maize chromosomes as observed for ectopic expression of cell fate alterations. Electronic Publication     

Quantitative trait loci for partial resistance to crown rust, Puccinia coronata, in cultivated oat, Avena sativa L.

To facilitate the detection of quantitative trait loci (QTLs) for partial resistance to oat crown rust, Puccinia coronata f. sp. avenae Eriks., a genetic map was generated in a population of 158 F(6)-derived oat recombinant inbred lines from a cross of a partial resistance line MN841801-1 by a susceptible cultivar selection 'Noble-2'. The map, developed using 230 marker loci, mostly restriction fragment length polymorphism and amplified fragment length polymorphism markers, spanned 1,509 cM (Haldane) arranged into 30 linkage groups of 2-18 markers each. Four consistently detected major QTLs for partial rust resistance, Prq1a, Prq1b, Prq2, and Prq7, and three minor QTLs, Prq3, Prq5, and Prq6, were found in tests involving three field and two greenhouse environments. In addition, two major QTLs for flowering time, Ftq1 and Ftq7, and five weaker QTLs, Ftq2, Ftq3, Ftq4, Ftq5, and Ftq6, were revealed. Overlapping of the map segments of Ftq1 and Prq1 and of Ftq7 and Prq7 suggested either linkage between the flowering time QTLs and resistance QTLs or a pleiotropic effect of the Ftq QTLs on rust resistance. Relatively low heritability estimates (0.30) obtained for partial resistance to crown rust in the field indicate a potential value for marker-assisted selection.     

Agronomic performance of lines derived from anther culture, maize pollination and single-seed descent in a spring wheat cross

Anther culture and maize hybridization are two frequently used techniques for doubled haploid production in wheat (Triticum aestivum L.). Information on the field performance of lines derived from these techniques is limited. This study was conducted to compare the performance of F_4:6 lines obtained by single-seed descent with lines obtained by anther culture and maize (Zea mays L.) pollination from the same cross of spring wheat, ’Chris’/MN 7529. Thirty-three lines derived from each of those techniques were evaluated in six environments for grain yield, protein content, test weight, heading date, kernel weight and plant height. Mean performance of the single-seed descent lines exceeded performance of the anther culture lines for grain yield, kernel weight and plant height with no apparent differences for grain protein content, test weight and heading date. No differences between trait means for the single-seed descent and maize pollination lines were found except for plant height. The best 5 lines from each method for grain yield, protein content and test weight were similar in performance except that the protein content was higher for the maize pollination lines than for the single-seed descent lines. Acceptable levels of agronomic performance could be found among lines from each method. Wide acceptance of the doubled haploid technique for pure line production in breeding programs may, however, be limited by the often poor efficiency of doubled haploid line production, resulting in smaller population sizes for selection of desirable traits in comparison to the single-seed descent method. Received: 31 July 1998 / Accepted: 28 November 1998     

Characterization of ‘Sun II’ oat monosomics through C-banding and identification of eight new ‘Sun II’ monosomics

 Monosomics are a powerful tool for genetic mapping in allopolyploid plant species such as oat (Avena sativa L., 2n=6x=42). A C-banded karyotype of the oat cultivar Sun II was compared with previously described oat karyotypes and was used to identify the missing chromosome in each line of Sun II aneuploids. These included new aneuploids, isolated among derivatives of oat haploids obtained from Sun II oat×maize crosses, along with the original Sun II aneuploid set which had been obtained by cytological screening of a Sun II population for spontaneous aneuploids. Eight new Sun II monosomics were identified among the derivatives of haploids from the oat×maize crosses, to give a total of 18 unique Sun II monosomic/nullisomic lines. All seven C-genome chromosomes are represented by Sun II monosomics. Chromosomes 13, 14 and 17 are not represented by Sun II aneuploids but are found in the Kanota monosomic series. Therefore, monosomics of some form are now available for all 21 oat chromosomes. A reciprocal translocation involving chromosomes 3C and 14, found in a portion of the original set of Sun II monosomic lines, was also described. No new translocations were detected in the Sun II×maize crosses. Received: 11 December 1996 / Accepted: 15 July 1997     

Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests.

With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non-denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for the RB1 , TP53 , MLH1 and BRCA1 genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.