Digestion Of Chromatin Research Articles

Chromatin organization in Entamoeba histolytica

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.

Gene expression and chromatin structure in the cellular slime mold, Dictyostelium discoideum.

Micrococcal nuclease digestion of chromatin from growing cells reveals a structural organization which differs for genes transcribed at diverse rates. The late cAMP dependent prespore genes which are not transcribed in growing cells are found in growing cells in a regular nucleosomal repeat with an average spacing of 168 nucleotides. By contrast genes expressed at a low level in growing cells show an irregular pattern of bands with an average distance between bands of 80 nucleotides. The sizes of the bands generated from the transcribed genes are consistent with the concept that transcription results in the loss of the linker region histone H1 with concomitant sliding of nucleosomes to generate close packed ("slipped") di, tri, and tetra nucleosomes lacking the linker region. Further analysis of dinucleosomes released by micrococcal nuclease digestion reveals that transcriptionally active genes are found associated with dinucleosomes species which may be lacking histone H1. The length of DNA protected by these dinucleosomes is heterogeneous, ranging from 250 to 300 nucleotides. Methodology is described which has been adapted to allow two dimensional hybridization mapping of nucleoprotein complexes on single copy Dictyostelium genes.

Location of nucleosomes in simian virus 40 chromatin

Over the past decade, the results of numerous indirect mappings analyses have not clarified whether or not nucleosomes occupy preferred positions in simian virus 40 (SV40) chromatin. To address this question more directly, we followed a shotgun cloning approach and determined the nucleotide sequences of over 400 cloned nucleosomal DNA fragments obtained from digestion of SV40 chromatin with micrococcal nuclease. Our results demonstrate and establish that nucleosomes do not occupy unique positions in SV40 minichromosomes and thus indicate the existence of at least several types of chromatin molecules having different nucleosome organization patterns. We developed two types of statistical analysis in order to examine the cloning data in greater detail. One type, overlap analysis, revealed the distribution of the cloned fragments with respect to SV40 DNA. The distribution exhibits an oscillating pattern, dividing the genome into regions of weak or strong nucleosome density. The other analysis determined the distribution of the midpoints of the cloned fragments and revealed potential strong and weak nucleosome location sites, and an early versus late distinction in organization of nucleosomes in SV40 chromatin. The late region appears to contain more strong nucleosome location sites (8) than the early region (4). The strongest nucleosome abuts the late side of the nuclease-hypersensitive region and includes the major transcription initiation site of the late genes. Another strong site precedes this nucleosome and includes sequences implicated in controlling the expression of the SV40 early and late genes. A strong or weak nucleosome location site is not apparent near the early side of the nucleosome-hypersensitive region. Only weak and overlapping nucleosome location sites are found in the region where replication terminates in the SV40 minichromosomes.

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN 145, and H1-containing, MN 165, MN 175, MN 185, MN 195, MN 205 and MN 215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca 2+) gave rise to nuclei-specific, H1-lacking MN 155. Digestion of H1-lacking chromatin produced only MN 145, MN 155 and MN 165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical “unfolded chromatin pattern”, digests of spermal nuclei contained primarily MN 145, MN 155, MN 235 and MN 245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN 155) or both sides of core DNA being a multiple of about 10 bp. In MN 235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.

