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1.
We have used salt extractions of nuclei and long agarose gels to dissect the chromatin fine structure of the histone gene repeat of Drosophila melanogaster. Extraction of nuclei with 0.35 M KCl removes many non-histone chromosomal proteins but does not significantly disturb the overall nucleosome arrangement of the repeat unit. After extraction of nuclei with 0.55 M KCl, which also removes histone Hl, the basic arrangement of nucleosome core particles in the repeat unit is not greatly disturbed and the exposed DNA segments near the 5' ends of the histone genes are also retained. Extraction of nuclei with 0.75 M or higher KCl concentrations causes extensive nucleosome sliding and rearrangement with accompanying changes in the nucleoprotein organization of the histone gene complex and loss of the 5' hypersensitive sites. Our results indicate that the histone gene repeat displays a highly organized chromatin structure in vivo.  相似文献   

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The nucleosomal organization of active and repressed alpha subtype histone genes has been investigated by micrococcal nuclease digestion of P. lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32P-labeled specific DNA probes. In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromatin. In contrast, both coding and spacer alpha subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed. Switching off of the alpha subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region. As probed by hybridization to D.melanogaster actin cDNA, actin genes retain a regular nucleosomal structure in all the investigated stages.  相似文献   

4.
Supercoiled plasmids Col E1 and cDm 506 (a Col E1 derivative carrying the D. melanogaster histone gene repeat) were treated with OsO4 in presence of pyridine and the reaction products were analyzed using different approaches. Gel electrophoresis showed that OsO4 binding to supercoiled DNA induced its relaxation without nicking. The amount of osmium bound to DNA (as determined electrochemically) increased with the extent of DNA relaxation. As a result of osmium modification of supercoiled cDm 506, a single denaturation "bubble" was observed in the electron microscope. Mapping of the osmium binding site by S1 nuclease cleavage followed by restriction enzyme digestion has revealed one major site in the intergenic spacer between the H1 and H3 histone genes of D. melanogaster. This site differs from the site cleaved by S1 nuclease in supercoiled DNA in the absence of osmium.  相似文献   

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Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.  相似文献   

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Chromatin structure of globin and ovalbumin genes in chicken erythrocyte nuclei has been investigated by means of the "nuclease criterion" (described earlier). In intact nuclei (i.e. in the presence of 3 mM MgCl2) DNase I cleaves chromatin of both genes generating fragments multiple of a double-nucleosome repeat (2N-periodicity). However, in the case of the globin gene, apart from the 2N-periodicity, fragments were observed that are multiple of 100 b.p. and are characteristic for partially unfolded chromatin. This distinction in nuclease cleavage patterns correlates with a higher sensitivity of the globin gene as compared with the inactive ovalbumin gene. At 0.5-0.7 mM MgCl2 the transition from dinucleosomal fragmentation with DNase I and DNase II to fragmentation via a 100 b.p. interval occurs and the difference in digestibility of both genes is dramatically increased. If chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer DNase Il generates an usual nucleosomal repeat, and in this ionic conditions one may not observe any difference in nuclease sensitivity of the analyzed genes. The data allow to suggest that the high nuclease sensitivity of potentially active genes can be conditioned by more relaxed arrangement of nucleosomes in higher order chromatin structure.  相似文献   

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The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.  相似文献   

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We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.  相似文献   

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The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.  相似文献   

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The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.  相似文献   

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At the late blastula stage of sea urchin development a changeover of histone synthesis and chromatin composition takes place. Synthesis of the early histone variants declines while another set, the late histone variants, begins to be detected. During subsequent development the late histones accumulate steadily. In the 9-day larva only late histone variants are detectable. Micrococcal nuclease acts differentially on early and late nuclei. There is a depressed release of acid-soluble DNA when chromatin containing the late histones is digested. Nucleosomal repeat lengths change systematically and in parallel with the changing histone composition. Blastula and preblastula chromatin have a significantly shorter major repeat length than does the chromatin of 9-, 11-, and 16-day larvae. Intermediate stages of development have chromatin with intermediate periodicities. These differences are observed when the determinations are made under denaturing conditions of electrophoresis. Repeat lengths were found to be independent of the extent of digestion at all stages examined except the pluteus, in which there is an increase of the apparent repeat length as digestion proceeds. Pancreatic DNase I digests nuclei from blastulae and 9-day larvae similarly. Changes in the histone composition of chromatin, in nuclease accessibility of chromatin, and in nucleosomal repeat length are all very closely correlated, implying that there are underlying causal relationships.  相似文献   

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Adaptive evolution of Cid, a centromere-specific histone in Drosophila   总被引:13,自引:0,他引:13  
Malik HS  Henikoff S 《Genetics》2001,157(3):1293-1298
Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, we find an excess of replacement changes that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. We propose that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery.  相似文献   

16.
The organization of oligonucleosomes in yeast   总被引:9,自引:2,他引:7       下载免费PDF全文
We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization. Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions. No nucleosomal repeat is seen early in digestion for the insoluble fraction. Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes. When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat. Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes.  相似文献   

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We have examined the role played by each histone in forming the structure of the ν-body. When DNAase I, DNAase II, trypsin, and chymotrypsin attack chromatin, characteristic discrete DNA and protein digest fragments are produced. Using this restriction of accessibility as diagnostic for chromatin structure, we have examined complexes of DNA with virtually all possible combinations of histones. The results strongly support our previous conclusion (Camerini-Otero, Sollner-Webb, and Felsenfeld, 1976) that the arginine-rich histones are unique in their ability to create, with DNA, a structure with many features of native chromatin. Acting together, slightly lysine-rich histones then modify this complex into one very similar to native chromatin. An analysis of the rate constants of staphylococcal nuclease digestion also confirms that the complex of H3, H4, and DNA is crucial to the structure of the ν-body.  相似文献   

19.
The structure of the Sipunculus erythrocyte chromatin has been characterized by electron microscopy and nuclease digestion (staphylococcal nuclease and pancreatic nuclease). Contrary to previous results [1], we were able to isolate and characterize a histone H2B in sipunculid nuclei. Though the histones H2A and H2B were markedly different from their vertebrate homologues, the subunit structure of the chromatin is the same. But the length of the repeat unit of DNA in the chromatin, is 177 ± 5 bp for the sipunculid erythrocyte nuclei, close to that reported for the chromatin of some lower eukaryotes.  相似文献   

20.
We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.   相似文献   

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