ADP-ribosylation induced changes in the conformation of the chromatin of the brain of developing rats

Conformational changes in the chromatin of the cerebral hemisphere of 3-, 14- and 30-day old developing rats were studied before and after its ADP-ribosylation using DNase I and micrococcal nuclease (MNase). The rate and extent of digestion of chromatin by DNase I are the highest at 3-day and decline progressively thereafter. The rate and extent of digestion by MNase do not change during development. ADP-ribosylation of chromosomal proteins was carried out by incubating nuclei with NAD+ for 30 min and was followed by endonuclease digestion. Both the rate and extent of digestion by DNase I and MNase were enhanced after ADP-ribosylation which was the maximum for 3-day rats.     

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: indications of a phase relation between restriction sites and chromatin subunits in African green monkey and calf nuclei.

The periodicities of the restriction enzyme cleavage sites in highly repetitive DNAs of six mammalian species (monkey, mouse, sheep, human, calf and rat) appear related to the length of DNA contained in the nucleosome subunit of chromatin. We suggest that the nucleosome structure is an essential element in the generation and evolution of repeated DNA sequences in mammals (Brown et al., 1978; Maio et al., 1977). The possibility of a phase relation between DNA repeat sequences and associated nucleosome proteins is consistent with this hypothesis and has been tested by restriction enzyme and micrococcal nuclease digestions of repetitive DNA sequences in isolated, intact nuclei.Sites for four different restriction enzyme activities, EcoRI, EcoRI¹, HindIII and HaeIII have been mapped within the repeat unit of component α DNA, a highly repetitive DNA fraction of the African green monkey. The periodicity of cleavage sites for each of the enzymes (176 ± 4 nucleotide base-pairs) corresponds closely to the periodicity (about 185 nucleotide base-pairs) of the sites attacked in the initial stages of micrococcal nuclease digestion of nuclear chromatin. In intact monkey nuclei, EcoRI-RI¹ sites are accessible to restriction enzyme cleavage; the HindIII and HaeIII sites are not. The results suggest (1) that, in component α chromatin, the EcoRI-RI¹ sites are found at the interstices of adjacent nucleosomes and (2) the HindIII and HaeIII sites are protected from cleavage by their location on the protein core of the nucleosome. This interpretation was confirmed by experiments in which DNA segments of mononucleosomes and nucleosome cores released from CV-1 nuclei by micrococcal nuclease were subsequently treated with EcoRI, EcoRI¹ and HindIII. A major secondary segment of component α, about 140 nucleotide base-pairs in length, was released only by treatment with HindIII, in keeping with the location of the HindIII sites in the restriction map and their resistance to cleavage in intact nuclei.EcoRI reduces calf satellite I DNA to a segment of about 1408 nucleotide basepairs. In contrast, restriction of calf satellite I DNA with EcoRI¹ produces six prominent segments ranging in size from 176 to 1408 nucleotide base-pairs. Treatment of isolated calf nuclei with either EcoRI or EcoRI¹ did not produce segments shorter than 1408 base-pairs, indicating that while canonical EcoRI sites are accessible to attack, the irregularly spaced EcoRI¹ sites are specifically blocked. The results are consistent with a phase relation between the repeat sequence of calf satellite I DNA and an octameric array of nucleosomes.     

In vitro ADP-ribosylation of chromosomal proteins of the brain of developing rats

In vitro ADP-ribosylation of chromosomal protein and its modulation by spermine, 3-aminobenzamide (3-AB) and benzamide were studied by incubating the nuclei of cerebral hemisphere of 3-, 14- and 30-day old rats with ³²P-NAD⁺. Histones get ADP-ribosylated more than the non-histone chromosomal (NHC) protein. H1 is the major target for ADP-ribosylation. Among the nucleosomal histones, H2B is ADP-ribosylated most. The other core histones also get ADP-ribosylated to a lesser extent. ADP-ribosylation of both histones and NHC proteins decreases during development. Spermine stimulates, whereas 3-AB and benzamide inhibit, ³²P-ADP-ribose incorporation into histones and NHC proteins. These effects decrease with development. Mild digestion of chromatin by micrococcal nuclease (MNase), EcoRI, and AluI prior to ADP-ribosylation stimulates incorporation of ³²P-ADP-ribose. The degree of stimulation decreases as development proceeds. Such alterations indicate progressive condensation of chromatin with development.     

