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Lifescience Database Archive (English)

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File list: Pol.Unc.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Unc.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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File list: Pol.Lng.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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File list: Pol.Pan.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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File list: Pol.Emb.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

Full Text Available Pol.Bon.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Bone SRX...,SRX351408 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bon.10.RNA_polymerase_II.AllCell.bed ...

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

Full Text Available Pol.YSt.20.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II Yeast... strain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.YSt.20.RNA_polymerase_II.AllCell.bed ...

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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Lifescience Database Archive (English)

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File list: Pol.Emb.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.CDV.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Neu.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.CDV.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Adp.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Adl.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Adl.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Neu.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Neu.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Utr.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.ALL.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Utr.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Oth.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Adp.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Kid.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Kid.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Oth.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Bld.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Kid.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Bld.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Kid.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Oth.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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Ubiquitylation and degradation of elongating RNA polymerase II

DEFF Research Database (Denmark)

Wilson, Marcus D; Harreman, Michelle; Svejstrup, Jesper Q

2013-01-01

During its journey across a gene, RNA polymerase II has to contend with a number of obstacles to its progression, including nucleosomes, DNA-binding proteins, DNA damage, and sequences that are intrinsically difficult to transcribe. Not surprisingly, a large number of elongation factors have....... In this review, we describe the mechanisms and factors responsible for the last resort mechanism of transcriptional elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation....

File list: Pol.YSt.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.YSt.10.RNA_Polymerase_II.AllCell sacCer3 RNA polymerase RNA Polymerase II Yeast... strain SRX092435,SRX360917,SRX360914,SRX497380,SRX497382,SRX497381,SRX360915 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.YSt.10.RNA_Polymerase_II.AllCell.bed ...

File list: Pol.Lar.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Lar.05.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Larvae SR...SRX151962,SRX182775,SRX661503,SRX013070,SRX013072,SRX013113,SRX013082,SRX151961 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Lar.05.RNA_polymerase_II.AllCell.bed ...

File list: Pol.Oth.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Pol.Lar.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Lar.20.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Larvae SR...SRX661503,SRX026742,SRX013070,SRX013072,SRX182775,SRX151961,SRX013082,SRX013113 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.Lar.20.RNA_polymerase_II.AllCell.bed ...

File list: Pol.EmF.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.EmF.05.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Embryonic...RX143288 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.EmF.05.RNA_Polymerase_II.AllCell.bed ...

File list: Pol.NoD.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.50.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.NoD.50.RNA_polymerase_II.AllCell.bed ...

File list: Pol.NoD.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.05.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II No descri...ption http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.NoD.05.RNA_polymerase_II.AllCell.bed ...

File list: Pol.NoD.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II No descr...iption http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.10.RNA_polymerase_II.AllCell.bed ...

File list: Pol.NoD.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.20.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II No descr...iption http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.NoD.20.RNA_polymerase_II.AllCell.bed ...

File list: Pol.NoD.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.10.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.10.RNA_polymerase_II.AllCell.bed ...

File list: Pol.NoD.05.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.05.RNA_Polymerase_II.AllCell sacCer3 RNA polymerase RNA Polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.05.RNA_Polymerase_II.AllCell.bed ...

File list: Pol.NoD.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.10.RNA_Polymerase_II.AllCell sacCer3 RNA polymerase RNA Polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.10.RNA_Polymerase_II.AllCell.bed ...

File list: Pol.NoD.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.20.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.20.RNA_polymerase_II.AllCell.bed ...

File list: Pol.NoD.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.NoD.50.RNA_polymerase_II.AllCell sacCer3 RNA polymerase RNA polymerase II No de...scription http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Pol.NoD.50.RNA_polymerase_II.AllCell.bed ...

File list: Pol.CeL.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.CeL.50.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Cell line...70,SRX749072,SRX749071,SRX749073,SRX017852,SRX529168 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.CeL.50.RNA_polymerase_II.AllCell.bed ...

File list: Pol.CeL.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.CeL.20.RNA_polymerase_II.AllCell dm3 RNA polymerase RNA polymerase II Cell line...70,SRX749072,SRX749071,SRX749073,SRX017852,SRX529168 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Pol.CeL.20.RNA_polymerase_II.AllCell.bed ...

RNA polymerase II collision interrupts convergent transcription

DEFF Research Database (Denmark)

Hobson, David J; Wei, Wu; Steinmetz, Lars M

2012-01-01

Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical...

Interaction of sigma 70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance.

Science.gov (United States)

Ferguson, A L; Hughes, A D; Tufail, U; Baumann, C G; Scott, D J; Hoggett, J G

2000-09-22

The interaction between the core form of bacterial RNA polymerases and sigma factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and sigma 70 has been investigated by surface plasmon resonance. The His-tagged form of sigma 70 factor was immobilised on a Ni2+-NTA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9x10(-7) M, and the dissociation rate constant for release of sigma from core, in the absence of DNA or transcription, was 4x10(-3) s(-1), corresponding to a half-life of about 200 s.

Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme*

Science.gov (United States)

Sato, Shigeo; Tomomori-Sato, Chieri; Tsai, Kuang-Lei; Yu, Xiaodi; Sardiu, Mihaela; Saraf, Anita; Washburn, Michael P.; Florens, Laurence; Asturias, Francisco J.; Conaway, Ronald C.

2016-01-01

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved “hinge” in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly. PMID:27821593

Evaluation of a new HTLV-I/II polymerase chain reaction

NARCIS (Netherlands)

Vrielink, H.; Zaaijer, H. L.; Cuypers, H. T.; van der Poel, C. L.; Woerdeman, M.; Lelie, P. N.; Winkel, C.; Reesink, H. W.

1997-01-01

AIM: Evaluation of a qualitative HTLV-I/II DNA polymerase chain reaction (PCR) test for the detection of HTLV-I/II DNA (Roche Diagnostic Systems, Branchburg, N.J., USA) in various panels. METHODS: The panels consisted of fresh EDTA blood samples from blood donors who were anti-HTLV-I/II ELISA

Relationship between RNA polymerase II and efficiency of vaccinia virus replication

International Nuclear Information System (INIS)

Wilton, S.; Dales, S.

1989-01-01

It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study the authors examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L 6 H 9 rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing cured enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with [ 35 S]methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rate of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, the authors suggest that mobilization of pol II depends on the efficiency of vaccinia virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

International Nuclear Information System (INIS)

Gilmour, D.S.; Lis, J.T.

1986-01-01

By using a protein-DNA cross-linking method, we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation

Single molecule imaging of RNA polymerase II using atomic force microscopy

International Nuclear Information System (INIS)

Rhodin, Thor; Fu Jianhua; Umemura, Kazuo; Gad, Mohammed; Jarvis, Suzi; Ishikawa, Mitsuru

2003-01-01

An atomic force microscopy (AFM) study of the shape, orientation and surface topology of RNA polymerase II supported on silanized freshly cleaved mica was made. The overall aim is to define the molecular topology of RNA polymerase II in appropriate fluids to help clarify the relationship of conformational features to biofunctionality. A Nanoscope III atomic force microscope was used in the tapping mode with oxide-sharpened (8-10 nm) Si 3 N 4 probes in aqueous zinc chloride buffer. The main structural features observed by AFM were compared to those derived from electron-density plots based on X-ray crystallographic studies. The conformational features included a bilobal silhouette with an inverted umbrella-shaped crater connected to a reaction site. These studies provide a starting point for constructing a 3D-AFM profiling analysis of proteins such as RNA polymerase complexes

CDK9-dependent RNA polymerase II pausing controls transcription initiation.

Science.gov (United States)

Gressel, Saskia; Schwalb, Björn; Decker, Tim Michael; Qin, Weihua; Leonhardt, Heinrich; Eick, Dirk; Cramer, Patrick

2017-10-10

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

Structural relationships among the multiple forms of DNA-dependent RNA polymerase II from cultured parsley cells

International Nuclear Information System (INIS)

Link, G.; Bogorad, L.; Kidd, G.H.; Richter, G.

1978-01-01

DNA-dependent RNA polymerase II (or B) was purified from cultured parsley cells, and its molecular structure was examined in detail. Upon centrifugation through glycerol gradients, RNA polymerase II sediments as a single band with an apparent sedimentation constant of 15S. No contamination with RNA polymerases I or III could be detected when the activity of purified RNA polymerase II was assayed in the presence of high concentrations of α-amanitin. Analysis of purified RNA polymerase II be nondenaturing and denaturing polyacrylamide gel electrophoresis revealed that this enzyme exists in multiple forms. They were designated II(O), II(A), and II(B). It is suggested that each form has a subunit of Mr = 140000 as well as smaller polypeptides in common. They differ, however, in the molecular weights of their largest subunits which is 220000 in form II(O), 200000 in form II(A), and 180000 in form II(B). These large subunits were labelled with 125 I, digested with trypsin, and tryptic digests were compared by two-dimensional analysis on thin-layer plates (Elder et al. (1977) J. Biol. Chem. 252, 6510-6515). Fingerprints of tryptic digests from the polypeptides with Mr = 220000, Mr = 200000, and Mr = 180000 were similar. It is, therefore, suggested that these subunits are stucturally related. A tryptic digest was also produced from the subunit with Mr = 140000. Its fingerprint was found to yield a considerably different distribution of peptides as compared to those from the three large subunits. (orig.) [de

JOYO MK-II core characteristics database

International Nuclear Information System (INIS)

Tabuchi, Shiro; Aoyama, Takafumi; Nagasaki, Hideaki; Kato, Yuichi

1998-12-01

The experimental fast reactor JOYO served as the MK-II irradiation bed core for testing fuel and material for FBR development for 15 years from 1982 to 1997. During the MK-II operation, extensive data were accumulated from the core characteristics tests conducted in thirty-one duty operations and thirteen special test operations. These core management data and core characteristics data were compiled into a database. The code system MAGI has been developed and used for core management of JOYO MK-II, and the core characteristics and the irradiation test conditions were calculated using MAGI on the basis of three dimensional diffusion theory with seven neutron energy groups. The core management data include extensive data, which were recorded on CD-ROM for user convenience. The data are specifications and configurations of the core, and for about 300 driver fuel subassemblies and about 60 uninstrumented irradiation subassemblies are core composition before and after irradiation, neutron flux, neutron fluences, fuel and control rod burn-up, and temperature and power distributions. MK-II core characteristics and test conditions were stored in the database for post analysis. Core characteristics data include excess reactivities, control rod worths, and reactivity coefficients, e.g., temperature, power and burn-up. Test conditions include both measured and calculated data for irradiation conditions. (author)

TAF(II)250: a transcription toolbox.

Science.gov (United States)

Wassarman, D A; Sauer, F

2001-08-01

Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

Interaction of amatoxins with plant cells and RNA polymerases II: selection of amanitin-resistant cell lines and synthesis of amanitin-based affinity ligands

International Nuclear Information System (INIS)

Little, M.C.

1984-01-01

A series of experiments directed toward deriving basic information regarding plant RNA polymerase II is presented. The experiments described relate to the potential of isolating RNA polymerase II mutants in plants, using carrot cell cultures as models. Additionally, the synthesis of amanitin-based affinity ligands to immobilize isolated plant RNA polymerase II and associated transcriptional complexes is described. RNA polymerase II activities have been isolated from suspension cultures of carrot and compared to other plant RNA polymerases II with respect to subunit analysis and inhibition with α-amanitin. RNA polymerase II purified by polymin P absorption, DE52, phosphocellulose, and RNA-agarose chromatography is shown to copurify with proteins of 175 (and 200), 135, 70, 43, 28, 22, and 17 kdaltons apparent molecular weights. Conditions for accurate determination of amanitin inhibition of the enzyme are established using 3 H-amanitin and are presented for the first time for plant RNA polymerase II; RNA polymerase II from these cultures is shown to be inhibited by 50% at 3-5 nM by α-amanitin, a value 10-50 times lower than previously reported

A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

Science.gov (United States)

Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

2012-01-01

Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

Science.gov (United States)

Robinson, Philip J; Trnka, Michael J; Bushnell, David A; Davis, Ralph E; Mattei, Pierre-Jean; Burlingame, Alma L; Kornberg, Roger D

2016-09-08

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

JOYO MK-II core characteristics database

International Nuclear Information System (INIS)

Ohkawachi, Yasushi; Maeda, Shigetaka; Sekine, Takashi; Aoyama, Takafumi

2003-04-01

The 'JOYO' MK-II core characteristics database was compiled and published in 1998. Comments and requests from many users led to the creation of a revised edition. The revisions include changes to the MAGI calculation code system to use the 70 group JFS-3-J3.2 constant set processed from the JENDL-3.2 library. Total control rod worth, reactor kinetic parameters and the MK-II core performance test results were included per user's requests. The core characteristics obtained from the 32 nd to 35 th operational cycles, which were conducted in the MK-III transition core, were newly added in this revised version. The MK-II core management data and core characteristics data were recorded to CD-ROM for user convenience. The Configuration Data' include the core arrangement and refueling record for each operational cycle. The 'Subassembly Library Data' include the atomic number density, neutron fluence, burn-up, integral power of 362 driver fuel subassemblies and 69 irradiation test subassemblies. The 'Output Data' contain the calculated neutron flux, gamma flux, power density, linear heat rate, coolant and fuel temperature distribution of all the fuel subassemblies at the beginning and end of each operational cycle. The 'Core Characteristics Data' include the measured excess reactivity, control rod worth calibration curve, and reactivity coefficients of temperature, power and burn-up. (author)

Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

Science.gov (United States)

Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

2016-05-03

The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression.

Heysham II/Torness AGR core integrity

International Nuclear Information System (INIS)

Birch, A.L.; Hampson, J.D.

1985-01-01

The design and construction process for the Heysham II/Torness AGR core structures is presented. The design intent utilizing all past experience in designing and building AGR core structures is described. The major aspects of the design criteria and the design conditions are outlined to demonstrate how the integrity of the Heysham II/Torness core is assured. Since no recognized codes of practice for graphite core design exist, the National Nuclear Corporation (NNC) have conceived design criteria utilizing reserve factors based on their design experience. Target reserve factors are defined for particular loading conditions including the ultimate 'safe-shutdown earthquake'. The substantial programme of computer analysis and RandD work to substantiate the design, including seismic qualification, is described. In keeping with their responsibility for the detailed core structure design and the fuel path geometry (guide tube system), NNC attach great importance to design/manufacture/construction liaison, which is demonstrated in the quality assurance section. (author)

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Science.gov (United States)

Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

2001-07-06

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Real-time dynamics of RNA Polymerase II clustering in live human cells

Science.gov (United States)

Cisse, Ibrahim

2014-03-01

Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

Genome-wide analysis of KAP1, the 7SK snRNP complex, and RNA polymerase II

Directory of Open Access Journals (Sweden)

Ryan P. McNamara

2016-03-01

Full Text Available The transition of RNA polymerase II (Pol II from transcription initiation into productive elongation in eukaryotic cells is regulated by the P-TEFb kinase, which phosphorylates the C-terminal domain of paused Pol II at promoter-proximal regions. Our recent study found that P-TEFb (in an inhibited state bound to the 7SK snRNP complex interacts with the KAP1/TRIM28 transcriptional regulator, and that KAP1 and the 7SK snRNP co-occupy most gene promoters containing paused Pol II. Here we provide a detailed experimental description and analysis of the ChIP-seq datasets that have been deposited into Gene Expression Omnibus (GEO: GS72622, so that independent groups can replicate and expand upon these findings. We propose these datasets would provide valuable information for researchers studying mechanisms of transcriptional regulation including Pol II pausing and pause release. Keywords: P-TEFb/7SK snRNP, KAP1, RNA polymerase II, ChIP-seq, Transcription elongation

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

Science.gov (United States)

Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

2016-01-01

Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry

DEFF Research Database (Denmark)

Chen, Zhuo Angel; Jawhari, Anass; Fischer, Lutz

2010-01-01

Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to ex...

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

DEFF Research Database (Denmark)

Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

2006-01-01

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

Cloning and identification of the gene coding for the 140-kd subunit of Drosophila RNA polymerase II

OpenAIRE

Faust, Daniela M.; Renkawitz-Pohl, Renate; Falkenburg, Dieter; Gasch, Alexander; Bialojan, Siegfried; Young, Richard A.; Bautz, Ekkehard K. F.

1986-01-01

Genomic clones of Drosophila melanogaster were isolated from a λ library by cross-hybridization with the yeast gene coding for the 150-kd subunit of RNA polymerase II. Clones containing a region of ∼2.0 kb with strong homology to the yeast gene were shown to code for a 3.9-kb poly(A)+-RNA. Part of the coding region was cloned into an expression vector. A fusion protein was obtained which reacted with an antibody directed against RNA polymerase II of Drosophila. Peptide mapping of the fusion p...

Microprocessor Recruitment to Elongating RNA Polymerase II Is Required for Differential Expression of MicroRNAs

Directory of Open Access Journals (Sweden)

Victoria A. Church

2017-09-01

Full Text Available The cellular abundance of mature microRNAs (miRNAs is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor’s differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha directly associates with the C-terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb. When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.

Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II

NARCIS (Netherlands)

B. Steurer (Barbara); J.A. Marteijn (Jurgen)

2016-01-01

textabstractThe faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The

SAF-A forms a complex with BRG1 and both components are required for RNA polymerase II mediated transcription.

Directory of Open Access Journals (Sweden)

Dzeneta Vizlin-Hodzic

Full Text Available BACKGROUND: Scaffold attachment factor A (SAF-A participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II. METHODOLOGY: Here we use co-localization, co-immunoprecipitation (co-IP and in situ proximity ligation assay (PLA to identify Brahma Related Gene 1 (BRG1, the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction. PRINCIPAL FINDINGS: We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected. CONCLUSIONS: We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.

Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I

International Nuclear Information System (INIS)

Bernges, F.; Holler, E.

1988-01-01

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in K/sub m/ values and a decrease in V/sub max/ values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [ 3 H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II)

Development of JOYO MK-II core characteristics database

International Nuclear Information System (INIS)

Tabuchi, Shiro; Aoyama, Takafumi

2000-01-01

The MK-II core of the experimental fast reactor JOYO served as the irradiation bed for testing fuels and materials for FBR development since 1982 for 15 years. During the MK-II operation, extensive data were accumulated from the core management calculations and characteristics tests conducted in thirty-one duty operations and thirteen special test operations. These core management data and core characteristics data were compiled into a database recorded on CD-ROM for user convenience. The calculated core management data are the text style data. The 'Configuration Data' include the history of the fuel exchange and core arrangement for each cycle. The Subassembly Library Data' include the atomic number density, neutron fluence, burn-up, integral power of about 300 fuel subassemblies, and 60 irradiation subassemblies. The 'Output Data' include the neutron fluxes, gamma fluxes, power density, linear heat rates, coolant and fuel temperature distributions of each core position at the beginning and end of each cycle. The measured core characteristics data, such as the excess reactivity, control rod worths, temperature coefficient, power coefficient, and burn-up coefficient are also included along with the measurement conditions. (J.P.N.)

Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription.

Science.gov (United States)

Bauer, David L V; Tellier, Michael; Martínez-Alonso, Mónica; Nojima, Takayuki; Proudfoot, Nick J; Murphy, Shona; Fodor, Ervin

2018-05-15

Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop.

Directory of Open Access Journals (Sweden)

Chenxi Qiu

2016-11-01

Full Text Available The active sites of multisubunit RNA polymerases have a "trigger loop" (TL that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

Functional conservation of RNA polymerase II in fission and budding yeasts.

Science.gov (United States)

Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P

2000-02-04

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

Comparison of facility characteristics between SCTF Core-I and Core-II

International Nuclear Information System (INIS)

Adachi, Hiromichi; Iwamura, Takamichi; Sobajima, Makoto; Ohnuki, Akira; Abe, Yutaka; Murao, Yoshio.

1990-08-01

The Slab Core Test Facility (SCTF) was constructed to investigate two-dimensional thermal-hydraulics in the core and fluid behavior of carryover water out of the core including its feed-back effect to the core behavior mainly during the reflood phase of a large break loss-of-coolant accident (LOCA) of a pressurized water reactor (PWR). Since three simulated cores are used in the SCTF Test Program and the design of these three cores are slightly different one by one, repeatability test is required to justify a direct comparison of data obtained with different cores. In the present report, data of Test S2-13 (Run 618) obtained with SCTF Core-II were compared with those of Test S1-05 (Run 511) obtained with the Core-I, which were performed under the forced-flooding condition. Thermal-hydraulic behaviors in these two tests showed quite similar characteristics of both system behavior and two-dimensional core behaviors. Therefore, the test data obtained from the two cores can be compared directly with each other. After the turnaround of clad temperatures, however, some differences were found in upper plenum water accumulation and resultant two-dimensional core cooling behaviors such as quench front propagation from bottom to top of the core. (author)

DNA structure in human RNA polymerase II promoters

DEFF Research Database (Denmark)

Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

1998-01-01

with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low...... protein in a manner reminiscent of DNA in a nucleosome. This notion is further supported by the finding that the periodic bendability is caused mainly by the complementary triplet pairs CAG/CTG and GGC/GCC, which previously have been found to correlate with nucleosome positioning. We present models where......The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters...

Evaluation of CCTF Core-II second acceptance Test C2-AC2 (Run 052)

International Nuclear Information System (INIS)

Okubo, Tsutomu; Murao, Yoshio

1984-03-01

In order to investigate the thermo-hydrodynamic behavior in a PWR during the reflood phase of the LOCA, large scale reflooding tests have been conducted at JAERI using the CCTF Core-I and Core-II facilities. This report presents the investigation on the difference in the thermo-hydrodynamic behavior observed between in the CCTF Core-I and Core-II facilities. For this purpose the test data of the second CCTF Core-II acceptance test C2-AC2 (Run 052) were evaluated by using the data of the Test CL-21 (Run 040) in the Core-I test series. The experimental conditions for these two tests were almost identical. Comparing the data of those two tests, the following is obtained. 1. The system behavior observed in the Core-II facility was nearly identical to that observed in the Core-I facility. 2. The core behavior observed in the Core-II facility was also nearly identical to that observed in the Core-I facility except for the top quenching behavior. 3. The differences in the top quenching behavior between the two facilities were as follows: (1) The selective occurrence of top quenching below the open holes of the upper core support plate observed in the Core-I facility was not observed in the Core-II facility. (2) Top quenching tended to occur less in the Core-II facility in the region where the initial average linear power density was over 1.69 kW/m. (author)

RecQL5 promotes genome stabilization through two parallel mechanisms--interacting with RNA polymerase II and acting as a helicase.

Science.gov (United States)

Islam, M Nurul; Fox, David; Guo, Rong; Enomoto, Takemi; Wang, Weidong

2010-05-01

The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

Irradiation of microphones in the EBR-II core

International Nuclear Information System (INIS)

Gavin, A.P.; Anderson, T.T.; Bobis, J.P.

1976-06-01

Six ANL developed high temperature microphone (acoustic detectors) have been exposed in flowing sodium in the In-Core Instrument Test Facility (INCOT) in the Experimental Breeder Reactor-II (EBR-II) for seven months without any indications of serious degradation of signal output due to the exposure. The YY05 experiment (EBR-II INCOT experiment designation) was performed to obtain data which would be useful in evaluating the ability of the microphones whose active elements are lithium niobate to serve as sensors for acoustic surveillance of fast breeder reactors. The reactor was at full power for 136 days of the experiment exposure period. The microphone temperatures varied from 371 0 C (700 0 F) to 621 0 C (1150 0 F). Neutron exposure varied from 2.64 x 10 22 nvt for the microphone at the elevation of the bottom of the EBR-II core to 0.24 x 10 22 nvt for the microphone at the elevation of the top of an EBR-II fuel assembly. The maximum gamma dose was 5 x 10 12 rads

Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

DEFF Research Database (Denmark)

Galli, Giorgio Giacomo; Honnens de Lichtenberg, Kristian; Carrara, Matteo

2012-01-01

and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining...

Real-time observation of the initiation of RNA polymerase II transcription.

Science.gov (United States)

Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

2015-09-10

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

Science.gov (United States)

Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-02-04

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Recognition of Core- and Polymerase-derived immunogenic peptides included in novel therapeutic vaccine by T cells from Chinese chronic hepatitis B patients.

Science.gov (United States)

Huang, D; Sansas, B; Jiang, J H; Gong, Q M; Jin, G D; Calais, V; Yu, D M; Zhu, M Y; Wei, D; Zhang, D H; Inchauspé, G; Zhang, X X; Zhu, R

2017-11-01

Chronic hepatitis B (CHB) is one of the major public health challenges in the world. Due to a strong interplay between specific T-cell immunity and elimination of hepatitis B virus (HBV), efforts to develop novel immunotherapeutics are gaining attention. TG1050, a novel immunotherapy, has shown efficacy in an animal study. To support the clinical development of TG1050 in China, specific immunity to the fusion antigens of TG1050 was assessed in Chinese patients. One hundred and thirty subjects were divided into three groups as CHB patients, HBV spontaneous resolvers, and CHB patients with HBsAg loss after antiviral treatment. HBV-specific T-cell responses to pools of HBV Core or Polymerase genotype D peptides included in TG1050 were evaluated. HBV Core- or Polymerase-specific cells were detected in peripheral blood mononuclear cells (PBMCs) from the different cohorts. The frequencies and intensities of HBV Core-specific immune responses were significantly lower in CHB patients than in HBsAg loss subjects. In CHB patients, a dominant pool derived from Polymerase (Pol1) was the most immunogenic. CHB patients with low viral loads (Core peptide pool. Overall, genotype D-derived peptides included in TG1050 could raise broad and functional T-cell responses in PBMCs from Chinese CHB patients infected with genotype B/C isolates. Core-specific immunogenic domains appeared as "hot spots" with the capacity to differentiate between CHB vs HBsAg loss subjects. These observations support the extended application and associated immune monitoring of TG1050 in China. © 2017 The Authors. Journal of Viral Hepatitis Published by John Wiley & Sons Ltd.

A Caenorhabditis elegans RNA polymerase II gene, ama-1 IV, and nearby essential genes.

Science.gov (United States)

Rogalski, T M; Riddle, D L

1988-01-01

The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20 degrees but are arrested as larvae at 25 degrees, and two others are fertile at 20 degrees and sterile at 25 degrees. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25 degrees is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four gamma-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development.

Advancing Polymerase Ribozymes Towards Self-Replication

Science.gov (United States)

Tjhung, K. F.; Joyce, G. F.

2017-07-01

Autocatalytic replication and evolution in vitro by (i) a cross-chiral RNA polymerase catalyzing polymerization of mononucleotides of the opposite handedness; (ii) non-covalent assembly of component fragments of an existing RNA polymerase ribozyme.

Improvement of SSR core design for ABWR-II

International Nuclear Information System (INIS)

Moriwaki, Masanao; Aoyama, Motoo; Okada, Hiroyuki; Kitamura, Hideya; Sakurada, Koichi; Tanabe, Akira

2003-01-01

In order to enhance the spectral shift effect in the ABWR-II reactor, a novel core design to bring out better performance of spectral shift rods (SSRs) is studied. The SSR is a new type of water rod, in which the water level develops naturally during operation and changes according to the coolant flow rate through the channel. By using the SSR, the average moderator density, which is directly related to core reactivity, can be controlled over a wide range by the core flow rate. In the new SSR core design, two types of SSR bundles, in which settings for the SSR water levels are different, are utilized and loaded according to flow distribution in the core. This two-region SSR core design allows wide variation in the average SSR water level, thus improving fuel economy. Enhancement of SSR function in the two-region SSR core increases the uranium saving factor by about 25%, from the 6% of the conventional uniform SSR core to about 8%. (author)

RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

Directory of Open Access Journals (Sweden)

Isabel X. Wang

2014-03-01

Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle

DEFF Research Database (Denmark)

Büchel, Gabriele; Carstensen, Anne; Mak, Ka-Yan

2017-01-01

MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes...

Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite.

Science.gov (United States)

Cho, J M; Kimball, A P

1982-08-15

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.

Ups and Downs of Poised RNA Polymerase II in B-Cells.