Chromatin structure of transferred genes in transgenic plants

AbstractThe chromatin structure of foreign genes in transgenic tobacco plants was investigated by digestion of nuclei with DNase I and micrococcal nuclease, respectively, followed by restriction and Southern analysis of the digestion products. The results were compared to the differential expression of the different transgenes. Two model systems were used: plants harbouring vector DNA derived from the disarmed vector pGV 3850 and plants harbouring the light‐regulated and organ‐specifically expressed potato ST‐LS1 gene and the cotransferred ncpaline synthase (nos) reporter gene. Our results show that transferred genes are located in DNase l‐sensitive domains in all transformants. Slight variations of DNase l‐sensitivity of the transferred ST‐LS1 constructs in different transformants neither reflected the between‐transformant variability of expression nor the organ‐specific activity of the transgenes. A deletion event was found responsible for silencing the ST‐LS1 gene but not the nos gene in one of the transformants. Whereas no DNase l‐hypersensitive sites were found within the 3850‐T‐DNA and the ST‐LS1 gene, one prominent site was mapped to the nos promoter within the ST‐LS1 construct in all transformants. Digestion of chromatin harbouring 3850‐T‐DNA with micrococcal nuclease resulted in a blurred nucleosomal pattern as compared to nucleolar and bulk chromatin, the extent of blurring being independent of the expression of transferred genes. The present results favour the “permissive domain” hypothesis which capitalizes on the chromatin surrounding the integration site as the determining factor for the chromatin structure of incoming alien genes. However, between‐transformant variability of expression is not reflected by differential sensitivity to DNase I. Hence, other factors than chromatin structure must be involved in creating “position effects”.

Isolation and characterisation of a 167 bp core particle isolated from stripped chicken erythrocyte chromatin

We have digested chicken erythrocyte soluble chromatin, both unstripped and stripped of histones H1 and H5 with either 0.6 M NaCl or DNA-cellulose, with micrococcal nuclease (MNase). Digestion of unstripped chromatin to monomeric particles initially paused at 188 bp DNA; continued digestion resulted in another pause at 177 before the 167 bp chromatosome and 146 bp core particle were obtained. Digestion of stripped chromatin to monomeric particles paused transiently at 177 bp; continued digestion resulted in marked pauses at 167 and 156 before the 146 bp core particle was obtained. These results suggested that 167 bp DNA representing two complete turns are bound to the histone octamer. Histone H1/H5 binds an additional two helical turns of DNA, thereby protecting up to 188 bp DNA against nuclease digestion. Monomeric particles containing 167 bp DNA were isolated from stripped chromatin and found by DNase I digestion to be a homogeneous population with a 10 bp DNA extension to either end relative to the 146 bp core particle. Thermal denaturation and circular dichroism spectroscopy showed stronger histone-DNA interactions and increased DNA winding as the length of DNA attached to the core histone octamer was decreased. Thermal denaturation also showed three classes of histone-DNA interaction: the core particle containing 167 bp DNA had tight binding of ten helical turns of DNA, intermediate binding of two helical turns and looser binding of four helical turns.

Instability of the nuclear chromatin of procyclic Trypanosoma brucei brucei

Digestion of nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms with micrococcal nuclease yielded DNA fragments which formed DNA ladders in agarose gels, similar to those of rat liver. However, the chromatin of trypanosomes was digested more rapidly. The digestion of T. b. brucei chromatin yielded a large amount of DNA fragments of core-particle size. The numbers of base pairs per nucleosomal and linker DNA were identical in both species, if the digestion conditions were reduced in the case of T. b. brucei. Psoralen cross-linking of soluble chromatin of trypanosomes at 5 mM salt at pH 7 or pH 10 resulted in an irregular array of single-stranded (ss) bubbles separated by variable stretches of double-stranded (ds) DNA. The proportion of ss DNA was low compared with the ratio of ss/ds stretches in rat liver chromatin, which also showed regularly arranged nucleosomal DNA. Soluble chromatin of T. b. brucei, pre-treated with 500 mM NaCl to remove a potential H1 and psoralen cross-linked at 5 mM salt at pH 7 or pH 10 was to a great extent ds in both situations. The true nucleosome filament organization of T. b. brucei chromatin could only be shown by psoralen cross-linking the DNA in whole nuclei under physiological conditions. The results indicate that the chromatin of procyclic T. b. brucei differs significantly in its compaction pattern from rat liver chromatin; a typical histone H1 is not found, and the DNA-protein interactions are also less stable and can more easily be destabilized by experimental conditions.

