Prevention of lymphadenopathy in MRL-lpr/lpr mice by blocking peripheral lymph node homing with Mel-14 in vivo

MRL-lpr/lpr mice develop massive lymphadenopathy and autoimmunity. There is evidence that both migration and local proliferation contribute to the accumulation of Ly-2-, L3T4-, 6B2+ T cells in the peripheral lymph node (PLN). Mel-14 is an antibody which binds to the lymphocyte lymph node homing receptor (gp90Mel-14) and can block migration of lymphocytes to the PLN. Treatment of mice from birth to 11 wk of age with Mel-14 and another rat IgG2a mAb, 6B2, resulted in reduction (10- to 20-fold) in lymphadenopathy. Mel-14, but not 6B2, preferentially reduced the percentages of Thy-1+, 6B2+ lymphocytes in the lymph node. Treatment with a third antibody, anti-Ly-1, had no effect on lymphadenopathy. Mel-14 treatment resulted in diversion of the Ly-2-, L3T4-, 6B2+, gp90Mel-14 cells to the spleen and consequently induced marked splenomegaly. Thymocytes from MRL-lpr/lpr and MRL-+/+ mice were analyzed by two-color flow cytometry analysis after depletion of Ly-2+ and L3T4+ T cells. There was no difference in the percent of Ly-2-, L3T4-, 6B2+, gp90Mel-14 positive thymocytes comparing these two strains. Mel-14 treatment did not alter Ig levels or autoantibody production. These studies suggest Mel-14 reduced lymphadenopathy by interfering with homing to PLN, whereas 6B2 may have interfered with marrow production of precursor cells or killed 6B2+ cells after they exited the marrow. The data are consistent with the idea that lymphadenopathy occurs in MRL-lpr/lpr mice due to increased homing gp90-Mel-14 T cells to the PLN and that gp90Mel-14 is a necessary receptor for the abnormal 6B2+ T cells.     

Intracellular Mn (II)-associated superoxide scavenging activity protects Cu,Zn superoxide dismutase-deficient Saccharomyces cerevisiae against dioxygen stress

Three Cu,Zn superoxide dismutase (SOD-1)-deficient Saccharomyces cerevisiae mutants do not grow in 100% O2 in rich medium and require Met and Lys when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J. (1985) Biochem. Biophys. Res. Commun. 130, 533-539). We show herein that medium manganese (II) accumulated by the mutants rescues these O2-sensitive phenotypes; 2 mM medium Mn2+ represented the threshold required for cell growth. The accumulation of Mn2+ was not oxygen-inducible since mutants grown aerobically and anaerobically accumulated the same amount of Mn2+. Mn2+ accumulation is not unique to these mutants since wild type accumulated almost twice as much Mn2+ as did mutant. ESR spectra of the cell extracts and whole cells loaded with Mn2+ were typical of free Mn(II) ion. These spectra could not account quantitatively for the total cellular Mn2+, however. A screen for soluble antioxidant activities in the Mn2+-supplemented cells detected O2- (superoxide) scavenging activity, with no change in catalase or peroxidase activities. This O2- scavenging activity was CN- and heat-resistant. No achromatic bands were revealed in nondenaturing gels of Mn2+- containing cell extracts stained for O2- scavenging activity. The Mn2+-dependent O2- scavenging activity in the cell extracts was quenched by EDTA and dialyzable. More than 60% of both the intracellular Mn2+ and the O2- scavenging activity was removed by 2-h dialysis. Dialyzed cells were not viable in air unless resupplemented with either Met or Mn2+. Although Mn2+ supported the aerobic growth of these mutants, excess Mn2+, which correlated with an elevated O2- scavenging activity, was toxic to both mutant and wild type. The results indicate that free or loosely bound Mn2+ ion protects the mutants against oxygen stress by providing an intracellular, presumably cytosolic, O2- scavenging activity which replaces the absent SOD-1.     

Phosphorylated mixed isomers of L-dopa increase melanin content in skins of Skh-2 pigmented hairless mice

Dopa phosphates, a new class of compounds, contain phosphate-ester linkages at the 3- and/or 4- positions of the phenylalanine ring of L-dopa. Dopa phosphates have been shown to increase pigment production in the epidermis of hairless mice. Groups of Skh-2 pigmented hairless mice were treated topically with various concentrations of dopa phosphates daily for five weeks. Half of each group received suberythemal UVB radiation three times weekly for four weeks from a bank of filtered FS20 lamps. UVB and dopa phosphates alone each caused a modest increase in epidermal pigmentation. However, treatment of mice with dopa phosphates plus UVB radiation resulted in a marked increase in pigmentation, greater than with either treatment alone. The optimal concentration of dopa phosphates was 0.01% (100 micrograms/ml Tris-glycerol buffer) whether or not they were applied in conjunction with UVB radiation. Histological analyses revealed that dopa phosphates and UVB radiation each caused an increase in the number of pigmented melanocytes in the epidermis. Control groups treated with Tris-glycerol buffer alone, or buffer containing L-phenylalanine or L-dopa showed no significant changes in pigmentation. Our results indicate that dopa phosphates stimulate the production of melanin and affect the development and distribution of melanocytes in the skin of Skh-2 mice. By these criteria, dopa phosphates and UVB act in a similar manner to increase melanin content in the skin. The processes may be related to those recently observed in cultured mouse melanoma cells where dopa phosphates are incorporated into melanin, presumably following enzymatic hydrolysis by cellular phosphatases with the resultant production of L-dopa and inorganic phosphate.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

