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J M Sogo

Publications and source records attributed to J M Sogo.

At least 19 recordsLinked to original sources
PubMed1992

Chromatin replication.

Just as the faithful replication of DNA is an essential process for the cell, chromatin structures of active and inactive genes have to be copied accurately. Under certain circumstances, however, the activity pattern has to be changed in specific ways. Although analysis of specific aspects of these complex processes, by means of model systems, has led to their further elucidation, the mechanisms of chromatin replication in vivo are still controversial and far from being understood completely. Progress has been achieved in understanding: 1. The initiation of chromatin replication, indicating that a nucleosome-free origin is necessary for the initiation of replication; 2. The segregation of the parental nucleosomes, where convincing data support the model of random distribution of the parental nucleosomes to the daughter strands; and 3. The assembly of histones on the newly synthesized strands, where growing evidence is emerging for a two-step mechanism of nucleosome assembly, starting with the deposition of H3/H4 tetramers onto the DNA, followed by H2A/H2B dimers.

Animals↗
PubMed1992

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.

Animals↗
PubMed1991

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.

Bacillus subtilis↗
PubMed1991

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5.

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.

Bacillus subtilis↗
PubMed1991

Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5- to 7-fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR-1), consistent with at least 85% of replication forks within this region emanating from OBR-1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce 'imbalanced DNA synthesis'. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non-nucleosomal DNA. Second, the fraction of RNA-primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single-stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.

Animals↗
PubMed1990

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.

Animals↗
PubMed1989

An enhancer stimulates transcription in trans when attached to the promoter via a protein bridge.

Two principal models have been invoked to explain transcriptional stimulation of RNA polymerase II genes by enhancers/upstream promoter elements: in one, upstream regulatory sequences directly interact with proximal promoter elements via proteins bound to the DNA ("looping" model); in the other, RNA polymerase II (or a transcription factor) binds to distal sequences and then scans along the DNA until it reaches the promoter ("scanning" or "entry site" model). So far, it has been reported that enhancers or upstream promoter elements transmit their effect on a gene only via covalently closed DNA, i.e., in a cis configuration. The looping model predicts, however, that the effect can be transmitted also in certain trans configurations. Here we demonstrate that an enhancer from SV40 or cytomegalovirus can stimulate transcription in vitro even when noncovalently attached to the beta-globin promoter via the proteins streptavidin or avidin. These findings are consistent with the looping model rather than the scanning model. In addition, stimulation of transcription in trans, as shown by our experiments, may be found in nature in phenomena such as transvection, where one chromosome affects gene expression in the paired homolog.

Animals↗
PubMed1989

Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle.

The structure of ribosomal chromatin in exponentially growing Friend cells, in stationary cells, and in metaphase chromosomes was studied by psoralen photocrosslinking. It is shown that in intact cells, two distinct types of ribosomal chromatin coexist in Friend cells, one that contains nucleosomes and represents the inactive copies and one that lacks a repeating structure and corresponds to the transcribed genes. A single gene copy is either in one or the other chromatin state. The relative amounts of the two types of structures are similar in interphase and metaphase, however, their run-on activities differ significantly. This suggests that the two states of chromatin are maintained independently of the transcriptional process and that they are stably propagated through the cell cycle.

Animals↗
PubMed1989

Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo.

Initiation of simian virus 40 (SV40) DNA replication is facilitated by two auxiliary sequences that flank the minimally required origin (ori) core sequence. In monkey cells, the replication rate of each of the four ori configurations changed with time after transfection in a characteristic pattern. This pattern was reproduced in an extract from SV40-infected monkey cells by varying the ratio of DNA substrate to cell extract; DNA replication in vitro depended on ori auxiliary sequences to the same extent as they did in vivo. Facilitation by ori auxiliary sequences was lost at high ratios of DNA to cell extract, revealing that the activity of these sequences required either multiple initiation factors or a molar excess of one initiation factor bound to ori. This parameter, together with ionic strength and the method used to measure DNA replication, determined the level of facilitation by ori auxiliary sequences in vitro. The activity of ori auxiliary sequences was not diminished in vivo or in vitro by increasing amounts of large tumor antigen. Therefore, ori auxiliary sequences promoted initiation of replication at some step after tumor antigen binding to ori. Furthermore, although cellular factors could modulate the activity of ori auxiliary sequences in vitro, these factors did not appear to involve nucleosome assembly because no correlation was observed between the number of nucleosomes assembled per DNA molecule and facilitation by ori auxiliary sequences. These results demonstrate that SV40 ori auxiliary sequences can function in vitro as they do in vivo and begin to elucidate their role in initiating DNA replication.

Animals↗
PubMed1988

Analysis of the psoralen-crosslinking pattern in chromatin DNA by exonuclease digestion.

