Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1

Summary The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48×10⁶). Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 andClo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

Immune recognition of AIDS virus antigens by human and murine cytotoxic T lymphocytes

The CTL response to HIV was analyzed in humans and in mice. By using a novel and strictly autologous lymphocyte culture system, human CTL lines were established with PBL from seropositive asymptomatic donors and from patients suffering from AIDS or presenting AIDS-related complex. CTL from HLA-A2 donors recognize and kill murine P815 mastocytoma cells doubly transfected with the human HLA-A2 gene and the HIV env gene; they also kill HLA-compatible human macrophages infected with HIV. CTL specific for the HIV env Ag were also generated in BALB/c mice by immunization with syngeneic murine cells transfected with the HIV env gene. Human and murine HIV-immune CTL populations belong to the CD8 subset of T lymphocytes and are restricted by class I HLA or H-2 transplantation Ag, respectively, in the recognition of HIV env Ag. The two different experimental systems presented here can be used to study CD8 lymphocyte immunity against HIV. The murine model of CTL immunity offers the additional advantage of avoiding the manipulation of infectious virus isolates.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Grafting of genetically modified cells: Effects of acetylcholine release in vivo

In this study, microdialysis was used to investigate functional recovery of central cholinergic neurons in the forebrain of rats with cortical devascularizing lesions. Mature male rats were unilaterally lesioned by disruption of the pia arachnoid vessels and genetically modified fibroblasts secreting nerve growth factor (NGF) were placed at the site of the lesion. One month following surgery, microdialysis probes were installed in the remaining cortex and were perfused with artificial cerebrospinal fluid (csf) containing neostigmine (5 nM) and/or KCl (100 mM). The basal (non-stimulated) release of acetylcholine (ACh) in the cortex was similar in all experimental groups, whereas KCl stimulated release of ACh was significantly augmented (P < 0.05) in the ipsilateral remaining cortex in lesioned animals that have been implanted with fibroblasts secreting NGF. These results suggest that NGF secreted by genetically engineered fibroblasts modulates neuroplasticity in the adult mammalian CNS and may favour recovery of cortical function following injury.     

Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity