Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).     

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon

Intergeneric somatic hybrid plants between Hamlin sweet orange [Citrus sinensis (L.) Osbeck] and Flying Dragon trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. Hamlin protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with Flying Dragon protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the Hamlin protoplasts with the subsequent expression of the trifoliate leaf character of Flying Dragon. Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.Abbreviations MDH malate dehydrogenase - CTV citrus tristeza virus - MT Murashige and Tucker basal medium - BH3 protoplast culture medium, Grosser and Chandler, 1987 - PEG polyethylene glycol - GA3 giberellic acid - BA N-(phenylmethyl)-1 H-purin-6-amine - HCl hydrochloric acid Florida Agricultural Experiment Station Journal Series No. 7972     

Transposase-induced excision and circularization of the bacterial insertion sequence IS911.

We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type.     

Sequence, Genomic Distribution and DNA Modification of a Mu1 Element from Non-Mutator Maize Stocks

The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F2 individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization

Pteroylpolyglutamate hydrolase was solubilized with Triton X-100 from human jejunal mucosal brush borders and purified approximately 5,000-fold using organomercurial affinity chromatography, DEAE-cellulose chromatography, and gel filtration. The apparent molecular weight of the purified enzyme in the Triton micelle was estimated as 700,000 using Bio-Gel A-1.5m gel filtration. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis followed by Coomassie stain demonstrated two polypeptide bands at 145,000 and 115,000 daltons. The purified enzyme had an isoelectric point of 7.2, was maximally active at pH 5.5, and was stable above pH 6.5 and at temperatures up to 65 degrees C for at least 90 min. Human jejunal brush-border pteroylpolyglutamate hydrolase is an exopeptidase which liberated [14C]Glu as the sole labeled product of PteGlu2[14C]Glue (where PteGlun represents pteroylpolyglutamate), failed to liberate a radioactive product from PteGlu2[14C]GluLeu2, and released all possible labeled PteGlun products during incubation with Pte[14C]GluGlu6 with the accumulation of Pte[14C]Glu. PteGlu2, PteGlu3, and PteGlu7 were substrates, each with Km = 0.6 microM, whereas PteGlu was a weak inhibitor of the hydrolysis of PteGlu3 with Ki = 20 microM. Components of the pteroyl moiety, Glu, and short chain Glun in alpha or gamma linkages were not inhibitory. The enzyme was activated by Zn2+ or Co2+. The properties of brush-border pteroylpolyglutamate hydrolase are different from those described for the soluble intracellular pteroylpolyglutamate hydrolase in other species and in human mucosa, yet are consistent with previous data on the process of hydrolysis of PteGlun in the intact human intestine.     

A monoclonal antibody which inhibits epidermal growth factor binding has opposite effects on the biological action of epidermal growth factor in different cells

An epidermal growth factor (EGF) receptor-interactive monoclonal antibody (151-IgG) that inhibits EGF binding to PC12 rat pheochromocytoma cells and to various other cell types has been produced. The hybridoma clone was obtained by fusing Sp2/O-Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside-solubilized protein from isolated PC12 cell plasma membranes. The antibody is an IgG which binds to protein A. 151-IgG did not bind EGF. At 0.5 degrees C 151-IgG was directly competitive for EGF binding to PC12 cells. It also inhibited EGF binding to bovine corneal endothelial cells, rabbit corneal fibroblasts, human foreskin fibroblasts, and normal rat kidney cells, and it slightly enchanced EGF binding to SW 3T3 cells. PC12 cells have the same number of binding sites for 151-IgG as for EGF (approximately 27,000 sites/cell). 151-IgG inhibited the photoactivatable cross-linking of EGF to a protein of Mr 170,000 in PC12 cells. 151-IgG inhibited the EGF-stimulated incorporation of [3H]thymidine into quiescent bovine corneal endothelial cells, rabbit corneal endothelial cells, epithelial normal rat kidney cells, and SW 3T3 cells while it enhanced the EGF-stimulated [3H]thymidine incorporation into quiescent human foreskin fibroblasts. 151-IgG by itself possessed intrinsic EGF-like activity for human fibroblasts but not for the other cells tested. This suggests that there is a difference in EGF receptors and/or processing in these normal cell types.     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.     

Intracellular or extracellular calcium can be used to trigger C5a-stimulated enzyme secretion from rabbit neutrophils

The ability of C5a to stimulate lysosomal enzyme release and 45Ca2+ efflux from rabbit neutrophils was studied. C5a stimulated beta-glucuronidase release from cytochalasin B-treated neutrophils either in the presence or absence of extracellular calcium. Depletion of cell calcium by pretreatment with the calcium ionophore A23187 blocked both the ability of C5a to elicit enzyme release in the absence of extracellular calcium and its ability to stimulate 45Ca2+ efflux. Both actions were dose-dependent over the same concentration range (10(-8)-10(-6) M ionophore A23187). In contrast, ionophore pretreatment had no effect on C5a-stimulated enzyme release in the presence of extracellular calcium. These results suggest that (a) release of cell calcium is required for enzyme secretion in the absence of extracellular calcium, and (b) C5a can trigger near-maximal enzyme release by using calcium from either of two sources: the extracellular space or an intracellular site.