Replication of Adenovirus 12 Deoxyribonucleic Acid in Association with the Nuclear Membrane

Analysis of nuclei of adenovirus 12-infected cells revealed that viral DNA replicated in association with the nuclear membrane and that complete viral DNA was liberated from the nuclear membrane. Analysis of isolated nuclei in vitro showed that DNA polymerase activity increased in the nuclear membrane of adenovirus 12-infected cells without addition of primer DNA.     

Membrane association and role in DNA uptake of the Bacillus subtilis PriA anaiogue ComF1

The late competence protein ComF1 is required for genetic transformation in Bacillus subtilis. Because of the sequence similarities of ComF1 to known ATP-dependent DNA helicases and translocases, we have hypothesized that this protein either unwinds bound double-stranded DNA or helps in the translocation of the transforming single-stranded DNA across the cell membrane. Two important implications of this hypothesis (the association of ComF1 with the membrane and its specific requirement for DNA uptake) have been tested in this report. Using cell fractionation techniques and Western blotting analysis, we show that ComF1 is located almost exclusively on the cell membrane and that it is membrane-targeted independently of other competence proteins. Moreover, ComF1 behaves like an integral membrane protein in extractability and detergent partition assays. We also show that this protein is required for the DNA-uptake step during transformation but not for DNA binding to the ceil surface. DNA uptake is blocked in strains with null mutations or in-frame deletions in comF1 but also in strains that overproduce the ComF1 protein under competence conditions. This last observation suggests that ComF1 expression must be balanced with that of other competence proteins, with which it may interact to form a multisubunit complex for DNA uptake.     

Conjugal transfer replication of R64drd11 plasmid DNA in the donor cells of Escherichia coli K-12

Summary Conjugation, the process of genetic transfer requiring cell-to-cell contact, has been the focus of many investigations. In recent years, the molecular aspect of conjugation has been questioned. Since it has been shown that during exponential growth plasmid DNA forms a complex with the folded chromosomal complex (FCC), the relationship of R64drd11 plasmid DNA to the FCC (chromosome plus membrane) during conjugal replication was examined. A cell system was used which allowed specific observation of conjugal events as they occurred in the donor cell. Evidence is presented to show that conjugally replicating R64drd11 covalently closed circular molecules co-sediment with the FCC in neutral sucrose gradients. The use of density gradients to separate DNA from membrane-bound DNA from free membrane, indicate that the membrane is the preferential structure for conjugally replicating plasmid DNA association.     

What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues

Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what are the actual limits of the DNA delivery into cells. Jean-Michel Escoffre and Thomas Portet have contributed equally to this work.     

Membrane damage as first and DNA as the secondary target for anti‐candidal activity of antimicrobial peptide P7 derived from cell‐penetrating peptide ppTG20 against Candida albicans

P7, a peptide analogue derived from cell‐penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti‐Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l ‐phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin‐treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC‐P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Plasmid replication and partition in Escherichiacoli: is the cell membrane the key?

The DNA–membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329–348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.     

Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane

To proliferate efficiently, cells must co‐ordinate division with chromosome segregation. In Bacillus subtilis, the nucleoid occlusion protein Noc binds to specific DNA sequences (NBSs) scattered around the chromosome and helps to protect genomic integrity by coupling the initiation of division to the progression of chromosome replication and segregation. However, how it inhibits division has remained unclear. Here, we demonstrate that Noc associates with the cell membrane via an N‐terminal amphipathic helix, which is necessary for function. Importantly, the membrane‐binding affinity of this helix is weak and requires the assembly of nucleoprotein complexes, thus establishing a mechanism for DNA‐dependent activation of Noc. Furthermore, division inhibition by Noc requires recruitment of NBS DNA to the cell membrane and is dependent on its ability to bind DNA and membrane simultaneously. Indeed, Noc production in a heterologous system is sufficient for recruitment of chromosomal DNA to the membrane. Our results suggest a simple model in which the formation of large membrane‐associated nucleoprotein complexes physically occludes assembly of the division machinery.     

DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles

We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 μm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. “Short” DNAs (oligonucleotide 21b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (C _endo) of d-erythro-sphingosine (Sph⁺) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below C _endo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. C _endo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph⁺ GUVs and about 20 mol% for SOPC/Sph⁺ or eggPC/Sph⁺ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes. Received: 18 April 1998 / Revised version: 6 August 1998 / Accepted: 7 August 1998     

Evidence for a functional association of DNA synthesis with the membrane in mitochondria of Saccharomyces cerevisiae

We have studied the effect of membrane fatty acid composition on replicative DNA synthetic activity in mitochondria isolated from Saccharomyces cerevisiae. Cells containing different levels of membrane unsaturated fatty acids were obtained by growth of a fatty acid desaturase mutant of Saccharomyces cerevisiae in glucose-limited chemostat cultures supplemented with various concentrations of Tween 80. Arrhenius plots of DNA synthetic activity in isolated mitochondria show a discrete discontinuity at specific temperature which are dependent on the membrane unsaturated fatty acid content of the mitochondria. This indicates a functional association of DNA replication with the mitochondrial membrane in Saccharomyces cerevisiae.     

