Barley seedlings homozygous both for the xantha-1(35) and tigrina-d(12) mutation accumulate magnesium protopophyrins and other precursors of chlorophyllide constitutively in darkness. The homozygous double mutant xantha-f(10), tigrina-O(34) produces protoporphyrin constitutively. These results provide evidence for the control of chlorophyllide synthesis in higher plants through the products of regulatory genes in the nucleus.

and 1Gesellschaft fir Biotechnologische Forschung (GBF), D-3300 Braunschweig, FRG Communicated by U.K.LaemmliPreviously, we have demonstrated that in Tetrahymena DNA topoisomerase I has a strong preference in situ for a hexadecameric sequence motif AZACTTAGAZAAATTA present in the non-transcribed spacers of r-chromatin. Here we characterize more extensively the interaction of purified topoisomerase I with specific hexadecameric sequences in cloned DNA. Treatment of topoisomerase I-DNA complexes with strong protein denaturants results in single strand breaks and covalent linkage of DNA to the 3' end of the broken strand. By mapping the position of the resulting nicks, we have analysed the sequence-specific interaction of topoisomerase I with the DNA. The experiments demonstrate that: (i) the enzyme cleaves specifically between the sixth and seventh bases in the hexadecameric sequence; (ii) a single base substitution in the recognition sequence may reduce the cleavage extent by 95%; (iii) the sequence specific cleavage is stimulated 8-fold by divalent cations; (iv) 30% of the DNA molecules are cleaved at the hexadecameric sequence while no other cleavages can be detected in the 1.6-kb fragment investigated;(v) the sequence specific cleavage is increased 2-to 3-fold in the presence of the antitumor drug camptothecin; (vi) at high concentrations of topoisomerase I, the cleavage pattern is altered by camptothecin; (vii) the equilibrium dissociation constant for interaction of topoisomerase I and the hexadecameric sequence can be estimated as -10-10 M.

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.

Topoisomerase I is in situ associated with DNaseI hypersensitive sites located in the promotor and terminator regions of the extrachromosomal rDNA in Tetrahymena thermophila at sites with sequences fitting the motif (sequence in text) Reconstitution experiments with purified topoisomerase I and cloned fragments of rDNA demonstrate that the enzyme exhibits the same binding and cleavage properties on naked DNA. These observations are striking as topoisomerase I previously has been found to exhibit low sequence specificity. The specific binding of the enzyme has an absolute requirement for divalent cations with a preference for Ca2+. The strong binding to the hexadecamer has been characterized by competition experiments, and it has been used to determine the molecular weight of the enzyme.

ATR-FTIR, FT-NIR and near-FT-Raman spectroscopy were used to characterize the molecular composition of human skinin vivoand pig ear skinin vitro. Due to different measurement depths the spectroscopic techniques reveal the characteristics of different layers of the skin. Tape stripping was used with the ATR-FTIR technique. Spectral differences concerning lipid content and conformation, protein secondary structure or content of water were found with respect to both gender and species (i.e. human versus pig ear) at all measured skin depths. New assignments of so far unassigned lipid and protein peaks in the FT-NIR and ATR-FTIR spectra of skin were made. PCA and PLS models were used to investigate the division of the recorded spectra into groups. With respect to classification of male and female subjects, the PLS discriminant analysis provided a classification accuracy of 64–93% based on the ATR-FTIR spectra and 83–89% based on the Raman spectra. With respect to classification of human skinin vivoand pig ear skinin vitro, the PLS discriminant analysis provided a classification accuracy of 75–100% based on the Raman spectra and 100% based on the ATR-FTIR spectra.