Directed excision of a transgene from the plant genome

Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALS^r), were integrated into the genome of tobacco and Arabidopsis. The ALS^r gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALS^T gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F₁ progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F₁ progeny were chimeric and some F₂ progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.     

Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

Dynamics ofPanonychus ulmi andTyphlodromus pyri: factors contributing to persistence

We addressed the question of persistence of predator and prey in a biological control system by examining temporal patterns ofPanonychus ulmi (Koch) and its predator,Typhlodromus pyri Scheuten at two geographic locations and at two spatial scales. At the scale of an orchard, bothP. ulmi andT. pyri were persistent over the time frame of 6 years. At the scale of an individual tree,T. pyri appeared to be more persistent than its prey,P. ulmi. We used a simulation model of single populations ofP. ulmi andT. pyri to determine which of several aspects of the biology of the two species could contribute to such a pattern. Spatial incongruity between predator and prey was essential for persistence of both species. The generalist food habit ofT. pyri probably contributes to the persistence ofT. pyri on individual trees, and may cause occasional extinction ofP. ulmi at this spatial scale. The presence of alternate food is likely an essential element for successful biological control in this system. Cannibalism byT. pyri results in higher prey densities, that is, it is detrimental to the biological control ofP. ulmi, but has no effect on the relative persistence of the two species.     

Conditions for selective degradation of lignin by the fungus Ganoderma australis

Summary The white-rot fungus Ganoderma australis selectively degrades lignin in the ecosystem palo podrido. Using conditions that simulate those of palo podrido in the laboratory, it was found that low nitrogen content and low O₂ tension stimulate the productio of manganese peroxidase and lignin degradation, and depress cellulose degradation and cellulase production. The inverse is found at high nitrogen concentration and high O₂ tension. This agrees with previous results indicating that low O₂ tension and low nitrogen stimulate selective lignin degradation by this fungus. Correspondence to: J. Eyzaguirre     

Duramycin effects on the structure and function of heart mitochondria. II. Energy conversion reactions.

The polypeptide antibiotic duramycin has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (Navarro et al., 1985, Biochemistry 24, 4645-4650). PE is a major component of mitochondrial membranes. Duramycin was used to probe the role of PE in mitochondrial energy conversion reactions with the following results: (i) Duramycin uncoupled mitochondrial respiration, decreasing the respiratory control ratio to 1 at 5 microM. At concentrations of duramycin in excess of 10 microM, ADP addition inhibited electron transport. (ii) Duramycin inhibited oxidative phosphorylation (C50 less than 2 microM). (iii) Duramycin stimulated mitochondrial ATP hydrolysis modestly. The antibiotic was 7- to 16-fold less effective in this regard than concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (F-CCP) which produced comparable uncoupling. (iv) Duramycin inhibited uncoupled ATPase activity (C50 = 8 microM). Inhibition of the ATPase activity of intact mitochondria was blocked by 1 mM MgCl2 and 5 mM CaCl2; inhibition persisted in sub-mitochondrial particles assayed in the presence of 3 mM MgCl2. The effects on mitochondrial function of free fatty acids (FFA) and duramycin are similar in many respects. It is suggested that duramycin, like FFA, uncouples via a nonclassical mechanism, possibly by disrupting intramembrane H+ transfer between redox and ATPase complexes. In addition, interaction of duramycin, either direct or indirect, with the F0 moiety of the mitochondrial ATPase and with one or more components of the respiratory electron transport chain is proposed.     

Ultrastructural localization of arachidonic acid in stimulated macrophages

The distribution of 3[H] arachidonic acid incorporated into cultured mouse peritoneal macrophages was assessed upon stimulation of the cells with either the calcium ionophore A23187 or zymosan. After a labeling time of 24 h, cells were stimulated and processed for light and electron microscopic autoradiography. Grains were primarily localized over the plasma membrane and lipid-containing vesicles of both control and stimulated cells. In macrophages stimulated with ionophore, a decreased labeling density was evident in both of these cell compartments. Similar alterations in labeling pattern were observed in zymosan treated cells, although a larger decline in grain density occurred from the plasma membrane compartment. Immunocytochemical localization of PGE2, a major eicosanoid product released upon ionophore stimulation, revealed the presence of the prostaglandin in clear vesicular structures, many of which appear to be continuous with the plasma membrane. These results provide morphological evidence that different cellular pools of arachidonic acid may be differentially mobilized for eicosanoid production as a function of the mode of stimulation.     

THE MATING SYSTEM GENETICALLY AFFECTS OFFSPRING PERFORMANCE IN WOODHOUSE'S TOAD (BUFO WOODHOUSEI)

To determine if the nonrandom, non-resource-based mating system of Bufo woodhousei affects tadpole performance, I performed a series of controlled matings and reared the tadpoles to metamorphosis in the laboratory and field. I asked whether differences in paternal identity, mating status, or body size were related to differences in tadpole mass, larval period duration, metamorphic mass, or survival of offspring. Although both laboratory and field rearings indicated that male and female parentage affected most offspring traits, no correspondence existed between either laboratory and field metamorphic mass or laboratory and field survival of offspring sired by the same male. The lack of correspondence between sire breeding values in the laboratory and field for two of three traits raises doubts as to the validity of drawing conclusions concerning how evolution might be expected to work from laboratory studies. Paternal effects were more pronounced in the field than in the laboratory, despite what is usually presumed to be a greater amount of environmental variation in the field. In the laboratory neither sire body size nor mating status affected any trait, but in the field larger males produced offspring that were 10% heavier at transformation than offspring sired by small males. This predictable relationship between sire phenotype (body size) and offspring performance means that nonrandom mating based on male body size could have a directional effect on offspring performance. Because larger males mate disproportionately often in this population (Woodward, 1982a; Mitchell, unpubl.), the mating system may exert a directional effect on metamorphic body size.     

MICROSPOROGENESIS IN PEANUT (ARACHIS HYPOGAEA)

The stage of pollen development at the time of anther culture is an important factor in the production of haploids. The objectives of the current study were to develop a staining procedure for peanut (Arachis hypogaea L., ssp. hypogaea) microspores, to describe and document the stages of microsporogenesis in peanut, and to confirm a previous report concerning correlations of peanut floral bud shape with stage of microspore development. A staining procedure using propionic carmine provided adequate staining of pollen mother cells, microspores, and pollen. Pollen mother cells and microspores could easily be differentiated by their size and cell wall structure. Plants grown in a controlled environment were found to have highly synchronized microspore development, both within an anther and among anthers contained in the same bud. In addition, floral bud shape was confirmed as a reliable indicator of anther stage in peanuts.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA

Two major subclasses of simian virus 40 (SV40) large T antigen were separated by zone velocity sedimentation of crude extracts from productively infected cells. These subclasses, which have been shown to differ biologically and biochemically ( Fanning et al., 1981), sedimented at 5-6S and 14-16S. The amount of T antigen in each form was estimated by complement fixation and by immunoprecipitation of T antigen from extracts of cells chronically labeled with [35S]methionine. Each form of T antigen was tested for specific binding to end-labeled restriction fragments of SV40 DNA using an immunoprecipitation assay. The 5-6S and 14-16S forms of T antigen both bound specifically to DNA sequences in the SV40 HindIII C fragment. The sequences required for binding both forms were localized in the same 35-bp region of the origin. However, significant differences in binding activity and affinity for specific and nonspecific DNA were demonstrated. These properties suggest that T antigen subclasses may serve different functions in the lytically infected cell.