Locations of nucleosomes on the regulatory region of simian virus 40 chromatin

We have asked where the nucleosomes are located with respect to the replication origin and regulatory region of simian virus 40 DNA, what would be the possible functional consequences of the identified locations, and to what extent these locations correlate with the current views on mechanisms involved in establishing nucleosome-free regions in chromatin. To identify the precise location of nucleosomes, we have shot-gun cloned and sequenced nucleosomal DNA obtained from micrococcal nuclease digestion of wt776 chromatin prepared late in infection. Our results indicate that nucleosomes do not occupy unique positions over the replication origin or the elements involved in transcriptional control. However, it appears that the nucleosome distribution is not random, since several nucleosomes are represented by two or more independently generated clones. Two nearly identical cloned fragments map over the replication origin; five include 1.5 copies of the 72 base-pair enhancer sequences; and eight map to a region that spans a DNA bending locus and the major transcription initiation site of the late genes. The complex nucleosome distribution pattern observed in our direct analysis suggests that disparate nucleosome-free regions may be involved in controlling replication, and selective expression of the viral early or late genes.

Chromatin digestion with restriction endonucleases reveals 150-160 bp of protected DNA in the centromere of chromosome XIV in Saccharomyces cerevisiae.

Isolated nuclei of Saccharomyces cerevisiae were incubated with five restriction nucleases. Out of the twenty-one recognition sequences for these nucleases in the centromere region of chromosome XIV, only five are accessible to cleavage. These sites map 11 bp and 74 bp to the left and 27 bp, 41 bp and 290 bp to the right, respectively, of the boundaries of the 118 bp functional CEN14 DNA sequence. The distance between the sites accessible to cleavage and closest to CEN14 is 156 bp, suggesting this is the maximal size of DNA protected in CEN14 chromatin. The DNA in CEN14 chromatin protected against cleavage with DNase I and micrococcal nuclease overlaps almost completely with this region. Hypersensitive regions flanking both sides are approximately 60 bp long. Analyses of other S. cerevisiae centromeres with footprinting techniques in intact cells or nucleolytic cleavages in isolated nuclei are discussed in relation to our results. We conclude that structural data of chromatin obtained with restriction nucleases are reliable and that the structure of CEN14 chromatin is representative for S. cerevisiae centromeres.

A stable alpha-helical element in the carboxy-terminal domain of free and chromatin-bound histone H1 from sea urchin sperm.

The carboxy-terminal domain (residues 121-248) of sea urchin sperm-specific H1 is not random coil but partly alpha-helical, even in 1 mM sodium phosphate, pH 7. The helix resides in a 57 residue proline-free segment which, in the intact histone, immediately abuts the central globular domain. The proline-free region, which is rich in lysine and alanine, is relatively resistant to tryptic digestion when the carboxy-terminal domain is bound to DNA. Two (overlapping) resistant peptides are shown by circular dichroism measurements to be substantially alpha-helical in 1 mM sodium phosphate and to increase in helix content to approximately 70% in 1 M NaCLO4. Tryptic digestion of chromatin gives resistant fragments containing both the globular domain and the contiguous proline-free segment, strongly suggesting that the alpha-helical segment also exists in chromatin, where it would be ideally placed to direct the path of the linker DNA entering or leaving the nucleosome. The linker in sea urchin sperm chromatin is long (approximately 74 bp), and the unusually long alpha-helical segment in the carboxy-terminal tail of sperm H1 which has amphipathic character due to the alanine distribution, and is likely to be curved, may be a special feature tailored to organize it.

Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry.

A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of c-myb gene regulation in MRL-lpr/lpr mice. Identification of a 5' c-myb nuclear protein binding site and high levels of binding factors in nuclear extracts of lpr/lpr lymph node cells.

