Gene expression enhancement due to plasmid maintenance.

Analysis of the chromosomic beta-galactosidase activity in strains of Escherichia coli with and without plasmids indicated that plasmid maintenance enhances gene expression. Cyclic AMP (cAMP) determinations confirmed that the gene enhancement observed in strains carrying plasmids was due to a small increase in the intracellular concentration of cAMP. Also, cells carrying plasmids displayed higher specific glucose uptake rates than did cells without plasmids. The increases in the expression of beta-galactosidase and the glucose uptake rate suggest a cAMP-mediated release of the glucose effect due to plasmid maintenance. Our results suggested that this effect is independent of the host and type and number of plasmids.     

Construction and characterization of a replication-deficient adenovirus expressing rat-soluble interleukin-6 receptor.

BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity. Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown. MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R). RESULTS: Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media. The kinetics of transgene expression depends both on the cell type and the species. sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo. Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.     

Complementation of replication-deficient deletion derivatives of plasmid mini-F.

Deleted mini-F plasmids with defects in replication were constructed and tested to see whether they could be rescued through complementation by a helper plasmid. This allowed us to identify two genetic loci determining trans-acting functions required for stable maintenance of plasmid mini-F, one encoded by the PstI fragment from 45.7 to 47.3 F-coordinates (F) and the other most probably located in the region from 43.1 to 43.8 F. The smallest mini-F plasmid that could be established through complementation consists of the PstI fragment 44.0 to 45.7 F, encoding origin II and the incB locus.     

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.     

Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication.

To assess which residues of Oct-1 POU-specific (POUs) are important for DNA recognition and stimulation of adenovirus DNA replication we have mutated 10 residues of the POUs helix-turn-helix motif implicated in DNA contact. Seven of these turned out to have reduced DNA binding affinity. Of these, three alanine substituted proteins were found to have a changed specificity using a binding site selection procedure. Mutation of the first residue in the recognition helix, Gln44, to alanine led to a loss of specificity for the first two bases, TA, of the wild-type recognition site TATGC(A/T)AAT. Instead of the A, a T was selected, suggesting a new contact and a novel specificity. A change in specificity was also observed for the T45A mutant, which could bind to TATAC(A/T)AAT, a site hardly recognized by the wild-type protein. Mutation of residue Arg49 led to a relaxed specificity for three consecutive bases, TGC. This residue, which is critical for high affinity binding, is absent from the structurally homologous lambdoid helix-turn-helix motifs. Employing a reconstituted system all but two mutants could stimulate adenovirus DNA replication upon saturation. Mutation of residues Gln27 and Arg49 impairs the ability of the Oct-1 POU domain protein to enhance replication, with a concomitant loss of DNA contacts. Since the POU domain binds the precursor terminal protein-DNA polymerase complex and guides it to the origin, lack of stimulation may be caused by incorrect targetting of the DNA polymerase due to loss of specificity.     

Mechanism of plasmid pT181 DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. This nick presumably serves as the start-site of pT181 replication by extension synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. In vitro replication of a recombinant plasmid carrying two pT181 origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading-strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two PT181 origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Characterization of changes in PER.C6 cellular metabolism during growth and propagation of a replication-deficient adenovirus vector

PER.C6 cells were cultivated for propagation of a replication-defective adenovirus vector in serum-free suspension bioreactors. Cellular metabolism during cell growth and adenovirus propagation was fully characterized using on-line and off-line methods. The energy metabolism was found to accelerate transiently after adenovirus infection with increases in glucose and oxygen consumption rates. Similar to other mammalian cells, glucose utilization was highly inefficient and a high lactate:glucose yield was observed, both before and after virus infection. A higher consumption of most of the essential amino acids was observed transiently after the infection, likely due to increased protein synthesis requirements for virus propagation. To improve virus propagation, a medium exchange strategy was implemented to increase PER.C6 cell concentration for infection. During cell growth, a 50% increase in glucose consumption and lactate production rates was observed after initiation of the medium exchange in comparison to the batch phase. This decrease in medium capacity only affected the central carbon metabolism and no increase in amino acid consumption was observed. In addition, even though cell concentrations of up to 10 x 10(6) cells/mL were reproducibly obtained by medium exchange, infections at cell concentrations higher than 1 x 10(6) cells/mL did not proportionally improve volumetric adenovirus productivities. No measured nutrient limitation was observed at those high cell concentrations, indicating that adenovirus cell-specific productivity at higher cell concentrations is highly dependent on cell physiology. These results provide a better understanding of PER.C6 cellular metabolism and a basis for intensifying PER.C6 growth and adenovirus propagation.     

