Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-sigma). All three strategies revealed that higher -sigma in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -sigma stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -sigma of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.     

Ellipticine increases the superhelical density of intracellular SV40 DNA by intercalation.

We investigated the in vivo effect of ellipticine, a mammalian topoisomeraseII(topoII) inhibitor, on SV40 DNA topology. In contrast to epipodophyllotoxins, ellipticine did not cause significant double stranded cleavage of intracellular SV40 DNA. Furthermore, ellipticine reduced cleavage induced by epipodophyllotoxins, VP16 and VM26. Unexpectedly, ellipticine dramatically increased the superhelical density of a fraction of intracellular SV40 DNA. Several lines of evidence suggest that the formation of this highly supercoiled DNA species (Ih form DNA) is not due to the inhibition of topoII per se, but is the result of intercalation by ellipticine in a subfraction of the intracellular SV40 chromatin followed by the fixation of DNA linking number by a topoisomerase activity. Based on the linking number change and the known unwinding angle of ellipticine, the intercalation density was calculated as one ellipticine molecule per 10-20 bp in the Ih DNA. This result suggests the existence of different populations of intracellular SV40 chromatin with respect to the accessibility to ellipticine intercalation.     

The in vivo reproductive potential of density separated cells

Murine ascites cells (L1210, L5178Y, Ehrlich ascites) were labelled with ¹³¹I-iododeoxyuridine and subjected to buoyant density centrifugation on a continuous, linear Ficoll gradient. Cell losses sustained during density centrifugation were evaluated by recording the amount of ¹³¹I recovered in the final cell fractions. The viability and proliferative capacity of the density separated cells were tested by monitoring the rate of ¹³¹I excretion following inoculation of the recovered cells into new, non-radioactive hosts.Density separation in Ficoll appeared to cause few, if any, adverse effects. Cell recovery under properly regulated experimental conditions was virtually complete (97% or higher). The reproductive potential of density-separated cells was identical to that of control cells. However, considerable cell mortality could be induced by permitting cellular aggregation in medium free of antiagglutinin or by exposure of excessive quantities of cells to a density gradient.Viability indices obtained with trypan blue proved unsuitable for predicting long-term survival. In some experiments the trypan blue data provided a 90–100% viability reading when in fact the entire cell population had been inactivated by irradiation or heat incubation. Since the trypan blue test also did not reveal the full extent of mortality among aggregated cells or cells recovered from overloaded gradients, it was concluded that the dye exclusion test, in spite of its utility for monitoring immediate cell death and membrane destruction, was of limited value for evaluating the reproductive potential of mammalian cells.     

mRNA analysis of single living cells

Analysis of specific gene expression in single living cells may become an important technique for cell biology. So far, no method has been available to detect mRNA in living cells without killing or destroying them. We have developed here a novel method to examine gene expression of living cells using an atomic force microscope (AFM). AFM tip was inserted into living cells to extract mRNAs. The obtained mRNAs were analyzed with RT-PCR, nested PCR, and quantitative PCR. This method enabled us to examine time-dependent gene expression of single living cells without serious damage to the cells.     

In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.     

Near-field optical analysis of living cells in vitro

Near-field optical analysis (NOA) provides morphological nanoscale mappings of living cells in liquid cell culture media and nondestructive insight into cell functionality. Here we show for the first time the performance of NOA in imaging living cells. Unlabeled human endothelial cells attached to polished titanium disks were analyzed with hydrophobically coated optical biosensors mounted to a near-field scanning optical microscope (NSOM). Biosensors and titanium substrates could be simply implemented in standard NSOM and high-throughput NOA.     

Formation of cruciform structures in pAO3 plasmid DNA on increasing superhelical density

The dependence of the crusiciform structure formation on superhelical density was studied by means of high resolution gel-electrophoresis. A short pAO3 DNA plasmid (1683 b. p.) which is a quarter of the ColE1 DNA plasmid and contains the main palindrome of ColE1 DNA was used. The excellent resolution of all topoisomers of pAO3 DNA in gel-electrophoresis made it possible to observe a sharp abruption in the pattern of pAO3 DNA topoisomers separation. The two-dimensional gel-electrophoresis data showed that observed abruption is caused by a sharp decrease of writhing in the molecules with superhelical density--sigma approximately equal to 0,05. An analysis of S1-nuclease digestion products of DNA with different superhelical density was accomplished and these data showed that a sharp structural transition in supercoiled DNA pAO3 is caused by formation of a cruciform structure in the main palindrome.     

The effect of superhelical density on the yield of single-strand breaks in gamma-irradiated plasmid DNA.

Using agarose gel electrophoresis, the formation of DNA single-strand breaks (SSBs) by 137Cs gamma irradiation was quantified in negatively supercoiled topological isomers of plasmid pUC18. The G value for SSB formation falls slightly from 1 x 10(8) to 8 x 10(-9) SSB Gy-1 Da-1 as the superhelical density varies from 0.00 to -0.08. This result is not in agreement with recent observations by others which suggest that increasing the negative superhelical density of plasmid DNA increases its sensitivity to X irradiation.     

Pronase reduces intramembranous particle density in uterine epithelial cells in vivo

The proteolytic enzyme, pronase, was injected into the uterine lumen of rats. This treatment removed half the intramembranous particles (IMPs) from the apical plasma membrane of the uterine epithelial cells but tight junctions of these cells were unaffected. We conclude that at least some of the IMPs are proteinaceous in nature and suggest that IMPs not affected by pronase may be deeply embedded in the lipid bilayer.     

The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.     

Estimation of specific growth rate from cell density measurements

For modelling purposes it is of great importance to derive the specific growth rate as a function of time from biomass measurements. Traditional methods such as exponential or polynomial fitting do not give satisfactory results nor do these methods take the noise characteristics of the biomass measurements into account. Standard recursive techniques, such as Kalman filtering, use only the data up to the time under consideration and are dependent of a good initial estimation. This paper describes a technique based on combining subsequent backward and forward extended Kalman filtering to give a smoothing estimator for the specific growth rate. The estimator does not need an initial value and is shown to have a single tuning parameter. The applicability of the estimator is demonstrated on batch and fed-batch cultivations of two organisms: Bordetella pertussis and Neisseria meningitidis.