Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.     

Heteropycnosis of an underreplicating chromosome

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that condensed chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. InD. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from theX chromosome, via3 and2, to chromosome4. In the male, the order isX, 2, 3, Y, and4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. TheX chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where theX chromosomes shorten less than the autosomes, and why each of theX chromosomes is 15% shorter than theX chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome3 condense by shortening, while chromosomes2 and4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation:(1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.     

Continuous production of L-alanine from fumarate in a two-stage membrane reactor

Summary L-alanine was produced continuously from fumaric acid by means of soluble aspartase and L-aspartate--decarboxylase. The two reaction steps were carried out in two membrane reactors in series at different pH and temperature. The retention of the soluble enzymes within the reactor vessels was achieved by means of ultrafiltration membranes.     

Polar group interaction and molecular packing of membrane lipids: The crystal structure of lysophosphatidylethanolamine

The conformation and molecular packing of 3-palmitoyl-dl-glycerol-1-phosphoryl-ethanolamine has been determined by a single crystal analysis (R = 0.115); it crystallizes in the monoclinic space group $\text{P2}{}_1\text{a}$ with a unit cell of $\text{a = 7.66}\text{A}\text{?}\text{, b = 9.08}\text{A}\text{?}\text{, c = 37.08}\text{A}\text{?}\text{and}\text{β = 90.2 °}$, with four molecules per unit cell. The molecules exist as configurational and conformational enantiomers and pack in a bilayer arrangement. The phosphorylethanolamine groups have an orientation parallel to the layer surface. The hydrocarbon chains are arranged according to the T∥ chain packing mode and adopt an extreme tilt of 57.5 ° with respect to the layer normal. The free glycerol hydroxyl group forms an intramolecular hydrogen bond with, a phosphate oxygen and thus affects the conformation and orientation of the head group. The phosphorylethanolamine dipoles are oriented parallel to each other in double rows, while they are antiparallel and form a continuous network in dilauroylphosphatidylethanolamine (Elder et al., 1977). The area per molecule in 3-palmitoyl-dl-glycerol-1-phosphorylethanolamine (34.8 Ų) is less than in diacylphosphatidylethanolamine (38.6 Ų), indicating that in the latter the hydrocarbon chains determine the molecular cross-section. The significance of the interaction and space requirement of the phosphorylethanolamine group for the phase behaviour of phosphatidylethanolamine is discussed.     

Studies on the origin of the near-infrared (800–900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 (‘copper signal’) of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (?170 to ?190°C). For some sets of samples spectra were recorded in the range 500–1100 nm. A particular effort was made to study this correlation with what are called ‘mixed valence’ states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205–215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10–15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700–800 nm region did not appear to be more useful than the 800–900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

Clostridium thermoaceticum forms methanol from carbon monoxide in the presence of viologen dyes

Whole cells of Clostridium thermoaceticum, crude extracts of such cells as well as the supernatant of 100 000 × g centrifugations catalyse the reduction of carbon monoxide to methanol in the presence of viologens or cobalt sepulchrate. Without such a mediator methanol could not be detected. The reaction shows a marked optimum at pH 5. The incubation of [5-¹⁴C]methyltetrahydrofolate led only to the formation of ¹⁴C-labeled ethanol; the radioactivity in methanol was negligible. The reaction seems to be catalysed by carbon monoxide dehydrogenase.