Directory of Open Access Journals (Sweden)

Phuong Dao

2016-04-01

Full Text Available Recent genome-wide analyses have uncovered a high accumulation of RNA polymerase II (Pol II at the 5' end of genes. This elevated Pol II presence at promoters, referred to here as Poll II poising, is mainly (but not exclusively attributed to temporal pausing of transcription during early elongation which, in turn, has been proposed to be a regulatory step for processes that need to be activated "on demand". Yet, the full genome-wide regulatory role of Pol II poising is yet to be delineated. To elucidate the role of Pol II poising in B cell activation, we compared Pol II profiles in resting and activated B cells. We found that while Pol II poised genes generally overlap functionally among different B cell states and correspond to the functional groups previously identified for other cell types, non-poised genes are B cell state specific. Focusing on the changes in transcription activity upon B cell activation, we found that the majority of such changes were from poised to non-poised state. The genes showing this type of transition were functionally enriched in translation, RNA processing and mRNA metabolic process. Interestingly, we also observed a transition from non-poised to poised state. Within this set of genes we identified several Immediate Early Genes (IEG, which were highly expressed in resting B cell and shifted from non-poised to poised state after B cell activation. Thus Pol II poising does not only mark genes for rapid expression in the future, but it is also associated with genes that are silenced after a burst of their expression. Finally, we performed comparative analysis of the presence of G4 motifs in the context of poised versus non-poised but active genes. Interestingly we observed a differential enrichment of these motifs upstream versus downstream of TSS depending on poising status. The enrichment of G4 sequence motifs upstream of TSS of non-poised active genes suggests a potential role of quadruplexes in expression

Actin is closely associated with RNA polymerase II and involved in activation of gene transcription

International Nuclear Information System (INIS)

Zhu Xiaojuan; Zeng Xianlu; Huang Baiqu; Hao, Shui

2004-01-01

Biochemical and morphological studies have demonstrated the presence of actin in the nucleus of different eukaryotic cells, whereas its role remains unclear. In this work, we studied the interaction and the functional relationship between nuclear actin and RNA polymerase II (RNAP II). The immunofluorescence study demonstrated a clear co-localization of nuclear actin with RNAP II in HeLa cells. Meanwhile, actin can be immunoprecipitated by anti-RNAP II antibody, indicating that they could interact with each other. Treatment of cells with α-amanitin induced the formation of actin bundle network in the nucleoplasm. Blocking of the formation of filamentous actin (F-actin) by cytochalasin B modified the distribution of actin. Although the actin content remained unchanged in resting and concanavalinA stimulated mouse lymphocytes, the actin content in the nuclei showed a progressive increase after stimulation. Furthermore, the antibody against actin blocked RNA synthesis in a eukaryotic in vitro transcription system. These observations implicate that nuclear actin interacts with RNAP II and may have function on the RNAP II-mediated transcription

Test of In-core Flux Detectors in KNK II

CERN Document Server

Hoppe, P

1979-01-01

The development of in-core detectors for Liquid Metal Fast Breeder Reactors (LMFBRs) is still in an early stage, and little operation experience is available. Therefore self-powered neutron and gamma detectors and neutron sensitive ionization chambers -especially developed for LMFBRs- have been tested in the Fast Sodium Cooled Test Reactor KNK II. Seven flux detectors have been installed in the core of KNK II by means of a special test rig. Five of them failed already within the first week during operation in the reactor. Due to measurements of electrical resistances and capacities, sodium penetrating into the detectors or cables probably seems to be the cause. As tests prior to the installation in the core proved the tightness of all detectors, it is suspected that small cracks have developed in the detector casings or in the outer cable sheaths during their exposure to the hot coolant. Two ionization chambers did not show these faults. However, one of them failed because the saturation current plateau disap...

The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

Science.gov (United States)

Jeronimo, Célia; Robert, François

2017-10-01

Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

Science.gov (United States)

Scott, D J; Ferguson, A L; Gallegos, M T; Pitt, M; Buck, M; Hoggett, J G

2000-12-01

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed.

Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

KAUST Repository

Da, Lin-Tai; Pardo-Avila, Fá tima; Xu, Liang; Silva, Daniel-Adriano; Zhang, Lu; Gao, Xin; Wang, Dong; Huang, Xuhui

2016-01-01

The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

KAUST Repository

Da, Lin-Tai

2016-04-19

The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

Experimental programme and analysis, ZENITH II, Core 4

Energy Technology Data Exchange (ETDEWEB)

Ingram, G.; Sanders, J. E.; Sherwin, J.

1974-10-15

The Phase 3 program of reactor physics experiments on the HTR (or Mk 3 GCR) lattices continued during the first half of 1974 with a study of a series of critical builds in Zenith II aimed at testing predictions of shut-down margins in the local criticality-situations arising during power reactor refueling. The paper describes the experimental program and the subsequent theoretical analysis using methods developed in the United Kingdom for calculating low-enriched uranium HTR fuel systems. The importance of improving the accuracy of predictions of shut-down margins arises from the basic requirement that the core in its most reactive condition and with a specified number of absorbers removed from the array must remain sub-critical with a margin adequate to cover the total uncertainty of +/- 1 Nile (that is, 1 % delta-k). The major uncertainty is that in modelling the complex fuel/absorber configuration, and this is the aspect essentially covered in the Zenith II Core 4 studies.

Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

Directory of Open Access Journals (Sweden)

Kevin M. Harlen

2016-06-01

Full Text Available Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II C-terminal domain (CTD and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7, we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

Inference of RNA polymerase II transcription dynamics from chromatin immunoprecipitation time course data.

Directory of Open Access Journals (Sweden)

Ciira wa Maina

2014-05-01

Full Text Available Gene transcription mediated by RNA polymerase II (pol-II is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2. The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ERα and FOXA1 binding in their proximal promoter regions.

Linking Core Promoter Classes to Circadian Transcription.

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Pål O Westermark

2016-08-01

Full Text Available Circadian rhythms in transcription are generated by rhythmic abundances and DNA binding activities of transcription factors. Propagation of rhythms to transcriptional initiation involves the core promoter, its chromatin state, and the basal transcription machinery. Here, I characterize core promoters and chromatin states of genes transcribed in a circadian manner in mouse liver and in Drosophila. It is shown that the core promoter is a critical determinant of circadian mRNA expression in both species. A distinct core promoter class, strong circadian promoters (SCPs, is identified in mouse liver but not Drosophila. SCPs are defined by specific core promoter features, and are shown to drive circadian transcriptional activities with both high averages and high amplitudes. Data analysis and mathematical modeling further provided evidence for rhythmic regulation of both polymerase II recruitment and pause release at SCPs. The analysis provides a comprehensive and systematic view of core promoters and their link to circadian mRNA expression in mouse and Drosophila, and thus reveals a crucial role for the core promoter in regulated, dynamic transcription.

Monte Carlo analysis of Musashi TRIGA mark II reactor core

International Nuclear Information System (INIS)

Matsumoto, Tetsuo

1999-01-01

The analysis of the TRIGA-II core at the Musashi Institute of Technology Research Reactor (Musashi reactor, 100 kW) was performed by the three-dimensional continuous-energy Monte Carlo code (MCNP4A). Effective multiplication factors (k eff ) for the several fuel-loading patterns including the initial core criticality experiment, the fuel element and control rod reactivity worth as well as the neutron flux measurements were used in the validation process of the physical model and neutron cross section data from the ENDF/B-V evaluation. The calculated k eff overestimated the experimental data by about 1.0%Δk/k for both the initial core and the several fuel-loading arrangements. The calculated reactivity worths of control rod and fuel element agree well the measured ones within the uncertainties. The comparison of neutron flux distribution was consistent with the experimental ones which were measured by activation methods at the sample irradiation tubes. All in all, the agreement between the MCNP predictions and the experimentally determined values is good, which indicated that the Monte Carlo model is enough to simulate the Musashi TRIGA-II reactor core. (author)

Evaluation report on CCTF Core-II reflood test C2-4 (Run 62)

International Nuclear Information System (INIS)

Okubo, Tsutomu; Iguchi, Tadashi; Sugimoto, Jun; Akimoto, Hajime; Murao, Yoshio; Okabe, Kazuharu.

1985-03-01

This report presents a data evaluation of the CCTF Core-II test C2-4 (Run 62), which was conducted on May 12, 1983. This test was conducted to investigate the reproducibility of tests in the CCTF Core-II test series. Therefore, the initial and boundary conditions of the present test were determined to be the same as those for the previously performed base case test (Test C2-SH1). Comparing the data of the present test with those of Test C2-SH1, the following results are obtained. (1) The initial and boundary conditions for the two tests were nearly identical except the temperature of the core barrel and the lower plenum fluid. The difference in the latter is considered to result in the difference in the core inlet subcooling of about 6 K at most. (2) The system behavior was almost identical. (3) The core cooling behavior was also nearly identical except a little difference in the rod surface temperature in the upper part of the high power region. (4) Taking account that the difference mentioned above in item (3) is small and can be explained qualitatively to be caused by the difference in the core inlet subcooling mentioned above in item (1), it is considered practically that there is the reproducibility of the thermo-hydrodynamic behavior in the CCTF Core-II tests. (author)

Test of In-core Flux Detectors in KNK II

International Nuclear Information System (INIS)

Hoppe, P.; Mitzel, F.

1979-10-01

The development of in-core detectors for Liquid Metal Fast Breeder Reactors (LMFBRs) is still in an early stage, and little operation experience is available. Therefore self-powered neutron and gamma detectors and neutron sensitive ionization chambers -especially developed for LMFBRs- have been tested in the Fast Sodium Cooled Test Reactor KNK II. Seven flux detectors have been installed in the core of KNK II by means of a special test rig. Five of them failed already within the first week during operation in the reactor. Due to measurements of electrical resistances and capacities, sodium penetrating into the detectors or cables probably seems to be the cause. As tests prior to the installation in the core proved the tightness of all detectors, it is suspected that small cracks have developed in the detector casings or in the outer cable sheaths during their exposure to the hot coolant. Two ionization chambers did not show these faults. However, one of them failed because the saturation current plateau disappeared and the other one's sensitivity decreased by a factor of five during the test period. It is suspected that in both cases changes of the filling gas might be involved

DNA repair in DNA-polymerase-deficient mutants of Escherichia coli

International Nuclear Information System (INIS)

Smith, D.W.; Tait, R.C.; Harris, A.L.

1975-01-01

Escherichia coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III can efficiently and completely execute excision-repair and postreplication repair of the uv-damaged DNA at 30 0 C and 43 0 C when assayed by alkaline sucrose gradients. Repair by Pol I - and Pol I - , Pol II - cells is inhibited by 1-β-D-arabinofuranosylcytosine (araC) at 43 0 C but not at 30 0 C, whereas that by Pol III - cells is insensitive to araC at any temperature. Thus, either Pol I or Pol III is required for complete and efficient repair, and in their absence Pol II mediates a limited, incomplete dark repair of uv-damaged DNA

Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7.

Science.gov (United States)

Qiu, Zilong; Jiang, Rongrong

2017-01-01

Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae , which finally led to improvement in yeast ethanol tolerance and production.

Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

DEFF Research Database (Denmark)

Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer

DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples. In conclusion, our data shows that exchanging the DNA polymerase......Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity...... commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied...

Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters.

Science.gov (United States)

Dergai, Oleksandr; Cousin, Pascal; Gouge, Jerome; Satia, Karishma; Praz, Viviane; Kuhlman, Tracy; Lhôte, Philippe; Vannini, Alessandro; Hernandez, Nouria

2018-05-01

RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters. © 2018 Dergai et al.; Published by Cold Spring Harbor Laboratory Press.

Verification of MVP-II and SRAC2006 code to the core physics vera benchmark problem

International Nuclear Information System (INIS)

Jati Susilo

2014-01-01

In this research, verification calculation for VERA core physics benchmark on the Zero Power Physical Test (ZPPT) of the nuclear reactor Watts Bar 1. The reactor is a 1000 MWe class of PWR designed by. Westinghouse, arranged from 193 unit of 17 x 17 fuel assembly consisting 3 type enrichment of UO2 that are 2.1wt%, 2.619wt% and 3.1wt%. Core power factor distribution and k-eff calculation has been done for the first cycle operation of the core at beginning of cycle (BOC) and hot zero power (HZP). In this calculation, MVP-II and CITATION module of SRAC2006 computer code has been used with ENDF/B-VII.0. cross section data library. Calculation result showed that differences value of k-eff for the core at controlled and uncontrolled condition between reference with MVP-II (-0,07% and -0,014%) and SRAC2006 (0,92% and 0,99%) are very small or below 1%. Differences value of radial power peaking factor at controlled and uncontrolled of the core between reference value with MVP-II are 0,38% and 1,53%, even though with SRAC2006 are 1,13% and -2,45%. It can be said that the calculation result by both computer code showing suitability with reference value. In order to determinate of criticality of the core, the calculation result using MVP-II code is more conservative compare with SRAC2006 code. (author)

Transitions to improved core electron heat confinement in JT-II plasmas

International Nuclear Information System (INIS)

Estrada, T.; Medina, F.; Ascasibar, E.; Balbin, R.; Castejon, F.; Hidalgo, C.; Lopez-Bruna, D.; Petrov, S.

2008-01-01

Transitions to improved core electron heat confinement are triggered by low order rational magnetic surfaces in TJ-II ECH plasmas. Transitions triggered by the rational surface n=4/m=2 show an increase in the ion temperature synchronized with the increase in the electron temperature. SXR measurements demonstrate that, under certain circumstances, the rational surface positioned inside the plasma core region precedes and provides a trigger for the transition. (author)

Two-dimensional thermal-hydraulic behavior in core in SCTF Core-II forced feed reflood tests

International Nuclear Information System (INIS)

Iwamura, Takamichi; Sobajima, Makoto; Okubo, Tsutomu; Ohnuki, Akira; Abe, Yutaka; Adachi, Hiromichi

1987-01-01

Major purpose of the Slab Core Test Program is to investigate the two-dimensional thermal-hydraulic behavior in the core during the reflood phase of a PWR-LOCA. It was revealed in the previous Slab Core Test Facility (SCTF) Core-II test results that the heat transfer was enhanced in the higher power bundles and degraded in the lower power bundles in the non-uniform radial power profile tests. In order to separately evaluate the effect of the radial power (Q) distribution itself and the effect of the radial temperature (T) distribution, four tests were performed with steep Q and T, flat Q and T, steep Q and flat T, and flat Q and steep T. Based on the test results, it was concluded that the radial temperature distribution which accompanied the radial power distribution was the dominant factor of the two-dimensional thermal-hydraulic behavior in the core during the initial period. Selected data from these four tests are also presented in this report. Some data from Test S2-12 (steep Q, T) were compared with TRAC post-test calculations performed by the Los Alamos National Laboratory. (author)

Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

OpenAIRE

Sauguet , Ludovic; Raia , Pierre; Henneke , Ghislaine; Delarue , Marc

2016-01-01

International audience; Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has ...

Functional and structural analysis of photosystem II core complexes from spinach with high oxygen evolution capacity

NARCIS (Netherlands)

Haag, Elisabeth; Irrgang, Klaus-D.; Boekema, Egbert J.; Renger, Gernot

1990-01-01

Oxygen-evolving photo system II core complexes were prepared from spinach by solubilizing photosystem II membrane fragments with dodecyl-β-D-maltoside. The core complexes consist of the intrinsic 47-kDa, 43-kDa, D1 and D2 polypeptides, the two subunits of cytochrome b559 and the extrinsic 33-kDa

Chromosomal loop/nuclear matrix organization of transcriptionally active and inactive RNA polymerases in HeLa nuclei.

Science.gov (United States)

Roberge, M; Dahmus, M E; Bradbury, E M

1988-06-05

The relative distribution of transcriptionally active and inactive RNA polymerases I and II between the nuclear matrix/scaffold and chromosomal loops of HeLa cells was determined. Total RNA polymerase was assessed by immunoblotting and transcribing RNA polymerase by a photoaffinity labeling technique in isolated nuclei. Nuclear matrix/scaffold was isolated by three methods using high-salt, intermediate-salt or low-salt extraction. The distribution of RNA polymerases I and II were very similar within each of the methods, but considerable differences in distributions were found between the different preparation methods. Either intermediate-salt or high-salt treatment of DNase I-digested nuclei showed significant association of RNA polymerases with the nuclear matrix. However, intermediate-salt followed by high-salt treatment released all transcribing and non-transcribing RNA polymerases. Nuclear scaffolds isolated with lithium diiodosalicylate (low-salt) contained very little of the RNA polymerases. This treatment, however, caused the dissociation of RNA polymerase II transcription complexes. These results show unambiguously that RNA polymerases, both in their active and inactive forms, are not nuclear matrix proteins. The data support models in which the transcriptional machinery moves around DNA loops during transcription.

Overview of the TITAN-II reversed-field pinch aqueous fusion power core design

Energy Technology Data Exchange (ETDEWEB)

Wong, C.P.C.; Creedon, R.L.; Grotz, S.; Cheng, E.T.; Sharafat, S.; Cooke, P.I.H.

1988-03-01

TITAN-II is a compact, high power density Reversed-Field Pinch fusion power reactor design based on the aqueous lithium solution fusion power core concept. The selected breeding and structural materials are LiNO/sub 3/ and 9-C low activation ferritic steel, respectively. TITAN-II is a viable alternative to the TITAN-I lithium self-cooled design for the Reversed-Field Pinch reactor to operate at a neutron wall loading of 18 MWm/sup 2/. Submerging the complete fusion power core and the primary loop in a large pool of cool water will minimize the probability of radioactivity release. Since the protection of the large pool integrity is the only requirement for the protection of the public, TITAN-II is a passive safety assurance design. 13 refs., 3 figs., 1 tab.

Overview of the TITAN-II reversed-field pinch aqueous fusion power core design

Energy Technology Data Exchange (ETDEWEB)

Wong, C.P.C.; Creedon, R.L.; Cheng, E.T. (General Atomic Co., San Diego, CA (USA)); Grotz, S.P.; Sharafat, S.; Cooke, P.I.H. (California Univ., Los Angeles (USA). Dept. of Mechanical, Aerospace and Nuclear Engineering; California Univ., Los Angeles, CA (USA). Inst. for Plasma and Fusion Research); TITAN Research Group

1989-04-01

TITAN-II is a compact, high-power-density Reversed-Field Pinch fusion power reactor design based on the aqueous lithium solution fusion power core concept. The selected breeding and structural materials are LiNO/sub 3/ and 9-C low activation ferritic steel, respectively. TITAN-II is a viable alternative to the TITAN-I lithium self-cooled design for the Reversed-Field Pinch reactor to operate at a neutron wall loading of 18 MW/m/sup 2/. Submerging the complete fusion power core and the primary loop in a large pool of cool water will minimize the probability of radioactivity release. Since the protection of the large pool integrity is the only requirement for the protection of the public, TITAN-II is a level 2 of passive safety assurance design. (orig.).

Adaptive Core Simulation Employing Discrete Inverse Theory - Part II: Numerical Experiments

International Nuclear Information System (INIS)

Abdel-Khalik, Hany S.; Turinsky, Paul J.

2005-01-01

Use of adaptive simulation is intended to improve the fidelity and robustness of important core attribute predictions such as core power distribution, thermal margins, and core reactivity. Adaptive simulation utilizes a selected set of past and current reactor measurements of reactor observables, i.e., in-core instrumentation readings, to adapt the simulation in a meaningful way. The companion paper, ''Adaptive Core Simulation Employing Discrete Inverse Theory - Part I: Theory,'' describes in detail the theoretical background of the proposed adaptive techniques. This paper, Part II, demonstrates several computational experiments conducted to assess the fidelity and robustness of the proposed techniques. The intent is to check the ability of the adapted core simulator model to predict future core observables that are not included in the adaption or core observables that are recorded at core conditions that differ from those at which adaption is completed. Also, this paper demonstrates successful utilization of an efficient sensitivity analysis approach to calculate the sensitivity information required to perform the adaption for millions of input core parameters. Finally, this paper illustrates a useful application for adaptive simulation - reducing the inconsistencies between two different core simulator code systems, where the multitudes of input data to one code are adjusted to enhance the agreement between both codes for important core attributes, i.e., core reactivity and power distribution. Also demonstrated is the robustness of such an application

ABWR-II Core Design with Spectral Shift Rods for Operation with All Control Rods Withdrawn

International Nuclear Information System (INIS)

Moriwaki, Masanao; Aoyama, Motoo; Anegawa, Takafumi; Okada, Hiroyuki; Sakurada, Koichi; Tanabe, Akira

2004-01-01

An innovative reactor core concept applying spectral shift rods (SSRs) is proposed to improve the plant economy and the operability of the 1700-MW(electric) Advanced Boiling Water Reactor II (ABWR-II). The SSR is a new type of water rod in which a water level is naturally developed during operation and changed according to the coolant flow rate through the channel. By taking advantage of the large size of the ABWR-II bundle, the enhanced spectral shift operation by eight SSRs allows operation of the ABWR-II with all control rods withdrawn. In addition, the uranium-saving factor of 6 to 7% relative to the reference ABWR-II core with conventional water rods can be expected due to the greater effect of spectral shift. The combination of these advantages means the ABWR-II with SSRs should be an attractive alternative for the next-generation nuclear reactor

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

Science.gov (United States)

Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Radioimmunoassay of surface antigen and core antibody of hepatitis B virus. Comparison of kits AUSRIA/CORE; AUSRIA II-125 and CORAB

Energy Technology Data Exchange (ETDEWEB)

Kselikova, M; Urbankova, J [Ustav Hematologie a Krevni Transfuze, Prague (Czechoslovakia)

1984-08-01

The sensitivity is compared of determination of surface antigen HBsAg and nuclear antibody HBcAb of the hepatitis B virus using kits for separate (AUSRIA II-125, CORAB) and simultaneous (AUSRIAsup(R)/CORE) determinations of third generation tests in selected samples of medical personnel, HBsAg carriers, patients at a dialysis centre, blood donors and in sera of HBsAg carriers diluted in steps from 4x10/sup -2/ to 4x10/sup -7/. HBsAg is always determined using the RIA technique, HBcAb is determined using the technique of radioimmunoassay with the CORAB kit and with the AUSRIA sup(R)/CORE kit using enzymeimmunoassay. The sensitivity of determination using the AUSRIA sup(R)/CORE kit is at least as good for both investigated indicators of the hepatitis B virus and that obtained using separate determination of HBsAg (AUSRIA II-125) and HBcAb (CORAB), this also using modified photocolorimetric determination. Only one AUSRIA/sup R//CORE kit was available for the investigation and the informative character of the report is emphasized.

[Effect of core: dentin thickness ratio on the flexure strength of IPS Empress II heat-pressed all-ceramic restorative material].

Science.gov (United States)

Liu, Yi-hong; Feng, Hai-lan; Bao, Yi-wang; Qiu, Yan

2007-02-18

To evaluate the effect of core:dentin thickness ratio on the flexure strength, fracture mode and origin of bilayered IPS Empress II ceramic composite specimens. IPS Empress II core ceramic, dentin porcelain and bilayered composite specimens with core:dentin thickness ratio of 2:1 and 1:1 were tested in three-point flexure strength. Mean strengths and standard deviations were determined. The optical microscopy was employed for identification of the fracture mode and origin. The flexure strength of dentin porcelain was the smallest(62.7 MPa), and the strength of bilayered composite specimens was smaller than single-layered core ceramic(190.2 MPa). The core: dentin ratio did not influence the strength of bilayered composite specimens. The frequency of occurrence of bilayered specimen delaminations was higher in the group of core: dentin thickness ratio of 1:1 than in the group of 2:1. IPS Empress II core ceramic was significantly stronger than veneering dentin porcelain. Core:dentin thickness ratio could significantly influence the fracture mode and origin, and bilayered IPS Empress II ceramic composite specimens showed little influence in the fracture strength.

Two-dimensional thermal-hydraulic behavior in core in SCTF Core-II cold leg injection tests

International Nuclear Information System (INIS)

Iwamura, Takamichi; Sobajima, Makoto; Okubo, Tsutomu; Ohnuki, Akira; Abe, Yutaka; Adachi, Hiromichi

1985-07-01

Major purpose of the Slab Core Test Program is to investigate the two-dimensional thermal-hydraulic behavior in the core during the reflood phase in a PWR-LOCA. In order to investigate the effects of radial power profile, three cold leg injection tests with different radial power profiles under the same total heating power and core stored energy were performed by using the Slab Core Test Facility (SCTF) Core-II. It was revealed by comparing these three tests that the heat transfer was enhanced in the higher power bundles and degraded in the lower power bundles in the non-uniform radial power profile tests. The turnaround temperature in the high power bundles were evaluated to be reduced by about 40 to 120 K. On the other hand, a two-dimensional flow in the core was also induced by the non-uniform water accumulation in the upper plenum and the quench was delayed resultantly in the bundles corresponding to the peripheral bundles of a PWR. However, the effect of the non-uniform upper plenum water accumulation on the turnaround temperature was small because the effect dominated after the turnaround of the cladding temperature. Selected data from Tests S2-SH1, S2-SH2 and S2-O6 are also presented in this report. Some data from Tests S2-SH1 and S2-SH2 were compared with TRAC post-test calculations performed by the Los Alamos National Laboratory. (author)

Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

Directory of Open Access Journals (Sweden)

Kathrin Davari

2017-04-01

Full Text Available Summary: Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP, and RNA polymerase II (RNA Pol II ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD and activation of the positive transcription elongation factor (pTEFb. Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation. : Davari et al. visualize global changes in RNA Pol II binding, transcription, splicing, and translation. T cells change their functional program by rapid de novo recruitment of RNA Pol II and coupled changes in transcription and translation. This coincides with fluctuations in RNA Pol II phosphorylation and a temporary reduction in cotranscriptional splicing. Keywords: RNA Pol II, cotranscriptional splicing, T cell activation, ribosome profiling, 4sU, H3K36, Ser-5 RNA Pol II, Ser-2 RNA Pol II, immune response, immediate-early genes

Fuel Management Strategies for a Possible Future LEU Core of a TRIGA Mark II Vienna

Energy Technology Data Exchange (ETDEWEB)

Khan, R.; Villa, M.; Steinhauser, G.; Boeck, H. [Vienna University of Technology-Atominstitut (Austria)

2011-07-01

The Vienna University of Technology/Atominstitut (VUT/ATI) operates a TRIGA Mark II research reactor. It is operated with a completely mixed core of three different types of fuel. Due to the US fuel return program, the ATI have to return its High Enriched Uranium (HEU) fuel latest by 2019. As an alternate, the Low Enrich Uranium (LEU) fuel is under consideration. The detailed results of the core conversion study are presented at the RRFM 2011 conference. This paper describes the burn up calculations of the new fuel to predict the future burn up behavior and core life time. It also develops an effective and optimized fuel management strategy for a possible future operation of the TRIGA Mark II with a LEU core. This work is performed by the combination of MCNP5 and diffusion based neutronics code TRIGLAV. (author)

Understanding variability of the Southern Ocean overturning circulation in CORE-II models

Science.gov (United States)

Downes, S. M.; Spence, P.; Hogg, A. M.

2018-03-01

The current generation of climate models exhibit a large spread in the steady-state and projected Southern Ocean upper and lower overturning circulation, with mechanisms for deep ocean variability remaining less well understood. Here, common Southern Ocean metrics in twelve models from the Coordinated Ocean-ice Reference Experiment Phase II (CORE-II) are assessed over a 60 year period. Specifically, stratification, surface buoyancy fluxes, and eddies are linked to the magnitude of the strengthening trend in the upper overturning circulation, and a decreasing trend in the lower overturning circulation across the CORE-II models. The models evolve similarly in the upper 1 km and the deep ocean, with an almost equivalent poleward intensification trend in the Southern Hemisphere westerly winds. However, the models differ substantially in their eddy parameterisation and surface buoyancy fluxes. In general, models with a larger heat-driven water mass transformation where deep waters upwell at the surface ( ∼ 55°S) transport warmer waters into intermediate depths, thus weakening the stratification in the upper 2 km. Models with a weak eddy induced overturning and a warm bias in the intermediate waters are more likely to exhibit larger increases in the upper overturning circulation, and more significant weakening of the lower overturning circulation. We find the opposite holds for a cool model bias in intermediate depths, combined with a more complex 3D eddy parameterisation that acts to reduce isopycnal slope. In summary, the Southern Ocean overturning circulation decadal trends in the coarse resolution CORE-II models are governed by biases in surface buoyancy fluxes and the ocean density field, and the configuration of the eddy parameterisation.

Experience with lifetime limits for EBR-II core components

International Nuclear Information System (INIS)

Lambert, J.D.B.; Smith, R.N.; Golden, G.H.

1987-01-01

The Experimental Breeder Reactor No. 2 (EBR-II) is operated for the US Department of Energy by Argonne National Laboratory and is located on the Idaho National Engineering Laboratory where most types of American reactor were originally tested. EBR-II is a complete electricity-producing power plant now in its twenty-fourth year of successful operation. During this long history the reactor has had several concurrent missions, such as demonstration of a closed Liquid-Metal Reactor (LMR) fuel cycle (1964-69); as a steady-state irradiation facility for fuels and materials (1970 onwards); for investigating effects of operational transients on fuel elements (from 1981); for research into the inherent safety aspects of metal-fueled LMR's (from 1983); and, most recently, for demonstration of the Integral Fast Reactor (IFR) concept using U-Pu-Zr fuels. This paper describes experience gained at EBR-II in defining lifetime limits for LMR core components, particularly fuel elements

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

Directory of Open Access Journals (Sweden)

Überla Klaus

2007-06-01

Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

RNA Polymerase II–The Transcription Machine

Indian Academy of Sciences (India)

Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 3. RNA Polymerase II – The Transcription Machine - Nobel Prize in Chemistry 2006. Jiyoti Verma Aruna Naorem Anand Kumar Manimala Sen Parag Sadhale. General Article Volume 12 Issue 3 March 2007 pp 47-53 ...