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability

In Agrobacterium tumefaciens, the stability of Ti plasmids differs depending on the strain. So far, little is known about genes that cause the difference in stability. The repABC operon is responsible for replication and incompatibility of Ti plasmids. We constructed recombinant plasmids carrying the repABC operon and different portions of pTi-SAKURA. Cells having the recombinant plasmids that harbored a 2.6-kbp NheI fragment of pTi-SAKURA were found to be transformed via conjugation 100-fold less frequently with a small incompatible repABC plasmid than cells having the recombinant plasmids lacking the 2.6-kbp NheI fragment. Since the phenomenon occurred only when the resident and incoming plasmids belonged to the same incompatibility group, it was suggested that the 2.6-kbp NheI fragment bears the potential enhancing incompatibility. The fragment contained an operon consisting of two open reading frames, tiorf24 and tiorf25. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a group of plasmid stability genes. Removal of the 2.6-kbp fragment from the resident pTi-SAKURA increased the resident plasmid ejection ratio by the incoming repABC plasmid, whereas addition of the fragment to pTiC58 decreased the ejection ratio, and the loss ratio during growth at 37 degrees C. These data suggest that tiorf24 and tiorf25 are responsible for the stability of pTi-SAKURA, and reduce, in the host bacterium, the frequency of ejection of the resident plasmid, presumably through an incompatibility mechanism.     

Identification of a protein linked to the ends of adenovirus DNA.

A DNA-protein complex from human adenoviruses has been further characterized by electron microscopy, radiochemical labeling and analytical ultracentrifugation. Preparations of the complex contain a large percentage of forms of DNA which are either circular or oligomeric and are readily distinguishable from preparations of pronase-treated adenovirus DNA by analytical ultracentrifugation in cesium chloride gradients containing 4 M guanidinium chloride. The protein component has been iodinated in vitro with ¹²⁵I using Bolton and Hunter reagent, and SDS-polyacrylamide gel analysis of the labeled protein indicates that it has an apparent molecular weight of 55,000 daltons. DNAase I digestion of the DNA-protein complex labeled with ³²PO₄ results in release of a ³²PO₄-labeled protein which remains labeled even after boiling in 1% SDS and 1% mercaptoethanol. Subsequent digestion of this entity with snake venom phosphodiesterase leads to release of ³²P₄-labeled 5′-phosphate deoxynucleotides. Digestion of the DNA-protein complex with Eco R1 and analysis of the isolated restriction fragments indicates that the protein is present on each terminal fragment. We conclude that there is a protein of 55,000 daltons directly attached to each 5′ end of molecule probably via a covalent linkage. We propose that the protein functions during DNA replication by facilitating priming of the progeny strands, thus allowing the 5′ ends of the DNA to be replicated.     

Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

Mechanism of DNA flexibility enhancement by HMGB proteins

The mechanism by which sequence non-specific DNA-binding proteins enhance DNA flexibility is studied by examining complexes of double-stranded DNA with the high mobility group type B proteins HMGB2 (Box A) and HMGB1 (Box A+B) using atomic force microscopy. DNA end-to-end distances and local DNA bend angle distributions are analyzed for protein complexes deposited on a mica surface. For HMGB2 (Box A) binding we find a mean induced DNA bend angle of 78°, with a standard error of 1.3° and a SD of 23°, while HMGB1 (Box A+B) binding gives a mean bend angle of 67°, with a standard error of 1.3° and a SD of 21°. These results are consistent with analysis of the observed global persistence length changes derived from end-to-end distance measurements, and with results of DNA-stretching experiments. The moderately broad distributions of bend angles induced by both proteins are inconsistent with either a static kink model, or a purely flexible hinge model for DNA distortion by protein binding. Therefore, the mechanism by which HMGB proteins enhance the flexibility of DNA must differ from that of the Escherichia coli HU protein, which in previous studies showed a flat angle distribution consistent with a flexible hinge model.     

Translin binding to DNA: recruitment through DNA ends and consequent conformational transitions

The human translin protein binds a variety of sequences (chromosomal breakpoint consensus sequences, their sequence variants, as well as nonbreakpoint sequences such as simple AT and GC repeats) at nanomolar protein concentration when short single strands ( approximately 20-30mers) are used as DNA targets. The protein, which is known to exist as an octamer in its free state, undergoes a conformational transition upon binding to short single strands leading either to a compaction or to the dissociation of the oligomer. Moreover, the protein oligomers tend to aggregate into complexes that get progressively larger as the length of the single-stranded DNA target increases. The protein loads onto duplexes via the free ends of DNA, generating higher oligomeric complexes as a function of protein concentration. Interestingly, the conformation of DNA targets encased by translin oligomer is significantly altered such that the single strand is rendered hypersensitive to DNase I. Furthermore, the loading of translin oligomers leads to tighter clamping of duplex ends. All of these observations, taken together, suggest that translin is a bona fide binder of DNA ends, thereby subjecting the DNA to a conformation conducive for repair steps during translocation events. We discuss the results in the perspective of translin biology.     

Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin

BACKGROUND: The hostile environment found in acute and chronic wounds decreases the physiological half-life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds. METHODS: Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication-deficient adenovirus (Ad5) containing the reporter gene for beta-galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine 2000, (2) FuGENE 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5-LacZ alone; (2) Ad5-LacZ precoated with Polybrene, or (3) carrier control (phosphate-buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation. RESULTS: Transfection efficacy was significantly improved in vitro (p < 0.001) as well as in partial thickness burned (p = 0.015) and unburned skin (p > 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10-fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein). CONCLUSIONS: It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene.