Junghuei Chen

The topology of the kinetoplast DNA network

Kinetoplast DNA (kDNA) of trypanosomatid parasites is a network of approximately 5000 catenated DNA minicircles and approximately 25 maxicircles. We developed the following strategy to deduce the topological linkage of the minicircles of the Crithidia fasciculata network. First, we used graph theory to provide precise models of possible network structures. Second, on the basis of these models, we predicted the frequencies of minicircle oligomers expected from random network breakage. Third, we determined the fragmentation pattern of kDNA networks as a function of the extent of digestion. Fourth, by comparison of the results with the predictions, we identified the model that best represents the network. We conclude that each minicircle is linked on average to three other minicircles. A honeycomb arrangement probably results, with each minicircle typically at the vertex of a hexagonal cell. This topology has implications for the assembly, structure, and function of kDNA networks.

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli.

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.

New motifs in DNA nanotechnology

Recently, we have invested a great deal of effort to construct molecular building blocks from unusual DNA motifs. DNA is an extremely favorable construction medium. The sticky-ended association of DNA molecules occurs with high specificity, and it results in the formation of B-DNA, whose structure is well known. The use of stable-branched DNA molecules permits one to make stick-figures. We have used this strategy to construct a covalently closed DNA molecule whose helix axes have the connectivity of a cube, and a second molecule, whose helix axes have the connectivity of a truncated octahedron. In addition to branching topology, DNA also yields control of linking topology, because double helical half-turns of B-DNA or Z-DNA can be equated, respectively, with negative or positive crossings in topological objects. Consequently, we have been able to use DNA to make trefoil knots of both signs and figure of 8 knots. By making RNA knots, we have discovered the existence of an RNA topoisomerase. DNA-based topological control has also led to the construction of Borromean rings, which could be used in DNA-based computing applications. The key feature previously lacking in DNA construction has been a rigid molecule. We have discovered that DNA double crossover molecules can provide this capability. We have incorporated these components in DNA assemblies that use this rigidity to achieve control on the geometrical level, as well as on the topological level. Some of these involve double crossover molecules, and others involve double crossovers associated with geometrical figures, such as triangles and deltahedra.

A PCR-based Protocol for In Vitro Selection of Non-crosshybridizing Oligonucleotides

DNA computing often requires oligonucleotides that do not produce erroneous cross-hybridizations. By using in vitro evolution, huge libraries of non-crosshybridizing oligonucleotides might be evolved in the test tube. As a first step, a fitness function that corresponds to noncrosshybridization has to be implemented in an experimental protocol. Therefore, a modified version of PCR that selects non-crosshybridizing oligonucleotides was designed and tested. Experiments confirmed that the PCR-based protocol did amplify maximally mismatched oligonucleotides selectively over those that were more closely matched. In addition, a reaction temperature window was identified in which discrimination between matched and mismatched might be obtained. These results are a first step toward practical manufacture of very large libraries of non-crosshybridizing oligonucleotides in the test tube.

A Software Tool for Generating Non-crosshybridizing Libraries of DNA Oligonucleotides

Under an all or nothing hybridization model, the problem of finding a library of non-crosshybridizing DNA oligonucleotides is shown to be equivalent to finding an independent set of vertices in a graph. Individual oligonucleotides or Watson-Crick pairs are represented as vertices. Indicating a hybridization, an edge is placed between vertices (oligonu-cleotides or pairs) if the minimum free energy of hybridization, according to the nearest-neighbor model of duplex thermal stability, is less than some threshold value. Using this equivalence, an algorithm is implemented to find maximal libraries. Sequence designs were generated for a test of a modified PCR protocol. The results indicated that the designed structures formed as planned, and that there was little to no secondary structure present in the single-strands. In addition, simulations to find libraries of 10-mers and 20-mers were done, and the base composition of the non-crosshybridizing libraries was found to be 2/3 A-T and 1/3 G-C under high salt conditions, and closer to uniform for lower salt concentrations.

Design and test of noncrosshybridizing oligonucleotide building blocks for DNA computers and nanostructures

DNA oligonucleotides that anneal to form duplexes in specific, planned configurations are a basic construction material for DNA-based computers and nanotechnology. Unplanned duplex configurations introduce errors in computations and defects in structures, and thus, the sequences must be designed to minimize these effects. A software design tool has been developed that uses thermodynamic models of DNA duplex thermal stability and algorithms from graph theory to select good sets of oligonucleotides. An example set was tested in the laboratory, and the designed sequences formed no unplanned duplexes and had no detectable secondary structure.

Changes in network topology during the replication of kinetoplast DNA.

Kinetoplast DNA of Crithidia fasciculata is a network containing several thousand topologically interlocked DNA minicircles. In the prereplicative Form I network, each of the 5000 minicircles is intact and linked to an average of three neighbors (i.e. the minicircle valence is 3). Replication involves the release of minicircles from the interior of the network, the synthesis of nicked or gapped progeny minicircles and the attachment of the progeny to the network periphery. The ultimate result is a Form II network of 10,000 nicked or gapped minicircles. Our measurements of minicircle valence and density, and the networks surface area, revealed striking changes in network topology during replication. During the S phase, the peripheral newly replicated minicircles have a density twice that of minicircles in Form I networks, which suggests that the valence might be as high as 6. Most of the holes in the central region that occur from the removal of intact minicircles are repaired so that the central density and valence remain the same, as in prereplicative networks. When minicircle replication is complete at the end of the S phase, the isolated network has the surface area of a prereplicative network, despite having twice the number of minicircles. During the G2 phase, the Form II network undergoes a remodeling in which the area doubles and the valence is reduced to 3. Finally, the interruptions in the minicircles are repaired and the double‐sized network splits in two.

Disruption of the Crithidia fasciculata KAP1 gene results in structural rearrangement of the kinetoplast disc

The mitochondrial DNA (kinetoplast DNA) in trypanosomatids exists as a highly organized nucleoprotein structure with the DNA consisting of thousands of interlocked circles. Four H1 histone-like proteins (KAP1, 2, 3 and 4) are associated with the kinetoplast DNA in the trypanosomatid Crithidia fasciculata. We have disrupted both alleles of the KAP1 gene in this diploid protozoan and shown that expression of the KAP1 protein is eliminated. The mutant strain is viable but has substantial rearrangement of the kinetoplast structure. Expression of the KAP1 protein from an episome restored expression of the KAP1 protein in the mutant strain and also restored a normal kinetoplast structure. These studies provide evidence that the KAP1 protein is involved in kinetoplast DNA organization in vivo but is nonessential for cell viability.

Origin of antibacterial stasis by polymyxin B in Escherichia coli

We show that blockage of hyperosmotic shock induced plasmolysis by polymyxin B (PxB) is related to its selective antimicrobial action against Gram-negative organisms. The rapid wrinkling of the cytoplasmic membrane induced by the hyperosmotic shrinkage of cytoplasmic volume due to the water efflux is monitored as an increase in the 90 degrees light scattering. The rapid scattering response is complete within 1 min after the addition of hyperosmolar NaCl. PxB decreases the amplitude of the rapid increase in the light scattering due to the shrinkage of the cytoplasmic volume by hyperosmotic shock. The amplitude is highest with cells in the early log phase of growth. The effect of PxB is induced rapidly and the maximum effect is seen within 1 min preincubation of cells. The effect of PxB is concentration dependent, and about 50% decrease in the amplitude is seen in the range of the growth inhibitory concentrations of PxB. The effect of PxB is not seen if added after the onset of the up-shock. As a heuristic model we suggest that PxB forms contacts between the two phospholipid interfaces that enclose the periplasmic space. The plasmolytic response results with osmY(-) mutant suggest that, like PxB, the osmY gene product in the periplasmic space prevents the shrinkage of the cytoplasmic compartment. Since PxB induces osmY transcription, we propose that, as a possible locus for the origin of the PxB induced stress, a contact between the phospholipid interfaces surrounding the periplasmic space triggers the metabolic changes leading to bacterial stasis.

Characterization of Non-crosshybridizing DNA Oligonucleotides Manufactured in vitro

Libraries of DNA oligonucleotides manufactured by an in vitro selection protocol were characterized for their non-crosshybridizing properties. Cloning and sequencing after several iterations of the protocol showed that the sequences, in general, became more non-crosshybridizing. Gel electrophoresis of protocol product, also, indicated non-crosshybridization, and showed evolution in the population of molecules under the non-crosshybridization selection pressure. Melting curves of protocol product also indicated non-crosshybridization when compared to control samples. Thus, it appears that the protocol does select populations of non-crosshybridizing sequences.