The Core Collapse Supernova Rate from the SDSS-II Supernova Survey

Energy Technology Data Exchange (ETDEWEB)

Taylor, Matt; Cinabro, David; Dilday, Ben; Galbany, Lluis; Gupta, Ravi R.; Kessler, R.; Marriner, John; Nichol, Robert C.; Richmond, Michael; Schneider, Donald P.; Sollerman, Jesper

2014-08-26

We use the Sloan Digital Sky Survey II Supernova Survey (SDSS-II SNS) data to measure the volumetric core collapse supernova (CCSN) rate in the redshift range (0.03 < z < 0.09). Using a sample of 89 CCSN, we find a volume-averaged rate of 1.06 ± 0.19 × 10(–)(4)((h/0.7)(3)/(yr Mpc(3))) at a mean redshift of 0.072 ± 0.009. We measure the CCSN luminosity function from the data and consider the implications on the star formation history.

An RNA polymerase II-and AGO4-associated protein acts in RNA-directed DNA methylation

KAUST Repository

Gao, Zhihuan

2010-04-21

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm. © 2010 Macmillan Publishers Limited. All rights reserved.

Kin28 regulates the transient association of Mediator with core promoters.

Science.gov (United States)

Jeronimo, Célia; Robert, François

2014-05-01

Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-terminal domain is mutated to replace Ser5 with alanine, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is released quickly from promoters after phosphorylation of Ser5 by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

Science.gov (United States)

Wong, J M; Ingles, C J

2001-02-01

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Budd, M.E.; Wittrup, K.D.; Bailey, J.E.; Campbell, J.L.

1989-01-01

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I

Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

DEFF Research Database (Denmark)

Helbo, Alexandra Søgaard; Lay, Fides D; Jones, Peter A

2017-01-01

Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high...

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

Science.gov (United States)

Nelson, Emily V; Pacheco, Jennifer R; Hume, Adam J; Cressey, Tessa N; Deflubé, Laure R; Ruedas, John B; Connor, John H; Ebihara, Hideki; Mühlberger, Elke

2017-10-01

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research. Copyright © 2017 Elsevier B.V. All rights reserved.

[RNA polymerase II and pre-mRNA splicing factors in diplotene oocyte nuclei of the giant African gastropod Achatina fulica].

Science.gov (United States)

Stepanova, I S; Bogoliubov, D S

2003-01-01

The nuclear distribution of pre-mRNA splicing factors (snRNPs and SR-protein SC35) and unphosphorylated from of RNA polymerase II (Pol II) was studied using fluorescent and immunoelectron cytochemistry in diplotene oocytes of the gastropod Achatina fulica. Association of Pol II and splicing factors with oocyte nuclear structures was analysed. The antibodies against splicing factors and Pol II were shown to label perichromatin fibrils at the periphery of condensed chromatin blocks as well as those in interchromatin regions of nucleoplasm. The revealed character of distribution of snRNPs, SC35 protein, and Pol II, together with the decondensed chromatin and absence of karyosphere, enable us to suggest that oocyte chromosomes maintain their transcriptional activity at the diplotene stage of oogenesis. In A. fulica oocytes, sparse nuclear bodies (NBs) of a complex morphological structure were revealed. These NBs contain snRNPs rather than SC35 protein. NBs are associated with a fibrogranular material (FGM), which contains SC35 protein. No snRNPs were revealed in this material. Homology of A. fulica oocyte nuclear structures to Cajal bodies and interchromatin granule clusters is discussed.

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

Science.gov (United States)

Tsai, Kuang-Lei; Sato, Shigeo; Tomomori-Sato, Chieri; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2013-01-01

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism. PMID:23563140

Traveling Rocky Roads: The Consequences of Transcription-Blocking DNA Lesions on RNA Polymerase II.

Science.gov (United States)

Steurer, Barbara; Marteijn, Jurgen A

2017-10-27

The faithful transcription of eukaryotic genes by RNA polymerase II (RNAP2) is crucial for proper cell function and tissue homeostasis. However, transcription-blocking DNA lesions of both endogenous and environmental origin continuously challenge the progression of elongating RNAP2. The stalling of RNAP2 on a transcription-blocking lesion triggers a series of highly regulated events, including RNAP2 processing to make the lesion accessible for DNA repair, R-loop-mediated DNA damage signaling, and the initiation of transcription-coupled DNA repair. The correct execution and coordination of these processes is vital for resuming transcription following the successful repair of transcription-blocking lesions. Here, we outline recent insights into the molecular consequences of RNAP2 stalling on transcription-blocking DNA lesions and how these lesions are resolved to restore mRNA synthesis. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Phosphine-free synthesis and characterization of type-II ZnSe/CdS core-shell quantum dots

Science.gov (United States)

Ghasemzadeh, Roghayyeh; Armanmehr, Mohammad Hasan; Abedi, Mohammad; Fateh, Davood Sadeghi; Bahreini, Zaker

2018-01-01

A phosphine-free route for synthesis of type-II ZnSe/CdS core-shell quantum dots, using green, low cost and environmentally friendly reagents and phosphine-free solvents such as 1-octadecene (ODE) and liquid paraffin has been reported. Hot-injection technique has been used for the synthesis of ZnSe core quantum dots. The CdS shell quantum dots prepared by reaction of CdO precursor and S powder in 1-octadecene (ODE). The ZnSe/CdS core-shell quantum dots were synthesized via successive ion layer adsorption and reaction (SILAR) technique. The characterization of produced quantum dots were performed by absorption and fluorescence spectroscopy, X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX) and transmission electron microscopy (TEM). The results showed the formation of type-II ZnSe/CdS core-shell quantum dots with FWHM 32 nm and uniform size distribution.

A CATALOG OF LOW-MASS STAR-FORMING CORES OBSERVED WITH SHARC-II AT 350 μ m

Energy Technology Data Exchange (ETDEWEB)

Suresh, Akshaya; Arce, Héctor G. [Department of Astronomy, Yale University, P.O. Box 208101, New Haven, CT 06520 (United States); Dunham, Michael M.; Bourke, Tyler L. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, MS 78, Cambridge, MA 02138 (United States); II, Neal J. Evans [Department of Astronomy, The University of Texas at Austin, 2515 Speedway, Stop C1400, Austin, TX 78712-1205 (United States); Merello, Manuel [Istituto di Astrofisica e Planetologia Spaziali-INAF, Via Fosso del Cavaliere 100, I-00133 Roma (Italy); Wu, Jingwen, E-mail: mdunham@cfa.harvard.edu [National Astronomical Observatories, Chinese Academy of Sciences, 20A Datun Road, Chaoyang District, Beijing 100012 (China)

2016-08-01

We present a catalog of low-mass dense cores observed with the SHARC-II instrument at 350 μ m. Our observations have an effective angular resolution of 10″, approximately 2.5 times higher than observations at the same wavelength obtained with the Herschel Space Observatory , albeit with lower sensitivity, especially to extended emission. The catalog includes 81 maps covering a total of 164 detected sources. For each detected source, we tabulate basic source properties including position, peak intensity, flux density in fixed apertures, and radius. We examine the uncertainties in the pointing model applied to all SHARC-II data and conservatively find that the model corrections are good to within ∼3″, approximately 1/3 of the SHARC-II beam. We examine the differences between two array scan modes and find that the instrument calibration, beam size, and beam shape are similar between the two modes. We also show that the same flux densities are measured when sources are observed in the two different modes, indicating that there are no systematic effects introduced into our catalog by utilizing two different scan patterns during the course of taking observations. We find a detection rate of 95% for protostellar cores but only 45% for starless cores, and demonstrate the existence of a SHARC-II detection bias against all but the most massive and compact starless cores. Finally, we discuss the improvements in protostellar classification enabled by these 350  μ m observations.

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

OpenAIRE

Wright, Kevin J.; Marr, Michael T.; Tjian, Robert

2006-01-01

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNA...

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein

Directory of Open Access Journals (Sweden)

RODRIGO VÁSQUEZ-DEL CARPIÓ

2006-01-01

Full Text Available Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp, which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

Directory of Open Access Journals (Sweden)

Masashi Miura

2013-12-01

Full Text Available SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

Science.gov (United States)

Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

2015-03-31

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Review of the SIMMER-II analyses of liquid-metal-cooled fast breeder reactor core-disruptive accident fuel escape

International Nuclear Information System (INIS)

DeVault, G.P.; Bell, C.R.

1985-01-01

Early fuel removal from the active core of a liquid-metal-cooled fast breeder reactor undergoing a core-disruptive accident may reduce the potential for large energetics resulting from recriticalities. This paper presents a review of analyses with the SIMMER-II computer program of the effectiveness of possible fuel escape paths. Where possible, how SIMMER-II compares with or is validated against experiments that simulated the escape paths also is discussed

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

Science.gov (United States)

Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

2010-01-01

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

International Nuclear Information System (INIS)

Nishida, C.; Reinhard, P.; Linn, S.

1988-01-01

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

Adaptive control method for core power control in TRIGA Mark II reactor

Science.gov (United States)

Sabri Minhat, Mohd; Selamat, Hazlina; Subha, Nurul Adilla Mohd

2018-01-01

The 1MWth Reactor TRIGA PUSPATI (RTP) Mark II type has undergone more than 35 years of operation. The existing core power control uses feedback control algorithm (FCA). It is challenging to keep the core power stable at the desired value within acceptable error bands to meet the safety demand of RTP due to the sensitivity of nuclear research reactor operation. Currently, the system is not satisfied with power tracking performance and can be improved. Therefore, a new design core power control is very important to improve the current performance in tracking and regulate reactor power by control the movement of control rods. In this paper, the adaptive controller and focus on Model Reference Adaptive Control (MRAC) and Self-Tuning Control (STC) were applied to the control of the core power. The model for core power control was based on mathematical models of the reactor core, adaptive controller model, and control rods selection programming. The mathematical models of the reactor core were based on point kinetics model, thermal hydraulic models, and reactivity models. The adaptive control model was presented using Lyapunov method to ensure stable close loop system and STC Generalised Minimum Variance (GMV) Controller was not necessary to know the exact plant transfer function in designing the core power control. The performance between proposed adaptive control and FCA will be compared via computer simulation and analysed the simulation results manifest the effectiveness and the good performance of the proposed control method for core power control.

Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle

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Gabriele Büchel

2017-12-01

Full Text Available MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II. To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

Dendritic chelating agents. 1. Cu(II) binding to ethylene diamine core poly (amidoamine) denderimers in aqueous solutions

International Nuclear Information System (INIS)

Diallo, Mamadou S.; Christie, Simone; Swaminathan, Pirabalini; Balogh, Lajos; Shi, XIANGYANG; Um, Wooyong; Papelis, Charalambos; Goddard, William A.; Johnson, J. H.

2004-01-01

The overall results of the proton and metal ion binding measurements suggest that the uptake of Cu(II) by EDA core PAMAM dendrimers involves both the dendrimer tertiary amine and terminal groups. However, the extents of protonation of these groups control the ability of the dentrimers to bind Cu(II). Analysis of the EXAFS spectra suggests that Cu(II) forms octahedral complexes involving the tertiary amine groups of Gx-NH2 EDA core PAMAM dendrimers at pH 7.0. The central Cu(II) metal ion of each of these complexes appears to be coordinated to 2-4 dendrimer tertiary amine groups located in the equatorial plane and 2 axial water molecules. Finally, we combine the results of our experiments with literature data to formulate and evaluate a phenomenological model of Cu(II) uptake by Gx-NH2 PAMAM dendrimers in aqueous solutions. At low metal ion-dendrimer loadings, the model provides a good fit of the measured extent of binding of Cu(II) in aqueous solutions of G4-NH2 PAMAM dendrimers at pH 7.0

Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography.

Science.gov (United States)

Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc

2016-08-22

Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi β-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

Phylogenetic analysis of partial RNA-polymerase blocks II and III of Rabies virus isolated from the main rabies reservoirs in Brazil.

Science.gov (United States)

Carnieli, Pedro; de Novaes Oliveira, Rafael; de Oliveira Fahl, Willian; de Carvalho Ruthner Batista, Helena Beatriz; Scheffer, Karin Corrêa; Iamamoto, Keila; Castilho, Juliana Galera

2012-08-01

This study describes the results of the sequencing and analysis of segments of Blocks II and III of the RNA polymerase L gene of Rabies virus isolates from different reservoir species of Brazil. The phylogenetic relations of the virus were determined and a variety of species-specific nucleotides were found in the analyzed areas, but the majority of these mutations were found to be synonymous. However, an analysis of the putative amino acid sequences were shown to have some characteristic mutations between some reservoir species of Brazil, indicating that there was positive selection in the RNA polymerase L gene of Rabies virus. On comparing the putative viral sequences obtained from the Brazilian isolates and other Lyssavirus, it was determined that amino acid mutations occurred in low-restriction areas. This study of the L gene of Rabies virus is the first to be conducted with samples of virus isolates from Brazil, and the results obtained will help in the determination of the phylogenetic relations of the virus.

Endoplasmic reticulum stress-responsive transcription factor ATF6α directs recruitment of the Mediator of RNA polymerase II transcription and multiple histone acetyltransferase complexes.

Science.gov (United States)

Sela, Dotan; Chen, Lu; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan Weliky; Conaway, Ronald C

2012-06-29

The basic leucine zipper transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. Previous studies have established that, in response to ER stress, ATF6α translocates to the nucleus and activates transcription of ER stress response genes upon binding sequence specifically to ER stress response enhancer elements in their promoters. In this study, we investigate the biochemical mechanism by which ATF6α activates transcription. By exploiting a combination of biochemical and multidimensional protein identification technology-based mass spectrometry approaches, we have obtained evidence that ATF6α functions at least in part by recruiting to the ER stress response enhancer elements of ER stress response genes a collection of RNA polymerase II coregulatory complexes, including the Mediator and multiple histone acetyltransferase complexes, among which are the Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes. Our findings shed new light on the mechanism of action of ATF6α, and they outline a straightforward strategy for applying multidimensional protein identification technology mass spectrometry to determine which RNA polymerase II transcription factors and coregulators are recruited to promoters and other regulatory elements to control transcription.

JOYO MK-II core characteristics database. Update to JFS-3-J3.2R

International Nuclear Information System (INIS)

Ohkawachi, Yasushi; Maeda, Shigetaka; Sekine, Takashi

2003-04-01

The 'JOYO' MK-II core characteristics database was compiled and published in 1998. Comments and requests from many users led to the creation of a revised edition in 2001. The revisions include changes to the MAGI calculation code system to use the 70 group JFS-3-J3.2 constant set processed from the JENDL-3.2 library. However, after the database was published, it was recently found that there were errors in the process of making the group constant set JFS-3-J3.2, and it was revised at JFS-3-J3.2R. Then, the group constant set was updated at JFS-3-J3.2R in this database. The MK-II core management data nad core characteristics data were recorded on CD-ROM for user convenience. The structure of the database is the same as in the first edition. The 'Configuration Data' include the core arrangement and refueling record for each operational cycle. The 'Subassembly Library Data' include the atomic number density, neutron fluence, burn-up, integral power of 362 driver fuel subassemblies and 69 irradiation test subassemblies. The 'Output Data' contain the calculated neutron flux, gamma flux, power density, linear heat rate, coolant and fuel temperature distribution of all the fuel subassemblies at the beginning and end of each operational cycle. The 'Core Characteristics Data' include the measured excess reactivity, control rod worth calibration curve, and reactivity coefficients of temperature, power and burn-up. The effect of updating the group constant set, the calculation results of excess reactivity decreased by about 0.15Δk/kk', and the effects to other core characteristics were negligible. (author)

Towards the molecular bases of polymerase dynamics

International Nuclear Information System (INIS)

Chela Flores, J.

1991-03-01

One aspect of the strong relationship that is known to exist between the processes of DNA replication and transcription is manifest in the coupling of the rates of movement of the replication fork (r f ) and RNA polymerase (r t ). We address two issues concerning the largely unexplored area of polymerase dynamics: (i) The validity of an approximate kinematic formula linking r f and r t suggested by experiments in which transcription is initiated in some prokaryotes with the antibiotic streptolydigin, and (ii) What are the molecular bases of the kinematic formula? An analysis of the available data suggests possible molecular bases for polymerase dynamics. In particular, we are led to a hypothesis: In active chromatin r t may depend on the length (λ t ) of the transcript of the primary messenger RNA (pre-mRNA). This new effect is subject to experimental verification. We discuss possible experiments that may be performed in order to test this prediction. (author). Refs, 6 tabs

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

Science.gov (United States)

Pinkney, M; Hoggett, J G

1988-03-15

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

Evaluation report on SCTF Core-II test S2-08

International Nuclear Information System (INIS)

Ohnuki, Akira; Iwamura, Takamichi; Abe, Yutaka; Murao, Yoshio; Adachi, Hiromichi.

1991-01-01

The present report investigates the effects of the difference of the core inlet subcooling during reflood in a PWR-LOCA on the thermal-hydraulic behaviors including two-dimensional behaviors in the pressure vessel in the Slab Core Test Facility (SCTF) Core-II tests under gravity feed mode. The following test results are examined: Tests S2-02 (Reference test) and Test S2-08 (High subcooling test). The degree of the difference of the subcooling between the two tests was about 20 to 35 K in the LPCI period. The following conclusions were obtained from this study: (1) Higher the subcooling gave larger amount of water accumulation in the core and gave better core cooling. These tendencies were also recognized in comparisons under the same distance from the quench front. Since the same tendencies can be predicted in the analyses with REFLA code because of the lower steam generation rate below quench front in the high subcooling test, the differences in the tests are supposed to be caused by the same reason. (2) Higher the subcooling gave larger amount of water accumulation in upper plenum. The carry-over liquid mass into hot leg became smaller in the later period in the higher subcooling test. These differences for carry-over and de-entrainment characteristics can be explained by the differences of quench velocity and of steam mass flow rate generated in the core. (3) No significant influence of the different degree of the subcooling was observed on the two-dimensional thermal-hydraulic behaviors in the pressure vessel. Namely, radial differences of sectional void fraction, heat transfer coefficient and the pressure among bundles at the same elevation were almost the same amount for the two tests. Radial differences of liquid levels in the upper plenum was also almost the same amount for the two tests. (J.P.N.)

Increasing the neutron flux study for the TRR-II core design

International Nuclear Information System (INIS)

Chen, C.-H.; Yang, J.-T.; Chou, Y.-C.

1999-01-01

The maximum unperturbed thermal flux of the originally proposed core design, which is a 6x6 square arrangement with power level of 20 MW and has been presented at the 6th Meeting of IGORR, for the TRR-II reactor is about 2.0x10 14 n/cm 2 -sec. However, it is no longer satisfied the user's requirement, that is, it must reach at least 2.5x10 14 n/cm 2 -sec. In order to enhance the thermal neutron flux, one of the most effective ways is to increase the average power density. Therefore, two new designs with more compact cores are then proposed and studied. One is 5x6 rectangular arrangement with power of 20 MW; the other one is 5x5 square arrangement with power of 16 MW. It is for sure that both core designs can satisfy thermal hydraulic safety limits. The designed parameters related to neutronics are listed and compared fundamentally. According to our calculation, although both cores have similar average power density, the results show that the 5x6/20 MW design has the maximum unperturbed thermal flux in the D 2 O region about 2.7x10 14 n/cm 2 -sec, and the 5x5/16 MW design has 2.5x10 14 n/cm 2 -sec. The maximum thermal flux in the neighborhood of the longer side of the 5x6 core is about 7% higher than the one in the neighborhood of any side of the 5x5 core. This 'long-side effect' gives the 5x6/20 MW core design an advantage of the utilization of the thermal neutron flux in the D 2 O region. In addition, the 5x5 core is also more sensitive to the reactivity change on account of in-core irradiation test facilities. Therefore, under overall considerations the 5x6/20 MW core design is chosen for further detailed design. (author)

Compact sodium cooled nuclear power plant with fast core (KNK II- Karlsruhe), Safety Report

International Nuclear Information System (INIS)

1977-09-01

After the operation of the KNK plant with a thermal core (KNK I), the installation of a fast core (KNK II) had been realized. The planning of the core and the necessary reconstruction work was done by INTERATOM. Owner and customer was the Nuclear Research Center Karlsruhe (KfK), while the operating company was the Kernkraftwerk-Betriebsgesellschaft mbH (KBG) Karlsruhe. The main goals of the KNK II project and its special experimental test program were to gather experience for the construction, the licensing and operation of future larger plants, to develop and to test fuel and absorber assemblies and to further develop the sodium technology and the associated components. The present safety report consists of three parts. Part 1 contains the description of the nuclear plant. Hereby, the reactor and its components, the handling facilities, the instrumentation with the plant protection, the design of the plant including the reactor core and the nominal operation processes are described. Part 2 contains the safety related investigation and measures. This concerns the reactivity accidents, local cooling perturbations, radiological consequences with the surveillance measures and the justification of the choice of structural materials. Part three finally is the appendix with the figures, showing the different buildings, the reactor and its components, the heat transfer systems and the different auxiliary facilities [de

Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

International Nuclear Information System (INIS)

Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

2011-01-01

The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

It's fun to transcribe with Fun30: A model for nucleosome dynamics during RNA polymerase II-mediated elongation.

Science.gov (United States)

Lee, Junwoo; Choi, Eun Shik; Lee, Daeyoup

2018-01-01

The ability of elongating RNA polymerase II (RNAPII) to regulate the nucleosome barrier is poorly understood because we do not know enough about the involved factors and we lack a conceptual framework to model this process. Our group recently identified the conserved Fun30/SMARCAD1 family chromatin-remodeling factor, Fun30 Fft3 , as being critical for relieving the nucleosome barrier during RNAPII-mediated elongation, and proposed a model illustrating how Fun30 Fft3 may contribute to nucleosome disassembly during RNAPII-mediated elongation. Here, we present a model that describes nucleosome dynamics during RNAPII-mediated elongation in mathematical terms and addresses the involvement of Fun30 Fft3 in this process.

Probing dark matter with star clusters: a dark matter core in the ultra-faint dwarf Eridanus II

Science.gov (United States)

Contenta, Filippo; Balbinot, Eduardo; Petts, James A.; Read, Justin I.; Gieles, Mark; Collins, Michelle L. M.; Peñarrubia, Jorge; Delorme, Maxime; Gualandris, Alessia

2018-05-01

We present a new technique to probe the central dark matter (DM) density profile of galaxies that harnesses both the survival and observed properties of star clusters. As a first application, we apply our method to the `ultra-faint' dwarf Eridanus II (Eri II) that has a lone star cluster ˜45 pc from its centre. Using a grid of collisional N-body simulations, incorporating the effects of stellar evolution, external tides and dynamical friction, we show that a DM core for Eri II naturally reproduces the size and the projected position of its star cluster. By contrast, a dense cusped galaxy requires the cluster to lie implausibly far from the centre of Eri II (>1 kpc), with a high inclination orbit that must be observed at a particular orbital phase. Our results, therefore, favour a DM core. This implies that either a cold DM cusp was `heated up' at the centre of Eri II by bursty star formation or we are seeing an evidence for physics beyond cold DM.

The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

Science.gov (United States)

Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

2014-01-01

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced.

An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

Science.gov (United States)

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of

In-core flow rate distribution measurement test of the JOYO irradiation core

International Nuclear Information System (INIS)

Suzuki, Toshihiro; Isozaki, Kazunori; Suzuki, Soju

1996-01-01

A flow rate distribution measurement test was carried out for the JOYO irradiation core (the MK-II core) after the 29th duty cycle operation. The main object of the test is to confirm the proper flow rate distribution at the final phase of the MK-II core. The each flow rate at the outlet of subassemblies was measured by the permanent magnetic flowmeter inserted avail of fuel exchange hole in the rotating plug. This is third test in the MK-II core, after 10 years absence from the final test (1985). Total of 550 subassemblies were exchanged and accumulated reactor operation time reached up to 38,000 hours from the previous test. As a conclusion, it confirmed that the flow rate distribution has been kept suitable in the final phase of the MK-II core. (author)

An evaluation of multigroup flux predictions in the EBR-II core

International Nuclear Information System (INIS)

Hill, R.N.; Fanning, T.H.; Finck, P.J.

1991-01-01

The unique physics characteristics of EBR-II which are difficult to model with conventional neutronic methodologies are identified; the high neutron leakage fraction and importance of neutron reflection cause errors when conventional calculational approximations are utilized. In this paper, various conventional and higher-order group constant evaluations and flux computation methods are compared for a simplified R-Z model of the EBR-II system. Although conventional methods do provide adequate predictions of the flux in the core region, significant mispredictions are observed in the reflector and radial blanket regions. Calculational comparisons indicate that a fine energy group structure is required for accurate predictions of the eigenvalue and flux distribution; greater detail is needed in the iron resonance scattering treatment. Calculational comparisons also indicate that transport theory with detailed anisotropic scattering treatment is required

An evaluation of multigroup flux predictions in the EBR-II core

Energy Technology Data Exchange (ETDEWEB)

Hill, R.N.; Fanning, T.H.; Finck, P.J.

1991-12-31

The unique physics characteristics of EBR-II which are difficult to model with conventional neutronic methodologies are identified; the high neutron leakage fraction and importance of neutron reflection cause errors when conventional calculational approximations are utilized. In this paper, various conventional and higher-order group constant evaluations and flux computation methods are compared for a simplified R-Z model of the EBR-II system. Although conventional methods do provide adequate predictions of the flux in the core region, significant mispredictions are observed in the reflector and radial blanket regions. Calculational comparisons indicate that a fine energy group structure is required for accurate predictions of the eigenvalue and flux distribution; greater detail is needed in the iron resonance scattering treatment. Calculational comparisons also indicate that transport theory with detailed anisotropic scattering treatment is required.

An evaluation of multigroup flux predictions in the EBR-II core

Energy Technology Data Exchange (ETDEWEB)

Hill, R.N.; Fanning, T.H.; Finck, P.J.

1991-01-01

The unique physics characteristics of EBR-II which are difficult to model with conventional neutronic methodologies are identified; the high neutron leakage fraction and importance of neutron reflection cause errors when conventional calculational approximations are utilized. In this paper, various conventional and higher-order group constant evaluations and flux computation methods are compared for a simplified R-Z model of the EBR-II system. Although conventional methods do provide adequate predictions of the flux in the core region, significant mispredictions are observed in the reflector and radial blanket regions. Calculational comparisons indicate that a fine energy group structure is required for accurate predictions of the eigenvalue and flux distribution; greater detail is needed in the iron resonance scattering treatment. Calculational comparisons also indicate that transport theory with detailed anisotropic scattering treatment is required.

Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.

Directory of Open Access Journals (Sweden)

Gwendal Le Martelot

Full Text Available Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.

Calculation analysis of TRIGA MARK II reactor core composed of two types of fuel elements

International Nuclear Information System (INIS)

Ravnik, M.

1988-11-01

The most important properties of mixed cores are treated for TRIGA MARK II reactor, composed of standard (20% enriched, 8.5w% U content) and FLIP (70% enriched, 8.5w% U content) fuel elements. Large difference in enrichment and presence of burnable poison in FLIP fuel have strong influence on the main core characteristics, such as: fuel temperature coefficient, power defect, Xe and Sm worth, power and flux distributions, etc. They are significantly different for both types of fuel. Optimal loading of mixed cores therefore strongly depends on the loading pattern of both types of fuel elements. Results of systematic calculational analysis of mixed cores are presented. Calculations on the level of fuel element are performed with WIMSD-4 computer code with extended cross-section library. Core calculations are performed with TRIGAP two-group 1-D diffusion code. Results are compared to measurements and physical explanation is provided. Special concern is devoted to realistic mixed cores, for which optimal in-core fuel management is derived. Refs, figs and tabs

Electron internal transport barrier in the core of TJ-II ECH plasmas

Energy Technology Data Exchange (ETDEWEB)

Estrada, T.; Hidalgo, C. [Laboratorio Nacional de Fusion por Confinamiento Magnetico. Asociacion EURATOM CIEMAT, Madrid (Spain); Dreval, N. [and others

2003-07-01

The influence of the magnetic topology on the formation of electron internal transport barriers (e-ITB) has been experimentally studied in the stellarator TJ-II. The formation of e-ITBs in electron cyclotron heated plasmas can be triggered by positioning a low order rational surface close to the plasma core region, while in configurations without any low order rational there are no indications of barrier formation within the available heating power. The e-ITB formation is characterized by an increase in the core electron temperature and plasma potential. Positive radial electric field increases in a factor of three in the plasma central region when the e-ITB forms. The results demonstrate that low order rational surfaces modify radial electric fields and electron heat transport. (orig.)

FINDING THE FIRST COSMIC EXPLOSIONS. II. CORE-COLLAPSE SUPERNOVAE

International Nuclear Information System (INIS)

Whalen, Daniel J.; Joggerst, Candace C.; Fryer, Chris L.; Stiavelli, Massimo; Heger, Alexander; Holz, Daniel E.

2013-01-01

Understanding the properties of Population III (Pop III) stars is prerequisite to elucidating the nature of primeval galaxies, the chemical enrichment and reionization of the early intergalactic medium, and the origin of supermassive black holes. While the primordial initial mass function (IMF) remains unknown, recent evidence from numerical simulations and stellar archaeology suggests that some Pop III stars may have had lower masses than previously thought, 15-50 M ☉ in addition to 50-500 M ☉ . The detection of Pop III supernovae (SNe) by JWST, WFIRST, or the TMT could directly probe the primordial IMF for the first time. We present numerical simulations of 15-40 M ☉ Pop III core-collapse SNe performed with the Los Alamos radiation hydrodynamics code RAGE. We find that they will be visible in the earliest galaxies out to z ∼ 10-15, tracing their star formation rates and in some cases revealing their positions on the sky. Since the central engines of Pop III and solar-metallicity core-collapse SNe are quite similar, future detection of any Type II SNe by next-generation NIR instruments will in general be limited to this epoch.

Stability Analysis of the EBR-I Mark-II Core Meltdown Accident

Energy Technology Data Exchange (ETDEWEB)

Lim, Jae-Yong; Kang, Chang Mu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2016-10-15

The purpose of this paper is to analyze the stability of the EBR-I core meltdown accident using the NuSTAB code. The result of NuSTAB analysis is compared with previous stability analysis by Sandmeier using the root locus method. The Experimental Breeder Reactor I (EBR-1) at Argonne National Laboratory was designed to demonstrate fast reactor breeding and to prove the use of liquid-metal coolant for power production and reached criticality in August 1951. The EBR-I reactor was undergoing a series of physics experiments and the Mark-II core was melted accidentally on Nov. 29, 1955. The experiment was going to increase core temperature to 500C to see if the reactor loses reactivity, and scram when the power reached 1500 kW or doubling of fission rate per second. However the operator scrammed with a slow moving control and missed the shutdown by two seconds and caused the core meltdown. The NuSTAB code has an advantage of analyzing space-dependent fast reactors and predicting regional oscillations compared to the point kinetics. Also, NuSTAB can be useful when the coupled neutronic-thermal-hydraulic codes cannot be used for stability analysis. Future work includes analyses of the PGSFR for various operating conditions as well as further validation of the NuSTAB calculations against SFR stability experiments when such experiments become available.

Genome-wide association of mediator and RNA polymerase II in wild-type and mediator mutant yeast.

Science.gov (United States)

Paul, Emily; Zhu, Z Iris; Landsman, David; Morse, Randall H

2015-01-01

Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstrating the modular nature of the Mediator complex in vivo. In addition, our data indicate that promoters exhibiting strong and stable occupancy by Mediator have a wide range of activity and are enriched for targets of the Tup1-Cyc8 repressor complex. We also identify a number of strong Mediator occupancy peaks that overlap dubious open reading frames (ORFs) and are likely to include previously unrecognized upstream activator sequences. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1976-01-01

Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation. In cells possessing all three DNA polymerases, both a greater amount of repair synthesis (''exaggerated'' repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited. In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated. In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5' → 3' exonuclease activity, exaggerated repair synthesis is minimal. After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis, but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not. These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining. DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action

Core map generation for the ITU TRIGA Mark II research reactor using Genetic Algorithm coupled with Monte Carlo method

Energy Technology Data Exchange (ETDEWEB)

Türkmen, Mehmet, E-mail: tm@hacettepe.edu.tr [Nuclear Engineering Department, Hacettepe University, Beytepe Campus, Ankara (Turkey); Çolak, Üner [Energy Institute, Istanbul Technical University, Ayazağa Campus, Maslak, Istanbul (Turkey); Ergün, Şule [Nuclear Engineering Department, Hacettepe University, Beytepe Campus, Ankara (Turkey)

2015-12-15

Highlights: • Optimum core maps were generated for the ITU TRIGA Mark II Research Reactor. • Calculations were performed using a Monte Carlo based reactor physics code, MCNP. • Single-Objective and Multi-Objective Genetic Algorithms were used for the optimization. • k{sub eff} and ppf{sub max} were considered as the optimization objectives. • The generated core maps were compared with the fresh core map. - Abstract: The main purpose of this study is to present the results of Core Map (CM) generation calculations for the İstanbul Technical University TRIGA Mark II Research Reactor by using Genetic Algorithms (GA) coupled with a Monte Carlo (MC) based-particle transport code. Optimization problems under consideration are: (i) maximization of the core excess reactivity (ρ{sub ex}) using Single-Objective GA when the burned fuel elements with no fresh fuel elements are used, (ii) maximization of the ρ{sub ex} and minimization of maximum power peaking factor (ppf{sub max}) using Multi-Objective GA when the burned fuels with fresh fuels are used. The results were obtained when all the control rods are fully withdrawn. ρ{sub ex} and ppf{sub max} values of the produced best CMs were provided. Core-averaged neutron spectrum, and variation of neutron fluxes with respect to radial distance were presented for the best CMs. The results show that it is possible to find an optimum CM with an excess reactivity of 1.17 when the burned fuels are used. In the case of a mix of burned fuels and fresh fuels, the best pattern has an excess reactivity of 1.19 with a maximum peaking factor of 1.4843. In addition, when compared with the fresh CM, the thermal fluxes of the generated CMs decrease by about 2% while change in the fast fluxes is about 1%.Classification: J. Core physics.

Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli.

Science.gov (United States)

Corzett, Christopher H; Goodman, Myron F; Finkel, Steven E

2013-06-01

Escherichia coli DNA polymerases (Pol) II, IV, and V serve dual roles by facilitating efficient translesion DNA synthesis while simultaneously introducing genetic variation that can promote adaptive evolution. Here we show that these alternative polymerases are induced as cells transition from exponential to long-term stationary-phase growth in the absence of induction of the SOS regulon by external agents that damage DNA. By monitoring the relative fitness of isogenic mutant strains expressing only one alternative polymerase over time, spanning hours to weeks, we establish distinct growth phase-dependent hierarchies of polymerase mutant strain competitiveness. Pol II confers a significant physiological advantage by facilitating efficient replication and creating genetic diversity during periods of rapid growth. Pol IV and Pol V make the largest contributions to evolutionary fitness during long-term stationary phase. Consistent with their roles providing both a physiological and an adaptive advantage during stationary phase, the expression patterns of all three SOS polymerases change during the transition from log phase to long-term stationary phase. Compared to the alternative polymerases, Pol III transcription dominates during mid-exponential phase; however, its abundance decreases to SOS induction by exogenous agents and indicate that cell populations require appropriate expression of all three alternative DNA polymerases during exponential, stationary, and long-term stationary phases to attain optimal fitness and undergo adaptive evolution.

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Science.gov (United States)

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Directory of Open Access Journals (Sweden)

Tom eKillelea

2014-05-01

Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit.

Science.gov (United States)

Cevher, Murat A; Shi, Yi; Li, Dan; Chait, Brian T; Malik, Sohail; Roeder, Robert G

2014-12-01

The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.

NSGA-II Algorithm with a Local Search Strategy for Multiobjective Optimal Design of Dry-Type Air-Core Reactor

Directory of Open Access Journals (Sweden)

Chengfen Zhang

2015-01-01

Full Text Available Dry-type air-core reactor is now widely applied in electrical power distribution systems, for which the optimization design is a crucial issue. In the optimization design problem of dry-type air-core reactor, the objectives of minimizing the production cost and minimizing the operation cost are both important. In this paper, a multiobjective optimal model is established considering simultaneously the two objectives of minimizing the production cost and minimizing the operation cost. To solve the multi-objective optimization problem, a memetic evolutionary algorithm is proposed, which combines elitist nondominated sorting genetic algorithm version II (NSGA-II with a local search strategy based on the covariance matrix adaptation evolution strategy (CMA-ES. NSGA-II can provide decision maker with flexible choices among the different trade-off solutions, while the local-search strategy, which is applied to nondominated individuals randomly selected from the current population in a given generation and quantity, can accelerate the convergence speed. Furthermore, another modification is that an external archive is set in the proposed algorithm for increasing the evolutionary efficiency. The proposed algorithm is tested on a dry-type air-core reactor made of rectangular cross-section litz-wire. Simulation results show that the proposed algorithm has high efficiency and it converges to a better Pareto front.

Phylogenetic analysis of the core histone doublet and DNA topo II genes of Marseilleviridae: evidence of proto-eukaryotic provenance.

Science.gov (United States)

Erives, Albert J

2017-11-28

While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like "doublets" in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of

The Arabidopsis Mediator Complex Subunits MED16, MED14, and MED2 Regulate Mediator and RNA Polymerase II Recruitment to CBF-Responsive Cold-Regulated Genes[C][W][OPEN

Science.gov (United States)

Hemsley, Piers A.; Hurst, Charlotte H.; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R.; De Cothi, Elizabeth A.; Steele, John F.; Knight, Heather

2014-01-01

The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation–induced freezing tolerance. In addition, these three subunits are required for low temperature–induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

Directory of Open Access Journals (Sweden)

Jiqin Wu

2015-06-01

Full Text Available RNA-dependent RNA polymerases (RdRPs from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

Chlorine-induced assembly of a cationic coordination cage with a μ5-carbonato-bridged Mn(II)24 core.

Science.gov (United States)

Xiong, Ke-Cai; Jiang, Fei-Long; Gai, Yan-Li; Yuan, Da-Qiang; Han, Dong; Ma, Jie; Zhang, Shu-Quan; Hong, Mao-Chun

2012-04-27

Chlorine caged in! The chlorine-induced assembly of six shuttlecock-like tetranuclear Mn(II) building blocks generated in situ based on p-tert-butylthiacalix[4]arene and facial anions gave rise to a novel truncated distorted octahedral cationic coordination cage with a μ(5)-carbonato-bridged Mn(II)(24) core. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generation and comprehensive analysis of an influenza virus polymerase cellular interaction network.

Science.gov (United States)

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent

2011-12-01

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response.

Two emissive-magnetic composite platforms for Hg(II) sensing and removal: The combination of magnetic core, silica molecular sieve and rhodamine chemosensors

Science.gov (United States)

Mao, Hanping; Liu, Zhongshou

2018-01-01

In this paper, a composite sensing platform for Hg(II) optical sensing and removal was designed and reported. A core-shell structure was adopted, using magnetic Fe3O4 nanoparticles as the core, silica molecular sieve MCM-41 as the shell, respectively. Two rhodamine derivatives were synthesized as chemosensor and covalently immobilized into MCM-41 tunnels. Corresponding composite samples were characterized with SEM/TEM images, XRD analysis, IR spectra, thermogravimetry and N2 adsorption/desorption analysis, which confirmed their core-shell structure. Their emission was increased by Hg(II), showing emission turn on effect. High selectivity, linear working curves and recyclability were obtained from these composite samples.

Preliminary investigations of a mixed standard-flip core for a TRIGA Mark II

International Nuclear Information System (INIS)

Ringle, John C.; Johnson, A.G.; Anderson, T.V.

1974-01-01

Several years ago it became apparent that due to our rapidly- increasing use rate, we would need a substantial amount of new fuel by late 1974 or early 1975. After investigations and discussions with GA, we decided that FLIP fuel would best meet our requirements for maximum fuel economy and high peak pulsing power. A proposal was submitted to the AEC for fuel assistance, and late in 1973 we were awarded a grant of $61,875. This will allow us to buy 3 FLIP-fueled-follower control rods, 1 instrumented FLIP fuel element, and 26 standard FLIP elements, giving us then a mixed core of approximately one-third FLIP and two-thirds standard elements. License amendments to accommodate this change are rather straightforward; modifications to the Technical Specifications will be somewhat more involved. The largest revisions which we envision are to our Safety Analysis Report. Although a few reactors have operated with a full FLIP core, and a few others have converted to mixed standard-FLIP cores, none of these has a standard Mark II core configuration. Those who have already converted to a mixed core have data and calculations which may be helpful to us, but the extent to which we can use these remains to be seen. The present status of our investigations into the analysis of a mixed standard-FLIP core will be presented. Any problems in calculational methods, finding appropriate data, modifications to Technical Specifications, etc., will be identified, and suggestions and help in these areas will be welcomed. (author)

Preliminary investigations of a mixed standard-flip core for a TRIGA Mark II

Energy Technology Data Exchange (ETDEWEB)

Ringle, John C; Johnson, A G; Anderson, T V [Oregon State University (United States)

1974-07-01

Several years ago it became apparent that due to our rapidly- increasing use rate, we would need a substantial amount of new fuel by late 1974 or early 1975. After investigations and discussions with GA, we decided that FLIP fuel would best meet our requirements for maximum fuel economy and high peak pulsing power. A proposal was submitted to the AEC for fuel assistance, and late in 1973 we were awarded a grant of $61,875. This will allow us to buy 3 FLIP-fueled-follower control rods, 1 instrumented FLIP fuel element, and 26 standard FLIP elements, giving us then a mixed core of approximately one-third FLIP and two-thirds standard elements. License amendments to accommodate this change are rather straightforward; modifications to the Technical Specifications will be somewhat more involved. The largest revisions which we envision are to our Safety Analysis Report. Although a few reactors have operated with a full FLIP core, and a few others have converted to mixed standard-FLIP cores, none of these has a standard Mark II core configuration. Those who have already converted to a mixed core have data and calculations which may be helpful to us, but the extent to which we can use these remains to be seen. The present status of our investigations into the analysis of a mixed standard-FLIP core will be presented. Any problems in calculational methods, finding appropriate data, modifications to Technical Specifications, etc., will be identified, and suggestions and help in these areas will be welcomed. (author)

Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB.

Science.gov (United States)

Magill, C P; Jackson, S P; Bell, S D

2001-12-14

Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro. These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB. Thus, the archaeal and eucaryal transcription machineries are fundamentally related. In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter. However the subunit(s) directly contacted by these factors has not been identified. Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK. Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

Science.gov (United States)

Bastian, Thomas W; Rice, Stephen A

2009-01-01

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

Reconstitution of active human core Mediator complex reveals a pivotal role of the MED14 subunit

Science.gov (United States)

Cevher, Murat A.; Shi, Yi; Li, Dan; Chait, Brian T.; Malik, Sohail; Roeder, Robert G.

2014-01-01

The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here, we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to mass spectrometry (CX-MS). Whereas the reconstituted head and middle modules can stably associate, only with incorporation of MED14 into the bi-modular complex does it acquire basal and coactivator functions. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematically dissecting the multiple layers of functionalities associated with the Mediator complex. PMID:25383669

Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop

International Nuclear Information System (INIS)

Kolonko, Nadine; Bannach, Oliver; Aschermann, Katja; Hu, Kang-Hong; Moors, Michaela; Schmitz, Michael; Steger, Gerhard; Riesner, Detlev

2006-01-01

Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta

Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family.

Science.gov (United States)

Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S

1998-02-01

RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast*

Science.gov (United States)

Ansari, Suraiya A.; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z.; Rode, Kara A.; Barber, Wesley T.; Ellis, Laura C.; LaPorta, Erika; Orzechowski, Amanda M.; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H.

2014-01-01

Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. PMID:24727477

Efficient simultaneous removal of U(VI) and Cu(II) from aqueous solution using core-shell nZVI@SA/CMC-Ca beads

International Nuclear Information System (INIS)

Shuhong Hu; Xiaoyan Lin; Wenhui Zhao; Ministry of Education, Sichuan; Xuegang Luo

2018-01-01

Core-shell nanoscale zero-valent iron@alginate/carboxymethyl cellulose sodium composite loaded with calcium (nZVI@SA/CMC-Ca) beads were synthesized in this study using coaxial electronic injection method. The adsorbent structure was characterized via FT-IR, SEM, EDX and XPS. The adsorption behavior of U(VI) and Cu(II) on core-shell nZVI@SA/CMC-Ca beads was studied under various experimental parameters like pH, contact time and temperature. The isotherm and the kinetic data, pertaining to the adsorption of U(VI) and Cu(II) by core-shell nZVI@SA/CMC-Ca beads obeyed both the Langmuir and Freundlich isotherms model and the pseudo-second-order kinetics model, respectively. The thermodynamic parameters revealed the spontaneous and endothermic nature of the adsorption. The experiment of regeneration and reusability suggested core-shell nZVI@SA/CMC-Ca bead was a regenerated material. (author)

The SOS and RpoS Regulons Contribute to Bacterial Cell Robustness to Genotoxic Stress by Synergistically Regulating DNA Polymerase Pol II.

Science.gov (United States)

Dapa, Tanja; Fleurier, Sébastien; Bredeche, Marie-Florence; Matic, Ivan

2017-07-01

Mitomycin C (MMC) is a genotoxic agent that induces DNA cross-links, DNA alkylation, and the production of reactive oxygen species (ROS). MMC induces the SOS response and RpoS regulons in Escherichia coli SOS-encoded functions are required for DNA repair, whereas the RpoS regulon is typically induced by metabolic stresses that slow growth. Thus, induction of the RpoS regulon by MMC may be coincidental, because DNA damage slows growth; alternatively, the RpoS regulon may be an adaptive response contributing to cell survival. In this study, we show that the RpoS regulon is primarily induced by MMC-induced ROS production. We also show that RpoS regulon induction is required for the survival of MMC-treated growing cells. The major contributor to RpoS-dependent resistance to MMC treatment is DNA polymerase Pol II, which is encoded by the polB gene belonging to the SOS regulon. The observation that polB gene expression is controlled by the two major stress response regulons that are required to maximize survival and fitness further emphasizes the key role of this DNA polymerase as an important factor in genome stability. Copyright © 2017 by the Genetics Society of America.

Reference core design Mark-I and -II of the experimental, multi-purpose, high-temperature, gas-cooled reactor

International Nuclear Information System (INIS)

Shindo, Ryuiti; Hirano, Mitsumasa; Aruga, Takeo; Yasukawa, Sigeru

1977-10-01

Reactivity worth of the control rods and power distribution in the initial hot-clean core of reference core design Mark-I and -II have been studied. The need for burnable poison was confirmed, because of the limitations in number, diameter and reactivity worth of the control rods due to structures of pressure vessel and fuel element and to safety of the core. While the initial excess reactivity is reduced by use of the burnable poison, the recovery of core reactivity with burnup of the burnable poison requires a complicated withdrawal sequence of the control rods. The radial power gradient in the core is not large, due to orifice control of the coolant helium flow, effectiveness of the reflector in the small core and continuous distribution of burnup in the core by one-batch refuelling scheme. The local peaking factor in unit orifice regions, therefore, is the most important core design. Control of the axial power distribution is necessary to reduce the maximum fuel temperature and the exponential power distribution peaked toward the inlet of the core is most suitable. However, insertion of the control rods from top of the core disturbs the axial power distribution, so this effect must be considered in design of the withdrawal sequence of control rods. Nuclear properties of the core were revealed from results of the study for the initial hot-clean core. (auth.)

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Science.gov (United States)

Cline, J; Braman, J C; Hogrefe, H H

1996-09-15

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

International Nuclear Information System (INIS)

Vázquez-Iglesias, Lorena; Lostalé-Seijo, Irene; Martínez-Costas, José; Benavente, Javier

2012-01-01

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

Energy Technology Data Exchange (ETDEWEB)

Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

2012-10-25

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

PWR Core II blanket fuel disposition recommendation of storage option study

International Nuclear Information System (INIS)

Dana, C.M.

1995-01-01

After review of the options available for current storage of T Plant Fuel the recommended option is wet storage without the use of chillers. A test has been completed that verifies the maximum temperature reached is below the industrial standard for storage of spent fuel. This option will be the least costly and still maintain the fuel in a safe environment. The options that were evaluated included dry storage with and without chillers, and wet storage with and without chillers. Due to the low decay heat of the Shippingport Core II Blanket fuel assemblies the fuel pool temperature will not exceed 100 deg. F

Heysham II/Torness power stations: Seismic qualification of core structures and boilers

International Nuclear Information System (INIS)

Shepherd, D.J.

1990-01-01

For the advanced gas cooled reactors at Heysham II and Torness the seismic qualification of the core and support structures and boilers posed special problems. In each case the response was highly non-linear due to impacting. Within the core itself there are many thousands of degrees of freedom each dominated by impacting during the seismic event and these impact forces are transmitted to the support structure. The boilers, although supported and located in the design case by linear systems, have their motion during the seismic event controlled by seismic restraints and other components which introduce substantial impacting during seismic excitation. For both these important components a substantial programme of testing was carried out to validate an analysis approach. This testing and correlation with analysis is described in detail for both components. In the case of the core the qualification was based upon a non-linear code AGRCORE which was specifically developed to handle the large number of impact degrees of freedom for this component. The implementation of this code is also described together with a brief summary of results. The boiler analysis was ultimately carried out using conventional finite difference codes and the implementation of these together with a summary of results is also presented. (author). 13 figs, 1 tab

Interfacial charge separation and recombination in InP and quasi-type II InP/CdS core/shell quantum dot-molecular acceptor complexes.

Science.gov (United States)

Wu, Kaifeng; Song, Nianhui; Liu, Zheng; Zhu, Haiming; Rodríguez-Córdoba, William; Lian, Tianquan

2013-08-15

Recent studies of group II-VI colloidal semiconductor heterostuctures, such as CdSe/CdS core/shell quantum dots (QDs) or dot-in-rod nanorods, show that type II and quasi-type II band alignment can facilitate electron transfer and slow down charge recombination in QD-molecular electron acceptor complexes. To explore the general applicability of this wave function engineering approach for controlling charge transfer properties, we investigate exciton relaxation and dissociation dynamics in InP (a group III-V semiconductor) and InP/CdS core/shell (a heterostructure beween group III-V and II-VI semiconductors) QDs by transient absorption spectroscopy. We show that InP/CdS QDs exhibit a quasi-type II band alignment with the 1S electron delocalized throughout the core and shell and the 1S hole confined in the InP core. In InP-methylviologen (MV(2+)) complexes, excitons in the QD can be dissociated by ultrafast electron transfer to MV(2+) from the 1S electron level (with an average time constant of 11.4 ps) as well as 1P and higher electron levels (with a time constant of 0.39 ps), which is followed by charge recombination to regenerate the complex in its ground state (with an average time constant of 47.1 ns). In comparison, InP/CdS-MV(2+) complexes show similar ultrafast charge separation and 5-fold slower charge recombination rates, consistent with the quasi-type II band alignment in these heterostructures. This result demonstrates that wave function engineering in nanoheterostructures of group III-V and II-VI semiconductors provides a promising approach for optimizing their light harvesting and charge separation for solar energy conversion applications.

Composition and strain effects in Type I and Type II heterostructure ZnSe/Cd(Zn)S and ZnSe/Cd1-xZnxS core/shell quantum dots

Science.gov (United States)

Gheshlaghi, Negar; Pisheh, Hadi Sedaghat; Ünlü, Hilmi

2017-11-01

We investigated the effect of ternary shell alloy composition on the bandgap and diameter of core of ZnSe / Cd1 - x Znx S heterostructure core/shell quantum dots, which were synthesized by using a simple colloidal technique. Characterization by using the x-ray diffraction (XRD), transmission electron microscopy (TEM), UV-Vis absorption and fluorescence emission spectroscopic techniques indicate that (i) there is a transition of ZnSe / Cd0.6 Zn0.4 S Type-I heterostructure (electrons and holes tend to localize in core) to ZnSe / Cd0.75 Zn0.25 S quasi-Type-II heterostructures (holes tend to localized in the core and electrons are delocalized) and (ii) then after large red shift and Stock-shift in PL emission spectra but not a distinct absorption peak in UV spectra become noticeable in ZnSe/Cd0.75Zn0.25 S quasi-Type II and ZnSe/CdS Type II heterostructures (electrons are localized in core and holes are localized in shell). Furthermore, the increase of Cd:S ratio in shell alloy composition shifts the XRD peaks to lower 2θ degrees which corresponds to tensile strain in the ZnSe core. Finally, the hydrostatic interfacial strain has effect on the squeezing or stretching the capped core: A decrease of compressive force on core from ZnSe/ZnS to tensile force in ZnSe/CdS with increase in Cd:S ratio indicates that transition of compressive strain to tensile strain takes place with the transition from Type-I to II heterostructure.

redD and actII-ORF4, Pathway-Specific Regulatory Genes for Antibiotic Production in Streptomyces coelicolor A3(2), Are Transcribed In Vitro by an RNA Polymerase Holoenzyme Containing σhrdD

NARCIS (Netherlands)

Fujii, T.; Gramajo, H.C.; Takano, E.; Bibb, M.J.

1996-01-01

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing σhrdD. Disruption of hrdD had no effect on antibiotic production, indicating that

Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

Science.gov (United States)

Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

2005-03-01

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

Directory of Open Access Journals (Sweden)

Shrividhya Srinivasan

2008-10-01

Full Text Available Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II. Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene

International Nuclear Information System (INIS)

Heilbronn, T.; Jahn, G.; Buerkle, A.; Freese, U.K.; Fleckenstein, B.; Zur Hausen, H.

1987-01-01

The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSF-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at T/sub m/ - 25/degrees/C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Esptein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein

Multireversible redox processes in pentanuclear bis(triple-helical) manganese complexes featuring an oxo-centered triangular {Mn(II)2Mn(III)(μ3-O)}5+ or {Mn(II)Mn(III)2(μ3-O)}6+ core wrapped by two {Mn(II)2(bpp)3}-.

Science.gov (United States)

Romain, Sophie; Rich, Jordi; Sens, Cristina; Stoll, Thibaut; Benet-Buchholz, Jordi; Llobet, Antoni; Rodriguez, Montserrat; Romero, Isabel; Clérac, Rodolphe; Mathonière, Corine; Duboc, Carole; Deronzier, Alain; Collomb, Marie-Noëlle

2011-09-05

A new pentanuclear bis(triple-helical) manganese complex has been isolated and characterized by X-ray diffraction in two oxidation states: [{Mn(II)(μ-bpp)(3)}(2)Mn(II)(2)Mn(III)(μ-O)](3+) (1(3+)) and [{Mn(II)(μ-bpp)(3)}(2)Mn(II)Mn(III)(2)(μ-O)](4+) (1(4+)). The structure consists of a central {Mn(3)(μ(3)-O)} core of Mn(II)(2)Mn(III) (1(3+)) or Mn(II)Mn(III)(2) ions (1(4+)) which is connected to two apical Mn(II) ions through six bpp(-) ligands. Both cations have a triple-stranded helicate configuration, and a pair of enantiomers is present in each crystal. The redox properties of 1(3+) have been investigated in CH(3)CN. A series of five distinct and reversible one-electron waves is observed in the -1.0 and +1.50 V potential range, assigned to the Mn(II)(4)Mn(III)/Mn(II)(5), Mn(II)(3)Mn(III)(2)/Mn(II)(4)Mn(III), Mn(II)(2)Mn(III)(3)/Mn(II)(3)Mn(III)(2), Mn(II)Mn(III)(4)/Mn(II)(2)Mn(III)(3), and Mn(III)(5)/Mn(II)Mn(III)(4) redox couples. The two first oxidation processes leading to Mn(II)(3)Mn(III)(2) (1(4+)) and Mn(II)(2)Mn(III)(3) (1(5+)) are related to the oxidation of the Mn(II) ions of the central core and the two higher oxidation waves, close in potential, are thus assigned to the oxidation of the two apical Mn(II) ions. The 1(4+) and 1(5+) oxidized species and the reduced Mn(4)(II) (1(2+)) species are quantitatively generated by bulk electrolyses demonstrating the high stability of the pentanuclear structure in four oxidation states (1(2+) to 1(5+)). The spectroscopic characteristics (X-band electron paramagnetic resonance, EPR, and UV-visible) of these species are also described as well as the magnetic properties of 1(3+) and 1(4+) in solid state. The powder X- and Q-band EPR signature of 1(3+) corresponds to an S = 5/2 spin state characterized by a small zero-field splitting parameter (|D| = 0.071 cm(-1)) attributed to the two apical Mn(II) ions. At 40 K, the magnetic behavior is consistent for 1(3+) with two apical S = 5/2 {Mn(II)(bpp)(3)}(-) and one S

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

Directory of Open Access Journals (Sweden)

Elisa Mentegari

2016-08-01

Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

Science.gov (United States)

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

JOYO coolant sodium and cover gas purity control database (MK-II core)

International Nuclear Information System (INIS)

Ito, Kazuhiro; Nemoto, Masaaki

2000-03-01

The experimental fast reactor 'JOYO' served as the MK-II irradiation bed core for testing fuel and material for FBR development for 15 years from 1982 to 1997. During the MK-II operation, impurities concentrations in the sodium and the argon gas were determined by 67 samples of primary sodium, 81 samples of secondary sodium, 75 samples of primary argon gas, 89 samples of secondary argon gas (the overflow tank) and 89 samples of secondary argon gas (the dump tank). The sodium and the argon gas purity control data were accumulated from in thirty-one duty operations, thirteen special test operations and eight annual inspections. These purity control results and related plant data were compiled into database, which were recorded on CD-ROM for user convenience. Purity control data include concentration of oxygen, carbon, hydrogen, nitrogen, chlorine, iron, nickel and chromium in sodium, concentration of oxygen, hydrogen, nitrogen, carbon dioxide, methane and helium in argon gas with the reactor condition. (author)

Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast.

