scholarly journals In vitro transcription of the avian oncornavirus genome by the RNA-directed DNA polymerase: effect of actinomycin D on the extent of transcription.

1977 ◽  
Vol 23 (1) ◽  
pp. 209-212 ◽  
Author(s):  
C H Clayman ◽  
M S Collett ◽  
A J Faras
Keyword(s):  
Dna Polymerase ◽  
Actinomycin D ◽  
In Vitro Transcription

Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors

1985 ◽  
Vol 5 (7) ◽  
pp. 1582-1590
Author(s):  
V C Culotta ◽  
R J Wides ◽  
B Sollner-Webb
Keyword(s):  
Transcription Factors ◽  
Dna Polymerase ◽  
In Vitro Transcription ◽  
Cell Extracts ◽  
S 100 ◽  
Major Late Promoter ◽  
Transcription Complexes ◽  
Polymerase I

RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.

scholarly journals Effects of acetaldehyde-induced DNA lesions on DNA metabolism

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Haruka Tsuruta ◽  
Yuina Sonohara ◽  
Kosuke Tohashi ◽  
Narumi Aoki Shioi ◽  
Shigenori Iwai ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Plasmid Dna ◽  
Restriction Enzymes ◽  
In Vitro Transcription ◽  
Dna Lesions ◽  
Dna Metabolism ◽  
Structural Distortions ◽  
Reactive Compound ◽  
Synthesis Reactions

Abstract Background Acetaldehyde, produced upon exposure to alcohol, cigarette smoke, polluted air and sugar, is a highly reactive compound that is carcinogenic to humans and causes a variety of DNA lesions in living human cells. Previously, we reported that acetaldehyde reacts with adjacent deoxyguanosine residues on oligonucleotides, but not with single deoxyguanosine residues or other deoxyadenosine, deoxycytosine, or thymidine residues, and revealed that it forms reversible intrastrand crosslinks with the dGpdG sequence (GG dimer). Results Here, we show that restriction enzymes that recognize a GG sequence digested acetaldehyde-treated plasmid DNA with low but significant efficiencies, whereas restriction enzymes that recognize other sequences were able to digest such DNA. This suggested that acetaldehyde produced GG dimers in plasmid DNA. Additionally, acetaldehyde-treated oligonucleotides were efficient in preventing digestion by the exonuclease function of T4 DNA polymerase compared to non-treated oligonucleotides, suggesting structural distortions of DNA caused by acetaldehyde-treatment. Neither in vitro DNA synthesis reactions of phi29 DNA polymerase nor in vitro RNA synthesis reactions of T7 RNA polymerase were observed when acetaldehyde-treated plasmid DNA was used, compared to when non-treated plasmid DNA was used, suggesting that acetaldehyde-induced DNA lesions inhibited replication and transcription in DNA metabolism. Conclusions Acetaldehyde-induced DNA lesions could affect the relative resistance to endo- and exo-nucleolytic activity and also inhibit in vitro replication and in vitro transcription. Thus, investigating the effects of acetaldehyde-induced DNA lesions may enable a better understanding of the toxicity and carcinogenicity of acetaldehyde.

scholarly journals Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors.

1985 ◽  
Vol 5 (7) ◽  
pp. 1582-1590 ◽  
Author(s):  
V C Culotta ◽  
R J Wides ◽  
B Sollner-Webb
Keyword(s):  
Transcription Factors ◽  
Dna Polymerase ◽  
In Vitro Transcription ◽  
Cell Extracts ◽  
S 100 ◽  
Major Late Promoter ◽  
Transcription Complexes ◽  
Polymerase I

RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.

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