A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.     

New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system

We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

Toxic effects of putrescine in rat brain: Polyamines can be involved in the action of excitotoxins

Summary After treatment with putrescine (PUT) 200 mg/kg, i.p., male rats displayed a behavioural pattern that included wet dog shakes and motor inco-ordination. The concentration of PUT in the brain paralleled the severity of clinical signs. Histological examination showed the presence of perivascular edema and moderate spongiosis. These biochemical and histological features were present 2 h after treatment. At 24 h PUT levels in frontal cortex decreased but the histological status of brain tissue remained. Pretreatment with hyperosmolal glycerol did not modify the effect of PUT on the brain content of polyamine or the histological condition at 2 h. These results support a neurotoxic role for putrescine. Such effects were similar to those of kainic acid at convulsant doses, suggesting a role for putrescine in the action of this excitotoxin.     

Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli

Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS-PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface.     

Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector.

Summary The isolation and properties of a hybrid plasmid carrying the Y gene of the lac operon of Escherichia coli are described. The lactose carrier protein, coded for by the Y gene, is readily identified upon lac operon induction in strains carrying the plasmid. The protein comprises about 15% of the cytoplasmic membrane protein synthesized in the first generation after induction, compared with a wild type strain induced under the same conditions where lactose carrier protein comprises 1.4% of the cytoplasmic membrane protein.     

A plasmid vector system for the expression of a triprotein consisting of beta-galactosidase, a collagenase recognition site and a foreign gene product

A plasmid-cloning vector system has been constructed which allows the production of fusion proteins with beta-galactosidase at the N terminus, followed by a recognition sequence for the site-specific protease, collagenase, and the foreign protein at the C terminus. A multicloning site allows the insertion of foreign genes in any translational reading frame. Fusion proteins were isolated by affinity chromatography on APTG-Sepharose. The foreign protein was released from the fusion product by collagenase cleavage. The vector was successfully utilized for the production of Escherichia coli single-stranded (ss) DNA-binding protein (SSB protein). The proteolytically released SSB protein resisted elution from an ss DNA-cellulose column with 1 M NaCl.     

Controlled expression of tagged proteins in Drosophila using a new modular P-element vector system

We have developed a new modular vector system that facilitates the combination of various DNA fragments as functional modules for P element-mediated transformation of Drosophila melanogaster. The basic vector pP?GS? contains unique sites for 17 restriction enzymes, including three 8-bp cutters, that allow one to combine various promoter elements, cDNAs and genomic DNA fragments, as well as protein tags and selectable marker genes, for a wide spectrum of transgene analyses. With this new vector system we analysed the chromosomal distribution of the Drosophila SU(VAR)3-9 protein tagged with EGFP, using hsp70-cDNA and genomic Su(var)3-9 constructs. We found preferential association of the tagged SU(VAR)3-9 with centric heterochromatin.     

Biochemical determination of insecticides via cholinesterases. 1. Acetylcholinesterase from rat brain: functional expression using a baculovirus system, and biochemical characterization

Using a baculovirus-insect cell system, acetylcholinesterase from rat brain (rbAChE) was actively expressed and biochemically characterized and compared to the non-recombinant, tissue-derived rbAChE, establishing biochemical identity between both enzymes. Inhibition kinetics were similar for both enzymes using reversible inhibitors specific to the active and peripheral site. Inhibition rate constants of both enzymes treated with paraoxon as a reference insecticide were identical for both enzymes. As neither the catalytic parameters nor inhibition kinetics revealed any significant difference among the tissue derived and the recombinant enzyme, the baculovirus-insect expression system can be used for the preparation of recombinant rbAChE and mutants thereof.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Endogenous zinc can be a modulator of glycinergic signaling pathway in the rat retina

Summary Zinc is a modulator of glutamatergic inputs in the hippocampus. In the retina, however, we previously reported that endogenous zinc is present in the non-glutamatergic neural processes and earlier electrophysiological studies suggest that zinc is a modulator of inhibitory signaling pathways, which are mediated by glycine and GABA. AII amacrine cells, a subpopulation of glycinergic amacrine cells, are identified by selective immunoreactivity for parvalbumin in the rat retina. In the present study, therefore, we focused on whether zinc is present in AII amacrine cells using silver amplification combined with immunohistochemistry in the rat retina. We also examined whether zinc modulate glycine response in the rat retina by the patch clamp technique. Association of silver precipitates with the parvalbumin-immunoreactive neural processes was observed at the ultrastructural level. We also found that zinc existed in the neural processes which were not parvalbumin-immunoreactive. Glycine-induced responses were augmented when the concentration of Zn²⁺ was below 10 M, but inhibited at Zn²⁺ concentrations of 50 M or more. Our results suggest the notion that zinc in neural processes of retinal neurons modulates the inhibitory signaling pathway, particularly that mediated by glycine receptors in AII amacrine cells.