Science.gov (United States)

Ansari, Suraiya A; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z; Rode, Kara A; Barber, Wesley T; Ellis, Laura C; LaPorta, Erika; Orzechowski, Amanda M; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H

2014-05-23

Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a standard data base for FBR core nuclear design. 10. Reevaluation of atomic number density of JOYO Mk-II core

Energy Technology Data Exchange (ETDEWEB)

Numata, Kazuyuki; Sato, Wakaei [Japan Nuclear Cycle Development Inst., Oarai, Ibaraki (Japan). Oarai Engineering Center; Ishikawa, Makoto; Arii, Yoshio [Nuclear Energy System Incorporation, Tokyo (Japan)

1999-07-01

The material composition of JOYO Mk-II core components in its initial core was reevaluated as a part of the effort for developing a standard data base for FBR core nuclear design. The special feature of the reevaluation is to treat the decay of Pu-241 isotope, so that the atomic number densities of Pu-241 and Am-241 in fuel assemblies can be exactly evaluated on the initial critical date, Nov. 22nd, 1982. Further, the atomic number densities of other core components were also evaluated to improve the analytical accuracy. Those include the control rods which were not so strictly evaluated in the past, and the dummy fuels and the neutron sources which were not treated in the analytical model so far. The results of the present reevaluation were as follows: (1) The changes of atomic number densities of the major nuclides such as Pu-239, U-235 and U-238 were about {+-}0.2 to 0.3%. On the other hand, the number density of Pu-241, which was the motivation of the present work, was reduced by 12%. From the fact, the number densities in the past analysis might be based on the isotope measurement of the manufacturing point of time without considering the decay of Pu-241. (2) As the other core components, the number densities of control rods and outer reflector-type A were largely improved. (author)

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

Science.gov (United States)

Taladriz, Soraya; Hanke, Tobias; Ramiro, María J.; García-Díaz, Miguel; Lacoba, Mario García de; Blanco, Luis; Larraga, Vicente

2001-01-01

We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. PMID:11557814

Interaction of gold nanoparticles with Pfu DNA polymerase and effect on polymerase chain reaction.

Science.gov (United States)

Sun, L-P; Wang, S; Zhang, Z-W; Ma, Y-Y; Lai, Y-Q; Weng, J; Zhang, Q-Q

2011-03-01

The interaction of gold nanoparticles with Pfu DNA polymerase has been investigated by a number of biological, optical and electronic spectroscopic techniques. Polymerase chain reaction was performed to show gold nanoparticles' biological effect. Ultraviolet-visible and circular dichroism spectra analysis were applied to character the structure of Pfu DNA polymerase after conjugation with gold nanoparticles. X-ray photoelectron spectroscopy was used to investigate the bond properties of the polymerase-gold nanoparticles complex. The authors demonstrate that gold nanoparticles do not affect the amplification efficiency of polymerase chain reaction using Pfu DNA polymerase, and Pfu DNA polymerase displays no significant changes of the secondary structure upon interaction with gold nanoparticles. The adsorption of Pfu DNA polymerase to gold nanoparticles is mainly through Au-NH(2) bond and electrostatic interaction. These findings may have important implications regarding the safety issue as gold nanoparticles are widely used in biomedical applications.

Evaluation report on CCTF Core-II reflood Test C2-15 (Run 75)

International Nuclear Information System (INIS)

Okubo, Tsutomu; Iguchi, Tadashi; Akimoto, Hajime; Murao, Yoshio

1992-01-01

This report presents an evaluation on the CCTF Core-II Test C2-15 (Run 75). The purpose of the test is to investigate whether the thermo-hydrodynamic behavior is different between the CCTF and the FLECHT-SET reflooding tests. For this purpose, test conditions of the present test were set as close as possible to those of concerned FLECHT-SET 2714B experiment, taking account of differences in facility design. Investigating results of both the tests, the following conclusions are obtained: (1) Some discrepancies were observed in the measured test conditions between the two tests. Out of them, difference in the Acc injection duration was large and affected test results, such as the water accumulation in the downcomer and the core and the core cooling, during the initial period. However, this effect was found to become small with time. (2) Taking account of this difference and the difference in the broken cold leg pressure loss coefficient between the two facilities, the overall reflooding behavior is judged to be similar in the two facilities. (3) The CCTF test results showed the core heat transfer enhancement in the higher power region due to its steep radial power distribution, whereas the FLECHT-SET did not due to its rather flat radial power distribution. This enhancement was observed significantly at 1.83 m but was smaller at the higher elevation. (4) The heat transfer was nearly identical between the two tests and an existing correlation could well predict the heat transfer coefficients of both the tests at the location where the heat transfer enhancement mentioned above (3) were small, during the time period when the effect of the difference in the Acc injection mentioned above (1) were small. (5) Therefore, the core cooling is expected to be almost the same in the CCTF and the FLECHT-SET under the same core boundary conditions and core radial power distribution. (author)

Optimal Implantation Depth and Adherence to Guidelines on Permanent Pacing to Improve the Results of Transcatheter Aortic Valve Replacement With the Medtronic CoreValve System: The CoreValve Prospective, International, Post-Market ADVANCE-II Study.

Science.gov (United States)

Petronio, Anna S; Sinning, Jan-Malte; Van Mieghem, Nicolas; Zucchelli, Giulio; Nickenig, Georg; Bekeredjian, Raffi; Bosmans, Johan; Bedogni, Francesco; Branny, Marian; Stangl, Karl; Kovac, Jan; Schiltgen, Molly; Kraus, Stacia; de Jaegere, Peter

2015-05-01

The aim of the CoreValve prospective, international, post-market ADVANCE-II study was to define the rates of conduction disturbances and permanent pacemaker implantation (PPI) after transcatheter aortic valve replacement with the Medtronic CoreValve System (Minneapolis, Minnesota) using optimized implantation techniques and application of international guidelines on cardiac pacing. Conduction disturbances are a frequent complication of transcatheter aortic valve replacement. The rates of PPI in the published reports vary according to bioprosthesis type and the indications for PPI. The primary endpoint was the 30-day incidence of PPI with Class I/II indications when the Medtronic CoreValve System was implanted at an optimal depth (≤6 mm below the aortic annulus). The timing and resolution of all new-onset conduction disturbances were analyzed. A total of 194 patients were treated. The overall rate of PPI for Class I/II indications was 18.2%. An optimal depth was reached in 43.2% of patients, with a nonsignificantly lower incidence of PPI in patients with depths ≤6 mm, compared with those with deeper implants (13.3% vs. 21.1%; p = 0.14). In a paired analysis, new-onset left bundle branch block and first-degree atrioventricular block occurred in 45.4% and 39.0% of patients, respectively, and resolved spontaneously within 30 days in 43.2% and 73.9%, respectively. In patients with new PPI, the rate of intrinsic sinus rhythm increased from 25.9% at 7 days to 59.3% at 30 days (p = 0.004). Optimal Medtronic CoreValve System deployment and adherence to international guidelines on cardiac pacing are associated with a lower rate of new PPI after transcatheter aortic valve replacement, compared with results reported in previous studies. (CoreValve Advance-II Study: Prospective International Post-Market Study [ADVANCE II]; NCT01624870). Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Generation and Comprehensive Analysis of an Influenza Virus Polymerase Cellular Interaction Network▿†§

Science.gov (United States)

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E.; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent

2011-01-01

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response. PMID:21994455

A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells.

Directory of Open Access Journals (Sweden)

Ayan Banerjee

2009-07-01

Full Text Available Making the correct choice between transcription elongation and transcription termination is essential to the function of RNA polymerase II, and fundamental to gene expression. This choice can be influenced by factors modifying the transcription complex, factors modifying chromatin, or signals mediated by the template or transcript. To aid in the study of transcription elongation and termination we have developed a transcription elongation reporter system that consists of tandem luciferase reporters flanking a test sequence of interest. The ratio of expression from the reporters provides a measure of the relative rates of successful elongation through the intervening sequence.Size matched fragments containing the polyadenylation signal of the human beta-actin gene (ACTB and the human beta-globin gene (HBB were evaluated for transcription termination using this new ratiometric tandem reporter assay. Constructs bearing just 200 base pairs on either side of the consensus poly(A addition site terminated 98% and 86% of transcription for ACTB and HBB sequences, respectively. The nearly 10-fold difference in read-through transcription between the two short poly(A regions was eclipsed when additional downstream poly(A sequence was included for each gene. Both poly(A regions proved very effective at termination when 1100 base pairs were included, stopping 99.6% of transcription. To determine if part of the increased termination was simply due to the increased template length, we inserted several kilobases of heterologous coding sequence downstream of each poly(A region test fragment. Unexpectedly, the additional length reduced the effectiveness of termination of HBB sequences 2-fold and of ACTB sequences 3- to 5-fold.The tandem construct provides a sensitive measure of transcription termination in human cells. Decreased Xrn2 or Senataxin levels produced only a modest release from termination. Our data support overlap in allosteric and torpedo mechanisms

How to switch the motor on: RNA polymerase initiation steps at the single-molecule level

NARCIS (Netherlands)

Marchetti, M.; Malinowska, A.; Heller, I.; Wuite, G. J. L.

RNA polymerase (RNAP) is the central motor of gene expression since it governs the process of transcription. In prokaryotes, this holoenzyme is formed by the RNAP core and a sigma factor. After approaching and binding the specific promoter site on the DNA, the holoenzyme-promoter complex undergoes

Xenon oscillation in a large PHWR core (Atucha II type): TRISIC code applicability

International Nuclear Information System (INIS)

Solanilla, Roberto

2000-01-01

A three dimensional nuclear reactor simulation code (TRISIC) was developed many years ago to design a PHWR (pressurizer heavy water reactors - Atucha type) based in the 'source-sink model' (heterogeneous theory). The limited processor computational performance available at that time was the constraint of the code when a detailed reactor description was necessary. A modern PC (pentium) code version with a full reactor core representation (461 fuel channels) including diagonal control rod banks and flux-reading detectors with theirs tube guide was used in the present paper for simulation of the Xenon transient when a local asymmetric perturbation was produced in a large core (Atucha II type). The results obtained and the computer time required for the 70 hour's simulation with an adequate time step, established the potential of the code to deal with this kind of transients. The paper shows that the method of TRISIC allows to detect and control azimuthal, radial and axial oscillation. This code is a proper way to elaborate a program of control rods movement from the flux reading detectors to damp the oscillation. TRISIC could also be a accurate tool to supervise the full core flux distribution in real time during the operation of the reactor. (author)

A structural role for the PHP domain in E. coli DNA polymerase III.

Science.gov (United States)

Barros, Tiago; Guenther, Joel; Kelch, Brian; Anaya, Jordan; Prabhakar, Arjun; O'Donnell, Mike; Kuriyan, John; Lamers, Meindert H

2013-05-14

In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination.

Science.gov (United States)

Marasco, Michelle; Li, Weiyi; Lynch, Michael; Pikaard, Craig S

2017-11-02

All eukaryotes have three essential nuclear multisubunit RNA polymerases, abbreviated as Pol I, Pol II and Pol III. Plants are remarkable in having two additional multisubunit RNA polymerases, Pol IV and Pol V, which synthesize noncoding RNAs that coordinate RNA-directed DNA methylation for silencing of transposons and a subset of genes. Based on their subunit compositions, Pols IV and V clearly evolved as specialized forms of Pol II, but their catalytic properties remain undefined. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is considerably more error-prone than Pols II or V, which may be tolerable in its synthesis of short RNAs that serve as precursors for siRNAs targeting non-identical members of transposon families. By contrast, Pol V exhibits high fidelity transcription, similar to Pol II, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci to which siRNA-Argonaute silencing complexes are recruited. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transitions to improved core electron heat confinement triggered by low order rational magnetic surfaces in the stellarator TJ-II

International Nuclear Information System (INIS)

Estrada, T.; Medina, F.; Lopez-Bruna, D.; AscasIbar, E.; BalbIn, R.; Cappa, A.; Castejon, F.; Eguilior, S.; Fernandez, A.; Guasp, J.; Hidalgo, C.; Petrov, S.

2007-01-01

Transitions to improved core electron heat confinement are triggered by low order rational magnetic surfaces in TJ-II electron cyclotron heated (ECH) plasmas. Experiments are performed changing the magnetic shear around the rational surface n = 3/m = 2 to study its influence on the transition; ECH power modulation is used to look at transport properties. The improvement in the electron heat confinement shows no obvious dependence on the magnetic shear. Transitions triggered by the rational surface n = 4/m = 2 show, in addition, an increase in the ion temperature synchronized with the increase in the electron temperature. Ion temperature changes had not been previously observed either in TJ-II or in any other helical device. SXR measurements demonstrate that, under certain circumstances, the rational surface positioned inside the plasma core region precedes and provides a trigger for the transition

Genetic and structural study of DNA-directed RNA polymerase II of Trypanosoma brucei, towards the designing of novel antiparasitic agents

Directory of Open Access Journals (Sweden)

Louis Papageorgiou

2017-03-01

Full Text Available Trypanosoma brucei brucei (TBB belongs to the unicellular parasitic protozoa organisms, specifically to the Trypanosoma genus of the Trypanosomatidae class. A variety of different vertebrate species can be infected by TBB, including humans and animals. Under particular conditions, the TBB can be hosted by wild and domestic animals; therefore, an important reservoir of infection always remains available to transmit through tsetse flies. Although the TBB parasite is one of the leading causes of death in the most underdeveloped countries, to date there is neither vaccination available nor any drug against TBB infection. The subunit RPB1 of the TBB DNA-directed RNA polymerase II (DdRpII constitutes an ideal target for the design of novel inhibitors, since it is instrumental role is vital for the parasite’s survival, proliferation, and transmission. A major goal of the described study is to provide insights for novel anti-TBB agents via a state-of-the-art drug discovery approach of the TBB DdRpII RPB1. In an attempt to understand the function and action mechanisms of this parasite enzyme related to its molecular structure, an in-depth evolutionary study has been conducted in parallel to the in silico molecular designing of the 3D enzyme model, based on state-of-the-art comparative modelling and molecular dynamics techniques. Based on the evolutionary studies results nine new invariant, first-time reported, highly conserved regions have been identified within the DdRpII family enzymes. Consequently, those patches have been examined both at the sequence and structural level and have been evaluated in regard to their pharmacological targeting appropriateness. Finally, the pharmacophore elucidation study enabled us to virtually in silico screen hundreds of compounds and evaluate their interaction capabilities with the enzyme. It was found that a series of chlorine-rich set of compounds were the optimal inhibitors for the TBB DdRpII RPB1 enzyme. All

Are There Mutator Polymerases?

Directory of Open Access Journals (Sweden)

Miguel Garcia-Diaz

2003-01-01

Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

Science.gov (United States)

Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

2015-01-01

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

s-core network decomposition: A generalization of k-core analysis to weighted networks

Science.gov (United States)

Eidsaa, Marius; Almaas, Eivind

2013-12-01

A broad range of systems spanning biology, technology, and social phenomena may be represented and analyzed as complex networks. Recent studies of such networks using k-core decomposition have uncovered groups of nodes that play important roles. Here, we present s-core analysis, a generalization of k-core (or k-shell) analysis to complex networks where the links have different strengths or weights. We demonstrate the s-core decomposition approach on two random networks (ER and configuration model with scale-free degree distribution) where the link weights are (i) random, (ii) correlated, and (iii) anticorrelated with the node degrees. Finally, we apply the s-core decomposition approach to the protein-interaction network of the yeast Saccharomyces cerevisiae in the context of two gene-expression experiments: oxidative stress in response to cumene hydroperoxide (CHP), and fermentation stress response (FSR). We find that the innermost s-cores are (i) different from innermost k-cores, (ii) different for the two stress conditions CHP and FSR, and (iii) enriched with proteins whose biological functions give insight into how yeast manages these specific stresses.

Measurements of neutron flux distributions in the core of the Ljubljana TRIGA Mark II Reactor

International Nuclear Information System (INIS)

Rant, J.; Ravnik, M.; Mele, I.; Dimic, V.

2008-01-01

Recently the Ljubljana TRIGA Mark II Reactor has been refurbished and upgraded to pulsed operation. To verify the core design calculations using TRIGAP and PULSTR1 codes and to obtain necessary data for future irradiation and neutron beam experiments, an extensive experimental program of neutron flux mapping and neutron field characterization was carried out. Using the existing neutron measuring thimbles complete axial and radial distributions in two radial directions were determined for two different core configurations. For one core configuration the measurements were also carried out in the pulsed mode. For flux distributions thin Cu (relative measurements) and diluted Au wires (absolute values) were used. For each radial position the cadmium ratio was determined in two axial levels. The core configuration was rather uniform, well defined (fresh fuel of a single type, including fuelled followers) and compact (no irradiation channels or gaps), offering unique opportunity to test the computer codes for TRIGA reactor calculations. The neutron flux measuring procedures and techniques are described and the experimental results are presented. The agreement between the predicted and measured power peaking factors are within the error limits of the measurements (<±5%) and calculations (±10%). Power peaking occurs in the B ring, and in the A ring (centre) there is a significant flux depression. (authors)

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

International Nuclear Information System (INIS)

Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung; Park, Sun; Shin, Ho-Joon; Kim, Kyongmin

2008-01-01

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate 32 P-ribonucleotides, but not HBV core particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems

Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis.

Science.gov (United States)

Smith, Alias J; Chudnovsky, Lorissa; Simoes-Barbosa, Augusto; Delgadillo-Correa, Maria G; Jonsson, Zophonias O; Wohlschlegel, James A; Johnson, Patricia J

2011-04-01

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

Novel Core Promoter Elements and a Cognate Transcription Factor in the Divergent Unicellular Eukaryote Trichomonas vaginalis▿

Science.gov (United States)

Smith, Alias J.; Chudnovsky, Lorissa; Simoes-Barbosa, Augusto; Delgadillo-Correa, Maria G.; Jonsson, Zophonias O.; Wohlschlegel, James A.; Johnson, Patricia J.

2011-01-01

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5′ untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif. PMID:21245378

Transient behaviour in the plasma core of TJ-II stellarator and its relation with rational surfaces

International Nuclear Information System (INIS)

Estrada, T.; Luna, E. de la; Ascasibar, E; Jimenez, J.A.; Castejon, F.; Garcia-Cortes, I.; Lopez-Fraguas, A.; Sanchez, J.; Tribaldos, V.

2002-01-01

A transient behaviour is observed in the plasma core of TJ-II stellarator with fast drops in the electron temperature. Changes in the line-averaged density are observed synchronized with temperature drops. This phenomenon appears in plasmas created and heated using 300 kW of electron cyclotron heating with high power density. The transient behaviour resembles both, the electric pulsation discovered in CHS and the 'electron root' feature reported by the W7-AS team. The flexibility and low magnetic shear of TJ-II have permitted the identification of the plasma current as the control parameter for the appearance of this phenomenon. The results obtained during the magnetic configuration scans carried out in TJ-II points to the hypothesis that the transient behaviour is connected with the presence of a rational surface close to the plasma centre. Equilibrium calculations performed with the VMEC code reinforce this hypothesis. (author)

GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein

DEFF Research Database (Denmark)

Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara

2011-01-01

in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear...... transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin a/ß pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O......-(thio)triphosphate (GTP¿S). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which...

Affinity isolation and I-DIRT mass spectrometric analysis of the Escherichia coli O157:H7 Sakai RNA polymerase complex.

Science.gov (United States)

Lee, David J; Busby, Stephen J W; Westblade, Lars F; Chait, Brian T

2008-02-01

Bacteria contain a single multisubunit RNA polymerase that is responsible for the synthesis of all RNA. Previous studies of the Escherichia coli K-12 laboratory strain identified a group of effector proteins that interact directly with RNA polymerase to modulate the efficiency of transcription initiation, elongation, or termination. Here we used a rapid affinity isolation technique to isolate RNA polymerase from the pathogenic Escherichia coli strain O157:H7 Sakai. We analyzed the RNA polymerase enzyme complex using mass spectrometry and identified associated proteins. Although E. coli O157:H7 Sakai contains more than 1,600 genes not present in the K-12 strain, many of which are predicted to be involved in transcription regulation, all of the identified proteins in this study were encoded on the "core" E. coli genome.

Neutronics analysis of the TRIGA Mark II reactor core and its experimental facilities

International Nuclear Information System (INIS)

Khan, R.

2010-01-01

The neutronics analysis of the current core of the TRIGA Mark II research reactor is performed at the Atominstitute (ATI) of Vienna University of Technology. The current core is a completely mixed core having three different types of fuels i.e. aluminium clad 20 % enriched, stainless steel clad 20 % enriched and SS clad 70 % enriched (FLIP) Fuel Elements (FE(s)). The completely mixed nature and complicated irradiation history of the core makes the reactor physics calculations challenging. This PhD neutronics research is performed by employing the combination of two best and well practiced reactor simulation tools i.e. MCNP (general Monte Carlo N-particle transport code) for static analysis and ORIGEN2 (Oak Ridge Isotop Generation and depletion code) for dynamic analysis of the reactor core. The PhD work is started to develop a MCNP model of the first core configuration (March 1962) employing fresh fuel composition. The neutrons reaction data libraries ENDF/B-VI is applied taking the missing isotope of Samarium from JEFF3.1. The MCNP model of the very first core has been confirmed by three different local experiments performed on the first core configuration. These experiments include the first criticality, reactivity distribution and the neutron flux density distribution experiment. The first criticality experiment verifies the MCNP model that core achieves its criticality on addition of the 57th FE with a reactivity difference of about 9.3 cents. The measured reactivity worths of four FE(s) and a graphite element are taken from the log book and compared with MCNP simulated results. The percent difference between calculations and measurements ranges from 4 to 22 %. The neutron flux density mapping experiment confirms the model completely exhibiting good agreement between simulated and the experimental results. Since its first criticality, some additional 104-type and 110-type (FLIP) FE(s) have been added to keep the reactor into operation. This turns the current

Structural and magnetic characterization of three tetranuclear Cu(II) complexes with face-sharing-dicubane/double-open-cubane like core framework

International Nuclear Information System (INIS)

Paul, Aparup; Bertolasi, Valerio; Figuerola, Albert; Manna, Subal Chandra

2017-01-01

Three novel tetranuclear copper(II) complexes namely [Cu 4 (L 1 ) 4 ]∙2(dmf) (1), [Cu 4 (L 1 ) 4 ] (2) and [Cu 4 (L 2 ) 2 (HL 2 ) 2 (H 2 O) 2 ]∙2(ClO 4 )·6(H 2 O) (3) (H 2 L 1 , (E)−2-((1-hydroxybutan-2-ylimino)methyl)phenol; H 2 L 2 , (E)−2-((1-hydroxybutan-2-ylimino)methyl)−6-methoxyphenol)) were synthesized from the self-assembly of copper(II) perchlorate and the tridentate Schiff base ligands. The structural determination reveals that crystallizes in the monoclinic system with space group C2/c, whereas both the and crystallize in the triclinic system with space group P-1. and possess face-sharing dicubane core, on the other hand complex 3 has double open cubane core structure. The copper(II) ions in the cubane core are in distorted square planar geometries, and weak π…π and C–H…π interactions lead to formation of a 2D supramolecular architecture for and . At room temperature and , exhibit fluorescence with a quantum yield (Φ s ) of 0.47, 0.49 and 0.38, respectively. Variable temperature magnetic susceptibility measurements in the range 2–300 K indicate an overall weak antiferromagnetic exchange coupling in all complexes. The PHI program was used to study their magnetic behaviour. In agreement with their face-sharing dicubane structure, a Hamiltonian of the type H =– J 1 (S 1 S 2 +S 1 S 2’ +S 1' S 2 +S 1' S 2’ ) – J 2 S 1 S 1’ , where S 1 = S 1’ = S 2 = S 2’ = S Cu =1/2, was used for studying and . Simulations performed suggest magnetic exchange constants with values close to J 1 =−20 cm −1 and J 2 =0 cm -1 for these complexes. On the other hand, the spin Hamiltonian H =– J 1 (S 1 S 4 +S 2 S 3 ) – J 2 (S 1 S 3 +S 2 S 4 ) – J 3 S 1 S 2 , where S 1 = S 2 = S 3 = S 4 = S Cu =1/2, was used to study the magnetic behaviour of the double open cubane core of and a good agreement between the experimental and simulated results was found by using the parameters g 1 = g 2 =2.20, g 3 = g 4 =2.18, J 1 =−36 cm -1 , J 2

Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

Directory of Open Access Journals (Sweden)

Uchechi E Ukaegbu

2014-01-01

Full Text Available Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2 has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1977-01-01

An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

Material correlations and models for the irradiation behavior of fissile and fertile material in SNR-300, Mark-II and KNK II, third core

International Nuclear Information System (INIS)

Fenneker; Steinmetz; Toebbe

1986-07-01

The report contains the material correlations and models used in the fuel pin design code IAMBUS for the irradiation behavior of PuO 2 -UO 2 fissile materials and UO 2 fertile materials of the SNR-300 Mark-II reload and the KNK II third core. They are applicable for pellet densities of more than 90 % of the theoretical density. The presented models of the fuel behavior and the applied material correlations have been derived either from single experiments or from the comparison of theoretically predicted integral fuel behavior with the results of fuel pin irradiation experiments. The material correlations have been examined and extended in the frame of the collaborations INTERATOM/KWU and INTERATOM/KfK. French and British results were included, when available from the European fast reactor knowledge exchange [de

The KNK II instrumentation for global and local supervision of the reactor core

International Nuclear Information System (INIS)

Steiger, W.O.

1990-01-01

After an introduction into the KNK plant itself, their historical development and their present situation, the instrumentation of the global and local supervision of the KNK II-core as well as the main safety-related i- and c-systems are described. Special emphasis is laid on the instrumentation of the reactor protection systems and the shutdown systems. After that some practices are reported about instrumentation behavior and lessons learned from the operation and maintenance of the above mentioned systems. At last follows a short description of the special instrumentation for the detection of failed fuel subassemblies and of the plant data processing system. (orig.)

The KNK II instrumentation for global and local supervision of the reactor core

International Nuclear Information System (INIS)

Steiger, W.O.

1991-01-01

After an introduction into the KNK plant itself, their historical development and their present situation, the instrumentation of the global and local supervision of the KNK II-core as well as the main safety-related instrumentation and control systems is described. Special emphasis is laid on the instrumentation of the reactor protection systems and the shut down systems. After that some practices are reported about instrumentation behavior and lessons learned from the operation and maintenance of the above mentioned systems. At last follows a short description of the special instrumentation for the detection of failed fuel subassemblies and of the plant data processing system. (author). 4 refs, 18 tabs

SMART core preliminary nuclear design-II

Energy Technology Data Exchange (ETDEWEB)

Lee, Jeong Chan; Ji, Seong Kyun; Chang, Moon Hee

1997-06-01

Three loading patterns for 330 MWth SMART core are constructed for 25, 33 and 29 CRDMs, and one loading pattern for larger 69-FA core with 45 CRDMs is also constructed for comparison purpose. In this study, the core consists of 57 reduced height Korean Optimized Fuel Assemblies (KOFAs) developed by KAERI. The enrichment of fuel is 4.95 w/o. As a main burnable poison, 35% B-10 enriched B{sub 4}C-Al{sub 2}O{sub 3} shim is used. To control stuck rod worth, some gadolinia bearing fuel rods are used. The U-235 enrichment of the gadolinia bearing fuel rods is 1.8 w/o as used in KOFA. All patterns return cycle length of about 3 years. Three loading patterns except 25-CRDM pattern satisfy cold shutdown condition of keff {<=} 0.99 without soluble boron. These three patterns also satisfy the refueling condition of keff {<=} 0.95. In addition to the construction of loading pattern, an editing module of MASTER PPI files for rod power history generation is developed and rod power histories are generated for 29-CRDM loading pattern. Preliminary Fq design limit is suggested as 3.71 based on KOFA design experience. (author). 9 tabs., 45 figs., 16 refs.

Transcription-factor occupancy at HOT regions quantitatively predicts RNA polymerase recruitment in five human cell lines.

