Endonuclease-sensitive regions in SV40 chromatin from cells infected with duplicated mutants

Wigmore, Dianne J.; Eaton, Richard W.; Scott, Walter A.

doi:10.1016/0042-6822(80)90348-7

Cited by 39 publications

(28 citation statements)

References 24 publications

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“…Chromatin from fractions 7 and 10 was also digested with DNase I, and the frequency of cleavage within the nucleasesensitive region was determined as described previously (33,44). DNase I cleaved preferentially within the 400-bp segment of the late side of the origin of replication in both chromatin subfractions; however, the preference was substantially greater in the more slowly sedimenting chromatin (ca.…”

Section: Resultsmentioning

confidence: 99%

“…Isolated chromatin was dialyzed into TENT buffer (10 mM Trishydrochloride [pH 7.4], 0.5 mM Na2EDTA, 50 mM NaCl, 0.17% Triton X-100). For all restriction enzyme digests of chromatin, the following conditions were used (final concentrations): 19 mM Tris-hydrochloride (pH 7.4), 44 Digest products were fractionated by electrophoresis in 1.4% agarose (4) or in 4% polyacrylamide (11). DNA fragments were detected by exposing the dried gel to Kodak XRP-1 X-ray film.…”

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confidence: 99%

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Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

Scott¹,

Walter²,

Cryer³

1984

Mol. Cell. Biol.

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A portion of the nucleoprotein containing viral DNA extracted from cells infected by simian virus (SV40) is preferentially cleaved by endonucleases in a region of the genome encompassing the origin of replication and early and late promoters. To explore this nuclease-sensitive structure, we cleaved SV40 chromatin molecules with restriction enzymes and digested the exposed termini with nuclease Bal3l. Digestion proceeded only a short distance in the late direction from the MspI site, but some molecules were degraded 400 to 500 base pairs in the early direction. By comparison, BglI-cleaved chromatin was digested for only a short distance in the early direction, but some molecules were degraded 400 to 450 base pairs in the late direction. These barriers to Bal3l digestion (bracketing the BglI and the MspI sites) define the borders of the same open region in SV40 chromatin that is preferentially digested by DNase I and other endonucleases. In a portion of the SV40 chromatin, Bal3l could not digest through the nuclease-sensitive region and reached barriers after digesting only 50 to 100 base pairs from one end or the other. Chromatin molecules that contain barriers in the BglI to MspI region are physically distinct from molecules that are open in this region as evidenced by partial separation of the two populations on sucrose density gradients.Viral nucleoprotein complex extracted from cells infected by simian virus 40 (SV40) resembles cellular chromatin (1,10,15,27). A short segment of the viral genome (ca. 400 base pairs [bp] extending from the SV40 origin of replication in the late direction) is hypersensitive to endonuclease cleavage (33,(38)(39)(40) and appears, in the electron microscope, as a region which is free of nucleosomes (19,32). Recently, more detailed studies have shown that, within the nuclease-sensitive site, there are subregions with differential sensitivity to DNase 1 (7,8,31,32). Presumably, these features reflect the structural details of the nucleoprotein within this region.Nuclease-hypersensitive sites usually occur over regions which contain genetic signals. Such a chromatin structure over a eucaryotic promoter may be a necessary (but not sufficient) condition for transcription from that promoter (6,12,43). In addition, hypersensitive sites have been reported which are probably not related to promoter function (3,22,28). SV40-infected cells provide a good opportunity to investigate the details of the nuclease-sensitive chromatin structure, since SV40 chromatin is present in high concentration by comparison with other genes and can easily be separated from bulk cellular chromatin.We have developed a new approach for probing nucleoprotein structure which should be applicable to the study of chromatin structure in other minichromosomes. SV40 is cleaved with a restriction enzyme which cuts at a unique position, and the exposed termini are digested with Bal3l, an exonuclease which can degrade from either 3' or 5' ends. MATERIALS AND METHODSGrowth of cells and virus infections. African green monk...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

Scott¹,

Walter²,

Cryer³

1984

Mol. Cell. Biol.

