An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis

A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.     

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus

Summary A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both×chromosomes, whereas only one×chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.