KAUST Repository

Foley, Joseph W; Sidow, Arend

2013-01-01

BACKGROUND: High-occupancy target (HOT) regions are compact genome loci occupied by many different transcription factors (TFs). HOT regions were initially defined in invertebrate model organisms, and we here show that they are a ubiquitous feature of the human gene-regulation landscape. RESULTS: We identified HOT regions by a comprehensive analysis of ChIP-seq data from 96 DNA-associated proteins in 5 human cell lines. Most HOT regions co-localize with RNA polymerase II binding sites, but many are not near the promoters of annotated genes. At HOT promoters, TF occupancy is strongly predictive of transcription preinitiation complex recruitment and moderately predictive of initiating Pol II recruitment, but only weakly predictive of elongating Pol II and RNA transcript abundance. TF occupancy varies quantitatively within human HOT regions; we used this variation to discover novel associations between TFs. The sequence motif associated with any given TF's direct DNA binding is somewhat predictive of its empirical occupancy, but a great deal of occupancy occurs at sites without the TF's motif, implying indirect recruitment by another TF whose motif is present. CONCLUSIONS: Mammalian HOT regions are regulatory hubs that integrate the signals from diverse regulatory pathways to quantitatively tune the promoter for RNA polymerase II recruitment.

Transcription-factor occupancy at HOT regions quantitatively predicts RNA polymerase recruitment in five human cell lines.

KAUST Repository

Foley, Joseph W

2013-10-20

BACKGROUND: High-occupancy target (HOT) regions are compact genome loci occupied by many different transcription factors (TFs). HOT regions were initially defined in invertebrate model organisms, and we here show that they are a ubiquitous feature of the human gene-regulation landscape. RESULTS: We identified HOT regions by a comprehensive analysis of ChIP-seq data from 96 DNA-associated proteins in 5 human cell lines. Most HOT regions co-localize with RNA polymerase II binding sites, but many are not near the promoters of annotated genes. At HOT promoters, TF occupancy is strongly predictive of transcription preinitiation complex recruitment and moderately predictive of initiating Pol II recruitment, but only weakly predictive of elongating Pol II and RNA transcript abundance. TF occupancy varies quantitatively within human HOT regions; we used this variation to discover novel associations between TFs. The sequence motif associated with any given TF\\'s direct DNA binding is somewhat predictive of its empirical occupancy, but a great deal of occupancy occurs at sites without the TF\\'s motif, implying indirect recruitment by another TF whose motif is present. CONCLUSIONS: Mammalian HOT regions are regulatory hubs that integrate the signals from diverse regulatory pathways to quantitatively tune the promoter for RNA polymerase II recruitment.

Analysis of energy released from core disruptive accident of sodium cooled fast reactor using CDA-ER and VENUS-II codes

Energy Technology Data Exchange (ETDEWEB)

Kang, S. H.; Ha, K. S. [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2013-05-15

The fast reactor has a unique feature in that rearranged core materials can produce a large increase in reactivity and recriticality. If such a rearrangement of core materials should occur rapidly, there would be a high rate of reactivity increase producing power excursions. The released energy from such an energetic recriticality might challenge the reactor vessel integrity. An analysis of the hypothetical excursions that result in the disassembly of the reactor plays an important role in a liquid metal fast reactor (LMFR) safety analysis. The analysis of such excursions generally consists of three phases (initial or pre-disassembly phase, disassembly phase, energy-work conversion phase). The first step is referred to as the 'accident initiation' or 'pre-disassembly' phase. In this phase, the accident is traced from some initiating event, such as a coolant pump failure or control rod ejection, up to a prompt critical condition where high temperatures and pressures rapidly develop in the core. Such complex processes as fuel pin failure, sodium voiding, and fuel slumping are treated in this phase. Several computer programs are available for this type of calculation, including SAS4A, MELT-II and FREADM. A number of models have been developed for this type of analysis, including the REXCO and SOCOOL-II computer programs. VENUS-II deals with the second phase (disassembly analysis). Most of the models used in the code have been based on the original work of Bethe and Tait. The disassembly motion is calculated using a set of two-dimensional hydrodynamics equations in the VENUS code. The density changes can be explicitly calculated, which in turn allows the use of a more accurate density dependent equation of state. The main functional parts of the computational model can be summarized as follows: Power and energy (point kinetics), Temperature (energy balance), Internal pressure (equation of state), Material displacement (hydrodynamics), Reactivity

Analysis of energy released from core disruptive accident of sodium cooled fast reactor using CDA-ER and VENUS-II codes

International Nuclear Information System (INIS)

Kang, S. H.; Ha, K. S.

2013-01-01

The fast reactor has a unique feature in that rearranged core materials can produce a large increase in reactivity and recriticality. If such a rearrangement of core materials should occur rapidly, there would be a high rate of reactivity increase producing power excursions. The released energy from such an energetic recriticality might challenge the reactor vessel integrity. An analysis of the hypothetical excursions that result in the disassembly of the reactor plays an important role in a liquid metal fast reactor (LMFR) safety analysis. The analysis of such excursions generally consists of three phases (initial or pre-disassembly phase, disassembly phase, energy-work conversion phase). The first step is referred to as the 'accident initiation' or 'pre-disassembly' phase. In this phase, the accident is traced from some initiating event, such as a coolant pump failure or control rod ejection, up to a prompt critical condition where high temperatures and pressures rapidly develop in the core. Such complex processes as fuel pin failure, sodium voiding, and fuel slumping are treated in this phase. Several computer programs are available for this type of calculation, including SAS4A, MELT-II and FREADM. A number of models have been developed for this type of analysis, including the REXCO and SOCOOL-II computer programs. VENUS-II deals with the second phase (disassembly analysis). Most of the models used in the code have been based on the original work of Bethe and Tait. The disassembly motion is calculated using a set of two-dimensional hydrodynamics equations in the VENUS code. The density changes can be explicitly calculated, which in turn allows the use of a more accurate density dependent equation of state. The main functional parts of the computational model can be summarized as follows: Power and energy (point kinetics), Temperature (energy balance), Internal pressure (equation of state), Material displacement (hydrodynamics), Reactivity feedback (Doppler and

Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing.

Science.gov (United States)

Mallory, Melanie A; Lucic, Danijela X; Sears, Mitchell T; Cloherty, Gavin A; Hillyard, David R

2014-05-01

HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method. Copyright © 2014 Elsevier B.V. All rights reserved.

The largest subunit of RNA polymerase II from the Glaucocystophyta: functional constraint and short-branch exclusion in deep eukaryotic phylogeny

Directory of Open Access Journals (Sweden)

Stiller John W

2005-12-01

Full Text Available Abstract Background Evolutionary analyses of the largest subunit of RNA polymerase II (RPB1 have yielded important and at times provocative results. One particularly troublesome outcome is the consistent inference of independent origins of red algae and green plants, at odds with the more widely accepted view of a monophyletic Plantae comprising all eukaryotes with primary plastids. If the hypothesis of a broader kingdom Plantae is correct, then RPB1 trees likely reflect a persistent phylogenetic artifact. To gain a better understanding of RNAP II evolution, and the presumed artifact relating to green plants and red algae, we isolated and analyzed RPB1 from representatives of Glaucocystophyta, the third eukaryotic group with primary plastids. Results Phylogenetic analyses incorporating glaucocystophytes do not recover a monophyletic Plantae; rather they result in additional conflicts with the most widely held views on eukaryotic relationships. In particular, glaucocystophytes are recovered as sister to several amoebozoans with strong support. A detailed investigation shows that this clade can be explained by what we call "short-branch exclusion," a phylogenetic artifact integrally associated with "long-branch attraction." Other systematic discrepancies observed in RPB1 trees can be explained as phylogenetic artifacts; however, these apparent artifacts also appear in regions of the tree that support widely held views of eukaryotic evolution. In fact, most of the RPB1 tree is consistent with artifacts of rate variation among sequences and co-variation due to functional constraints related to C-terminal domain based RNAP II transcription. Conclusion Our results reveal how subtle and easily overlooked biases can dominate the overall results of molecular phylogenetic analyses of ancient eukaryotic relationships. Sources of potential phylogenetic artifact should be investigated routinely, not just when obvious "long-branch attraction" is encountered.

Piezo-phototronic Effect Enhanced UV/Visible Photodetector Based on Fully Wide Band Gap Type-II ZnO/ZnS Core/Shell Nanowire Array.

Science.gov (United States)

Rai, Satish C; Wang, Kai; Ding, Yong; Marmon, Jason K; Bhatt, Manish; Zhang, Yong; Zhou, Weilie; Wang, Zhong Lin

2015-06-23

A high-performance broad band UV/visible photodetector has been successfully fabricated on a fully wide bandgap ZnO/ZnS type-II heterojunction core/shell nanowire array. The device can detect photons with energies significantly smaller (2.2 eV) than the band gap of ZnO (3.2 eV) and ZnS (3.7 eV), which is mainly attributed to spatially indirect type-II transition facilitated by the abrupt interface between the ZnO core and ZnS shell. The performance of the device was further enhanced through the piezo-phototronic effect induced lowering of the barrier height to allow charge carrier transport across the ZnO/ZnS interface, resulting in three orders of relative responsivity change measured at three different excitation wavelengths (385, 465, and 520 nm). This work demonstrates a prototype UV/visible photodetector based on the truly wide band gap semiconducting 3D core/shell nanowire array with enhanced performance through the piezo-phototronic effect.

Whole-core damage analysis of EBR-II driver fuel elements following SHRT program

International Nuclear Information System (INIS)

Chang, L.K.; Koenig, J.F.; Porter, D.L.

1987-01-01

In the Shutdown Heat Removal Testing (SHRT) program in EBR-II, fuel element cladding temperatures of some driver subassemblies were predicted to exceed temperatures at which cladding breach may occur. A whole-core thermal analysis of driver subassemblies was performed to determine the cladding temperatures of fuel elemnts, and these temperatures were used for fuel element damage calculation. The accumulated cladding damage of fuel element was found to be very small and fuel element failure resulting from SHRT transients is unlikely. No element breach was noted during the SHRT transients. The reactor was immediately restarted after the most severe SHRT transient had been completed and no driver fuel breach has been noted to date. (orig.)

Evaluation report on CCTF Core-II reflood tests C2-AC1 (run 51) and C2-4 (run 62)

International Nuclear Information System (INIS)

Sugimoto, Jun; Iguchi, Tadashi; Murao, Yoshio

1984-02-01

A reflood test program has been conducted at Japan Atomic Energy Research Institute (JAERI) using large scale test facilities named Cylindrical Core Test Facility (CCTF) and Slab Core Test Facility (SCTF). The present report describes the effect of the initial clad temperature i.e., the initial stored energy on reflood phenomena observed in CCTF Core-II tests C2-ACl and C2-4. The peak clad temperatures of tests C2-ACl and C2-4 were 863 K and 1069 K, respectively at reflood initiation. With higher initial clad temperature, obtained were lower water accumulation in the core and upper plenum, and higher loop mass flow rate in an early reflood transient due to larger heat release of the stored energy in the core. Core inlet flow conditions were only affected shortly after the reflood initiation, causing the suppressed flooding rate and the larger U-tube flow oscillation between the core and the downcomer. In the core, with higher initial clad temperature, slower quench front propagation and higher turnaround temperature were observed. Responses to a higher initial clad temperature were similar to those observed in CCTF Core-I and FLECHT tests. Thus, the lower temperature rise with higher initial clad temperature was experimentally confirmed. The importance of higher flooding rate at initial period was analytically shown for further decreasing the temperature rise. (author)

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

Energy Technology Data Exchange (ETDEWEB)

Janowska, Beata [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Kurpios-Piec, Dagmara [Department of Biochemistry, Medical University of Warsaw, Banacha 1, 02-097 Warsaw (Poland); Prorok, Paulina [Institute of Genetics and Biotechnology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw (Poland); Szparecki, Grzegorz [Medical University of Warsaw, Zwirki i Wigury 61, 02-097 Warsaw (Poland); Komisarski, Marek [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Kowalczyk, Pawel [Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University, Pawinskiego 5a, 02-106 Warsaw (Poland); Janion, Celina [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Tudek, Barbara, E-mail: tudek@ibb.waw.pl [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw (Poland); Institute of Genetics and Biotechnology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw (Poland)

2012-01-03

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C Much-Greater-Than A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZ{alpha} gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA{sup -}Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II. Mutation spectrum established for strains expressing only Pol V, showed that in uvrA{sup -} bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T {yields} C:G, A:T {yields} G:C, G:C {yields} A:T and G:C {yields} T:A prevailed. The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.

Femtosecond visible/visible and visible/mid-IR pump-probe study of the photosystem II core antenna complex CP47

NARCIS (Netherlands)

Groot, M.L.; Breton, J.; van Wilderen, L.; Dekker, J.P.; van Grondelle, R.

2004-01-01

CP47 is one of the two core antenna proteins of Photosystem II involved in the transfer of solar energy toward the photochemically active reaction center, the D1D2cytb559 complex. We have performed vis/vis and vis/mid-IR pump-probe experiments at room temperature as a first step in linking the

Structural and magnetic characterization of three tetranuclear Cu(II) complexes with face-sharing-dicubane/double-open-cubane like core framework

Energy Technology Data Exchange (ETDEWEB)

Paul, Aparup [Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal (India); Bertolasi, Valerio [Dipartimento di Scienze Chimiche e Farmaceutiche, Centro di Strutturistica Diffrattometrica, Università di Ferrara, Via L. Borsari, 46, 44100 Ferrara (Italy); Figuerola, Albert [Departament de Química Inorgànica i Orgànica (Secció de Química Inorgànica) and Institut de Nanociència i Nanotecnologia (IN2UB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona (Spain); Manna, Subal Chandra, E-mail: scmanna@mail.vidyasagar.ac.in [Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal (India)

2017-05-15

Three novel tetranuclear copper(II) complexes namely [Cu{sub 4}(L{sup 1}){sub 4}]∙2(dmf) (1), [Cu{sub 4}(L{sup 1}){sub 4}] (2) and [Cu{sub 4}(L{sup 2}){sub 2}(HL{sup 2}){sub 2}(H{sub 2}O){sub 2}]∙2(ClO{sub 4})·6(H{sub 2}O) (3) (H{sub 2}L{sup 1}, (E)−2-((1-hydroxybutan-2-ylimino)methyl)phenol; H{sub 2}L{sup 2}, (E)−2-((1-hydroxybutan-2-ylimino)methyl)−6-methoxyphenol)) were synthesized from the self-assembly of copper(II) perchlorate and the tridentate Schiff base ligands. The structural determination reveals that crystallizes in the monoclinic system with space group C2/c, whereas both the and crystallize in the triclinic system with space group P-1. and possess face-sharing dicubane core, on the other hand complex 3 has double open cubane core structure. The copper(II) ions in the cubane core are in distorted square planar geometries, and weak π…π and C–H…π interactions lead to formation of a 2D supramolecular architecture for and . At room temperature and , exhibit fluorescence with a quantum yield (Φ{sub s}) of 0.47, 0.49 and 0.38, respectively. Variable temperature magnetic susceptibility measurements in the range 2–300 K indicate an overall weak antiferromagnetic exchange coupling in all complexes. The PHI program was used to study their magnetic behaviour. In agreement with their face-sharing dicubane structure, a Hamiltonian of the type H =– J{sub 1}(S{sub 1}S{sub 2}+S{sub 1}S{sub 2’}+S{sub 1'}S{sub 2}+S{sub 1'}S{sub 2’}) – J{sub 2}S{sub 1}S{sub 1’}, where S{sub 1} = S{sub 1’} = S{sub 2} = S{sub 2’} = S{sub Cu} =1/2, was used for studying and . Simulations performed suggest magnetic exchange constants with values close to J{sub 1} =−20 cm{sup −1} and J{sub 2} =0 cm{sup -1} for these complexes. On the other hand, the spin Hamiltonian H =– J{sub 1}(S{sub 1}S{sub 4}+S{sub 2}S{sub 3}) – J{sub 2}(S{sub 1}S{sub 3}+S{sub 2}S{sub 4}) – J{sub 3}S{sub 1}S{sub 2}, where S{sub 1} = S{sub 2} = S{sub 3} = S{sub 4

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

Science.gov (United States)

Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide

2018-05-05

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of an Unfolding Intermediate for a DNA Lesion Bypass Polymerase

Science.gov (United States)

Sherrer, Shanen M.; Maxwell, Brian A.; Pack, Lindsey R.; Fiala, Kevin A.; Fowler, Jason D.; Zhang, Jun; Suo, Zucai

2012-01-01

Sulfolobus solfataricusDNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C. PMID:22667759

Reflood behavior at low initial clad temperature in Slab Core Test Facility Core-II

International Nuclear Information System (INIS)

Akimoto, Hajime; Sobajima, Makoto; Abe, Yutaka; Iwamura, Takamichi; Ohnuki, Akira; Okubo, Tsutomu; Murao, Yoshio; Okabe, Kazuharu; Adachi, Hiromichi.

1990-07-01

In order to study the reflood behavior with low initial clad temperature, a reflood test was performed using the Slab Core Test Facility (SCTF) with initial clad temperature of 573 K. The test conditions of the test are identical with those of SCTF base case test S2-SH1 (initial clad temperature 1073 K) except the initial clad temperature. Through the comparison of results from these two tests, the following conclusions were obtained. (1) The low initial clad temperature resulted in the low differential pressures through the primary loops due to smaller steam generation in the core. (2) The low initial clad temperature caused the accumulated mass in the core to be increased and the accumulated mass in the downcomer to be decreased in the period of the lower plenum injection with accumulator (before 50s). In the later period of the cold leg injection with LPCI (after 100s), the water accumulation rates in the core and the downcomer were almost the same between both tests. (3) The low initial clad temperature resulted in the increase of the core inlet mass flow rate in the lower plenum injection period. However, the core inlet mass flow rate was almost the same regardless of the initial clad temperature in the later period of the cold leg injection period. (4) The low initial clad temperature resulted in the low turnaround temperature, high temperature rise and fast bottom quench front propagation. (5) In the region apart from the quench front, low initial clad temperature resulted in the lower heat transfer. In the region near the quench front, almost the same heat transfer coefficient was observed between both tests. (6) No flow oscillation with a long period was observed in the SCTF test with low initial clad temperature of 573 K, while it was remarkable in the Cylindrical Core Test Facility (CCTF) test which was performed with the same initial clad temperature. (J.P.N.)

Impact of advanced BWR core physics method on BWR core monitoring

Energy Technology Data Exchange (ETDEWEB)

Moon, H; Wells, A [Siemens Power Corporation, Richland (United States)

2000-07-01

Siemens Power Corporation recently initiated development of POWERPLEX{sup TM}-III for delivery to the Grand Gulf Nuclear Power Station. The main change introduced in POWERPLEX{sup TM}-III as compared to its predecessor POWERPLEX{sup TM}-II is the incorporation of the advances BWR core simulator MICROBURN-B2. A number of issues were identified and evaluated relating to the implementation of MICROBURN-B2 and its impact on core monitoring. MICROBURN-B2 demands about three to five times more memory and two to three times more computing time than its predecessor MICROBURN-B in POWERPLEX {sup TM}-II. POWERPLEX{sup TM}-III will improve thermal margin prediction accuracy and provide more accurate plant operating conditions to operators than POWERPLEX{sup TM}-II due to its improved accuracy in predicted TIP values and critical k-effective. The most significant advantage of POWERPLEX{sup TM}-III is its capability to monitor a relaxed rod sequence exchange operation. (authors)

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

OpenAIRE

Phutikanit, Nawapen; Suwimonteerabutr, Junpen; Harrison, Dion; D'Occhio, Michael; Carroll, Bernie; Techakumphu, Mongkol

2010-01-01

Abstract Background The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Methods Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic ...

Functional roles of DNA polymerases β and γ

International Nuclear Information System (INIS)

Huebscher, U.; Kuenzle, C.C.; Spadari, S.

1979-01-01

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC2.7.7.7)β and γ were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. uv irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase β which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this was probably due to the inability of brain tissue to excise alkylated bases from DNA. The role of DNA polymerase γ was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that under these conditions, DNA replication occurs in miatochondria, and exploiting the fact that DNA polymerase γ is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase γ in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase α, we conclude that DNA polymerase α is mainly responsible for DNA replication in nuclei, DNA polymerase β is involved in nuclear DNA repair, and DNA polymerase γ is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase α in DNA repair or for DNA polymerase β in DNA replication cannot be excluded

DNA polymerase preference determines PCR priming efficiency.

Science.gov (United States)

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially

The Gpn3 Q279* cancer-associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting.

Science.gov (United States)

Barbosa-Camacho, Angel A; Méndez-Hernández, Lucía E; Lara-Chacón, Bárbara; Peña-Gómez, Sonia G; Romero, Violeta; González-González, Rogelio; Guerra-Moreno, José A; Robledo-Rivera, Angélica Y; Sánchez-Olea, Roberto; Calera, Mónica R

2017-11-01

Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting. © 2017 Federation of European Biochemical Societies.

Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

OpenAIRE

Davidson, John F.; Fox, Richard; Harris, Dawn D.; Lyons-Abbott, Sally; Loeb, Lawrence A.

2003-01-01

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

Prospects for future uranium savings through LWRs with high performance cores

International Nuclear Information System (INIS)

Mochida, T.; Yamamoto, T.; Sasaki, M.; Matsuura, H.; Ueji, M.; Murata, T.; Kanda, K.; Oka, Y.; Kondo, S.

1995-01-01

Since 1986, Nuclear Power Engineering Cooperation (NUPEC) has been studying four types of LWR high performance core concepts (i.e., the uranium saving core I (USC-I), the uranium saving core II (USC-II), the high moderation core (HMC) and the low moderation core (LMC)), which aim at improvement of uranium and plutonium utilization. After the evaluation of fundamental core performance and uranium and plutonium material balance for each reactor, potential uranium savings with different reactor strategies are evaluated for the Japanese scenario with assumption of the growth of future nuclear power plant generation, annual reprocessing capacity and schedules for the introduction of high performance core. At 2030, about 3-6% savings in uranium demand are expected by USC-I or USC-II strategy, while about 14% savings by HMC strategy and about 8% by LMC strategy. (author)

Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

Science.gov (United States)

Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

2017-03-31

In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G 1 , S, and G 2 ), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

Directory of Open Access Journals (Sweden)

Sallie Richard

2005-08-01

Full Text Available Abstract Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNApol generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNApol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNApol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.

Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC

Directory of Open Access Journals (Sweden)

Engelen Stefan

2012-02-01

Full Text Available Abstract Background Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.

Electron internal transport barrier formation and dynamics in the plasma core of the TJ-II stellarator

Energy Technology Data Exchange (ETDEWEB)

Estrada, T [Laboratorio Nacional de Fusion por Confinamiento Magnetico, Asociacion Euratom-CIEMAT, 28040 Madrid (Spain); Krupnik, L [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine); Dreval, N [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine); Melnikov, A [Institute of Nuclear Fusion, RRC ' Kurchatov Institute' , Moscow, Russia (Russian Federation); Khrebtov, S M [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine); Hidalgo, C [Laboratorio Nacional de Fusion por Confinamiento Magnetico, Asociacion Euratom-CIEMAT, 28040 Madrid (Spain); Milligen, B van [Laboratorio Nacional de Fusion por Confinamiento Magnetico, Asociacion Euratom-CIEMAT, 28040 Madrid (Spain); Castejon, F [Laboratorio Nacional de Fusion por Confinamiento Magnetico, Asociacion Euratom-CIEMAT, 28040 Madrid (Spain); AscasIbar, E [Laboratorio Nacional de Fusion por Confinamiento Magnetico, Asociacion Euratom-CIEMAT, 28040 Madrid (Spain); Eliseev, L [Institute of Nuclear Fusion, RRC ' Kurchatov Institute' , Moscow, Russia (Russian Federation); Chmyga, A A [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine); Komarov, A D [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine); Kozachok, A S [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine); Tereshin, V [Institute of Plasma Physics, NSC ' KIPT' , Kharkov (Ukraine)

2004-01-01

The influence of magnetic topology on the formation of electron internal transport barriers (e-ITBs) has been studied experimentally in electron cyclotron heated plasmas in the stellarator TJ-II. e-ITB formation is characterized by an increase in core electron temperature and plasma potential. The positive radial electric field increases by a factor of 3 in the central plasma region when an e-ITB forms. The experiments reported demonstrate that the formation of an e-ITB depends on the magnetic configuration. Calculations of the modification of the rotational transform due to plasma current lead to the interpretation that the formation of an e-ITB can be triggered by positioning a low order rational surface close to the plasma core region. In configurations without any central low order rational, no barrier is formed for any accessible value of heating power. Different mechanisms associated with neoclassical/turbulent bifurcations and kinetic effects are put forward to explain the impact of magnetic topology on radial electric fields and confinement.

Core power capability verification for PWR NPP

International Nuclear Information System (INIS)

Xian Chunyu; Liu Changwen; Zhang Hong; Liang Wei

2002-01-01

The Principle and methodology of pressurized water reactor nuclear power plant core power capability verification for reload are introduced. The radial and axial power distributions of normal operation (category I or condition I) and abnormal operation (category II or condition II) are simulated by using neutronics calculation code. The linear power density margin and DNBR margin for both categories, which reflect core safety, are analyzed from the point view of reactor physics and T/H, and thus category I operating domain and category II protection set point are verified. Besides, the verification results of reference NPP are also given

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity.

Science.gov (United States)

Tajaddod, Mansoureh; Tanzer, Andrea; Licht, Konstantin; Wolfinger, Michael T; Badelt, Stefan; Huber, Florian; Pusch, Oliver; Schopoff, Sandy; Janisiw, Michael; Hofacker, Ivo; Jantsch, Michael F

2016-10-25

Short interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels. Here, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels. Our study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

Archaeal RNA polymerase arrests transcription at DNA lesions.

Science.gov (United States)

Gehring, Alexandra M; Santangelo, Thomas J

2017-01-01

Transcription elongation is not uniform and transcription is often hindered by protein-bound factors or DNA lesions that limit translocation and impair catalysis. Despite the high degree of sequence and structural homology of the multi-subunit RNA polymerases (RNAP), substantial differences in response to DNA lesions have been reported. Archaea encode only a single RNAP with striking structural conservation with eukaryotic RNAP II (Pol II). Here, we demonstrate that the archaeal RNAP from Thermococcus kodakarensis is sensitive to a variety of DNA lesions that pause and arrest RNAP at or adjacent to the site of DNA damage. DNA damage only halts elongation when present in the template strand, and the damage often results in RNAP arresting such that the lesion would be encapsulated with the transcription elongation complex. The strand-specific halt to archaeal transcription elongation on modified templates is supportive of RNAP recognizing DNA damage and potentially initiating DNA repair through a process akin to the well-described transcription-coupled DNA repair (TCR) pathways in Bacteria and Eukarya.

EBR-II Data Digitization

Energy Technology Data Exchange (ETDEWEB)

Yoon, Su-Jong [Idaho National Lab. (INL), Idaho Falls, ID (United States); Rabiti, Cristian [Idaho National Lab. (INL), Idaho Falls, ID (United States); Sackett, John [Idaho National Lab. (INL), Idaho Falls, ID (United States)

2014-08-01

1. Objectives To produce a validation database out of those recorded signals it will be necessary also to identify the documents need to reconstruct the status of reactor at the time of the beginning of the recordings. This should comprehends the core loading specification (assemblies type and location and burn-up) along with this data the assemblies drawings and the core drawings will be identified. The first task of the project will be identify the location of the sensors, with respect the reactor plant layout, and the physical quantities recorded by the Experimental Breeder Reactor-II (EBR-II) data acquisition system. This first task will allow guiding and prioritizing the selection of drawings needed to numerically reproduce those signals. 1.1 Scopes and Deliverables The deliverables of this project are the list of sensors in EBR-II system, the identification of storing location of those sensors, identification of a core isotopic composition at the moment of the start of system recording. Information of the sensors in EBR-II reactor system was summarized from the EBR-II system design descriptions listed in Section 1.2.

Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity.

Science.gov (United States)

Mulligan, M E; Hawley, D K; Entriken, R; McClure, W R

1984-01-11

We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

Improving the calculated core stability by the core nuclear design optimization

International Nuclear Information System (INIS)

Partanen, P.

1995-01-01

Three different equilibrium core loadings for TVO II reactor have been generated in order to improve the core stability properties at uprated power level. The reactor thermal power is assumed to be uprated from 2160 MW th to 2500 MW th , which moves the operating point after a rapid pump rundown where the core stability has been calculated from 1340 MW th and 3200 kg/s to 1675 MW th and 4000 kg/s. The core has been refuelled with ABB Atom Svea-100 -fuel, which has 3,64% w/o U-235 average enrichment in the highly enriched zone. PHOENIX lattice code has been used to provide the homogenized nuclear constants. POLCA4 static core simulator has been used for core loadings and cycle simulations and RAMONA-3B program for simulating the dynamic response to the disturbance for which the stability behaviour has been evaluated. The core decay ratio has been successfully reduced from 0,83 to 0,55 mainly by reducing the power peaking factors. (orig.) (7 figs., 1 tab.)

The expanding polymerase universe.

Science.gov (United States)

Goodman, M F; Tippin, B

2000-11-01

Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

Science.gov (United States)

Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

2017-05-11

Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols.

Science.gov (United States)

Angers, M; Cloutier, J F; Castonguay, A; Drouin, R

2001-08-15

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.

CALTECH CORE-COLLAPSE PROJECT (CCCP) OBSERVATIONS OF TYPE II SUPERNOVAE: EVIDENCE FOR THREE DISTINCT PHOTOMETRIC SUBTYPES

Energy Technology Data Exchange (ETDEWEB)

Arcavi, Iair; Gal-Yam, Avishay; Yaron, Ofer [Department of Particle Physics and Astrophysics, Weizmann Institute of Science, Rehovot 76100 (Israel); Cenko, S. Bradley; Becker, Adam B. [Department of Astronomy, University of California, Berkeley, CA 94720-3411 (United States); Fox, Derek B. [Department of Astronomy and Astrophysics, Pennsylvania State University, University Park, PA 16802 (United States); Leonard, Douglas C. [Department of Astronomy, San Diego State University, San Diego, CA 92182 (United States); Moon, Dae-Sik [Department of Astronomy and Astrophysics, University of Toronto, Toronto, ON M5S 3H4 (Canada); Sand, David J. [Las Cumbres Observatory Global Telescope Network, Santa Barbara, CA 93117 (United States); Soderberg, Alicia M. [Harvard-Smithsonian Center for Astrophysics, Cambridge, MA 02138 (United States); Kiewe, Michael [Department of Physics, University of Wisconsin, Madison, WI 53706 (United States); Scheps, Raphael [King' s College, University of Cambridge, Cambridge CB2 1ST (United Kingdom); Birenbaum, Gali [12 Amos St, Ramat Chen, Ramat Gan 52233 (Israel); Chamudot, Daniel [20 Chen St, Petach Tikvah 49520 (Israel); Zhou, Jonathan, E-mail: iair.arcavi@weizmann.ac.il [101 Dunster Street, Box 398, Cambridge, MA 02138 (United States)

2012-09-10

We present R-band light curves of Type II supernovae (SNe) from the Caltech Core-Collapse Project (CCCP). With the exception of interacting (Type IIn) SNe and rare events with long rise times, we find that most light curve shapes belong to one of three apparently distinct classes: plateau, slowly declining, and rapidly declining events. The last class is composed solely of Type IIb SNe which present similar light curve shapes to those of SNe Ib, suggesting, perhaps, similar progenitor channels. We do not find any intermediate light curves, implying that these subclasses are unlikely to reflect variance of continuous parameters, but rather might result from physically distinct progenitor systems, strengthening the suggestion of a binary origin for at least some stripped SNe. We find a large plateau luminosity range for SNe IIP, while the plateau lengths seem rather uniform at approximately 100 days. As analysis of additional CCCP data goes on and larger samples are collected, demographic studies of core-collapse SNe will likely continue to provide new constraints on progenitor scenarios.

The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.

Science.gov (United States)

Fox, Melanie J; Gao, Hongyu; Smith-Kinnaman, Whitney R; Liu, Yunlong; Mosley, Amber L

2015-01-01

The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets.

Safety analysis calculations for a mixed and full FLIP core in a TRIGA Mark II

International Nuclear Information System (INIS)

Ringle, John C.; Hornyik, K.; Robinson, A.H.; Anderson, T.V.; Johnson, A.G.

1976-01-01

The Oregon State TRIGA Reactor will be reloading with FLIP fuel in August 1976. As we are the first Mark II TRIGA with a circular grid pattern and graphite reflector to utilize FLIP fuel, the safety analysis calculations performed at other facilities using FLIP were only of limited use to us. A multigroup, multiregion, one-dimensional diffusion theory code was used to calculate power densities in six different operational cores - mixed to full FLIP. Pulsing characteristics were obtained from a computer code based on point kinetics, with adiabatic heating of the fuel, linear temperature dependence of the specific heat, and prompt fuel temperature feedback coefficient. The results of all pertinent calculations will be presented. (author)

Safety analysis and optimization of the core fuel reloading for the Moroccan TRIGA Mark-II reactor

International Nuclear Information System (INIS)

Nacir, B.; Boulaich, Y.; Chakir, E.; El Bardouni, T.; El Bakkari, B.; El Younoussi, C.

2014-01-01

Highlights: • Additional fresh fuel elements must be added to the reactor core. • TRIGA reactor could safely operate around 2 MW power with 12% fuel elements. • Thermal–hydraulic parameters were calculated and the safety margins are respected. • The 12% fuel elements will have no influence on the safety of the reactor. - Abstract: The Moroccan TRIGA MARK II reactor core is loaded with 8.5% in weight of uranium standard fuel elements. Additional fresh fuel elements must periodically be added to the core in order to remedy the observed low power and to return to the initial reactivity excess at the End Of Cycle. 12%-uranium fuel elements are available to relatively improve the short fuel lifetime associated with standard TRIGA elements. These elements have the same dimensions as standards elements, but with different uranium weight. The objective in this study is to demonstrate that the Moroccan TRIGA reactor could safely operate, around 2 MW power, with new configurations containing these 12% fuel elements. For this purpose, different safety related thermal–hydraulic parameters have been calculated in order to ensure that the safety margins are largely respected. Therefore, the PARET model for this TRIGA reactor that was previously developed and combined with the MCNP transport code in order to calculate the 3-D temperature distribution in the core and all the most important parameters like the axial distribution of DNBR (Departure from Nucleate Boiling Ratio) across the hottest channel. The most important conclusion is that the 12% fuel elements utilization will have no influence on the safety of the reactor while working around 2 MW power especially for configurations based on insertions in C and D-rings

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

Science.gov (United States)

Mirzakhanyan, Yeva; Gershon, Paul D

2017-09-01

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

The human RNA polymerase II-associated factor 1 (hPaf1: a new regulator of cell-cycle progression.

Directory of Open Access Journals (Sweden)

Nicolas Moniaux

2009-09-01

Full Text Available The human PAF (hPAF complex is part of the RNA polymerase II transcription apparatus and regulates multiple steps in gene expression. Further, the yeast homolog of hPaf1 has a role in regulating the expression of a subset of genes involved in the cell-cycle. We therefore investigated the role of hPaf1 during progression of the cell-cycle.Herein, we report that the expression of hPaf1, a subunit of the hPAF complex, increases with cell-cycle progression and is regulated in a cell-cycle dependant manner. hPaf1 specifically regulates a subclass of genes directly implicated in cell-cycle progression during G1/S, S/G2, and G2/M. In prophase, hPaf1 aligns in filament-like structures, whereas in metaphase it is present within the pole forming a crown-like structure, surrounding the centrosomes. Moreover, hPaf1 is degraded during the metaphase to anaphase transition. In the nucleus, hPaf1 regulates the expression of cyclins A1, A2, D1, E1, B1, and Cdk1. In addition, expression of hPaf1 delays DNA replication but favors the G2/M transition, in part through microtubule assembly and mitotic spindle formation.Our results identify hPaf1 and the hPAF complex as key regulators of cell-cycle progression. Mutation or loss of stoichiometry of at least one of the members may potentially lead to cancer development.

RNA Polymerase II Second Largest Subunit Molecular Identification of Boletus griseipurpureus Corner From Thailand and Antibacterial Activity of Basidiocarp Extracts.

Science.gov (United States)

Aung-Aud-Chariya, Amornrat; Bangrak, Phuwadol; Lumyong, Saisamorn; Phupong, Worrapong; Aggangan, Nelly Siababa; Kamlangdee, Niyom

2015-03-01

Boletus griseipurpureus Corner, an edible mushroom, is a putative ectomycorrhizal fungus. Currently, the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties. The purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities. Basidiocarps were collected from 2 north-eastern provinces, Roi Et and Ubon Ratchathani, and from 2 southern provinces, Songkhla and Surat Thani, in Thailand. Genomic DNA was extracted and molecular structure was examined using the RNA polymerase II (RPB2) analysis. Antibacterial activities of basidiocarp extracts were conducted with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523 and methicillin-resistant Staphylococcus aureus (MRSA) 189 using the agar-well diffusion method. All the samples collected for this study constituted a monophyletic clade, which was closely related with the Boletus group of polypore fungi. For the antibacterial study, it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract. Basidomes collected from four locations in Thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria. The health benefits of this edible species should be evaluated further.

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Directory of Open Access Journals (Sweden)

Dominik M Meinel

2013-11-01

Full Text Available Messenger RNA (mRNA synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5 diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1 phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

Science.gov (United States)

Meinel, Dominik M; Burkert-Kautzsch, Cornelia; Kieser, Anja; O'Duibhir, Eoghan; Siebert, Matthias; Mayer, Andreas; Cramer, Patrick; Söding, Johannes; Holstege, Frank C P; Sträßer, Katja

2013-11-01

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.

Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

Energy Technology Data Exchange (ETDEWEB)

Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra; Tao, Yizhi Jane; Vasquez-Del Carpio, Rodrigo; Nibert, Max L.; Patton, John T.; Harrison, Stephen C. (Harvard-Med); (NIH); (CH-Boston)

2009-04-08

Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-07-14

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-01-01

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

RNA binding and replication by the poliovirus RNA polymerase

International Nuclear Information System (INIS)

Oberste, M.S.

1988-01-01

RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

Science.gov (United States)

Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C.; Nogales, Eva; Taatjes, Dylan J.

2011-01-01

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in

Unveiling the Hot Molecular Core in the Ultracompact H II Region with Extended Emission G12.21-0.10

Science.gov (United States)

de la Fuente, E.; Trinidad, M. A.; Porras, A.; Rodríguez-Rico, C.; Araya, E. D.; Kurtz, S.; Hofner, P.; Nigoche-Netro, A.

2018-04-01

We present a multiwavelength study of the cometary H II region G12.21-0.10 using the VLA and OVRO. Both radio continuum (0.3, 0.7, 2 and 3.6 cm) and spectral lines of H41α, 13CS(2-1) & (1-0), and NH3(2,2) & (4,4) observations are included. We find two 3 mm continuum peaks toward G12.21-0.10; one of them is spatially coincident with the UC H II region, while the other coincides spatially with a molecular clump. We also find that the 0.7, 2 and 3.6 cm continuum and H41α line are only detected toward the UC H II region, while the 13CS, and NH3 are spatially associated with the molecular clump. Based on the morphology, kinetic temperature (≍86 K), volumetric density (≍1.5×106 cm-3) and linear size (≍0.22 pc) of the molecular clump, we suggest this source is consistent with a hot molecular core.

Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity.

Science.gov (United States)

Parasuram, Ramya; Coulther, Timothy A; Hollander, Judith M; Keston-Smith, Elise; Ondrechen, Mary Jo; Beuning, Penny J

2018-02-20

The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue Pol III alpha subunit is responsible for the polymerase activity and copies DNA accurately, making one error per 10 5 nucleotide incorporations. The goal of this research is to determine the residues that contribute to the activity of the polymerase subunit. Homology modeling and the computational methods of THEMATICS and POOL were used to predict functionally important amino acid residues through their computed chemical properties. Site-directed mutagenesis and biochemical assays were used to validate these predictions. Primer extension, steady-state single-nucleotide incorporation kinetics, and thermal denaturation assays were performed to understand the contribution of these residues to the function of the polymerase. This work shows that the top 15 residues predicted by POOL, a set that includes the three previously known catalytic aspartate residues, seven remote residues, plus five previously unexplored first-layer residues, are important for function. Six previously unidentified residues, R362, D405, K553, Y686, E688, and H760, are each essential to Pol III activity; three additional residues, Y340, R390, and K758, play important roles in activity.

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

Science.gov (United States)

Freemont, P S; Ollis, D L; Steitz, T A; Joyce, C M

1986-09-01

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Evolving a polymerase for hydrophobic base analogues.

Science.gov (United States)

Loakes, David; Gallego, José; Pinheiro, Vitor B; Kool, Eric T; Holliger, Philipp

2009-10-21

Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

F.B.R. Core mock-up RAPSODIE - II - numerical models

International Nuclear Information System (INIS)

Brochard, D.; Hammami, L.; Gantenbein, F.

1990-01-01

To study the behaviour of LMFBR cores excited by a seism, tests have been performed on the RAPSODIE core mock-up. The aim of this paper is to present the numerical models used to interprete these tests and the comparisons between calculations and experimental results

[Three regions of Rpb10 mini-subunit of nuclear RNA polymerases are strictly conserved in all eukaryotes].

Science.gov (United States)

Shpakovskiĭ, G V; Lebedenko, E N

1996-12-01

The rpb10+ cDNA from the fission yeast Schizosaccharomyces pombe was cloned using two independent approaches (PCR and genetic suppression). The cloned cDNA encoded the Rpb10 subunit common for all three RNA polymerases. Comparison of the deduced amino acid sequence of the Sz. pombe Rbp10 subunit (71 amino acid residues) with those of the homologous subunits of RNA polymerases I, II, and III from Saccharomyces cerevisiae and Home sapiens revealed that heptapeptides RCFT/SCGK (residues 6-12), RYCCRRM (residues 43-49), and HVDLIEK (residues 53-59) were evolutionarily the most conserved structural motifs of these subunits. It is shown that the Rbp10 subunit from Sz. pombe can substitute its homolog (ABC10 beta) in the baker's yeast S. cerevisiae.

TRIGGERING COLLAPSE OF THE PRESOLAR DENSE CLOUD CORE AND INJECTING SHORT-LIVED RADIOISOTOPES WITH A SHOCK WAVE. II. VARIED SHOCK WAVE AND CLOUD CORE PARAMETERS

Energy Technology Data Exchange (ETDEWEB)

Boss, Alan P.; Keiser, Sandra A., E-mail: boss@dtm.ciw.edu, E-mail: keiser@dtm.ciw.edu [Department of Terrestrial Magnetism, Carnegie Institution, 5241 Broad Branch Road, NW, Washington, DC 20015-1305 (United States)

2013-06-10

A variety of stellar sources have been proposed for the origin of the short-lived radioisotopes that existed at the time of the formation of the earliest solar system solids, including Type II supernovae (SNe), asymptotic giant branch (AGB) and super-AGB stars, and Wolf-Rayet star winds. Our previous adaptive mesh hydrodynamics models with the FLASH2.5 code have shown which combinations of shock wave parameters are able to simultaneously trigger the gravitational collapse of a target dense cloud core and inject significant amounts of shock wave gas and dust, showing that thin SN shocks may be uniquely suited for the task. However, recent meteoritical studies have weakened the case for a direct SN injection to the presolar cloud, motivating us to re-examine a wider range of shock wave and cloud core parameters, including rotation, in order to better estimate the injection efficiencies for a variety of stellar sources. We find that SN shocks remain as the most promising stellar source, though planetary nebulae resulting from AGB star evolution cannot be conclusively ruled out. Wolf-Rayet (WR) star winds, however, are likely to lead to cloud core shredding, rather than to collapse. Injection efficiencies can be increased when the cloud is rotating about an axis aligned with the direction of the shock wave, by as much as a factor of {approx}10. The amount of gas and dust accreted from the post-shock wind can exceed that injected from the shock wave, with implications for the isotopic abundances expected for a SN source.

Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase

International Nuclear Information System (INIS)

Amin, Anthony; Zaccardi, Joe; Mullen, Stanley; Olland, Stephane; Orlowski, Mark; Feld, Boris; Labonte, Patrick; Mak, Paul

2003-01-01

A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K d values ranging from 30 to 60 μM. In addition, these peptides inhibited the NS5B activity in vitro with IC 50 ranging from 6 to 48 μM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase α, human polymerase β, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC 50 of 50 μM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis

Modeling of the core of Atucha II nuclear power plant

International Nuclear Information System (INIS)

Blanco, Anibal

2007-01-01

This work is part of a Nuclear Engineer degree thesis of the Instituto Balseiro and it is carried out under the development of an Argentinean Nuclear Power Plant Simulator. To obtain the best representation of the reactor physical behavior using the state of the art tools this Simulator should couple a 3D neutronics core calculation code with a thermal-hydraulics system code. Focused in the neutronic nature of this job, using PARCS, we modeled and performed calculations of the nuclear power plant Atucha 2 core. Whenever it is possible, we compare our results against results obtained with PUMA (the official core code for Atucha 2). (author) [es

Structural and magnetic characterization of three tetranuclear Cu(II) complexes with face-sharing-dicubane/double-open-cubane like core framework

Science.gov (United States)

Paul, Aparup; Bertolasi, Valerio; Figuerola, Albert; Manna, Subal Chandra

2017-05-01

Three novel tetranuclear copper(II) complexes namely [Cu4(L1)4]•2(dmf) (1), [Cu4(L1)4] (2) and [Cu4(L2)2(HL2)2(H2O)2]•2(ClO4)·6(H2O) (3) (H2L1, (E)-2-((1-hydroxybutan-2-ylimino)methyl)phenol; H2L2, (E)-2-((1-hydroxybutan-2-ylimino)methyl)-6-methoxyphenol)) were synthesized from the self-assembly of copper(II) perchlorate and the tridentate Schiff base ligands. The structural determination reveals that complex 1 crystallizes in the monoclinic system with space group C2/c, whereas both the complexes 2 and 3 crystallize in the triclinic system with space group P-1. Complexes 1 and 2 possess face-sharing dicubane core, on the other hand complex 3 has double open cubane core structure. The copper(II) ions in the cubane core are in distorted square planar geometries, and weak π…π and C-H…π interactions lead to formation of a 2D supramolecular architecture for complexes 1 and 2. At room temperature complexes 1, 2 and 3, exhibit fluorescence with a quantum yield (Φs) of 0.47, 0.49 and 0.38, respectively. Variable temperature magnetic susceptibility measurements in the range 2-300 K indicate an overall weak antiferromagnetic exchange coupling in all complexes. The PHI program was used to study their magnetic behaviour. In agreement with their face-sharing dicubane structure, a Hamiltonian of the type H =- J1(S1S2+S1S2'+S1'S2+S1'S2') - J2S1S1', where S1 = S1' = S2 = S2' = SCu =1/2, was used for studying complexes 1 and 2. Simulations performed suggest magnetic exchange constants with values close to J1 =-20 cm-1 and J2 =0 cm-1 for these complexes. On the other hand, the spin Hamiltonian H =- J1(S1S4+S2S3) - J2(S1S3+S2S4) - J3S1S2, where S1 = S2 = S3 = S4 = SCu =1/2, was used to study the magnetic behaviour of the double open cubane core of complex 3 and a good agreement between the experimental and simulated results was found by using the parameters g1 = g2 =2.20, g3 = g4 =2.18, J1 =-36 cm-1, J2 =-44 cm-1 and J3 =0 cm-1.

Structure and function of DNA polymerase μ

International Nuclear Information System (INIS)

Matsumoto, Takuro; Maezawa, So

2013-01-01

DNA polymerases are enzymes playing the central role in DNA metabolism, including DNA replication, DNA repair and recombination. DNA polymerase μ (pol μ DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) in X family DNA polymerases function in non-homologous end-joining (NHEJ), which is the predonmiant repair pathway for DNA double-strand breaks (DSBs). NHEJ involves enzymes that capture both ends of the broken DNA strand, bring them together in a synaptic DNA-protein complex, and repair the DSB. Pol μ and pol λ fill in the gaps at the junction to maintain the genomic integrity. TdT synthesizes N region at the junction during V(D)J recombination and promotes diversity of immunoglobulin or T-cell receptor gene. Among these three polymerases, the regulatory mechanisms of pol μ remain rather unclear. We have approached the mechanism of pol μ from both sides of structure and cellular dynamics. Here, we propose some new insights into pol μ and the probable NHEJ model including our findings. (author)

Graded core/shell semiconductor nanorods and nanorod barcodes

Science.gov (United States)

Alivisatos, A. Paul; Scher, Erik C.; Manna, Liberato

2010-12-14

Graded core/shell semiconductor nanorods and shaped nanorods are disclosed comprising Group II-VI, Group III-V and Group IV semiconductors and methods of making the same. Also disclosed are nanorod barcodes using core/shell nanorods where the core is a semiconductor or metal material, and with or without a shell. Methods of labeling analytes using the nanorod barcodes are also disclosed.

Fukushima Daiichi Unit 1 Uncertainty Analysis-Exploration of Core Melt Progression Uncertain Parameters-Volume II.

Energy Technology Data Exchange (ETDEWEB)

Denman, Matthew R. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Brooks, Dusty Marie [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

2015-08-01

Sandia National Laboratories (SNL) has conducted an uncertainty analysi s (UA) on the Fukushima Daiichi unit (1F1) accident progression wit h the MELCOR code. Volume I of the 1F1 UA discusses the physical modeling details and time history results of the UA. Volume II of the 1F1 UA discusses the statistical viewpoint. The model used was developed for a previous accident reconstruction investigation jointly sponsored by the US Department of Energy (DOE) and Nuclear Regulatory Commission (NRC). The goal of this work was to perform a focused evaluation of uncertainty in core damage progression behavior and its effect on key figures - of - merit (e.g., hydrogen production, fraction of intact fuel, vessel lower head failure) and in doing so assess the applicability of traditional sensitivity analysis techniques .

Inner Core Rotation from Geomagnetic Westward Drift and a Stationary Spherical Vortex in Earth's Core

Science.gov (United States)

Voorhies, C. V.

1999-01-01

The idea that geomagnetic westward drift indicates convective leveling of the planetary momentum gradient within Earth's core is pursued in search of a differentially rotating mean state, upon which various oscillations and secular effects might be superimposed. The desired state conforms to roughly spherical boundary conditions, minimizes dissipative interference with convective cooling in the bulk of the core, yet may aide core cooling by depositing heat in the uppermost core and lower mantle. The variational calculus of stationary dissipation applied to a spherical vortex within the core yields an interesting differential rotation profile akin to spherical Couette flow bounded by thin Hartmann layers. Four boundary conditions are required. To concentrate shear induced dissipation near the core-mantle boundary, these are taken to be: (i) no-slip at the core-mantle interface; (ii) geomagnetically estimated bulk westward flow at the base of the core-mantle boundary layer; (iii) no-slip at the inner-outer core interface; and, to describe magnetic locking of the inner core to the deep outer core, (iv) hydrodynamically stress-free at the inner-outer core boundary. By boldly assuming the axial core angular momentum anomaly to be zero, the super-rotation of the inner core is calculated to be at most 1.5 degrees per year.

Polymerase Gamma Disease through the Ages

Science.gov (United States)

Saneto, Russell P.; Naviaux, Robert K.

2010-01-01

The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 ("POLG"). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems…

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase.

Science.gov (United States)

Melissis, S; Labrou, N E; Clonis, Y D

2006-07-28

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

Science.gov (United States)

Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P; Lee, Kyung-Jong; Chen, David J; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F

2009-07-01

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

Coupled neutronics and thermal hydraulics of high density cores for FRM II

Energy Technology Data Exchange (ETDEWEB)

Breitkreutz, Harald

2011-03-04

According to the 'Verwaltungsvereinbarung zwischen Bund und Land vom 30.5.2003' and its updating on 13.11.2010, the Forschungs-Neutronenquelle Heinz Maier-Leibnitz, Frm II, has to convert its fuel element to an uranium enrichment which is significantly lower than the current 93%, in case this is economically reasonable and doesn't impact the reactor performance immoderate. In the framework of this conversion, new calculations regarding neutronics and thermal hydraulics for the anticipated core configurations have to be made. The computational power available nowadays allows for detailed 3D calculations, on the neutronic as well as on the thermal hydraulic side. In this context, a new program system, 'X{sup 2}', was developed. It couples the Monte Carlo code McnpX, the computational fluid dynamics code Cfx and the burn-up code sequence MonteBurns. The codes were modified and extended to meet the requirements of the coupled calculation concept. To verify the new program system, highly detailed calculations for the current fuel element were made and compared to simulations and measurements that were performed in the past. The results strengthen the works performed so far and show that the original, conservative approach overestimates all critical thermal hydraulic values. Using the CFD software, effects like the impact of the combs that fix the fuel plates and the pressure drop at the edges of the fuel plates were studied in great detail for the first time. Afterwards, a number of possible new fuel elements with lower enrichment, based on disperse and monolithic UMo (uranium with 8 wt.-% Mo) were analysed. A number of straight-forward conversion scenarios was discussed, showing that a further compaction of the fuel element, an extended cycle length or an increased reactor power is needed to compensate the flux loss, which is caused by the lower enrichment. This flux loss is in excess of 7%. The discussed new fuel elements include a 50

Coupled neutronics and thermal hydraulics of high density cores for FRM II

International Nuclear Information System (INIS)

Breitkreutz, Harald

2011-01-01

According to the 'Verwaltungsvereinbarung zwischen Bund und Land vom 30.5.2003' and its updating on 13.11.2010, the Forschungs-Neutronenquelle Heinz Maier-Leibnitz, Frm II, has to convert its fuel element to an uranium enrichment which is significantly lower than the current 93%, in case this is economically reasonable and doesn't impact the reactor performance immoderate. In the framework of this conversion, new calculations regarding neutronics and thermal hydraulics for the anticipated core configurations have to be made. The computational power available nowadays allows for detailed 3D calculations, on the neutronic as well as on the thermal hydraulic side. In this context, a new program system, 'X 2 ', was developed. It couples the Monte Carlo code McnpX, the computational fluid dynamics code Cfx and the burn-up code sequence MonteBurns. The codes were modified and extended to meet the requirements of the coupled calculation concept. To verify the new program system, highly detailed calculations for the current fuel element were made and compared to simulations and measurements that were performed in the past. The results strengthen the works performed so far and show that the original, conservative approach overestimates all critical thermal hydraulic values. Using the CFD software, effects like the impact of the combs that fix the fuel plates and the pressure drop at the edges of the fuel plates were studied in great detail for the first time. Afterwards, a number of possible new fuel elements with lower enrichment, based on disperse and monolithic UMo (uranium with 8 wt.-% Mo) were analysed. A number of straight-forward conversion scenarios was discussed, showing that a further compaction of the fuel element, an extended cycle length or an increased reactor power is needed to compensate the flux loss, which is caused by the lower enrichment. This flux loss is in excess of 7%. The discussed new fuel elements include a 50% enriched disperse UMo core with

Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

Directory of Open Access Journals (Sweden)

Ester Sesmero

2015-07-01

Full Text Available Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV, human immunodeficiency virus (HIV and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

Optimal implantation depth and adherence to guidelines on permanent pacing to improve the results of transcatheter aortic valve replacement with the medtronic corevalve system: The CoreValve prospective, international, post-market ADVANCE-II study

NARCIS (Netherlands)

A.S. Petronio (Anna); J.-M. Sinning (Jan-Malte); N.M. van Mieghem (Nicolas); G. Zucchelli (Giulio); G. Nickenig (Georg); R. Bekeredjian (Raffi); J. Bosmans (Johan); F. Bedogni (Francesco); M. Branny (Marian); K. Stangl (Karl); J. Kovac (Jan); M. Schiltgen (Molly); S. Kraus (Stacia); P.P.T. de Jaegere (Peter)

2015-01-01

textabstractObjectives The aim of the CoreValve prospective, international, post-market ADVANCE-II study was to define the rates of conduction disturbances and permanent pacemaker implantation (PPI) after transcatheter aortic valve replacement with the Medtronic CoreValve System (Minneapolis,

α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

Directory of Open Access Journals (Sweden)

Olivier Martínez

Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Science.gov (United States)

Sidstedt, Maja; Romsos, Erica L; Hedell, Ronny; Ansell, Ricky; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter; Hedman, Johannes

2017-02-07

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR).

Science.gov (United States)

Sang, Fuming; Zhang, Zhizhou; Yuan, Lin; Liu, Deli

2018-02-26

Multi-round PCR is an important technique for obtaining enough target DNA from rare DNA resources, and is commonly used in many fields including forensic science, ancient DNA analysis and cancer research. However, multi-round PCR is often aborted, largely due to the accumulation of non-specific amplification during repeated amplifications. Here, we developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs). Different PCR assays, DNA polymerases (Pfu and Taq), DNA sizes and GC amounts were compared in this study. In the presence of QDs, PCR specificity could be retained even in the ninth-round amplification. Moreover, the longer and more complex the targets were, the earlier the abortion happened in multi-round PCR. However, no obvious enhancement of specificity was found in multi-round PCR using Taq DNA polymerase. Significantly, the fidelity of Pfu polymerase based multi-round PCR was not sacrificed in the presence of QDs. Besides, pre-incubation at 50 °C for an hour had no impact on multi-round PCR performance, which further authenticated the hot start effect of QDs modulated in multi-round PCR. The findings of this study demonstrated that a cost-effective and promising multi-round PCR technique for large-scale and high-throughput sample analysis could be established with high specificity, sensibility and accuracy.

