Renzo Lucchini

Replication of Yeast rDNA Initiates Downstream of Transcriptionally Active Genes

In the yeast S. cerevisiae, ARS (autonomously replicating sequence) elements located in the intergenic spacers of the rRNA gene locus are infrequently activated as origins of replication. We analyzed the rARS activation with a combination of neutral/neutral (N/N) two-dimensional (2D) gel electrophoresis and either the intercalating drug psoralen, which in vivo specifically marks the transcribing gene copies, or the selective accessibility of restriction sites in transcriptionally active genes. We found that initiation of replication starts at those rARSs placed immediately downstream of transcribing rRNA genes. This correlation between transcription and replication is consistent with the presence of nucleosome-free enhancers at each transcriptionally active gene copy and suggests that the transcription factor Abf1p is involved in replication initiation at the ARS in the rDNA gene locus.

Nucleosome positioning at the replication fork

In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross‐linked replication intermediates purified from preparative two‐dimensional agarose gels were analysed by exonuclease digestion or primer extension. Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery. Therefore, both the ‘old’ randomly segregated nucleosomes as well as the ‘new’ assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus.

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6‐bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.

In vivo mapping of nucleosomes using psoralen-DNA crosslinking and primer extension.

By the use of psoralen crosslinking and primer extension, a method was developed which allows the analysis of chromatin structure in vivo. Using a yeast minichromosome, >9 nucleosomes were mapped with a resolution of at least +/-30 bp.

Histone composition and core histone acetylation of transcriptionally active ribosomal chromatin of Physarum polycephalum

Abstract In situ EcoRI digestion of ribosomal gene chromatin in nucleoli of Physarum polycephalum releases a fragment of the transcribed part of the rRNA gene as shown by hybridization and psoralen crosslinking. In contrast to whole nucleoli, the active ribosomal chromatin fragment does not contain H1, H2A and H2B, but only H3 and H4. Comparison of the acetylation of histones in nuclei, nucleoli and active ribosomal chromatin does not reveal significant differences. These results suggest that the lack of H1, H2A and H2B is a prerequisite for in vivo transcription of the ribosomal gene chromatin of Physarum.