Microscopic Analysis of DNA and DNA-Protein Assembly by Transmission Electron Microscopy, Scanning Tunneling Microscopy and Scanning Force Microscopy

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA super-coiling were analyzed using electron microscopy. In parallel, this Mg2+/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or in liquid by scanning force microscopy. An advantage of such a correlative approach is that parallel imaging can reveal complementary information. The benefit of such a combined approach in analysis of protein-induced DNA bending is discussed

Prochloron research

The purpose was to prepare Prochloron photosynthetic membranes for the isolation of the two major chlorophyll-proteins, the P700-chlorophyll a-protein and the light-harvesting chlorophyll a/b-protein, using SDS-polyacrylamide gel electrophoresis. The prepared proteins (purified) were examined for their cross-reactivity to polyclonal antibodies prepared from higher plant proteins. In addition, material was prepared for electron microscopy, and isolation of the DNA for determination of its general complexity (COT analysis) and similarity to barley chloroplast DNA and Anabaena DNA by using restriction-endonuclease analysis. Kleinschmidt spreads of the DNA were in the electron microscope to identify and measure the extent and size of the circlar DNA

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein–DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein–DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents

The process of infection with bacteriophage phiX174, XXX. Replication of double-stranded phiX DNA

Intermediates involved in the replication of double-stranded phiX174 RF DNA have been identified and partially characterized. Analysis of pulse-labeled RF DNA suggests that the synthesis of progeny RF molecules involves, in part, the addition of nucleotides to linear complementary strands on a circular parental strand as template, so as to produce intermediate DNA strands of greater than viral length. Electron microscopy reveals DNA rings with "tails" and "double rings," which could be the intermediate structures. A model is postulated for the replication process

Changes in the Topology of DNA Replication Intermediates: In vivo vs In vitro

Most of the methods used to analyze DNA, including electrophoresis, electron microscopy or atomic force microscopy, involve de-proteinization, and it is well known that the removal of proteins affects DNA topology. After de-proteinization in vitro, the topology of replication intermediates changes significantly. A comprehensive analysis of the topological changes introduced during DNA isolation (de-proteinization) is important to get a better understanding of DNA topology in vivo. The topology of replication intermediates examined by electrophoresis, electron microscopy or atomic force microscopy in vitro does not necessarily represent the situation in vivo.CONACYT - Consejo Nacional de Ciencias y TecnologíaPROCIENCI

Synthesis of Pyridinium Amphiphiles Used for Transfection and Some Characteristics of Amphiphile/DNA Complex Formation

Pyridinium amphiphiles have found practical use for the delivery of DNA into cells. Starting from 4-methylpyridine, a general synthesis has been devised for the production of pyridinium amphiphiles which allows variation in both the hydrophobic part and in the headgroup area of the compounds. By means of differential scanning microcalorimetry, zeta potential, particle size measurements and cryo electron microscopy, some characteristics of the pyridinium amphiphile/ DNA complexes have been determined.

Segregation of a microsporidian parasite during host cell mitosis

We investigated the segregation of an intracellular microsporidian parasite during host cell division. A time-course experiment was carried out to examine the distribution of parasites relative to host chromosomal DNA via light and electron microscopy. Fluorescent light microscopy and EM studies showed that the parasite lay in the perinuclear zone of the host cell during interphase and segregated to daughter cells at mitosis. At metaphase, the parasite was frequently closely associated with host microtubules and mitochondria. Electron-dense bridges were observed between the parasites and the host microtubules and also between host mitochondria and microtubules. The study suggests that both the parasite and the host cell organelles segregate in association with spindle microtubules

Development of a DNA-Liposome Complex for Gene Delivery Applications

The association structures formed by cationic liposomes and DNA(Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520 ± 12 nm to 464 ± 25 nm) while the PDI increased after lyophilization (0.094 ± 0.017 to 0.220 ± 0.004). In addition. The mean particle size of vesicles increases when DNA is incorporated to the liposomes (673 ± 27 nm). According to the Scanning Electron Microscopy(SEM) and transmission electron microscopy (TEM) images, the spherical shape of liposomes confirmed their successful preservation and reconstitution from the powder. It was found that liposomal formulation has enhanced transfection considerably compared to the naked DNA as negative control. Finally, liposomal formulation in this research had a better function than Lipofectamine® 2000 as a commercialized product because the cellular activity (cellular protein) was higher in the prepared lipoplex than Lipofectamine® 2000