Lymph node T cells of MRL-lpr/lpr mice are characterized by the production of very large amounts of c-myb mRNA. To study the control of c-myb expression, a search was made for sites on the 5' c-myb gene which could bind regulatory proteins. DNase I digestion of nuclear chromatin uncovered four DNase I hypersensitive sites in the first intron of the c-myb gene, and a single site approximately 300 bp 5' to the initiation codon. Lambda exonuclease digestion of a 5'-myb fragment in the presence of nuclear extracts from either MRL-lpr/lpr PLN or EL-4 thymoma revealed stop sites approximately 300 bp 5' (-271 to -322) to the ATG initiation codon. DNase I footprint analysis demonstrated a guanine-cytosine enriched region of potential binding sites (-274 to -319) in the region of the stop sites and a fifth potential binding site closer to the initiation codon (-163 to -168). Specific gel shift bands were detected by a 5'-myb fragment (-346 to -155) with extracts from a number of different lymphoid cell lines and the appropriate specific and non-specific competitor DNA. The DNA giving rise to these gel shift bands encompassed the region defined by the stop site and footprinting studies. To determine whether or not the protein binding to the 5' c-myb gene at -274 to -319 was associated with increased c-myb mRNA, we studied nuclear extracts of several cell lines and compared the amount of binding to the amount of c-myb mRNA found on Northern analyses. Among the cell lines, there was a correlation between c-myb expression and the amount of the 5'-myb DNA binding protein. In addition, MRL-lpr/lpr lymph node cells had high c-myb expression and large amounts of the 5'-myb binding protein. This result suggests that the binding may play some role in the c-myb expression. Moreover, the most immature cell lines had the greatest amount of the binding factor, suggesting that its regulatory effect on c-myb expression might be important in early differentiation events.

Selective production of autoantibodies in graft-vs-host-induced and spontaneous murine lupus. Predominant reactivity with histone regions accessible in chromatin.

The injection of (C57BL/6 X DBA/2)F1 mice with parental DBA/2 lymphoid cells leads to a lupus-like disease in which IgG autoantibodies are targeted to certain nuclear and cell surface antigens. To investigate further the extent of antibody diversity in this graft-vs-host (GVH) model, we studied the specificity of antihistone antibodies induced by the GVH reaction. High levels of IgG antibodies to histones H1 and H2B were detected whereas responses to H2A, H3, and H4 were only marginally elevated above pre-GVH levels. Immunoblotting analysis further revealed that the response to H2B was focused on epitopes that most likely reside in the N-terminal region. In contrast, F1 mice immunized with H2B/RNA complexes in adjuvant produced antibodies to the N terminus as well as to other regions of the H2B molecule. Thus, the antihistone response stimulated by the GVH reaction is only a fraction of the potentially activatable B cell repertoire. We also determined whether antibodies that arise spontaneously in genetically predisposed lupus strains were restricted in their histone reactivity. The response to core histones was highly variable among individual animals of the NZB/NZW and MRL-lpr/lpr strains despite their inbred nature. However, nearly all mice exhibited a preferential reactivity for epitopes in histone regions that are lost after partial trypsin digestion of chromatin. These data demonstrating autoantibody responses that are limited to particular histone regions support a mechanism by which B cells are selectively activated in murine lupus. The predominant production of antibodies to histone regions that are exposed in nucleosomes raises the possibility that chromatin is an antigenic stimulus for histone-specific B cells in this disease.

The DNase I generated disomal series is coherent to 16 N Implications for coiling models of chromatin structure

The disomal series generated by the digestion of chromatin with DNase I has been followed to its highest orders using Klenow end-labelling and field-inversion gel electrophoresis to maximise the resolution of large DNA fragments. The series is coherent to the 16 N level and as such is incompatible with the most structurally acceptable coiling models. We propose that this is evidence for the general unsuitability of coiling models and is support for the existence of simple “back-to-back” double-stranded structures within chromatin.

Characterization of DNA binding proteins released from sarcoma-180 chromatin during brief digestion with DNase-I.