Seismic responses of N-Reactor core. Independent review of Phase II work

International Nuclear Information System (INIS)

Chen, J.C.; Lo, T.; Chinn, D.J.; Murray, R.C.; Johnson, J.J.; Maslenikov, O.R.

1985-08-01

Seismic response of the N-Reactor core was independently analyzed to validate the results of Impell's analysis. The analysis procedure consists of two major stages: linear soil-structure interaction (SSI) analysis of the overall N-Reactor structure complex and nonlinear dynamic analysis of the reactor core. In the SSI analysis, CLASSI computer codes were used to calculate the SSI response of the structures and to generate the input motions for the nonlinear reactor core analysis. In addition, the response was compared to the response from the SASSI analysis under review. The impact of foundation modeling techniques and the effect of soil stiffness variation on SSI response were also investigated. In the core analysis, a nonlinear dynamic analysis model was developed. The stiffness representation of the model was calculated through a finite element analysis of several local core geometries. Finite element analyses were also used to study the block to block interaction characteristics. Using this nonlinear dynamic model along with the basemat time histories generated from CLASSI and SASSI, several dynamic analyses of the core were performed. A series of sensitivity studies was performed to investigate the discretization of the core, the effect of vertical acceleration, the effect of basemat rocking, and modeling assumptions. In general, our independent analysis of core response validates the order of magnitude of the displacement calculated by Impell. 11 refs., 110 figs., 12 tabs

KNK II, third core: Design Report of the breeder assemblies NR-230R, NR-231R, NR-233D and NR-234D

International Nuclear Information System (INIS)

Ricken, E.

1988-07-01

The report describes the blanket assemblies of the third core of KNK II and their operational behavior. The assemblies and their individual components are described and the design criteria, design methods and design results are presented. With the help of enveloping proofs the respectation of the design targets up to the foreseen residence time of 720 equivalent full-power days is evidenced [de

Recovery of infectious type Asia1 foot-and-mouth disease virus from suckling mice directly inoculated with an RNA polymerase I/II-driven unidirectional transcription plasmid.

Science.gov (United States)

Lian, Kaiqi; Yang, Fan; Zhu, Zixiang; Cao, Weijun; Jin, Ye; Li, Dan; Zhang, Keshan; Guo, Jianhong; Zheng, Haixue; Liu, Xiangtao

2015-10-02

We developed an RNA polymerase (pol) I- and II-driven plasmid-based reverse genetics system to rescue infectious foot-and-mouth disease virus (FMDV) from cloned cDNA. In this plasmid-based transfection, the full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences, which were arranged downstream of the two promoters (cytomegalovirus (CMV) and pol I promoter) and upstream of the terminators and polyadenylation signal, respectively. The utility of this method was demonstrated by the recovery of FMDV Asia1 HN/CHA/06 in BHK-21 cells transfected with cDNA plasmids. Furthermore, infectious FMDV Asia1 HN/CHA/06 could be rescued from suckling mice directly inoculated with cDNA plasmids. Thus, this reverse genetics system can be applied to fundamental research and vaccine studies, most notably to rescue those viruses for which there is currently an absence of a suitable cell culture system. Copyright © 2015 Elsevier B.V. All rights reserved.

Inert and stable erbium(III)-cored complexes based on metalloporphyrins bearing aryl-ether dendron for optical amplification: synthesis and emission enhancement

International Nuclear Information System (INIS)

Oh, Jae Buem; Kim, Yong Hee; Nah, Min Kook; Kim, Hwan Kyu

2005-01-01

We have developed novel inert and stable erbium (Er)(III)-cored complexes based on metalloporphyrins for optical amplification. The functionalized metalloporphyrin ligands have been designed and synthesized to provide enough coordination sites for the formation of inert and stable 9-coordinated Er(III)-cored complexes. Er 3+ ions were encapsulated by the metalloporphyrin ligands, such as Zn(II)- and Pt(II)-porphyrins. The near-infrared (IR) emission intensity of Er 3+ ion is much stronger in the Er(III)-cored complex based on Pt(II)-porphyrin than Er(III)-cored complex based on Zn(II)-porphyrin. Furthermore, we have incorporated a G2-aryl-ether functionalized dendron into the Er(III)-cored complex, yielding an Er(III)-cored dendrimer complex bearing the Pt(II)-porphyrin. The Er(III)-cored dendrimer complex shows the stronger near-IR emission intensity than the corresponding complex based on Pt(II)-porphyrin by seven times in solid state. The lifetimes of the emission band of Pt(II)-porphyrin ligands in the visible region were found to be 30 and 40 μs for the Er(III)-cored complex and the Er(III)-cored dendrimer complex based on Pt(II)-porphyrin in deoxygenated THF solution samples, respectively. Also, in both cases, the sensitized luminescence intensity is increased in deoxygenated solution. Therefore, it indicates that the energy transfer from the metalloporphyrins to Er 3+ ions takes places through the triplet state. In this paper, the synthesis and photophysical properties of novel Er(III)-cored complexes based on metalloporphyrins and Er(III)-cored dendrimer complex based on metalloporphyrin will be discussed

HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

Science.gov (United States)

Guo, Fang; Zhao, Qiong; Sheraz, Muhammad; Cheng, Junjun; Qi, Yonghe; Su, Qing; Cuconati, Andrea; Wei, Lai; Du, Yanming; Li, Wenhui; Chang, Jinhong; Guo, Ju-Tao

2017-09-01

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

Directory of Open Access Journals (Sweden)

Fang Guo

2017-09-01

Full Text Available Hepatitis B virus (HBV core protein assembles viral pre-genomic (pg RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs and sulfamoylbenzamides (SBAs, have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

Science.gov (United States)

Guo, Fang; Zhao, Qiong; Cheng, Junjun; Qi, Yonghe; Su, Qing; Wei, Lai; Li, Wenhui; Chang, Jinhong

2017-01-01

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. PMID:28945802

Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing

Directory of Open Access Journals (Sweden)

Kush Shrivastava

2015-10-01

Full Text Available Aim: To study the major histocompatibility complex (MHC Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and nucleotide sequencing techniques. Materials and Methods: DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR software. Results: TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE, however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison. Conclusion: The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI, both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

Evaluation of aseismic integrity in HTTR core-bottom structure. Pt. 1. Aseismic test for core-bottom structure

International Nuclear Information System (INIS)

Iyoku, T.; Futakawa, M.; Ishihara, M.

1994-01-01

The aseismic tests were carried out using (1)/(5)-scale and (1)/(3)-scale models of the core-bottom structure of the HTTR to quantitatively evaluate the response of acceleration, strain, impact load etc. The following conclusions are obtained. (i) The frequency response of the keyway strain is correlative with that of the impact acceleration on the hot plenum block. (ii) It was confirmed through (1)/(5)-scale and (1)/(3)-scale model tests that the applied similarity law is valid to evaluate the seismic response characteristics of the core-bottom structure. (ii) The stress of graphite components estimated from the scale model test using S 2 -earthquake excitation was sufficiently lower than the allowable stress used as the design criterion. ((orig.))

A rapid and convergent synthesis of the integrastatin core

KAUST Repository

Tadross, Pamela M.; Bugga, Pradeep; Stoltz, Brian M.

2011-01-01

The tetracyclic core of the integrastatin natural products has been prepared in a convergent and rapid manner. Our strategy relies upon a palladium(ii)-catalyzed oxidative cyclization to form the central [3.3.1]-dioxabicycle of the natural product core. Overall, the core has been completed in only 4 linear steps from known compounds. © 2011 The Royal Society of Chemistry.

Calculational and experimental experience on core management of experimental fast reactor 'JOYO'

International Nuclear Information System (INIS)

Yoshida, A.; Arii, Y.; Shono, A.; Suzuki, S.; Kinjo, K.

1992-01-01

For the core management of JOYO Mark-II, many core characteristics have been calculated with the core management code system 'MAGI', and measurements have also been carried out at each duty operation cycle. From the evaluation of these results, the characteristics of core parameters such as criticality, reactivity coefficients, and control rod worth can be predicted accurately as followings; excess reactivity: ± 0.1% Δk/k, outlet temperature of subassembly: ±10degC, fuel burn-up: ±5%, control rod worth: ±5%. As a result, we can not only get steady operation of JOYO but also perform various irradiation tests with satisfied conditions. This paper presents experience obtained until now through twenty three duty cycle operations of Mark-II core in JOYO. (author)

NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

International Nuclear Information System (INIS)

Shen Cenchao; Yang Wenjuan; Ji Qiaoli; Zhang Zhizhou; Maki, Hisaji; Dong Anjie

2009-01-01

Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

Southernmost carriers of HTLV-I/II in the world.

Science.gov (United States)

Cartier, L; Araya, F; Castillo, J L; Zaninovic, V; Hayami, M; Miura, T; Imai, J; Sonoda, S; Shiraki, H; Miyamoto, K

1993-01-01

To clarify the real distribution of HTLV-I and -II carriers among indigenous people in central and South America, blood samples collected from indigenous people in isolated regions of Southern Chile were examined. Among 199 inhabitants from Chiloe Island and Pitrufquen town, three cases (1.5%) showed positive anti-HTLV-I antibodies. Two out of the three (82-year-old male and 58-year-old female) reacted to HTLV-II-specific Gag and/or Env proteins but not to HTLV-I-specific ones. The latter case was confirmed as an HTLV-II carrier by polymerase chain reaction test.

Geriatric core competencies for family medicine curriculum and enhanced skills: care of elderly.

Science.gov (United States)

Charles, Lesley; Triscott, Jean A C; Dobbs, Bonnie M; McKay, Rhianne

2014-06-01

There is a growing mandate for Family Medicine residency programs to directly assess residents' clinical competence in Care of the Elderly (COE). The objectives of this paper are to describe the development and implementation of incremental core competencies for Postgraduate Year (PGY)-I Integrated Geriatrics Family Medicine, PGY-II Geriatrics Rotation Family Medicine, and PGY-III Enhanced Skills COE for COE Diploma residents at a Canadian University. Iterative expert panel process for the development of the core competencies, with a pre-defined process for implementation of the core competencies. Eighty-five core competencies were selected overall by the Working Group, with 57 core competencies selected for the PGY-I/II Family Medicine residents and an additional 28 selected for the PGY-III COE residents. The core competencies follow the CanMEDS Family Medicine roles. Both sets of core competencies are based on consensus. Due to demographic changes, it is essential that Family Physicians have the required skills and knowledge to care for the frail elderly. The core competencies described were developed for PGY-I/II Family Medicine residents and PGY-III Enhanced Skills COE, with a focus on the development of geriatric expertise for those patients that would most benefit.

FACT facilitates chromatin transcription by RNA polymerases I and III

DEFF Research Database (Denmark)

Birch, Joanna L; Tan, Bertrand C-M; Panov, Kostya I

2009-01-01

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle....... The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results...... in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III...

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

Science.gov (United States)

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

The involvement of topoisomerases and DNA polymerase I in the mechanism of induced thermal and radiation resistance in yeast

International Nuclear Information System (INIS)

Boreham, D.R.; Trivedi, A.; Weinberger, P.; Mitchel, R.E.

1990-01-01

Either an ionizing radiation exposure or a heat shock is capable of inducing both thermal tolerance and radiation resistance in yeast. Yeast mutants, deficient in topoisomerase I, in topoisomerase II, or in DNA polymerase I, were used to investigate the mechanism of these inducible resistances. The absence of either or both topoisomerase activities did not prevent induction of either heat or radiation resistance. However, if both topoisomerase I and II activities were absent, the sensitivity of yeast to become thermally tolerant (in response to a heat stress) was markedly increased. The absence of only topoisomerase I activity (top1) resulted in the constitutive expression of increased radiation resistance equivalent to that induced by a heat shock in wild-type cells, and the topoisomerase I-deficient cells were not further inducible by heat. This heat-inducible component of radiation resistance (or its equivalent constitutive expression in top1 cells) was, in turn, only a portion of the full response inducible by radiation. The absence of polymerase I activity had no detectable effect on either response. Our results indicate that the actual systems that confer resistance to heat or radiation are independent of either topoisomerase activity or DNA polymerase function, but suggest that topoisomerases may have a regulatory role during the signaling of these mechanisms. The results of our experiments imply that maintenance of correct DNA topology prevents induction of the heat-shock response, and that heat-shock induction of a component of the full radiation resistance in yeast may be the consequence of topoisomerase I inactivation

Tunable luminescent emission characterization of type-I and type-II systems in CdS-ZnSe core-shell nanoparticles: Raman and photoluminescence study.

Science.gov (United States)

Ca, Nguyen Xuan; Lien, V T K; Nghia, N X; Chi, T T K; Phan, The-Long

2015-11-06

We used wet chemical methods to synthesize core-shell nanocrystalline samples CdS(d)/ZnSe N , where d = 3-6 nm and N = 1-5 are the size of CdS cores and the number of monolayers grown on the cores, respectively. By annealing typical CdS(d)/ZnSe N samples (with d = 3 and 6 nm and N = 2) at 300 °C for various times t an = 10-600 min, we created an intermediate layer composed of Zn1-x Cd x Se and Cd1-x Zn x S alloys with various thicknesses. The formation of core-shell structures and intermediate layers was monitored by Raman scattering and UV-vis absorption spectrometers. Careful photoluminescence studies revealed that the as-prepared CdS(d)/ZnSe N samples with d = 5 nm and N = 2-4, and the annealed samples CdS(3 nm)/ZnSe2 with t an ≤ 60 min and CdS(6 nm)/ZnSe2 with t an ≤ 180 min, show the emission characteristics of type-II systems. Meanwhile, the other samples show the emission characteristics of type-I systems. These results prove that the partial separation of photoexcited carriers between the core and shell is dependent strongly on the engineered core-shell nanostructures, meaning the sizes of the core, shell, and intermediate layers. With the tunable luminescence properties, CdS-ZnSe-based core-shell materials are considered as promising candidates for multiple-exciton generation and single-photon sources.

Spectroscopic properties of reaction center pigments in photosystem II core complexes: revision of the multimer model.

Science.gov (United States)

Raszewski, Grzegorz; Diner, Bruce A; Schlodder, Eberhard; Renger, Thomas

2008-07-01

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., P(+)Pheo(-),P(+)Q(A)(-),(3)P) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His(198), the axial ligand of the special-pair chlorophyll P(D1), and D1-Thr(179), an amino-acid residue nearest to the accessory chlorophyll Chl(D1), on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl(D1). Compared to isolated reaction centers, the site energy of Chl(D1) is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl(D1) rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by approximately 10 nm than the low-energy exciton state of the two special-pair chlorophylls P(D1) and P(D2) which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl(D1) and P(D1), reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are

Evaluation Of Oxide And Silicide Mixed Fuels Of The RSG-GAS Core

International Nuclear Information System (INIS)

Tukiran; Sembiring, Tagor Malem; Suparlina, Lily

2000-01-01

Fuel exchange of the RSG-GAS reactor core from uranium oxide to uranium silicide in the same loading, density, and enrichment, that is 250 gr, 2.98 gr/cm 3 , and 19.75%, respectively, will be performed in-step wise. In every cycle of exchange with 5/1 mode, it is needed to evaluate the parameter of reactor core operation. The parameters of the reactor operation observed are criticality mass of fuels, reactivity balance, and fuel reactivity that give effect to the reactor operation. The evaluation was done at beginning of cycle of the first and second transition core with compared between experiment and calculation results. The experiments were performed at transition core I and II, BOC, and low power. At transition core I, there are 2 silicide fuels (RI-224 and R1-225) in the core and then, added five silicide fuels (R1-226, R1-252, R1-263, and R1-264) to the core, so that there are seven silicide fuels in the transition core II. The evaluation was done based on the experiment of criticality, control rod calibration, fuel reactivity of the RSG-GAS transition core. For inserting 2 silicide fuels in the transition core I dan 7 fuels in the transition core II, the operation of RSG-GAS core fulfilled the safety margin and the parameter of reactor operation change is not occur drastically in experiment and calculation results. So that, the reactor was operated during 36 days at 15 MW, 540 MWD at the first transition core. The general result showed that the parameter of reactor operation change is small so that the fuel exchange from uranium oxide to uranium silicide in the next step can be done

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

Science.gov (United States)

Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2009-04-01

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

Reverse transcriptase-quantitative polymerase chain reaction (RT ...

African Journals Online (AJOL)

The reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a highly specific polymerase chain reaction (PCR) method that allows one to detect very low transcription levels of functional gene(s) in soil. RT-qPCR helps us to know the active members of the microbial community, and their activities can be ...

Evaluation report on CCTF core-II reflood test C2-6 (Run 64)

International Nuclear Information System (INIS)

Akimoto, Hajime; Iguchi, Tadashi; Sugimoto, Jun; Okubo, Tsutomu; Murao, Yoshio; Okabe, Kazuharu.

1985-03-01

In order to evaluate the effect of the radial power profile on the system behavior and the core thermal hydraulic behavior during the reflood phase of a PWR LOCA, a test was performed using the Cylindrical Core Test Facility(CCTF) with the flat radial power profile. The test was conducted with the same total core power as that of the steep radial power test C2-5(Run 63). Through the comparisons of the results from these two tests, the following conclusions were obtained: (1) The radial power profile in the core has weak effect on the thermal hydraulic behavior in the primary system except the core. (2) Almost the same differential pressure was observed at various elevations in the periphery of the core regardless of different radial power profile. The result suggests that the core differential pressure is determined mainly by the total power and the total stored energy rather than by the local power and the local stored energy. (3) The test results support the single channel core model with the average power rod used in the reactor safety analysis codes such as REFLA-1DS, WREM for the evaluation of the overall system behavior. (4) In the steep radial power test, the heat transfer coefficient in the central(high power) region was higher than that in the peripheral(low power) region. The tendency was not explained by the estimation with the heat transfer correlation developed by Murao and Sugimoto assuming that the void fraction was uniform in a horizontal cross section. It is necessary to study more the dependency of core heat transfer on the radial power profile in the wide core. (author)

Enhancement of DNA polymerase activity in potato tuber slices

International Nuclear Information System (INIS)

Watanabe, Akira; Imaseki, Hidemasa

1977-01-01

DNA polymerase was extracted from potato (Soleum tuberosum L.) tuber discs and the temporal correlation of its activity change to DNA synthesis in vivo was examined during aging of the discs. Most of the DNA polymerase was recovered as a bound form in the 18,000 x g precipitate. Reaction with the bound-form enzyme was dependent on the presence of four deoxynucleoside triphosphates, Mg 2+ , and a template. ''Activated'' DNA and heat-denatured DNA, but not native DNA, were utilized as templates. The polymerase activity was sensitive to SH reagents. Fresh discs, which do not synthesize DNA in vivo, contained a significant amount of DNA polymerase and its activity increased linearly with time until 48 hr after slicing and became four times that of fresh discs after 72 hr, whereas the activity of DNA synthesis in vivo increased with time and decreased after reaching a maximum at 30 hr. Cycloheximide inhibited the enhancement of polymerase activity. DNA polymerase from aged and fresh discs had identical requirements for deoxynucleotides and a template in their reactions, sensitivity to SH reagent, and affinity to thymidine triphosphate. (auth.)

Progenitors of low-luminosity Type II-Plateau supernovae

Science.gov (United States)

Lisakov, Sergey M.; Dessart, Luc; Hillier, D. John; Waldman, Roni; Livne, Eli

2018-01-01

The progenitors of low-luminosity Type II-Plateau supernovae (SNe II-P) are believed to be red supergiant (RSG) stars, but there is much disparity in the literature concerning their mass at core collapse and therefore on the main sequence. Here, we model the SN radiation arising from the low-energy explosion of RSG stars of 12, 25 and 27 M⊙ on the main sequence and formed through single star evolution. Despite the narrow range in ejecta kinetic energy (2.5-4.2 × 1050 erg) in our model set, the SN observables from our three models are significantly distinct, reflecting the differences in progenitor structure (e.g. surface radius, H-rich envelope mass and He-core mass). Our higher mass RSG stars give rise to Type II SNe that tend to have bluer colours at early times, a shorter photospheric phase, and a faster declining V-band light curve (LC) more typical of Type II-linear SNe, in conflict with the LC plateau observed for low-luminosity SNe II. The complete fallback of the CO core in the low-energy explosions of our high-mass RSG stars prevents the ejection of any 56Ni (nor any core O or Si), in contrast to low-luminosity SNe II-P, which eject at least 0.001 M⊙ of 56Ni. In contrast to observations, Type II SN models from higher mass RSGs tend to show an H α absorption that remains broad at late times (due to a larger velocity at the base of the H-rich envelope). In agreement with the analyses of pre-explosion photometry, we conclude that low-luminosity SNe II-P likely arise from low-mass rather than high-mass RSG stars.

Some recriticality studies with SIMMER-II

International Nuclear Information System (INIS)

Bohl, W.R.

1979-01-01

The SIMMER-II code was applied to the problem of evaluating the potential for recriticality in an LMFBR HCDA. The phenomenology examined was based on the post SAS3D behavior in a CRBR LOF accident. The SIMMER-II results were found to be sensitive to the development of fuel blockages. If blockages were formed close to the core, the core-disruption phase of the behavior resulted from the significant energy required following SAS3D termination to raise the average steel temperature to saturation conditions; also material deposition near heat sinks eliminated condensation surfaces and induced core pressurization and fuel collapse. Excessive ramp rates (greater than $100/s) were judged to be unlikely, but improved knowledge and calculational treatments of the applicable phenomenology would be desirable

DNA Polymerase Fidelity: Beyond Right and Wrong.

Science.gov (United States)

Washington, M Todd

2016-11-01

Accurate DNA replication depends on the ability of DNA polymerases to discriminate between correctly and incorrectly paired nucleotides. In this issue of Structure, Batra et al. (2016) show the structural basis for why DNA polymerases do not efficiently add correctly paired nucleotides immediately after incorporating incorrectly paired ones. Copyright © 2016 Elsevier Ltd. All rights reserved.

76 FR 1213 - Core Principles and Other Requirements for Swap Execution Facilities

Science.gov (United States)

2011-01-07

... Part II Commodity Futures Trading Commission 17 CFR Part 37 Core Principles and Other Requirements... RIN Number 3038-AD18 Core Principles and Other Requirements for Swap Execution Facilities AGENCY... Compliance With the Core Principles III. Effective Date and Transition Period IV. Related Matters A...

Development and application of a T7 RNA polymerase-dependent expression system for antibiotic production improvement in Streptomyces.

Science.gov (United States)

Wei, Junhong; Tian, Jinjin; Pan, Guoqing; Xie, Jie; Bao, Jialing; Zhou, Zeyang

2017-06-01

To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces. A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h. The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.

Hydrogen-Bonding Capability of a Templating Difluorotoluene Nucleotide Residue in an RB69 DNA Polymerase Ternary Complex

Energy Technology Data Exchange (ETDEWEB)

Xia, Shuangluo; Konigsberg, William H.; Wang, Jimin (Yale)

2011-08-29

Results obtained using 2,4-difluorotoluene nucleobase (dF) as a nonpolar thymine isostere by Kool and colleagues challenged the Watson-Crick dogma that hydrogen bonds between complementary bases are an absolute requirement for accurate DNA replication. Here, we report crystal structure of an RB69 DNA polymerase L561A/S565G/Y567A triple mutant ternary complex with a templating dF opposite dTTP at 1.8 {angstrom}-resolution. In this structure, direct hydrogen bonds were observed between: (i) dF and the incoming dTTP, (ii) dF and residue G568 of the polymerase, and (iii) dF and ordered water molecules surrounding the nascent base pair. Therefore, this structure provides evidence that a templating dF can form novel hydrogen bonds with the incoming dTTP and with the enzyme that differ from those formed with a templating dT.

RNA Pol II promotes transcription of centromeric satellite DNA in beetles.

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Zeljka Pezer

Full Text Available Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera. This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80% are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A tails in a portion of the transcripts indicate RNA polymerase II-dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome.

Improved Rock Core Sample Break-off, Retention and Ejection System, Phase II

Data.gov (United States)

National Aeronautics and Space Administration — The proposed effort advances the design of an innovative core sampling and acquisition system with improved core break-off, retention and ejection features. Phase 1...

Tailoring Sandwich Face/Core Interfaces for Improved Damage Tolerance

DEFF Research Database (Denmark)

Lundsgaard-Larsen, Christian; Berggreen, Christian; Carlsson, Leif A.

2010-01-01

Various modifications of the face/core interface in foam core sandwich specimens are examined in a series of two papers. This paper constitutes part I and describes the finite element analysis of a sandwich test specimen, i.e. a DCB specimen loaded by uneven bending moments (DCB-UBM). Using...... this test almost any mode-mixity between pure mode I and mode II can be obtained. A cohesive zone model of the mixed mode fracture process involving large-scale bridging is developed. Results from the analysis are used in Part II, which describes methods and results of a series of experiments....

Competition between replicative and translesion polymerases during homologous recombination repair in Drosophila.

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Daniel P Kane

Full Text Available In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis.

Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome

Energy Technology Data Exchange (ETDEWEB)

DeFreitas, E.; Hilliard, B.; Cheney, P.R.; Bell, D.S.; Kiggundu, E.; Sankey, D.; Wroblewska, Z.; Palladino, M.; Woodward, J.P.; Koprowski, H. (Wistar Inst., Philadelphia, PA (United States))

1991-04-01

Chronic fatigue immune dysfunction syndrome (CFIDS) is a recently recognized illness characterized by debilitating fatigue as well as immunological and neurological abnormalities. Once thought to be caused by Epstein-Barr virus, it is now thought to have a different but unknown etiology. The authors evaluted 30 adult and pediatric CFIDS patients from six eastern states for the presence of human T-lymphotropic virus (HTLV) types I and II by Western immunoblotting, polymerase chain reaction, and in situ hybridization of blood samples. The majority of patients were positive for HTLV antibodies by Western blotting and for HTLV-II gag sequences by polymerase chain reaction and in situ hybridization. Twenty nonexposure healthy controls were negative in all assays. These data support an association between an HTLV-II-like virus and CFIDS.

Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome

International Nuclear Information System (INIS)

DeFreitas, E.; Hilliard, B.; Cheney, P.R.; Bell, D.S.; Kiggundu, E.; Sankey, D.; Wroblewska, Z.; Palladino, M.; Woodward, J.P.; Koprowski, H.

1991-01-01

Chronic fatigue immune dysfunction syndrome (CFIDS) is a recently recognized illness characterized by debilitating fatigue as well as immunological and neurological abnormalities. Once thought to be caused by Epstein-Barr virus, it is now thought to have a different but unknown etiology. The authors evaluted 30 adult and pediatric CFIDS patients from six eastern states for the presence of human T-lymphotropic virus (HTLV) types I and II by Western immunoblotting, polymerase chain reaction, and in situ hybridization of blood samples. The majority of patients were positive for HTLV antibodies by Western blotting and for HTLV-II gag sequences by polymerase chain reaction and in situ hybridization. Twenty nonexposure healthy controls were negative in all assays. These data support an association between an HTLV-II-like virus and CFIDS

Effect of γ-irradiated DNA on the activity of DNA polymerase

International Nuclear Information System (INIS)

Leadon, S.A.; Ward, J.F.

1981-01-01

A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Quencher-Free Fluorescence Method for the Detection of Mercury(II Based on Polymerase-Aided Photoinduced Electron Transfer Strategy

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Haisheng Liu

2016-11-01

Full Text Available A new quencher-free Hg2+ ion assay method was developed based on polymerase-assisted photoinduced electron transfer (PIET. In this approach, a probe is designed with a mercury ion recognition sequence (MRS that is composed of two T-rich functional areas separated by a spacer of random bases at the 3′-end, and a sequence of stacked cytosines at the 5′-end, to which a fluorescein (FAM is attached. Upon addition of Hg2+ ions into this sensing system, the MRS folds into a hairpin structure at the 3′-end with Hg2+-mediated base pairs. In the presence of DNA polymerase, it will catalyze the extension reaction, resulting in the formation of stacked guanines, which will instantly quench the fluorescence of FAM through PIET. Under optimal conditions, the limit of detection for Hg2+ ions was estimated to be 5 nM which is higher than the US Environmental Protection Agency (EPA standard limit. In addition, no labeling with a quencher was requiring, and the present method is fairly simple, fast and low cost. It is expected that this cost-effective fluorescence method might hold considerable potential in the detection of Hg2+ ions in real biological and environmental samples.

Quencher-Free Fluorescence Method for the Detection of Mercury(II) Based on Polymerase-Aided Photoinduced Electron Transfer Strategy.

Science.gov (United States)

Liu, Haisheng; Ma, Linbin; Ma, Changbei; Du, Junyan; Wang, Meilan; Wang, Kemin

2016-11-18