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.     

Site-specific phasing in the chromatin of the rDNA in Dictyostelium discoideum

The rDNA in Dictyostelium discoideum is organized in linear, extrachromosomal, palindromic dimers of approximately 88 X 10(3) bases in length. The dimers are repeated about 90 times per haploid genome. Using indirect end-labeling, we have mapped micrococcal nuclease and DNAase I-sensitive sites in the chromatin near the rDNA telomeres. This region is 3' to the 36 S rRNA coding region and contains a single 5 S rRNA cistron but is primarily non-coding. We have observed somewhat irregularly spaced but specific phasing of nuclease-sensitive sites relative to the underlying DNA sequence. Comparison of the sites in chromatin with those in naked DNA reveals an unusual and striking pattern: the sites in naked DNA that are attacked most readily by both nucleases, presumably because of the specificity of the nucleases for certain sequences or physical characteristics of the DNA, appear to be the same sites that are most protected in chromatin. This pattern extends over most of a 10(4) base region, from the sequence immediately distal to the 36 S rRNA coding region and extending to the terminus. Although much of the sequence-specific phasing is irregularly spaced, salt extraction data are consistent with the presence of nucleosomes. In addition, phasing in the terminal region may be directed partially by proteins that do not bind DNA as tightly as do core histones. We present a model for phasing in spacer regions in which the sequence preferences of nucleases such as micrococcal nuclease and DNAase I may be useful tools in predicting nucleosome placement.     

Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia.

Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication. Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding. The rDNA accounted for one half of the label incorporated into cellular DNA during this period. Electron microscopy of the purified rDNA showed 1% replicative intermediates. Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central nontranscribed region of the palindromic molecule. Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis. The electron microscopic data was consistent with an origin of replication located approximatley 600 base pairs from the center of the rDNA of T. thermophila, in contrast to a more central location in the rDNA of T. pyriformis. One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule.     

Regions in the ribosomal minichromosome of Physarum polycephalum are protected from restriction nucleases; protection is insensitive to high salt in the G phase and sensitive in the M phase of the cell cycle

In the central spacer region of the extrachromosomal ribosomal DNA of the slime mold Physarum polycephalum, four small regions of related sequence are completely inaccessible to restriction endonucleases (HinfI and MboI). In addition, some sequences neighboring the inaccessible ones, are partially inaccessible to restriction enzymes and micrococcal nuclease. Taking advantage of the natural synchrony of Physarum plasmodia, we found that this protection is present throughout the cell cycle. Treatment with high salt (2.5 M-NaCl) of nuclei from the G2 phase of the cell cycle left the protection essentially unchanged. When nuclei from the M phase were treated with salt, the protection was abolished. The inaccessible sites are located close to the origins of replication of the rDNA.     

Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species.

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA. Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized.     

Ribosomal DNA sequences attached to the nuclear matrix

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.     

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.     

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.     

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.