DNA rearrangements in Euplotes crassus coincide with discrete periods of DNA replication during the polytene chromosome stage of macronuclear development.

Macronuclear development in Euplotes crassus begins with polytenization of micronuclear chromosomes and is accompanied by highly precise excision of DNA sequences known as internal eliminated sequences and transposon-like elements (Tecs). Quantitation of radiolabeled-precursor incorporation into DNA indicates that DNA synthesis during formation of polytene chromosomes is not continuous and occurs during two distinct periods. We demonstrate that the timing of Tec excision coincides with these replication periods and that excision can occur during both periods even at a single locus. We also show that Tec and internal eliminated sequence excisions are coincident in the second replication period, thus providing further evidence for similarity in their excision mechanism. Inhibition of DNA synthesis with hydroxyurea diminishes Tec element excision, indicating that replication is an important aspect of the excision process.     

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.     

Metabolic pathways and energetics of the acetone-oxidizing,sulfate-reducing bacterium,Desulfobacterium cetonicum

Acetone degradation by cell suspensions of Desulfobacterium cetonicum was CO₂-dependent, indicating initiation by a carboxylation reaction. Degradation of butyrate was not CO₂-dependent, and acetate accumulated at a ratio of 1 mol acetate per mol butyrate degraded. In cultures grown on acetone, no CoA transfer apparently occurred, and no acetate accumulated in the medium. No CoA-ligase activities were detected in cell-free crude extracts. This suggested that the carboxylation of acetone to acetoacetate, and its activation to acetoacetyl-CoA may occur without the formation of free acetoacetate. Acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA, which were oxidized to CO₂ via the acetyl-CoA/carbon monoxide dehydrogenase pathway. The measured intracellular acyl-CoA ester concentrations allowed the calculation of the free energy changes involved in the conversion of acetone to acetyl-CoA. At in vivo concentrations of reactants and products, the initial steps (carboxylation and activation) must be energy-driven, either by direct coupling to ATP, or coupling to transmembrane gradients. The G of acetone conversion to two acetyl-CoA at the expense of the energetic equivalent of one ATP was calculated to lie very close to 0kJ (mol acetone)^-1. Assimilatory metabolism was by an incomplete citric acid cycle, lacking an activity oxidatively decarboxylating 2-oxoglutarate. The low specific activities of this cycle suggested its probable function in anabolic metabolism. Succinate and glyoxylate were formed from isocitrate by isocitrate lyase. Glyoxylate thus formed was condensed with acetyl-CoA to form malate, functioning as an anaplerotic sequence. A glyoxylate cycle thus operates in this strictly anaerobic bacterium. Phosphoenolpyruvate (PEP) carboxykinase formed PEP from oxaloacetate. No pyruvate kinase activity was detected. PEP presumably served as a precursor for polyglucose formation and other biosyntheses.Abbreviations MV ²⁺ Oxidized methyl viologen - PEP Phosphoenolpyruvate - PHB Poly--hydroxybutyrate     

Immunohistochemical analysis of muscle cytochromec oxidase deficiency in children

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochromec oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic iinvestigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seenn with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

Clostridium magnum sp. nov., a non-autotrophic homoacetogenic bacterium

A new mesophilic, sporeforming, strictly anaerboic bacterium was isolated form enrichments with 2,3-butanediol as sole substrate and pasteurized freshwater sediment as inoculum. Cells were large, motile rods, and elliptical spores were formed subterminally or centrally. They stained Gram-negative, but no typical outer membrane layer could be observed by electron microscopy of ultrathin sections. 2,3-Butanediol, acetoin, fructose, glucose, sucrose, xylose, malate and citrate served as substrates and were completely converted to acetate with concomitant reduction of carbon dioxide. Growth on glucose (t _dmin=1.4 h) was faster than on butanediol (t _dmin=3.6 h). No growth occurred on hydrogen/carbon dioxide, on formate or on methanol. The guanine plus cytosine content of the DNA was 29.1%. The new isolate is described as a new species, Clostridium magnum sp. nov.     

A C5'-centred deoxyribose radical in single crystals of 5-chloro-and 5-bromodeoxyuridine X-irradiated at low temperatures.

X-irradiation at 77 K and subsequent warming to 150 K of single crystals of 5-chloro-and 5-bromodeoxyuridine produces a radical located at position C5' of the deoxyribose moiety. The radical exhibits identical spectral properties in both crystal systems. It is characterized by interaction of the unpaired electron with an alpha-proton(-17-0, -8-7, -33-7G), a beta-proton (18-6, 21-3, 17-5 G) as well as an OH-proton (4-8, 7-9, 12-8 G). The principal values of the g-tensor are 2-0034, 2-0049 and 2-0042. The spectral parameters are discussed in relation to those of the same or similar radicals in other nucleosides (-tides).     

Repair rate in human fibroblasts measured by thymine dimer excorporation

Summary The UV photoproduct, thymine dimer ( ), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m² creates 0.045% (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m². The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.