Theo Koller

Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle

The structure of ribosomal chromatin in exponentially growing Friend cells, in stationary cells, and in metaphase chromosomes was studied by psoralen photocrosslinking. It is shown that in intact cells, two distinct types of ribosomal chromatin coexist in Friend cells, one that contains nucleosomes and represents the inactive copies and one that lacks a repeating structure and corresponds to the transcribed genes. A single gene copy is either in one or the other chromatin state. The relative amounts of the two types of structures are similar in interphase and metaphase, however, their run-on activities differ significantly. This suggests that the two states of chromatin are maintained independently of the transcriptional process and that they are stably propagated through the cell cycle.

Detection of genotoxic activity in native hospital waste water by the umuC test.

The genotoxic potential of the waste water of a hospital was evaluated by the umuC test. Within 2 years over 800 native waste water samples were analysed. Genotoxic activity was found in 13% of the samples. The highest genotoxic activity occurred in the morning hours, but genotoxic samples were detected also during the day and at night. 96% of the genotoxic waste water samples revealed a genotoxic potential without growth inhibition of test bacteria monitored as OD600, in the same way as antineoplastic drugs like mitomycin C or cisplatin. 4% of the genotoxic waste water samples showed combined cytotoxic and genotoxic activities as seen in control experiments using glutaraldehyde containing disinfectants and certain antibiotics.

Geometry and Physics of Catenanes Applied to the Study of DNA Replication

The concept of ideal geometric configurations was recently applied to the classification and characterization of various knots. Different knots in their ideal form (i.e., the one requiring the shortest length of a constant-diameter tube to form a given knot) were shown to have an overall compactness proportional to the time-averaged compactness of thermally agitated knotted polymers forming corresponding knots. This was useful for predicting the relative speed of electrophoretic migration of different DNA knots. Here we characterize the ideal geometric configurations of catenanes (called links by mathematicians), i.e., closed curves in space that are topologically linked to each other. We demonstrate that the ideal configurations of different catenanes show interrelations very similar to those observed in the ideal configurations of knots. By analyzing literature data on electrophoretic separations of the torus-type of DNA catenanes with increasing complexity, we observed that their electrophoretic migration is roughly proportional to the overall compactness of ideal representations of the corresponding catenanes. This correlation does not apply, however, to electrophoretic migration of certain replication intermediates, believed up to now to represent the simplest torus-type catenanes. We propose, therefore, that freshly replicated circular DNA molecules, in addition to forming regular catenanes, may also form hemicatenanes.

Transcriptional Activity and Chromatin Structure of Enhancer-Deleted rRNA Genes in Saccharomyces cerevisiae

ABSTRACT We used the psoralen gel retardation assay and Northern blot analysis in an in vivo yeast system to analyze effects of rDNA enhancer deletions on the chromatin structure and the transcription of tagged rDNA units. We found that upon deletion of a single enhancer element, transcription of the upstream and downstream rRNA gene was reduced by about 50%. Although removing both flanking enhancers of an rRNA gene led to a further reduction in transcription levels, a significant amount of transcriptional activity remained, either resulting from the influence of more distantly located enhancer elements or reflecting the basal activity of the polymerase I promoter within the nucleolus. Despite the reduction of transcriptional activity upon enhancer deletion, the activation frequency (proportion of nonnucleosomal to nucleosomal gene copies in a given cell culture) of the tagged rRNA genes was not significantly altered, as determined by the psoralen gel retardation assay. This is a strong indication that, within the nucleolus, the yeast rDNA enhancer functions by increasing transcription rates of active rRNA genes and not by activating silent transcription units.

DNA Structural Patterns and Nucleosome Positioning

There is no clear picture to date of the mechanisms determining nucleosome positioning. Generally, local DNA sequence signals (sequence-dependent positioning) or non-local signals (e.g. boundary effects) are possible. We have analyzed the DNA sequences of a series of positioned and mapped nucleosome cores in a systematic search for local sequence signals. The data set consists of 113 mapped nucleosome cores, mapped in vivo, in situ, or in reconstituted chromatin. The analysis focuses on the periodic distribution of sequence elements implied by each of six different published DNA structural models. We have also investigated the periodic distribution of all mono-, di-, and trinucleotides. An identical analysis was performed on a set of isolated chicken nucleosome cores (nucleosome data from the literature) that are presumably positioned due to local sequence signals. The results show that the sequences of the isolated nucleosome cores have a number of characteristic features that distinguish them clearly from randomly chosen reference DNA. This confirms that the positioning of these nucleosomes is mainly sequence-dependent (i.e., dependent on local octamer-DNA interactions) and that our algorithms are able to detect these patterns. Using the same algorithms, the sequences of the mapped nucleosome cores, however, are on average very similar to randomly chosen reference DNA. This suggests that the position of the majority of these nucleosomes can not be attributed to the sequence patterns implemented in our algorithms. The arrangement of positioned nucleosomes seems to be the result of a dynamic interplay of octamer-DNA interactions, nucleosome-nucleosome interactions and other positioning signals with varying relative contributions along the DNA.

A plasmid rescue to investigate mutagenesis in transgenic D. melanogaster

We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo. Transgenic Drosophila lines were established by transformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis. The target gene can be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation of the recircularized shuttle vectors. The resulting circular plasmids are then transformed back into E. coli lacZ- mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal. The number of inactivated versus intact lacZ genes directly indicates the mutation frequency. By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely. Spontaneous background mutation rates using this system are 2.6 +/- 0.6 x 10(-4). Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8 +/- 0.6 x 10(-4) for 0.5 mM and 6.9 +/- 1.2 x 10(-4) for 1 mM ENU, respectively.