@article{LandoEndesfelderBergeretal.2012, author = {Lando, David and Endesfelder, Ulrike and Berger, Harald and Subramanian, Lakxmi and Dunne, Paul D. and McColl, James and Klenerman, David and Carr, Antony M. and Sauer, Markus and Allshire, Robin C. and Heilemann, Mike and Laue, Ernest D.}, title = {Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast}, series = {Open Biology}, volume = {2}, journal = {Open Biology}, number = {120078}, doi = {10.1098/rsob.120078}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134682}, year = {2012}, abstract = {The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.}, language = {en} } @article{KreftBergerHaertleinetal.1983, author = {Kreft, J{\"u}rgen and Berger, Harald and H{\"a}rtlein, Michael and M{\"u}ller, Bodo and Weidinger, Gerhard and Goebel, Werner}, title = {Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60596}, year = {1983}, abstract = {From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertionwas recloned as aPstl fragment into pJKK3-1, a shuttle vector which rep{\"u}cates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no extemal or intemal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at Ievels comparable to those ofthe B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin 0-producing strain of Streptococcus pyogenes or from {\"u}steriolysin-producing strains of Usteria monoeytogenes, no positive hybridization signals were obtained. These data soggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.}, subject = {Biologie}, language = {en} } @article{BergerHackerJuarezetal.1982, author = {Berger, Harald and Hacker, J{\"o}rg and Juarez, Antonio and Hughes, Colin and Goebel, Werner}, title = {Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-40255}, year = {1982}, abstract = {We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes 04, 06, 018, and 075, The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further sub cloned either as Sail fragments in pACYC184 or as BamHI-SaLI fragments in a recombinant plasmid (pANN202) containing cistron C (hlye) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure, These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.}, language = {en} }