Using eggshell membranes as a DNA source for population genetic research

In the context of population genetic research, a faster and less invasive method of DNA sampling would allow large-scale assessments of genetic diversity and genetic differentiation with the help of volunteer observers. The aim of this study was to investigate the usefulness of eggshell membranes as a DNA source for population genetic research, by addressing eggshell membrane DNA quality, degeneration and cross-contamination. To this end, a comparison was made with blood-derived DNA samples. We have demonstrated 100% successful DNA extraction from post-hatched Black-tailed Godwit (Limosa limosa) eggshell membranes as well as from blood samples. Using 11 microsatellite loci, DNA amplification success was 99.1% for eggshell membranes and 97.7% for blood samples. Genetic information within eggshell membrane DNA in comparison to blood DNA was not affected (F _ST = −0.01735, P = 0.999) by degeneration or possible cross-contamination. Furthermore, neither degeneration nor cross-contamination was apparent in total genotypic comparison of eggshell membrane DNA and blood sample DNA. Our research clearly illustrates that eggshell membranes can be used for population genetic research.     

Heterospecific transformation in Bacillus subtilis: protein composition of a membrane-DNA complex containing unstable heterologous donor-recipient complex

Summary Previously it was demonstrated that, in contrast to the homologous donor-recipient complex, the unstable heterologous donor-recipient complex remains bound to the cellular membrane. To examine whether proteins known to be involved in the processing of transforming DNA in Bacillus subtilis are associated with membrane fragments which carry chromosomal DNA, a crude membrane-DNA complex was subjected to electrophoresis through a sucrose gradient. This resulted in the separation of membrane fragments associated with DNA and free membrane fragments. By means of two-dimensional gel electrophoresis several proteins, either uniquely present or considerably enriched in the purified membrane-DNA complex, were detected. Among these proteins we identified the 45 kD recE gene product, required for recombination, the 18 kD binding protein involved in the binding of transforming DNA and a 17 kD nuclease involved in the entry of transforming DNA.These results suggest that the membrane sites at which donor DNA integrates into the recipient chromosome are in the vicinity of the sites of entry of donor DNA through the membrane.Abbreviations DNAase I deoxyribonuclease I - DRC donor-recipient DNA complex - PEG polyethyleneglycol - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - TCA trichloroacetic acid     

Membrane binding of bacteriophage SPP1 DNA

Summary A fast sedimenting complex was isolated from B. subtilis cells infected with bacteriophage SPP1 by renografin centrifugation. This complex was identified as membrane bound parental and replicating SPP1 DNA. Synthesis of SPP1 DNA takes place in close association with the membrane. This newly synthesized DNA is then released into the cytoplasm. During release, concatemeric SPP1 DNA is sized into monomeric DNA molecules.Experiments reported were part of the Doctoral Thesis submitted by K.J. Burger to the Freie Universität Berlin     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

The character of protein-nucleic interaction in relation to the mtDNA-membrane complex

Summary Specific sites that interact with structural proteins of the mitochondrial inner membrane were found in mitochondrial DNA (mtDNA) of rat liver. Analysis of the isolated DNA fragments revealed their capacity to form a complex with membrane proteinsin vitro and allowed the detection of a protein with a molecular weight 40,000. The size of the fragments was found to be 12–18 nucleotide pairs with an average molecular weight 10,000 MtDNA sites recognized by membrane protein proved to be quite unique in having a secondary structure, a high content of AT sequences (82%) and oligopyrimidine blocks. It was shown that the light mtDNA strand, rich in adenine, is 60% more active in the binding with membrane mitochondria than the heavy one.     

Mechanism of dehydration inactivation of Lactobacillus plantarum

The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.Correspondence to: K. van't Riet     

Membrane association of NAD pyrophosphatase inSalmonella typhimurium

Salmonella typhimurium was fractionated into cytoplasmic, periplasmic, and membrane fractions to determine the cellular location of nicotinamide adenine dinucleotide pyrophosphatase. The results indicate that this enzyme is associated almost exclusively with the inner membrane. Studies utilizing porin mutants indicate that NAD probably transverses the outer membrane via pores and is degraded to NMN at the inner membrane by NAD pyrophosphatase. Based upon the lack of significant NAD pyrophosphatase activity in cytoplasmic fractions, we theorize that most if not all intracellular turnover of NAD is probably the result of DNA ligase activity.     

Arrangement of chromatin in the nucleus

Summary The factors responsible for producing some degree of order to the arrangement of chromatin in the nucleus are reviewed. They are the following: 1. Chromosomes are attached to the nuclear membrane, nucleolus and intranuclear matrix. As a result they have a relatively fixed position in the nucleus. 2. In some species somatic pairing results in alignment of homologs. This is rare in mammals. 3. The association of ribosomal DNA and 5S DNA with the nucleolous results in the close approximation of the chromosomes carrying these DNA sequences. In man and other animals the most obvious consequence is satellite association. 4. Heterochromatin is condensed onto the inner nuclear membrane and periphery of the nucleolous while genetically active chromatin occupies the more central portion of the nucleus. The results is a peripheral location of late replicating DNA and a central location of early relicating DNA. 5. The DNA replication points tend to be associated with the nuclear matrix. Autoradiography of briefly labelled cells shows a high frequency of grains associated with nuclear matrix material. 6. Heterochromatin association results in chromocenters and ectopic pairing. 7. In addition to all these is the Rabl orientation or alignment of centromeres with centromeres and telomeres with telomeres. This polarization of the chromosomes results from the traction on the centromeres by the spindle fibers. There is no firm evidence for any higher degrees of order that might bring specific functioning genes into close proximity.Supported by NIH Grant GM 15886     

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.     

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage λgtll called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997)191–198)found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.