theEscherichia Coli Research Articles

Inducible Gene Expression of the Human Immunodeficiency Virus LTR in a Replication-Incompetent Herpes Simplex Virus Vector

Although replication-incompetent herpes simplex virus (HSV) vectors have the capability to express foreign genes, successful development of these vectors for gene delivery would require that expression of the foreign gene be regulated. To investigate the feasibility of obtaining inducible expression of a foreign gene in such a vector, a replication-incompetent HSV vector, vd120/LTRβ, was developed that used the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) to express theEscherichia coli lacZgene. Examination oflacZexpression from the HIV-1 LTR in vd120/LTRβ-infected cells indicated that the LTR was active as a promoter under both replicating and nonreplicating conditions, although expression oflacZunder nonreplicating conditions was approximately 4-fold lower. In addition, the LTR expressedlacZin a manner distinct from that of well-characterized HSV-1 promoters of each temporal class. The effect of the HIV-1 regulatory protein Tat on expression from the LTR in vd120/LTRβ was examined by infection of two different HeLa-derived cell lines that constitutively expressed Tat, HL2/3, and HLtat. Compared to infection of HeLa cells,lacZexpression from vd120/LTRβ-infected HL2/3 and HLtat cells increased from 4- to 24-fold, depending on the multiplicity of vector infection. Sustained expression oflacZfrom the LTR in vd120/LTRβ-infected cells was not observed even in the continuous presence of Tat, although vector could be recovered for up to 5 days after infection. However, the amount of recoverable vector decreased during this time, suggesting that cellular cytotoxicity may account for some of the decrease in Tat-mediated expression from the LTR.

Early olfactory fiber projections and cell migration into the rat telencephalon

The formation and development of primary olfactory axons was studied in the rat embryo using acetylcholinesterase histochemistry, immunocytochemistry for neuron-specific β-tubulin (TuJ1) and growth associated protein 43 (GAP43), and a fluorescent tracer DiI. Olfactory axons extend from the olfactory receptor neurons localized in the olfactory epithelium. These fibers grow to reach and enter the olfactory bulbs, where they form the first relay and integrative synaptic station in the olfactory system: the olfactory glomerulus. In this communication we address the development of primary olfactory fibers: first from the olfactory placode and later from the olfactory epithelium. Olfactory fibers enter the olfactory bulbs apparently in a disordered manner but soon arrange themselves in hook shaped aggregates of fibers, with many boutons (inmature synaptic terminals), to form the glomeruli. We detected this kind of structure for the first time at embryonic day 16. The olfactory receptor cells are usually anchored in the basal lamina of the olfactory epithelium but some of them, after reaching their targets, lose their epithelial attachment, leave the olfactory epithelium and migrate to and enter the olfactory bulbs. The traffic of cells between the olfactory epithelium and the brain lasts late into embryonic development. We describe four types of migratory mechanism used by different populations of cells to reach their targets in the telencephalic vesicle and propose the existence of migrating cells that enter the telencephalon. These data were corroborated by injections into the olfactory epithelium a of murine retrovirus carrying theEscherichia coli lac-Z gene.

Toxicity of Replication-Defective Adenoviral Recombinants in Dissociated Cultures of Nervous Tissue

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neuronsin vitroandin vivo;however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time.In vitro,this may be due to direct toxicity of the protein product of the transgene or adenoviral molecule(s).In vivo,in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord–dorsal root ganglia were exposed to AVR containing theEscherichia coli lacZ(E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1+E3)-deleted replication-incompetent AVR at high titers [≥5×108viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, β-galactosidase, at titers that result in 55–75% transduction efficiency (5×107–5×108viral particles/ml or m.o.i. 100–1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.

Site-Directed Mutagenesis Techniques in the Study ofEscherichia coliSerine Hydroxymethyltransferase

The 3340-bp fragment containing theEscherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5′–3′ and 3′–5′). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on theglyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, theE. colistrain GS245 used to express recombinant serine hydroxymethyltransferase was maderecA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5′-phosphate-binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions ofl- andd-alanine confirmed the suggestion that the active site lysine is not the base that removes the α-proton from the substrate (Schirchet al.,1993,J. Biol. Chem.268, 23132–23138).

Direct Intracerebral Nerve Growth Factor Gene Transfer Using a Recombinant Adenovirus: Effect on Basal Forebrain Cholinergic Neurons during Aging

Gene therapy in the nervous system offers an attractive strategy for the administration of therapeutic factors in a potentially region-specific, sustained, and well-tolerated manner. We tested the trophic effect of a recombinant adenovirus encoding nerve growth factor (AdNGF)in vivoon basal forebrain cholinergic neurons of aged rats, a neuronal population affected during normal and pathological aging. Three weeks after unilateral injection of the recombinant adenovirus into the nucleus basalis magnocellularis, a significant increase in the somal areas of cholinergic neurons ipsilateral to the injection was observed. No increase was detected in animals receiving a recombinant adenovirus carrying theEscherichia coli Lac Zreporter gene. Injected animals did not lose weight, an adverse effect usually described after intracerebroventricular infusion of NGF, and no tissue loss or massive local inflammatory response was observed around injection sites. Thus, a single intracerebral injection of AdNGF produces trophic effects similar to those resulting from chronic intracerebroventricular high levels of NGF. These findings indicate that recombinant adenoviruses encoding growth factors are potentially powerful tools for improving neuronal deficits associated with degenerative processes.