When chromatin is photoreacted with psoralen, crosslinks occur preferentially in the linker DNA between nucleosomes. The pattern of these crosslinks can be analysed by exonuclease digestion of random DNA fragments, since the exonucleases tested stop at sites of psoralen-crosslinks. Further digestion of these fragments with S1-nuclease leads to DNA fragments of nucleosomal and polynucleosomal size, which presumably carry psoralen-crosslinks at both ends. This method of analysis of chromatin structure complements the classical micrococcal nuclease digestion analysis, since it can be performed in vitro as well as in vivo, and since it is independent of pH and ionic conditions.

Chemical Phenomena↗
PubMed1988

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.

Animals↗
PubMed1987

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.

Chromatin↗
PubMed1986

Structure of in-vivo transcribing chromatin as studied in simian virus 40 minichromosomes.

In order to study the structure of chromatin during transcription, individual in-vivo transcribing simian virus 40 (SV40) minichromosomes were analyzed in the electron microscope after crosslinking the nascent RNA strands with different psoralen derivatives to the template DNA. Since psoralen crosslinks the DNA between nucleosomes, spreading of the crosslinked DNA and DNA-RNA complexes reveals single-stranded bubbles at positions where nucleosomes were located. We found that the transcribing SV40 minichromosomes contained a similar number of nucleosomes as did the minichromosomes without crosslinked nascent RNA. The nascent RNA was crosslinked in about equal proportions either in single-stranded bubbles of nucleosomal length or in continuously crosslinked regions between bubbles, in contrast with control experiments with ribosomal chromatin of Dictyostelium. Treatment of SV40 minichromosomes with 1.2 M-NaCl before and during photocrosslinking with psoralen led to the disappearance of the single-stranded bubbles. Since no bubbles could be detected at the attachment sites of the RNA molecules when the nucleosomes were disrupted in high salt, and since in about half of the molecules the RNA was attached to fully crosslinked linker DNA, we assume that the single-stranded bubbles with crosslinked RNA are not due to protection by the elongating RNA polymerase II complex, but are rather due to nucleosome-like structures. At the resolution level of single nucleosomes, these results imply for the first time that nucleosome-like structures (perhaps modified compared with "normal" nucleosomes) on SV40 minichromosomes do not prevent transcription elongation by RNA polymerase II.

Chromatin↗
PubMed1986

Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures.

The structure of replicating simian virus 40 (SV40) minichromosomes was studied by DNA crosslinking with trimethyl-psoralen. The procedure was used both in vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected cells. Both procedures gave essentially the same results. Mature SV40 minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), except for those molecules with a nucleosome-free gap, which are interpreted to contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes are present in the unreplicated parental stem with the replication fork possibly penetrating into the nucleosomal DNA before the histone octamer is removed. Nucleosomes reassociate on the newly replicated DNA branches at distances from the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( +/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, daughter duplexes contained unequal numbers of nucleosomes, supporting dispersive and random segregation of parental nucleosomes. These were arranged in clusters with normal nucleosome spacing. We detected a novel type of interlocked dimer comprising two fully replicated molecules connected by a single-stranded DNA bridge. We cannot decide whether these dimers represent hemicatenanes or whether the two circles are joined by a Holliday-type structure. The joining site maps within the replication terminus. We propose that these dimers represent molecules engaged in strand segregation.

Cross-Linking Reagents↗
PubMed1985

Inhibition of DNA synthesis by aphidicolin induces supercoiling in simian virus 40 replicative intermediates.

Highly torsionally stressed replicative intermediate SV40 DNA molecules are produced when ongoing replicative DNA synthesis is inhibited by aphidicolin, a specific inhibitor of DNA polymerase alpha. The high negative superhelical density of these molecules can be partially released by intercalating drugs such as chloroquine or ethidium bromide. The torsionally stressed replicative intermediates bind to monoclonal anti-Z-DNA antibodies. Electron microscopy of anti-Z-DNA cross-linked to torsionally stressed replicative intermediates shows that the antibody specifically binds close to the replication forks. The superhelical structures are not formed when SV40 DNA replication is inhibited by both aphidicolin and novobiocin, suggesting that a topoisomerase type II-like enzyme is somehow involved in the introduction of torsional strain in replicative intermediate DNA. One interpretation of our data is that fork movement continues to some rather limited extent when SV40 DNA synthesis in replicative chromatin is blocked by aphidicolin. After deproteinization, the exposed single-stranded DNA branches reassociate to form paranemic DNA structures with left-handed helical stretches, while the reduced linking number of the parental strands induces a high negative superhelical density.

Antibodies, Monoclonal↗
PubMed1979

Comparison of the A-T rich regions and the Bacillus subtilis RNA polymerase binding sites in phage phi 29 DNA.

By using a modification of the BAC spreading method for mounting the DNA for electron microscopy, partial denaturation maps of protein-free phi 29 DNA and of phi 29 DNA containing protein p3 were obtained. In phi 29 p3-DNA1 the protein does not seem to influence the melting of the ends of the molecules. The comparison of the partial denaturation map and the B. subtilis RNA polymerase binding sites indicates that five of the seven early promoters (A1, A2, A3, B2 and C2) are located in A-T rich DNA regions whereas the other two early promoters (B1 and C1) are located in less A-T rich sites.

Bacillus subtilis↗