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Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly[Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio)propionyl residues in order to have each amino group of poly[Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond. As evidenced by agarose gel electrophoresis and ethidium bromide/pDNA fluorescence restoration, poly[Lys-(AEDTP)] polyplexes were decondensed and the plasmid released upon treatment with either dithiothreitol, glutathione in the presence of glutathione reductase, or the thioredoxin reductase. Electron microscopy showed that polyplexes exhibiting spherical particles of a mean size at about 100 nm were decondensed in the presence of glutathione and exhibited filamentous aggregates. Finally, we found that the transfection of 293T7 and HepG2 cells was 10- and 50-fold more efficient with poly[Lys-(AEDTP)] polyplexes, respectively, than with poly[Lys] polyplexes. These results indicate that disulfide-containing cationic polymers must be borne in mind for developing polymer-base gene delivery systems.

Sequence-specific interactions of 20-mer G,A-containing triple helix-forming oligonucleotides (TFOs) and bis-PNAs (peptide nucleic acids) with double-stranded DNA was visualized by electron (EM) and atomic force (AFM) microscopies. Triplexes formed by biotinylated TFOs are easily detected by both EM and AFM in which streptavidin is a marker. AFM images of the unlabeled triplex within a long plasmid DNA show a approximately 0.4-nm height increment of the double helix within the target site position. TFOs conjugated to a 74-nt-long oligonucleotide forming a 33-bp-long hairpin form extremely stable triplexes with the target site that are readily imaged by both EM and AFM as protruding DNA. The short duplex protrudes in a perpendicular direction relative to the double helix axis, either in the plane of the support or out of it. In the latter case, the apparent height of the protrusion is approximately 1.5 nm, when that of the triplex site is increased by 0.3-0.4 nm. Triplex formation by bis-PNA, in which two decamers of PNA are connected via a flexible linker, causes deformations of the double helix at the target site, which is readily detected as kinks by both EM and AFM. Moreover, AFM shows that these kinks are often accompanied by an increase in the DNA apparent height of approximately 35%. This work shows the first direct visualization of sequence-specific interaction of TFOs and PNAs, with their target sequences within long plasmid DNAs, through the measurements of the apparent height of the DNA double helix by AFM.

High resolution electron microscopy reveals that fully active alpha 2‐macroglobulin (α2M) from fresh human plasma presents a very characteristic tetrameric structure. This native conformation of the α2M molecule is described here for the first time, along with its various orientations in negatively stained preparations. Although the native form is sensitive to inactivation, glutaraldehyde fixation is not necessary for its observation except when ammonium salts are used. The tetrameric structure of α2M undergoes a drastic conformational change when the protein is treated either with trypsin, thrombin or methylamine, as evidenced by the appearance of the typical)+(structure already described in the literature. The various aspects of this second conformation correspond to different orientations of the molecules in the stain film, and depend upon the nature of the support.

To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. During the few last years, it has been shown that the plusstrand DNA synthesis step of lentiviruses differs significantly from that of other retroviruses. This step terminates by a strand displacement (SD) synthesis ofϳ99 nucleotides (nt) at the center of the genome, which generates a central DNA flap corresponding to a three-stranded structure with two overlapping positive-strand segments (6,44). This central flap synthesis appears to be important for the replication of human immunodeficiency virus type 1 (HIV-1) in nondividing cells, since mutants altering its formation are defective in their replication (5, 6, 22). Nuclear import of the proviral DNA is impaired with these mutants, while some HIV-derived vectors supporting the central flap synthesis are activated for genetic transduction (12, 53). With such an impact, the HIV-1 central DNA flap requires now a complete characterization (45).The different steps leading to the central flap formation during the lentiviral plus-strand DNA synthesis are summarized in Fig. 1. This model is based on ex vivo experiments that showed full-length nonintegrated linear DNA molecules extracted from HIV-1-infected cells containing a central discontinuity on the plus strand (4,6,21,44). The plus-strand DNA synthesis is initiated from a couple of canonical polypurine tracts (PPTs), which are resistant against the nucleolytic degradation activity of the reverse transcriptase (RT)-associated RNase H (37, 38, 39). The universal one flanking the 3Ј element U3R is referred to as the 3ЈPPT primer, and the other one, located at the center of the RNA chain, is referred to as the cPPT primer. These two RNA sequences are efficient primers for a discontinuous double initiation of the plus-strand synthesis (4,5,21,22,27). The upstream strand initiated at the 3ЈPPT is elongated through the cPPT, and by a mechanism of strand displacement, up to a nearby site located 80 to 100 nt downstream, referred as the central termination sequence (CTS). CTS is extremely efficient in terminating HIV-1 RTcatalyzed DNA elongation, whereas RT only pauses at this ...

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and Summary.-Circular kinetoplastic DNA was fractionated from Trypanosoma cruzi DNA by Hg-Cs2SO4 gradient. Covalantly closed circular DNA was isolated by buoyant density centrifugation in ethidium bromide CsCl density gradients. Electron We have previously described a circular DNA, 0.45 , long, extracted from the kinetoplast of Trypanosoma cruzi.1 In this paper we report the results of additional studies on this DNA: covalently closed circular duplexes and catenated forms of DNA circles sometimes associated with linear DNA. These different molecular forms of kinetoplastic DNA have been fractionated by buoyant density centrifugation with ethidium bromide in cesium chloride.Materials and Methods.-Culture of trypanosomes: Trypanosomes (Trypanosoma cruzi, strain "Institut Pasteur") were grown for 6 days at 27"C in 500-ml flasks containing 300 ml of Parker 199 medium supplemented with 10% of the liquid phase of Tobie medium; 250 IU of penicillin/ml and 50 ug of streptomycin/ml were also added to the medium.The trypanosomes (about 107 cells/ml) were washed five to six times with SSC (0.15 M NaCl-0.015 M sodium citrate) and collected by centrifugation at 1000 g.Preparation and fractionation of kinetoplastic DNA: Trypanosomes were lysed with 1% sodium dodecyl sulfate; DNA was extracted according to the method of Marmur;'0 two treatments with 50 jig RNase/ml were made under standard conditions. The material was subjected to preparative density gradient centrifugation in Cs2SO4 in the presence of mercuric ions. Kinetoplastic DNA, which has a base composition different from 210

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