Brief digestion of sarcoma-180 chromatin (less than or equal to 5%) by pancreatic DNase-I releases six non-histone proteins with Mol. wt. 21.5K, 26K, 72.5K, 77K, 90K and 120K dalton and pI values ranging from 4.7 to 12.4. The protein of Mol. wt. 77K dalton was obtained at high alkaline range of pH = 12.4. The antibodies against these proteins induce dose dependent inhibition in transcription of native chromatin. Results suggest a role of these proteins in positive control of gene transcription in sarcoma-180 cells.

Chromatin structure. Further evidence against the existence of a beaded subunit for the 30-nm fiber.

The size distribution of chromatin fragments released by micrococcal nuclease digestion of liver chromatin at various ionic strengths was examined. Below 20 mM ionic strength, gradient profiles with a peak centered at 6 nucleosomes are generated, whereas between 20 and 50 mM the peak is always centered on 12 nucleosomes, and above 50 mM ionic strength the 30-nm fiber becomes less accessible to the nuclease and there is a corresponding increase in the size distribution of fragments in the gradients. However, extensive digestions always give profiles with a peak of 12 nucleosomes as nuclease-resistant dodecamers accumulate. All of these observations are consistent with the winding of the 10-nm polynucleosome chain into a helical coil commencing at about 20 mM ionic strength. The helical turns are stabilized by histone H1 interactions between 20 and 50 mM ionic strength producing stable dodecamers. Above 50 mM ionic strength the coil condenses longitudinally and the profiles are consistent with a random attack of this fiber by the nuclease. Consequently it is not necessary to invoke the existence of a subunit bead to explain the profiles. We further define the conditions at which specific structural transitions take place and provide methodology for the preparation of chromatin at various levels of condensation.

ATP as an alternative inhibitor of bacterial and endogenous nucleases and its effect on native chromatin compaction.

The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp-1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nuclease-treated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10(-4) M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 bp in length. Comparative digestion of chromatin with staphylococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition.

DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features.

A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin.

Differentiation of nuclear structure during the sexual cycle in Tetrahymena thermophila: II. Degeneration and autolysis of macro- and micronuclei

During the postzygotic period of the sexual phase (conjugation) in the protozoon, Tetrahymena, the parental somatic macronucleus and one of the presumptive germinal micronuclei degenerate and eventually disintegrate within the cells. Electron microscopy was applied to reveal the fine-structural details of the degeneration and autolysis of these macro- and micronuclei. From detailed analysis of our data, we divided the different processes involved into several distinct cytological stages. An initial event of degeneration is the strong condensation of the nuclei, referred to as nuclear pyknosis. This process also involves transformation of the nuclear envelope, including the loss of pore complexes, attached ribosomes, and continuity with the endoplasmic reticulum. Following this event, large, flattened cisternae can occasionally be seen in the process of enveloping pyknotic nuclei ( autophagosome formation). A later stage is reached when the autophagosome has closed and its inner membrane has dissolved ( autophagosome type II according to the classification of Nilsson). At this stage, small, electron-lucent vesicles can be seen fusing with and/or ‘pinching off’ from the phagosome membrane around old macronuclei. Very dense old macronuclei are sometimes encountered in which the surface layer has become so compressed that the residual nuclear envelope underneath the autophagosome membrane is obscured ( compact autophagosome stage). In a later typical stage, the residual nuclear envelope reappears and separates from the autophagosome membrane ( ‘sickle’ stage). During this stage, there is the first clear morphological indication of chromatin digestion. The last stage that we were able to observe is characterized by the presence of a large autophagosome and a light ‘halo’ around the enclosed pyknotic nucleus ( ‘halo’ stage). During this stage, lysosomes fuse with the autophagosome, creating an auto(phago)lysosome. Within these organelles, progressive digestion and destruction of the pyknotic nuclei are observable.

Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

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.