Synthesis and biological activity of substituted 7-aza-8-aza(oxa)-bicyclo[4.3.0]-6,9-nonadienes

Hydroxylamine- and hydrazine-induced heterocyclization of readily available 5-hydroxy-2.4-diacetyl-5-methyl-3R-cyclohexanones (R = methyl, phenyl, m-nitrophenyl, α-furyl) yielded 6-acetyl-5-hydroxy-5.9-dimethyl-7R-1-aza(oxa)-2-azabicyclo[4.3.0]-2,8-nonadienes (R = methyl, phenyl,m-nitrophenyl. α-furyl). The biological activity of the latter was tested using theEscherichia coli phage T4, and was also tested on a stored lyophilized culture ofYersinia pestis EU.

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.

ThehupB gene of theAzotobacter chroococcum hydrogenase gene cluster is involved in nickel metabolism

InAzotobacter chroococcum the hydrogenase gene (hup) cluster spans about 14 kb of DNA. The genes coding for the small and large subunits,hupSL, are located at the 5′ end, and a cluster of genes,hupABYCDE, resembling theEscherichia coli hyp operon, is located at the 3′ end. In this study, we determined the effect of adding nickel to the medium used for the growth ofhup mutants. Hydrogenase activity was restored tohupA andhupB mutants, but nothupY, hupD, orhupE mutants, by the addition of nickel to the growth medium, suggesting that the products ofhupA andhupB are somehow involved in nickel metabolism. The restoration of hydrogenase activity to thehupB mutant required protein synthesis.

The Escherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system.

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and the Escherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.

CDNA cloning and expression of aTalaromyces emersonii amylase encoding genetic determinant inEscherichia coli

Intact mRNA has been isolated from the thermophilic fungusTalaromyces emersonii following growth on starch containing media. This has been used as template to synthesise cDNA. The cDNA was cloned into theEscherichia coli expression vector system, pUC18 and this was used to transformE. coli. Transformed colonies were screened for production of amylase activity and a number of positive recombinants were detected. One of those was found to contain a plasmid named pMH1, which harboured a 1.2 kb insert. Sub-cloning experiments verified that the amylase phenotype was encoded for by this fragment. The fragment was characterised using restriction enzyme cleavage site analysis. The origin of the insert was verified using both Northem and Southem blotting hybridisation analysis ofT. emersonii RNA and DNA respectively.

Molecular cloning, characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.

Free and integrated plasmid DNA in spiroplasmas

Spiroplasma citri was found to carry an 8.0 kb plasmid that differed from previously describedS. citri plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid inS. citri (R8A2, Maroc) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome. Significant quantities of integrated plasmid sequences were also observed in the corn stunt spiroplasma,S. kunkelii, while small quantities of free and/or integrated plasmid DNA could be detected in some spiroplasmas serologically and genotypically remote fromS. citri. Integrated plasmid sequences were cloned into theEscherichia coli plasmid pUC13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of fragments, rather than entire plasmid DNA, inserted into specific sites in the spiroplasma chromosome. Although the biological role of the pRA1 plasmid remains unclear, theS. citri subclones containing large quantities of free plasmid exhibited slower growth rates and a tendency to lyse.

Establishment of Cockayne syndrome fibroblast cell line belonging to complementation group B by SV40 transformation.

A permanent fibroblast cell line of Cockayne syndrome (CS) belonging to complementation group B (CS1BESV) was established by simian virus 40 (SV40) transformation. The ultraviolet light (UV)-sensitivity of CS1BESV was similar to that of parental primary CS1BE fibroblast. The recovery of the rate of semiconservative DNA synthesis after UV-irradiation (12 J/m2) was absent in CS1BESV. With respect to the rate of semiconservative DNA synthesis after UV-irradiation (12 J/m2), CS1BESV complemented the primary fibroblast of group A CS (CS3BE) but not group B CS (CS1BE), confirming that CS1BESV belongs to complementation group B. The CS1BESV cells were transfected with recombinant vectors (pSV2gpt) carrying theEscherichia coli xanthine-guanine phosphoribosyltransferase gene (Ecogpt). They exhibited stable transformation frequency of 1×10−3. The CS1BESV cells will be useful as a permanent source of supply of parental group B CS cells for genetic complementation tests of CS and as a DNA transfer recipient for cloning of the gene which is deficient in CS.

Consequences of losing ribonuclease III on the Escherichia coli cell.

An isogenic pair of Escherichia coli strains, one carrying an rnc+ and the other an rnc- allele (a mutation which reduces the level of ribonuclease III), was compared. The rnc- strain fails to grow at very elevated temperatures (for E. coli) while the rnc+ strain does grow exponentially. Assaying the residual RNase III like activity in extracts of the rnc- strain at different pHs and at different temperatures suggested that this residual RNase III like activity is not due to RNase III. This raised the possibility that the rnc- strain is devoid of any RNase III activity in the cell. Comparing the decay of newly synthesized RNA and functional decay of beta-galactosidase mRNA in such strains revealed that in both strains these parameters proceed in similar rates, which suggests that RNase III is not involved in the metabolism of mRNA. During carbon starvation preexisting total RNA, as well as 23S and 16S rRNA, decay faster in the rnc- strain, thus eliminating the possibility that RNase III is the endoribonuclease which initiates the decay of rRNA during starvation (Kaplan and Apirion, 1975a).

Mapping of the gene for cytosine deaminase on the Escherichia coli chromosome.

The gene coding for the enzyme cytosine deaminase (cod) was located on theEscherichia coli chromosome. The marker is cotransducible withlac and the map order isproB-cod-lac.

The adenosine triphosphate pool and the adenyl cyclase activity in Escherichia coli strains with different UV sensitivity.

Two genetically relatedEscherichia coli strains were tested for the ATP pool and for the adenyl cyclase activity in the cell membrane fractions. TheEscherichia coli strain BS1with a high UV sensitivity showed a higher ATP level than the wild typeEscherichia coli B strain. The adenyl cyclase activity was found to be lower in the sensitive strain than in the wild typeEscherichia coli.