Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.     

Structure of the extrachromosomal ribosomal RNA chromatin of Physarum polycephalum

Isolated nucleoli from exponentially growing microplasmodia of Physarum polycephalum were digested with micrococcal nuclease or DNAase I, or were photoreacted with trimethyl psoralen. In the coding region for the precursor of the ribosomal RNA, micrococcal nuclease and DNAase I digestions show predominantly a smear, and treatment with psoralen leads to a fairly continuous crosslinking of the DNA. All three assays are compatible with the absence of a typical nucleosomal array in most of the gene copies. In contrast, in the central non-transcribed spacer, except in the immediate 5'-flanking region, micrococcal nuclease and DNAase I digestions yield fragments that are multiples of a basic repeat, compatible with a nucleosomal packing of this region. The crosslinking pattern with psoralen confirms this conclusion. In addition, there are three sites over 400 base-pairs long that are inaccessible for psoralen crosslinking. Two of these sites have been mapped to the putative origins of replication. In the terminal non-transcribed spacer, except in the immediate 3'-flanking region, digestions with micrococcal nuclease and DNAase I give a smeared repeat. The crosslinking pattern after treatment with psoralen suggests that this region is packed in nucleosomes, except for about 900 base-pairs constituting the telomere regions of the linear extrachromosomal palindromic rDNA. Micrococcal nuclease digestion of the immediate 5'-flanking region shows a complete absence of any nucleosomal repeat, but digestion with DNAase I leads to a faint ten base-pair repeat. In contrast, in the 3'-flanking regions both nuclease assays indicate a chromatin structure similar to the coding region. Both flanking regions are unusual with respect to psoralen crosslinking, in that crosslinking is reduced both in chromatin and deproteinized DNA. On the basis of the known sequence-dependent psoralen crosslinking and the established sequences in these regions, crosslinking should be expected to occur. However, it does not and we therefore propose the presence of an unusual DNA conformation in these regions.