Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.     

Adaptation of Phytoplankton-Degrading Microbial Communities to Thermal Reactor Effluent in a New Cooling Reservoir

In water column and sediment inocula from a nuclear reactor cooling reservoir, natural phytoplankton substrate labeled with ¹⁴C was used to determine aerobic and anaerobic mineralization rates for a range of temperatures (25, 40, 55, and 70°C) expected during reactor operation. For experiments that were begun during reactor shutdown, aerobic decomposition occurred at temperatures of <55°C. After 2 months of reactor operation, aerobic rates increased substantially at 55 and 70°C, although maximum rates were observed at temperatures of ≤40°C. The temperature range for which maximum anaerobic mineralization (i.e., the sum of CH₄ and CO₂) was observed was 25 to 40°C when the reactor was off, expanding to 25 to 55°C during reactor operation. Increased rates at 55°C, but not 70°C, correlated with an increase in the ratio of cumulative methane to carbon dioxide produced over 21 days. When reduced reactor power lowered the maximum temperature of the reservoir to 42°C, aerobic decomposition at 70°C was negligible, but remained substantial at 55°C. Selection for thermophilic decomposers occurred rapidly in this system in both aerobic and anaerobic communities and did not require prolonged exposure to elevated temperatures.     

Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15 Pseudomonas syringae strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at −5 or −9°C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.     

Expression of human salivary protein genes

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamulaet al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44A;Biochem. Genet. 15:549) and later supported by biochemical studies (Karnet al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamulaet al., Fed. Proc. 43:1522, 1984; Maedaet al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland byin situ hybridization.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

The structure of the basement membrane of human lymph node high endothelial venules: An ultrastructural, histochemical and immunocytochemical study

Summary The structure of the basement membrane of the high endothelium of reactive human lymph nodes was investigated by techniques selective for carbohydrates (periodic acid-Schiff; critical electrolyte concentration staining with Alcian Blue; lectin histochemistry), specific proteins (immunohistochemistry for laminin and fibronectin) and by conventional techniques of light and transmission electron microscopy. Adjacent small lymphocytes were assigned to B and T cell subsets by use of monoclonal antibodies and they were analysed for non-specific esterase,-glucuronidase,-N-acetylglucaminidase and proteolytic activities. The basement membranes were shown to be distinctive and to contain three layers, of differing laminin, glycosaminoglycan and glycoprotein oligosaccharide content. Certain lymphocytes (probably T) contained enzymes potentially able to degrade some components of these basement membranes.     

Hexavalent capsomers of herpes simplex virus type 2: symmetry, shape, dimensions, and oligomeric status

The structures of the hexavalent capsomers of herpes simplex virus type 2 were analyzed by negative staining electron microscopy of capsomer patches derived from partially disrupted nucleocapsids. Optimally computer-averaged images were formed for each of the three classes of capsomer distinguished by their respective positions on the surface of the icosahedral capsid with a triangulation number of 16; in projection, each capsomer exhibited unequivocal sixfold symmetry. According to correspondence analysis of our set of capsomer images, no significant structural differences were detected among the three classes of capsomers, as visualized under these conditions. Taking into account information from images of freeze-dried, platinum-shadowed nucleocapsid fragments, it was established that each hexavalent capsomer is a hexamer of the 155-kilodalton major capsid protein. The capsomer has the form of a sixfold hollow cone approximately 12 nm in diameter and approximately 15 nm in depth, whose axial channel tapers in width from the outside towards the inner capsid surface.     

An X-autosome fusion chromosome of Caenorhabditis elegans

Summary The translocation mnT12(IV;X) is a fusion of holocentric chromosomes IV and X, the breakpoints occurring near the left end of IV and the right end of X. Animals homozygous for mnT12 are viable and fertile; they contain five pairs of chromosomes rather than the normal set of six pairs. The mnT12 chromosome is larger than all wild-type chromosomes and thus identifies linkage groups IV and X cytologically. Hermaphrodites heterozygous for mnT12 show high frequency meiotic nondisjunction both between mnT12 and the X chromosome, which results in a high incidence of male self progeny (27% compared to the wild-type incidence of 0.2%), and between mnT12 and chromosome IV, which results in a high incidence of self progeny essentially trisomic for chromosome IV (karyotype IV/mnT12/mnT12). The viability of chromosome IV trisomics has been confirmed by constructing animals trisomic for only normal copies of chromosome IV; these animals are morphologically wild type. Meiotic chromosome disjunction in mnT12 homozygotes appears to be normal, although the frequency of recombination between markers that are normally X-linked is significantly reduced. Males of genotype IV/mnT12/0 are fertile. They can be thought of as having a neo-X(mnT12) neo-Y(normal IV) karyotype since it is possible to maintain a male-hermaphrodite stock of C. elegans consisting of such males and hermaphrodites carrying two neo-X chromosomes and no neo-Y; the organism is thus converted from an XO:XX type of sex determination to an XX:XX system.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station