Conformational changes in the chromatin of the brain of developing rats and its modulation by zinc chloride

Conformational changes in the chromatin of the brain were studied during the development of the rat (3-, 14-and 30-day old) using microccoccal nuclease (MCN) and DNase I. The rate and extent of digestion of chromatin by MCN is not altered during development. However, pre-incubation of slices of the cerebral cortex with ZnCl₂ increases the initial rate of digestion by MCN by 2–3-fold, and also enhances the production of monomer DNA. The rate and extent of digestion of chromatin by DNase I is greater in an early stage of development. The initial rate of digestion by DNase I is stimulated by 3–4-fold after ZnCl₂ treatment. These data show that changes occur in the conformation of chromatin, particularly in the internucleosomal region of brain cells as they pass from dividing to the non-dividing state.     

Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with ¹⁴C-thymidine and the newly synthesized strands - with bromodeoxyu ridine and ³H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15–20% newly replica ted DNA Analysis of the distribution of ¹⁴C-labeled parental DNA fragments complementary to the ³H-nascent strand has shown that the ¹⁴C-labeled fragments prevail in the region of 30–50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5′-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems tobe induced by interactions in the chromatin.     

Analysis of chromatin of skeletal muscle of developing rats using micrococcal nuclease and DNase I

Conformational changes in the chromatin of skeletal muscle of 3-, 14-and 30 day-old developing rats have been studied using DNase I and micrococcal nuclease (MCN). Purified nuclei were digested separately by MCN and DNase I. The rate and extent of digestion by MCN decreases gradually as development proceeds. The electrophoretic pattern of MCN digested DNA, however, shows no change. The kinetics of digestion of nuclei by DNase I show no change with development. However, the electrophoretic pattern of DNase I digested DNA shows a gradual decrease in the amount of 10–30 bp fragments with progressive development. These studies show that the chromatin of the skeletal muscle undergoes certain conformational changes during postnatal development, and such changes in chromatin may be necessary for terminal differentiation of this tissue.     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Summary A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20.DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.     

Mosaicism for the Charcot-Marie-Tooth disease type 1A duplication suggests somatic reversion

A female patient with clinical signs and symptoms of a demyelinating neuropathy was shown to have a duplication of the 1.5-Mb region on chromosome 17p11.2, typical of the great majority of cases of Charcot-Marie-Tooth disease type 1A (CMT1A). However, analysis of DNA extracted from peripheral blood revealed a 2:2.4 instead of the usual 2:3 ratio between the 7.8- and 6.0-kb EcoRI fragments in the proximal and distal repetitive extragenic palindromic (REP) elements of CMT1A. Detection of a 3.2-kb EcoRI/SacI kb junction fragment with probe pLR7.8 confirmed the CMT1A duplication. The dosage of this junction fragment, compared with a 2.8-kb EcoRI/SacI fragment of the proximal REP elements of CMT1A, was 2:0.58 instead of the expected 2:1 dosage for heterozygous CMT1A duplications. We hypothesized that the lower dosages of these restriction fragments specific for the CMT1A duplication were due to mosaicism; this was confirmed by fluorescence in situ hybridization analysis with the D17S122-specific probe pVAW409R1. In peripheral blood lymphocytes the percentage of interphase nuclei with a duplication in 17p11.2 was 49%. In interphase nuclei extracted from buccal mucosa, hair-root cells or paraffin-embedded nervous tissue the duplication was detectable in 51%, 66% and 74%, respectively. This is the first report of mosaicism in a patient with a CMT1A duplication identified by three different and independent techniques. Received: 14 November 1995 / Revised: 13 February 1996     

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10–25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5 to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.     

Characterisation of plasmids coding for the restriction endonuclease EcoRI

Summary The properties of two plasmids coding for the EcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6x10⁶ daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor R1–19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7x10⁶ daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1–19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.     

