New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells

Carlson, Jonathan O.; Pfenninger, Oswald; Sinden, Richard R.; Lehman, John M.; Pettijohn, David E.

doi:10.1093/nar/10.6.2043

Cited by 25 publications

(11 citation statements)

References 31 publications

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“…The accessibility of DNA in the different ribosomal chromatin regions for psoralen was analyzed by using the gel retardation assay developed by our group (5,42,43). This method is based on the fact that the more a DNA fragment is cross-linked with psoralen, the slower it migrates in a native agarose gel (2,42). Isolated nuclei were extensively photoreacted with psoralen, and the purified, cross-linked DNA was digested with various restriction enzymes and electrophoresed alongside non-cross-linked control DNA.…”

Section: Resultsmentioning

confidence: 99%

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

Lucchini¹,

Sogo²

1992

Mol. Cell. Biol.

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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.In the last few years, much progress has been achieved in identifying the different sequence elements located in the intergenic spacers that are involved in the regulation of the rRNA genes. Despite the fact that the DNA sequence of the ribosomal spacers has diverged very rapidly between different organisms, recent studies indicate a general consensus in the function as well as in the arrangement of transcriptional regulatory elements in almost all higher eukaryotes (for reviews, see references 35, 44, and 45). However, some exceptions seem to exist even among closely related species. The most striking difference resides in the function of the spacer elements close to the 3' end of the rRNA precursor coding regions in the two frog species Xenopus laevis and Xenopus borealis. In fact, a series of transcriptional analyses failed to detect a true termination site at the 3' end of the X. laevis rRNA genes (9, 18), whereas the related sites in X. borealis behave like efficient terminators (21) (sites T2, see Fig.

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Section: Resultsmentioning

confidence: 99%

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

Lucchini¹,

Sogo²

1992

Mol. Cell. Biol.

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“…Sequence specific positioning of nucleosomes on DNA has been addressed by Carlson et al (148) utilizing TMP photoaddition together with Hind III restriction analysis. Hind III was used in this study because preliminary experiment:~ with TMP modified plasmid DNA showed that its recognition sequence was 15-fold more susceptible to psoralen inactivation than other restriction sites examined.…”

Section: Chromatin Structurementioning

confidence: 99%

Psoralens as Photoactive Probes of Nucleic Acid Structure and Function: Organic Chemistry, Photochemistry, and Biochemistry

Cimino

Gamper

Isaacs

et al. 1985

Annu. Rev. Biochem.

750

361

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“…Lanes 1 and 8 are untreated, EcoRI-digested naked DNA, and lane 2 is DNA from nuclei, mocktreated with psoralen by adding 37 pL of ethanol only, and irradiated with UVA for 30 min.fractions(Figure 2, lanes 3-7). As the length of UVA irradiation time increases, both active and inactive rDNAs accumulate cross-links, retarding their migration on the gel [e.g., seeCarlson et al (1982)l. As a control, nuclei were mock-treated with psoralen by adding ethanol alone (psoralen is dissolved in ethanol) and UV-irradiated for 30 min.…”

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confidence: 99%

Repair of UV Damage in Actively Transcribed Ribosomal Genes

Fritz¹,

Smerdon²

1995

Biochemistry

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Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active ribosomal genes in mouse Friend erythroleukemia cells. Ribosomal genes (rDNA) are multicopied, but only a fraction is transcriptionally active (or transcriptionally "poised"). Selective psoralen binding was used to separate the active fraction, which has an open chromatin structure, from inactive rDNA. EcoRI digestion was used to selectively release the active fraction from nuclei for DNA repair studies. UV dose response curves indicate there is no significant bias for CPD formation in either strand of both types of rDNA chromatin. More importantly, there was no evidence for transcription repair coupling in the individual strands of active and total rDNA. Indeed, over an 8 h period (one cell-cycle), repair of CPDs was almost nonexistent in either strand of active and total rDNA. Furthermore, the fraction of each chromatin structure remains constant during these repair times, suggesting that chromatin rearrangements observed during excision repair of nonnucleolar chromatin do not occur following UV damage of rDNA. However, CPDs are removed efficiently from the transcribed strand of the constitutively expressed c-abl gene (transcribed by Pol II), demonstrating that these cells are capable of transcription repair coupling.

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New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells

Cited by 25 publications

References 31 publications

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

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

Psoralens as Photoactive Probes of Nucleic Acid Structure and Function: Organic Chemistry, Photochemistry, and Biochemistry

Repair of UV Damage in Actively Transcribed Ribosomal Genes

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