Adenoassociated virus has a unique chromatin structure

1982 ◽  
Vol 60 (10) ◽  
pp. 1001-1005 ◽  
Author(s):  
Martha Brown ◽  
Joseph Weber
Keyword(s):  
Blot Hybridization ◽  
Adenovirus Type ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Eukaryotic Chromatin ◽  
Nuclease Digestion ◽  
Adenoassociated Virus ◽  
Cellular Dna ◽  
Oligomeric Dna

The organization of intranuclear adenoassociated virus DNA (AAV) was examined following micrococcal nuclease digestion of nuclei prepared from cells coinfected with AAV type 2 (AAV-2) and adenovirus type 2 (Ad2). Blot-hybridization analysis of the DNA with AAV-2, Ad2, and cellular DNA probes revealed that AAV-2 chromatin has a unique structure, which upon nuclease digestion gives rise to a smear of oligomeric DNA fragments from 600–2200 base pairs in length with only a very faint band about 160 base pairs and no discrete multimers. This structure was similar to, but distinguishable from, Ad2 chromatin and completely unrelated to eukaryotic chromatin.

scholarly journals Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

1985 ◽  
Vol 5 (11) ◽  
pp. 2936-2942 ◽  
Author(s):  
H T Liu ◽  
R Baserga ◽  
W E Mercer
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Histone H3 ◽  
Adenovirus Type ◽  
3T3 Cells ◽  
Cycle Dependent ◽  
Cellular Dna ◽  
Adenovirus Type 2

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum

1985 ◽  
Vol 5 (11) ◽  
pp. 2936-2942
Author(s):  
H T Liu ◽  
R Baserga ◽  
W E Mercer
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Histone H3 ◽  
Adenovirus Type ◽  
3T3 Cells ◽  
Cycle Dependent ◽  
Cellular Dna ◽  
Adenovirus Type 2

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

Adenovirus chromatin structure at different stages of infection

1981 ◽  
Vol 1 (12) ◽  
pp. 1094-1105
Author(s):  
E Daniell ◽  
D E Groff ◽  
M J Fedor
Keyword(s):  
Dna Synthesis ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Fragment Size ◽  
Human Adenovirus ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Viral Dna ◽  
Infected Cells ◽  
Cellular Dna

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins

1988 ◽  
Vol 8 (1) ◽  
pp. 105-113
Author(s):  
B Safer ◽  
R B Cohen ◽  
S Garfinkel ◽  
J A Thompson
Keyword(s):  
Uv Light ◽  
Promoter Sequence ◽  
Adenovirus Type ◽  
Micrococcal Nuclease ◽  
Cross Linking ◽  
Affinity Labeling ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence ◽  
Adenovirus Type 2

A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

scholarly journals Slaying the last unicorn - discovery of histones in the microalga Nanochlorum eucaryotum

2020 ◽  
Author(s):  
Valerie WC Soo ◽  
Tobias Warnecke
Keyword(s):  
Exceptional Case ◽  
Micrococcal Nuclease ◽  
Histone Fold ◽  
Histone Mrnas ◽  
Micrococcal Nuclease Digestion ◽  
Eukaryotic Chromatin ◽  
Nuclease Digestion ◽  
Eukaryotic Gene ◽  
Core Histones

ABSTRACTHistones are the principal constituents of eukaryotic chromatin. The four core histones (H2A, H2B, H3, and H4) are conserved across sequenced eukaryotic genomes and therefore thought to be universal to eukaryotes. In the early 1980s, however, a series of biochemical investigations failed to find evidence for histones or nucleosomal structures in the microscopic green alga Nanochlorum eucaryotum. If true, derived histone loss in this lineage would constitute an exceptional case that might help us further understand the principles governing eukaryotic gene regulation. To substantiate these earlier reports of histone loss in N. eucaryotum, we sequenced, assembled and quantified its transcriptome. Following a systematic search for histone-fold domains in the assembled transcriptome, we detect orthologs to all four core histones. We also find histone mRNAs to be highly expressed, comparable to the situation in other eukaryotes. Finally, we obtain characteristic protection patterns when N. eucaryotum chromatin is subjected to micrococcal nuclease digestion, indicating widespread formation of nucleosomal complexes in vivo. We conclude that previous reports of missing histones in N. eucaryotum were mistaken. By all indications, N. eucaryotum has histone-based chromatin characteristic of most eukaryotes.

scholarly journals DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins.

1988 ◽  
Vol 8 (1) ◽  
pp. 105-113 ◽  
Author(s):  
B Safer ◽  
R B Cohen ◽  
S Garfinkel ◽  
J A Thompson
Keyword(s):  
Uv Light ◽  
Promoter Sequence ◽  
Adenovirus Type ◽  
Micrococcal Nuclease ◽  
Cross Linking ◽  
Affinity Labeling ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence ◽  
Adenovirus Type 2

A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

Nuclease sensitivity of postreplicated chromatin from Ehrlich ascites tumour cells

1981 ◽  
Vol 59 (1) ◽  
pp. 22-29
Author(s):  
Brian L. A. Miki ◽  
John J. Heikkila ◽  
Ian R. Brown
Keyword(s):  
Structural Modification ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Selective Labelling ◽  
Dna Repeat ◽  
Ehrlich Ascites ◽  
Nuclease Digestion ◽  
Ehrlich Ascites Tumour ◽  
Ascites Tumour Cells ◽  
Ehrlich Ascites Tumour Cells

Chromatin appears to undergo structural modification after replication and before integration into bulk chromatin. In ascites cells, postreplicated chromatin displays a transient resistance to digestion with micrococcal nuclease. This resistance may be correlated with a shorter DNA repeat length (178 base pairs) than that found in bulk chromatin (187 base pairs). Selective labelling or selective digestion of DNA sequence classes could not account for these observations. In both bulk and postreplicated chromatin, three electrophoretic types of mononucleosomes were found. Postreplicated mononucleosome types showed selective sensitivities to nuclease digestion whereas bulk mononucleosome types did not.

An association between replicating adenovirus DNA and the nuclear matrix of infected HeLa cells

1985 ◽  
Vol 63 (6) ◽  
pp. 654-660 ◽  
Author(s):  
H. B. Younghusband
Keyword(s):  
Hela Cells ◽  
Nuclear Matrix ◽  
Close Association ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Viral Dna ◽  
Chase Period ◽  
Nuclease Digestion ◽  
Mature Virus Particle ◽  
Cellular Dna

An association between newly synthesized human adenovirus type 5 DNA and the nuclear matrix of infected HeLa cells is described. Adenovirus-infected cells were pulsed labeled with [3H]thymidine late in infection and the nuclear matrix was prepared. After a 1-min pulse more than 95% of the labeled viral DNA was matrix associated and, when compared with total cell DNA, was resistant to DNase I digestion. When the pulse is longer or is followed by a chase period, the viral DNA remains nuclear matrix associated and less nuclease sensitive than bulk cellular DNA. The resistance to nuclease digestion may result from the close association of viral DNA with the nuclear matrix or could be due to a number of viral-specific proteins which are nuclear matrix associated. It is concluded that viral DNA synthesis occurs in association with the nuclear matrix and the newly synthesized DNA remains matrix associated until it is incorporated into a mature virus particle.

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