Transduction of Plasmid Antibiotic Resistance Determinants with PseudoT-Even Bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 fromEscherichia coli recA ⁺ and recA ^– donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc–segE–uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages , T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limitedin vivo by modification–restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification–restriction systemsEcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification–restriction system.     

Overall changes in chromatin sensitivity to DNase I during differentiation

The DNase I sensitivity of total chromatin was studied in fixed cells and nuclei isolated from proliferating and terminally differentiated cells, by measuring the incorporation of labelled nucleotides into DNase-sensitive sites, and electrophoresis of DNA isolated from DNase-treated nuclei. The unfixed nuclei were sensitive to digestion at around 10 micrograms/ml, the fixed cells at 30 ng/ml DNase I concentration. Proliferating Rauscher leukemia cells were more digestible than normal spleen cells. The DNase I sensitivity of the human HL60 leukemia line decreased upon DMSO-induced differentiation but still exceeded the digestibility of nuclei from normal human peripheral blood. A novel flow-cytometric technique was developed to study DNase sensitivity at the cell level. It confirmed the relative resistance of differentiated cells to DNase I and ruled out the possibility that this could be due to an altered distribution of cell cycle phases. The overall DNase I sensitivity of chromatin was compared with the sensitivity of the c-myc gene and the myc-associated hypersensitive sites. The latter sites were detected at 1 microgram/ml DNase I in HL60 nuclei. They disappeared partially upon DMSO-induced differentiation. At 10 micrograms/ml, myc was degraded in both growing and differentiating HL60, but not in HPB cells. These data suggest that a progressive condensation of the chromatin occurs during terminal differentiation which gradually involves specific genes that need to be inactivated.     

Physical characterization of the plasmid pON5300

Summary The antibiotic resistance plasmid Rldrd19Km-which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the Rldrd19 and the fragments responsible for kanamycin resistance were found to be missing in Rldrd19Km ^– and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of Rldrd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.     

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

Summary 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 by and 74 by to the left and 27 bp, 41 by and 290 by to the right, respectively, of the boundaries of the 118 by 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 by 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.     

The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [¹⁴C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number of short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding.It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.     

Mapping of additional restriction enzyme cleavage sites on bacteriophage MB78 genome

A physical map of bacteriophage MB78 DNA indicating the cleavage sites for the enzymeBglII,ClaI,EcoRI,PvuII,SalI andSmaI comprising of a total of 34 cleavage sites have been constructed earlier. The cleavage sites for a few more restriction endonucleases likeApaI,AvaI,BglI,HindIII,KpnI andXhoI have now been mapped. A total of 72 cleavage sites on MB78 DNA are known by now. Relative positions ofEcoRI I and J fragments which could not be decided earlier has now been determined.     

Preferential sites of early DNA cleavage in apoptosis and the pathway of nuclear damage

 We have tested the specific hypothesis that the pathway of nuclear collapse in apoptosis is governed by the early attack on active chromatin at spatially restricted nuclear sites. Cell death in PC12 pheochromocytoma cells deprived of serum growth factors, in HL-60 leukemic cells treated with inhibitors of protein or RNA biosynthesis, and in U937 histiocytic lymphoma cells exposed to the cytokine tumor necrosis factor alpha showed a common mechanism in the targeting of DNA for degradation. An incorporation assay with labeled nucleotide revealed an early selective nicking in peripheral nuclear chromatin with concomitant diminution in the amount of immunoreactive lamin B protein. This was followed by a phase of more extensive cleavages, continued nuclear protein loss, chromatin collapse, and fragmentation of nuclei. The spatial restriction of early cleavages is similar to the nicking obtained by the application of exogenous DNase I to fixed nuclei of normal cells and to that obtained in the activation of the endogenous endonuclease of liver nuclei by Ca²⁺. These similarities suggest that, in apoptosis, activation of an endonuclease preferentially recognizing a specific chromatin configuration, such as that of active (DNase I-sensitive) genes, underlies the early spatial demarcation of cleavages. Accepted: 12 October 1998