An AFLP-markers based genetic linkage map of Heterobasidion annosum locating intersterility genes

A genetic linkage map of the basidiomycete Heterobasidion annosum, casual agent of root rot in conifers, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. In a population consisting of 102 progeny isolates, 358 AFLP markers were scored. The linkage analysis generated 19 large linkage groups, containing 6 or more markers, which covered 1468 cM. The physical size to genetic distance was approximately 11.1 kbp/cM. Segregation of three intersterility gene loci were analysed through mating of the progeny isolates with three tester strains carrying known intersterility genotypes. The loci for the two intersterility genes (S and P) were successfully located in the map. Segregation of the mating type locus was analysed by backcrossing the progeny isolates with their parental strains. The mating type locus could not be located in the map.     

Growth of heat-injured Vibrio parahaemolyticus in media supplemented with various cations.

Mid- to late logarithmic growth phase cells of Vibrio parahaemolyticus grown in tryptic soy broth (TSB) containing 0.5, 3.0, and 7.5% NaCl were heated for 8 min at 45 degrees C in 0.1 M phosphate buffer (pH 7.2) containing 3% NaCl. Colony formation on thiosulfate-citrate-bile salts-sucrose agar (TCBS) containing 2% NaCl was greatest for unheated cells that had been grown in 7.5% NaCl-TSB; cells grown in 0.5% NaCl-TSB formed a greater number of colonies on 1.0% NaCl-TCBS. Thermal injury was evident in heated cells, regardless of the NaCl concentration in TSB growth medium. The effects of Mg2+, K+, and Li+ added as chlorides to 0.5% NaCl-TSB on the growth of nonheated and heated V. parahaemolyticus were studied. Lower levels of Mg2+ and slightly higher levels of K+ were required to replace Na+ in TSB inoculated with thermally injured cells that had been originally grown in 3.0 and 7.5% NaCl-TSB. LiCl had an inhibitory effect on both nonheated and heated cells when present in the recovery medium (0.5% NaCl-TSB) at concentrations as low as 0.5%. Increased numbers of colonies were formed by heated cells plated in MgCl2-supplemented TCBS, regardless of the NaCl concentration in the original growth medium. Potassium had little, if any, effect on colony formation by nonheated V. parahaemolyticus recovered on TCBS and may have had a detrimental effect on heat-injured cells.     

Sodium/proton antiport is required for growth of Escherichia coli at alkaline pH

Evidence is presented indicating that Escherichia coli requires the Na+/H+ antiporter and external sodium (or lithium) ion to grow at high pH. Cells were grown in plastic tubes containing medium with a very low Na+ content (5-15 microM). Normal cells grew at pH 7 or 8 with or without added Na+, but at pH 8.5 external Na was required for growth. A mutant with low antiporter activity failed to grow at pH 8.5 with or without Na+. On the other hand, another mutant with elevated antiporter activity grew at a higher pH than normal (pH 9) in the presence of added Na+ or Li+. Amiloride, an inhibitor of the antiporter, prevented cells from growing at pH 8.5 (plus Na+), although it had no effect on growth in media of lower pH values.     

The Na+,K+,2Cl- -cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway

We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl- -cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+,K+,2Cl- -cotransport system. LK1 cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide-sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1 cells (increase in Vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)-bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)-bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1 cells present, 1.44 pmol/mg protein, specifically bound (3H)-bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)-ouabain binding sites as well as an increase in the activity of the Na+,K+-ATPase, expressed as a function of ouabain-sensitive 86Rb+ uptake. Furthermore, LK1 cells were different in the concentrations of intracellular Na+ (increases) and K+ (decreases) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.     

Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli

The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.     

