Sites in Simian Virus 40 Chromatin Which Are Preferentially Cleaved by Endonucleases

Scott, Walter A.; Wigmore, Dianne J.

doi:10.1016/b978-0-12-045450-1.50084-6

Cited by 39 publications

(58 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This is consistent with observations that only a fraction of the SV40 chromatin isolated from infected cells exhibits an open region as seen in the electron microscope (19,20,32) or by sensitivity to endonucleases (32,33 MspI. 32p_ labeled SV40 chromatin was fractionated by centrifugation into a 5 to 20% sucrose gradient, and the location of nucleoproteins containing SV40 DNA was determined by electrophoresis of a deproteinized sample from each gradient fraction followed by ethidium bromide staining.…”

Section: Discussionsupporting

confidence: 87%

“…Major barriers to Bal3l digestion appear to define the outer borders of the region of SV40 chromatin previously shown to be preferentially cleaved by endonucleases (7,8,(31)(32)(33)(38)(39)(40). These borders appear as clusters of Bal3l stop sites located between nucleotides 390 and 450 on the late side (Fig.…”

Section: Discussionmentioning

confidence: 90%

“…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%

“…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).…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Scott¹,

Walter²,

Cryer³

1984

Mol. Cell. Biol.

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Scott¹,

Walter²,

Cryer³

1984

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…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.…”

mentioning

confidence: 99%

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

Gerard¹,

Montelone²,

Walter³

et al. 1985

Mol. Cell. Biol.

View full text Add to dashboard Cite

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...

show abstract

Differential regulation by SV40 t-antigen binding at site I defines two distinct classes of nucleosome-free promoter

Kube

Milavetz

1996

Anat. Rec.

View full text Add to dashboard Cite

Background T‐antigen binding site I has been shown previously to play a role in regulating the proportion of Simian Virus 40 (SV40) chromosomes containing a nuclease hypersensitive promoter region. In order to determine whether these changes in nuclease hypersensitivity were a result of changes in the proportion of SV40 chromosomes, which contained a nucleosome‐free promoter region, SV40 chromosomes were visualized by electron microscopy. Methods SV40 chromosomes were prepared from cells infected with either wild‐type or mutant virus lacking T‐antigen binding site I, and the chromosomes were analyzed by electron microscopy for the presence of nucleosome‐free regions. Results Both the wild‐type and mutant chromatin were found to contain heterogeneous nucleosome‐free promoter regions consisting of small (3–4 times the normal internucleosomal distance) and large (greater than four times the normal internucleosomal distance) regions. Quantification of the proportion of chromosomes containing each type of region indicated that deletion of site I resulted in a 25% increase in the proportion of chromosomes containing a large nucleosome‐free region but had no effect on the proportion of chromosomes containing a small nucleosome‐free region. Conclusions The results indicate that there are two distinct classes of nucleosome‐free promoter region in SV40 chromatin and that T‐antigen binding to site I is specifically involved in the regulation of only the class that contains a large nucleosome‐free region. © 1996 Wiley‐Liss, Inc.

show abstract

Sites in Simian Virus 40 Chromatin Which Are Preferentially Cleaved by Endonucleases

Cited by 39 publications

References 0 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.

Differential regulation by SV40 t-antigen binding at site I defines two distinct classes of nucleosome-free promoter

Contact Info

Product

Resources

About