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“…Cleavage of viral chromatin by endogenous nuclease in the presence of MnCl2 has been described previously (11,23,30) (25), and hybridized in the presence of 50% formamide for 2 days at 37°C (28) with a 32P-labeled probe prepared by nick translation (21).…”

Section: Methodsmentioning

confidence: 99%

“…All essential information required for the hypersensitive site is contained within a 273-base-pair (bp) segment of DNA spanning the origin of replication (11,30 We have investigated the genetic elements responsible for the chromatin structure in this region by analyzing a number of additional mutants. No single segment is essential for nuclease sensitivity.…”

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confidence: 99%

“…All essential information required for the hypersensitive site is contained within a 273-base-pair (bp) segment of DNA spanning the origin of replication (11,30). Insertion of unrelated sequences between nucleotides 37 and 38 (SV numbering system of Buchman et al [4]) moves the pattern of nuclease cleavage sites in the late direction, indicating that sequences responsible for the positions of these sites lie on the side of nucleotide 37 which is distal from the origin of replication (15).…”

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Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin.

Gerard¹,

Montelone²,

Walter³

et al. 1985

Mol. Cell. Biol.

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A nuclease-sensitive region forms in chromatin containing a 273-base-pair (bp) segment of simian virus 40 DNA encompassing the viral origin of replication and early and late promoters. We have saturated this region with short deletion mutations and compared the nuclease sensitivity of each mutated segment to that of an unaltered segment elsewhere in the partially duplicated mutant. Although no single DNA segment is required for the formation of a nuclease-sensitive region, a deletion mutation (d145) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity. Deletion mutations limited to only one copy of the 21-bp repeats had little, if any, effect. A mutant (d1135) lacking all copies of the 21-and 72-bp repeats, while retaining the origin of replication and the TATA box, did not exhibit a nuclease-sensitive region. Mutants which showed reduced nuclease sensitivity had this effect throughout the nuclease-sensitive region, not just at the site of the deletion, indicating that although multiple determinants must be responsible for the nuclease-sensitive chromatin structure they do not function with complete independence. Mutant d19, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the Bgll site is 146 bp away from the site of the deletion.Simian virus 40 (SV40)-infected cells contain SV40 DNA in a nucleoprotein structure with many similarities to cellular chromatin. The availability of an amplified, homogeneous DNA species in a chromatin-like structure has provided convenient material for the study of specific features of chromatin. Of particular interest is the short region adjacent to the viral origin of replication (ori site) which is hypersensitive to cleavage by endonucleases (23,26,27,29) and which appears as a gap in the nucleosome pattern when visualized in the electron microscope (16,22). This region contains the early and late gene promoters, the transcriptional enhancer sequences, and the viral origin of replication. The distinctive chromatin structure may be relevant to any or all of these functions.To identify which sequences are responsible for the nuclease-sensitive region, partially duplicated SV40 mutants have been studied. All essential information required for the hypersensitive site is contained within a 273-base-pair (bp) segment of DNA spanning the origin of replication (11,30 We have investigated the genetic elements responsible for the chromatin structure in this region by analyzing a number of additional mutants. No single segment is essential for nuclease sensitivity. However, overall accessibility to nuclease was dramatically decreased by a mutant affecting only the two exact copies of the 21-bp repeated sequences. Insertion of a foreign DNA segment into this region also substantially diminished nuclease sensitivity. By contrast, deletion of only one of the two exact copies of the 21-bp repeats had little effect on the pattern of nuclease cleavage.These results imply that the region of the genome e...

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Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Cereghini¹,

Yaniv²

1984

The EMBO Journal

170

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Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin‐promoter‐enhancer region of SV40, when introduced into CV‐1 or Cos‐1 monkey cells by DEAE‐dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin‐specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non‐replicating and post‐replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein‐DNA interactions during viral infection or upon cell differentiation.

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Endonuclease-sensitive regions in SV40 chromatin from cells infected with duplicated mutants

Cited by 39 publications

References 24 publications

Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin.

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

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