Detection of phenotypic differences on human malignant melanoma lines and their variant sublines with monoclonal antibodies

The hybridoma technique was used to generate monoclonal antibodies against a wide spectrum of melanoma-associated surface antigens. Mice were immunized against the human melanoma lines Mel A-375, SK Mel-25, and Mel S-5 (subclone of SK Mel-25), which differ with respect to a number of biological and biochemical properties. Spleen cells were fused with P3 X 63-AG8.653 myeloma cells. Twenty hybridomas producing antibodies that were negative on platelets, leukocytes, and monocytes but positive on melanoma cells were isolated and recloned. The specificity of antibodies was investigated on 30 human melanoma and nonmelanoma lines. Five groups of antibodies could be distinguished by their reactivity (1) with few melanoma lines and embryonic fibroblasts; (2) with melanoma, neuroblastoma, and teratoma; (3) with melanoma, neuroblastoma, glioblastoma, teratoma, and carcinoma; (4) with melanoma, teratoma, and carcinoma; and (5) with melanoma, neuroblastoma, teratoma, glioblastoma, carcinoma, embryonic fibroblasts, and B-lymphoblastoid cells. The antigen expression was qualitatively and quantitatively different from cell line to cell line. No evidence for melanoma-specific antigens was found. Eight antibodies were isolated detecting phenotypic differences on sublines of SK Mel-25.     

Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype

Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13(-/-) mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose.

Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked alpha-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV1-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The alpha-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, YX/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, YX/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkage of the Dao-1 gene for D-amino-acid oxidase to the pgm-1 gene for phosphoglucomutase-1 on the mouse chromosome 5

Linkage of the Dao-1 gene controlling D-amino-acid oxidase to the Pgm-1 gene controlling phosphoglucomutase-1 on the mouse chromosome 5 was examined. Mutant ddY/DAO- mice carrying a null Dao-1c allele had a Pgm-1a allele. Mutant mice were crossed to C3H/HeN and NZB/Kl mice carrying the Dao-1+ and Pgm-1b alleles. The hybrid F1 mice were backcrossed to the ddY/DAO- and their progeny were examined for alleles for D-amino-acid oxidase and phosphoglucomutase-1. In both backcrosses, the progeny with recombinant-type combinations of the alleles were significantly less than the progeny with the parental-type combinations, indicating the linkage of the Dao-1 and Pgm-1 genes. The recombination frequency between these loci was estimated to be 19.8 +/- 4.0%.     

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.     

Effect of a water soluble derivative of alpha-tocopherol on radiation response of Saccharomyces cerevisiae

The radioprotection conferred by a highly water soluble glucose derivative of alpha-tocopherol, namely, 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) in Saccharomyces cerevisiae was studied. Cells grown in standard YEPD-agar medium and irradiated in the presence of TMG showed a concentration dependent higher survival up to 10 mM of TMG in comparison to cells irradiated in distilled water. Treatment of TMG to cells given either before or immediately after irradiation but not during irradiation, had no effect on their radiation response. S. cerevisiae strain LP1383 (rad52) which is defective in recombination repair showed enhanced radioresistance only when subjected to irradiation in presence of TMG. Cells of rad52 strain grown in the medium containing TMG showed a radiation response similar to that of cells grown in the medium without TMG. The nature of TMG dependent enhanced radioresistance was studied by scoring the mutations in the strain D-7, which behaved like wild type strain in complete medium, at trp and ilv loci. Our study indicated that TMG confers radioresistance in S. cerevisiae possibly by two mechanisms viz. (i), by eliminating radiation induced reactive free radicals when the irradiation is carried out in the presence of TMG and (ii), by activating an error prone repair process involving RAD52 gene, when the cells are grown in the medium containing TMG.     

Ev 2, a genetic locus containing structural genes for endogenous virus, codes for Rous-associated virus type 0 produced by line 72 chickens.

ev 2 is one of seven recently described genetic loci of chickens which contain structural genes for endogenous virus. ev 2 is present exclusively in line 72 chickens, an inbred strain of white Leghorns which is homozygous for the capacity to produce Rous-associated virus type 0 (RAV-0), a subgroup E virus. This phenotype is known as V+ and has been assigned a genetic allele designated V-E7. The segregation of ev 2 was followed in a genetic cross in which the V-E7+ phenotype was also segregating. The progeny of the cross were analyzed for endogenous viral loci by cleavage of embryo DNA with restriction endonuclease SstI, electrophoretic separation of the resulting fragments, and identification of bands containing viral sequences by hybridization of the DNA to radiolabeled viral RNA. Four endogenous viral loci, ev 1, ev 2, ev 4, and ev 5, were identified in the progeny of the cross. One of the progeny contained no detectable endogenous viral sequences. ev 1, ev 4, and ev 5 were present in progeny of both the V-E7+ and V-E7- phenotypes. ev 2 was present exclusively in progeny of the V-E7+ phenotype, and all V-E7+ progeny contained ev 2. In addition, one of the V-E7+ progeny contained only ev 2. FRom these data, we conclude that ev 2 codes for RAV-0 virus produced by the cells of line 72 chickens.     

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.