Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome

To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T-DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the β-glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene. For both Nicotiana species kanamycin-resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T-DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T-DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T-DNA integration and deletion in the plant genome are discussed.     

Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering.

The substrate fermentation range of the ethanologenic bacterium Zymomonas mobilis was expanded to include the pentose sugar, L-arabinose, which is commonly found in agricultural residues and other lignocellulosic biomass. Five genes, encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB), and transketolase (tktA), were isolated from Escherichia coli and introduced into Z. mobilis under the control of constitutive promoters that permitted their expression even in the presence of glucose. The engineered strain grew on and produced ethanol from L-arabinose as a sole C source at 98% of the maximum theoretical ethanol yield, based on the amount of consumed sugar. This indicates that arabinose was metabolized almost exclusively to ethanol as the sole fermentation product, with little by-product formation. Although no diauxic growth pattern was evident, the microorganism preferentially utilized glucose before arabinose, apparently reflecting the specificity of the indigenous facilitated diffusion transport system. This microorganism may be useful, along with the previously developed xylose-fermenting Z. mobilis (M. Zhang, C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio, Science 267:240-243, 1995), in a mixed culture for efficient fermentation of the predominant hexose and pentose sugars in agricultural residues and other lignocellulosic feedstocks to ethanol.     

The human mineralocorticoid receptor gene (MLR) is located on chromosome 4 at q31.2

The gene for the human mineralocorticoid receptor (MLR) was previously localized to chromosome 4. Here, we have localized this gene to 4q31.2 by in situ hybridization. This precise mapping of MLR will assist in the linkage analysis and genetic characterization of pseudohypoaldosteronism, an autosomal recessive disorder which likely results from a defect in the MLR gene.     

Sequential biochemical and morphological events during assembly of the fertilization membrane of the sea urchin.

The fertilization membrane of Strongylocentrotus purpuratus undergoes changes in morphology, solubility, and permeability during the process of hardening. As the fertilization membrane elevates from the egg surface, it retains casts of the tips of the microvillous processes of the plasma membrane. The dome-shaped microvillar casts become angular at the same time that the fertilization membrane becomes resistant to solubilization in mercaptan solutions. 2-4 min after this morphological and chemical transition, the fertilization membrane becomes impermeable to the lectin conconavalin A, as monitored by binding of 125I- or fluorescein-labeled concanavalin A. Glycine ethyl ester inhibits the changes in morphology, solubility, and permeability, whereas sodium sulfite inhibits only the permeability block and resistance to solubilization by mercaptans. Parthenogenetic activation with the divalent ionophore, A23187, elicits fertilization membrane elevation more rapidly than does activation by fertilization; however, the morphological and permeability changes characteristic of hardening proceed more slowly. Elevation and hardening of the fertilization membrane thus appear to be discrete, multiple-step assembly processes that occur in fixed sequence, with kinetics that are affected by the mechanism of cortical granule exocytosis.     

Rates of small-scale species mobility in alvar limestone grassland

Abstract. Small-scale species frequency and cumulative species frequency were studied in four plots in limestone grassland of the Veronica spicata-Avenula pratensis association on Stora Alvaret on the Baltic island of Öland, Sweden. Species mobility was expressed as increase in cumulative species frequency in 20 subplots of 100 cm². Observed cumulative frequencies from 1985–1989 in all four plots, and from 1985–1995 in one plot were compared with values following from two null models, a ‘minimal mobility’ model and a random mobility model. In ca. 50 % of the cases the observed cumulative frequency was not significantly different from the random expectation. However, in many such cases the mean annual frequency was either very high or very low. Three ways of calculating the mobility rate are presented though only one is used: (observed cumulative frequency -lowest annual frequency) / expected cumulative frequency. Values × 100 range from 0 to 100. There were slight differences between the four plots which were interpreted in terms of differences in grazing intensity and soil depth. It is stressed that the idea of the Carousel model has never been meant to suggest that all species would show random mobility, which we now quantify, but that species differ in their mobility rate and that the mean rate is much higher than generally realized.     

Design of a gas chromatograph with parallel radioactivity and mass spectrometric detection : Application to the identification of the major metabolite of d-limonene associated with α2u-globulin

A Perkin Elmer 3920 gas chromatograph, equipped with a versatile inlet system (i.e. an injector/trap), was interfaced to a radioactivity detector and a mass-selective detector (H/P 5970B) to identify ¹⁴C-labeled compounds. The use of a pre-trap as a demountable, programmable-temperature injector, in conjunction with the injector/trap, allowed the introduction of 0.5-ml samples of rat kidney cytosol extracts to 0.32 mm I.D. capillary columns. The instrumentation greatly facilitated the identification of the major radiolabeled metabolite of d-limonene associated with the male rat-specific protein α2u-globulin as 1,2-cis-d-limonene oxide.