Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin

1982 ◽  
Vol 2 (7) ◽  
pp. 782-788
Author(s):  
R D Gerard ◽  
M Woodworth-Gutai ◽  
W A Scott
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Deletion Mutants ◽  
Sequence Information ◽  
Base Pairs ◽  
Deletion Mutations ◽  
Sensitive Site ◽  
Late Side ◽  
Nuclease Sensitivity

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.

scholarly journals Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin.

1982 ◽  
Vol 2 (7) ◽  
pp. 782-788 ◽  
Author(s):  
R D Gerard ◽  
M Woodworth-Gutai ◽  
W A Scott
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Deletion Mutants ◽  
Sequence Information ◽  
Base Pairs ◽  
Deletion Mutations ◽  
Sensitive Site ◽  
Late Side ◽  
Nuclease Sensitivity

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.

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

1985 ◽  
Vol 5 (1) ◽  
pp. 52-58
Author(s):  
R D Gerard ◽  
B A Montelone ◽  
C F Walter ◽  
J W Innis ◽  
W A Scott
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Viral Origin ◽  
Deletion Mutations ◽  
Sensitive Region ◽  
Complete Independence ◽  
Sensitive Structure ◽  
Nuclease Sensitivity

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 (dl45) 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 (dl135) 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 dl9, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the BglI site is 146 bp away from the site of the deletion.

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

1985 ◽  
Vol 5 (1) ◽  
pp. 52-58 ◽  
Author(s):  
R D Gerard ◽  
B A Montelone ◽  
C F Walter ◽  
J W Innis ◽  
W A Scott
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Viral Origin ◽  
Deletion Mutations ◽  
Sensitive Region ◽  
Complete Independence ◽  
Sensitive Structure ◽  
Nuclease Sensitivity

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 (dl45) 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 (dl135) 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 dl9, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the BglI site is 146 bp away from the site of the deletion.

scholarly journals A REFINED MAP OF THE hisG GENE OF SALMONELLA TYPHIMURIUM

Genetics ◽  
1979 ◽  
Vol 92 (1) ◽  
pp. 17-26
Author(s):  
I Hoppe ◽  
H M Johnston ◽  
D Biek ◽  
J R Roth
Keyword(s):  
Salmonella Typhimurium ◽  
Transposable Element ◽  
Structural Gene ◽  
Biosynthetic Pathway ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Deletion Mutations ◽  
Insertion Sites

ABSTRACT The hisG gene is the most operator-proximal structural gene of the histidine operon; it encodes the feedback-inhibitable first enzyme of the biosynthetic pathway. Previously, hisG mutants were mapped into seven intervals defined by the available deletion mutations having endpoints in the hisG gene. The map has been refined using over 60 new deletion mutants. The new map divides the gene into 41) deletion intervals, which average approximately 30 base pairs in length. The map has been used to analyze the distribution of insertion sites for the transposable element Tn10 and has permitted conclusions on the distribution of duplication endpoints. The map promises to be useful in analysis of his regulation and, more particularly, in the determination of the possible role of the hisG enzyme in this mechanism.

Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function

1983 ◽  
Vol 3 (7) ◽  
pp. 1295-1309
Author(s):  
R W Scott ◽  
S M Tilghman
Keyword(s):  
Transient Expression ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Alpha Fetoprotein ◽  
Tata Box ◽  
Deletion Mutants ◽  
Promoter Function ◽  
Flanking Region

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.

How damaged is the biologically active subpopulation of transfected DNA?

1984 ◽  
Vol 4 (3) ◽  
pp. 387-398
Author(s):  
C T Wake ◽  
T Gudewicz ◽  
T Porter ◽  
A White ◽  
J H Wilson
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Double Strand Breaks ◽  
Biologically Active ◽  
Exact Nature ◽  
Dna Molecules ◽  
Base Pairs ◽  
Strand Breaks ◽  
Dna Ends ◽  
Prevalent Form

Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of double-strand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.

scholarly journals Chromatin Structure of the Simian Virus 40 Late Promoter: a Deletional Analysis

1999 ◽  
Vol 73 (3) ◽  
pp. 1990-1997 ◽  
Author(s):  
Michael Friez ◽  
RaeJean Hermansen ◽  
Barry Milavetz
Keyword(s):  
Dna Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Late Promoter ◽  
Reporter System ◽  
Free Region ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Minimal Sequence ◽  
Nuclease Sensitivity

ABSTRACT The goal of this study was to determine the minimal sequence within the simian virus 40 (SV40) late promoter region, nucleotides (nt) 255 to 424, capable of phasing nucleosomes as measured by its ability to confer the greatest endonuclease sensitivity on adjacent DNA sequences. To identify the minimal sequence, a deletional analysis of the late region was performed by utilizing a SV40 recombinant reporter system. The reporter system consisted of a series of unique restriction sites introduced into SV40 at nt 2666. The unique restriction sites allowed the insertion of test sequences as well as measurement of conferred endonuclease sensitivity. The results of the deletional analysis demonstrated that constructs capable of conferring the greatest nuclease sensitivities consistently included nt 255 to 280. The activator protein 4 (AP-4) and GTIIC transcription factor binding sequences lie within this region and were analyzed individually. Their abilities to confer nuclease sensitivity upon the reporter nearly matched that of the entire late domain. These results suggest that transcription factors AP-4 and transcription-enhancing factor which binds the GTIIC sequence are able to confer significant levels of nuclease sensitivity and are likely involved in the formation of the SV40 nucleosome-free region.

scholarly journals A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

2019 ◽  
Author(s):  
Samuel J. Dobson ◽  
Jamel Mankouri ◽  
Adrian Whitehouse
Keyword(s):  
Ion Channels ◽  
Broad Spectrum ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Asymptomatic Infection ◽  
Endocytic Pathway ◽  
Endosomal Trafficking ◽  
Nuclear Entry ◽  
Novel Target

ABSTRACTFollowing internalisation viruses have to escape the endocytic pathway and deliver their genomes to initiate replication. Members of the Polyomaviridae transit through the endolysosomal network and through the endoplasmic reticulum (ER), from which heavily degraded capsids escape into the cytoplasm prior to nuclear entry. Acidification of endosomes and ER entry are essential in the lifecycle of polyomaviruses, however many mechanistic requirements are yet to be elucidated. Alteration of endocytic pH relies upon the activity of ion channels. Using two polyomaviruses with differing capsid architecture, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we firstly describe methods to rapidly quantify infection using an IncuCyte ZOOM instrument, prior to investigating the role of K+ and Ca2+ channels during early stages of infection. Broad spectrum inhibitors identified that MCPyV, but not SV40, is sensitive to K+ channel modulation. In contrast, both viruses are restricted by the broad spectrum Ca2+ channel inhibitor verapamil, however specific targeting of transient or long lasting Ca2+ channel subfamilies had no detrimental effect. Further investigation revealed that tetrandrine blockage of two-pore channels (TPCs), the activity of which is essential for endolysosomal-ER fusion, ablates infectivity of both MCPyV and SV40 by preventing disassembly of the capsid, which is required for the exposure of minor capsid protein nuclear signals necessary for nuclear transport. This study therefore identifies a novel target to restrict the entry of polyomaviruses.IMPORTANCEPolyomaviruses establish ubiquitous, asymptomatic infection in their host. However, in the immunocompromised these viruses can cause a range of potentially fatal diseases. Through the use of SV40 and MCPyV, two polyomaviruses with different capsid organisation, we have investigated the role of ion channels during infection. Here, we show that Ca2+ channel activity is essential for both polyomaviruses and that MCPyV is also sensitive to K+ channel blockage, highlighting new mechanistic requirements of ion channels during polyomavirus infection. In particular, tetrandrine blockage of endolysosomal-ER fusion is highlighted as an essential modulator of both SV40 and MCPyV. Given that the role of ion channels in disease have been well characterised, there is a large panel of clinically available therapeutics that could be repurposed to restrict persistent polyomavirus infection and may ultimately prevent polyomavirus-associated disease.

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