Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Thelander, M; Thelander, Lars

doi:10.1002/j.1460-2075.1989.tb08383.x

Cited by 41 publications

(23 citation statements)

References 36 publications

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“…A somewhat similar arrangement is found in the serum-stimulated ␤-actin gene promoter, where the serum response element and the CCAAT box are closely located within a 50-base pair region centered at nt Ϫ75 with the NF-Y binding situated upstream from the serum response element (22,23). However, there are no direct similarities between the reported serum response element consensus sequence 5Ј-CC(AϩT) 6 GG-3Ј and the R2 promoter ␤, ␥, and ␦ sequences. Furthermore, in the thrombospondin case, deletion of the upstream serum response element or the CCAAT both resulted in decreased serum response.…”

Section: Discussionmentioning

confidence: 87%

“…6 and Footnote 1, where ϩ1 indicates the position of the transcription start) ligated into the unique SmaI site in the polylinker of the vector p19luc (16). The plasmid p19lucR2 1.0 contains a ClaI-PvuII (nt Ϫ1006 to ϩ17) fragment of the R2 promoter ligated into the polylinker of the p19luc vector cleaved with HindIII (made blunt end by filling in using the Klenow fragment) and SmaI.…”

Section: Methodsmentioning

confidence: 99%

“…Reporter gene constructs containing the R2 promoter-1st exon/1st intron indicate that the transcriptional block is active also in vivo, and S phase-specific protein binding was identified to a DNA region just upstream from the block. 1 The mouse R2 promoter contains a TTTAAA motif at position nt 2 Ϫ24 and a CCAAT motif at position nt Ϫ75 upstream from the transcription start (6). DNase I footprinting analyses revealed four DNA-protein binding regions within the R2 promoter.…”

mentioning

confidence: 99%

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Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Filatov

Thelander

1995

Journal of Biological Chemistry

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Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide ؊672 to nucleotide ؊527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.Ribonucleotide reductase (EC 1.17.4.1) is a key enzyme in DNA precursor synthesis reducing all four ribonucleotides to the corresponding deoxyribonucleotides (1, 2). Mouse ribonucleotide reductase is a heterodimer composed of the two homodimeric subunits, proteins R1 and R2, each inactive alone. Enzyme activity is cell cycle regulated with low or undetectable levels in G 0 /G 1 and maximal activity in the S phase of the cell cycle (3).The mouse R1 and R2 mRNA expression is S phase specific with very low or undetectable levels in G 0 /G 1 cells, a pronounced increase as cells progress into S phase, and a decline when cells progress into G 2 ϩ M (4). Reporter gene constructs show that the R2 promoter is activated almost immediately after quiescent G 0 /G 1 synchronized cells are released by serum readdition. Promoter activity then increases steadily, reaching its maximum at around 12 h after serum readdition (5). From the early promoter activation, the R2 gene could be classified as an immediate early response gene. However, in vitro studies demonstrated that this early activation only results in the synthesis of immature short R2 mRNA transcripts due to a G 1 -specific transcriptional block located in the first intron of the R2 gene (5). This block is not released until cells reach S phase when full-length transcripts are synthesized. Reporter gene constructs containing the R2 promoter-1st exon/1st intron indicate that the transcriptional block is active also in vivo, and S phase-specific protein binding was identified to a DNA region just upstream from the block. The mouse R2 promoter contains a TTTAAA motif at position nt 2 Ϫ24 and a CCAAT motif at position nt Ϫ75 upstream from the transcription start (6). DNase I footprinting analyses revealed four DNA-protein binding regions within the R2 promoter. The region most proximal to the transcription start (nt Ϫ93 to Ϫ56) includes the CCAAT box and is called ␣. The other three DNA-protein binding re...

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Section: Discussionmentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Filatov

Thelander

1995

Journal of Biological Chemistry

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“…A vector construct containing the genomic R2 gene in pUC18 was obtained by partial digestion of the pM2Hind13 plasmid with ClaI (at nucleotide Ϫ1006) and NdeI (25). The long DNA fragment containing the entire R2 genomic gene including 1,000 bp of promoter was isolated, blunted with the Klenow fragment of DNA polymerase, and religated (D construct).…”

Section: Methodsmentioning

confidence: 99%

Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis

Chabes

Pfleger

Kirschner

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

146

139

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Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1-anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an in vitro ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosis͞G 1 in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G1, may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G 0͞G1.

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“…This transcriptional behavior of the DHFR promoter suggests a mechanism of inhibition of gene expression in which folate cofactors participate . The structure of the promoter for DHFR with multiple Sp 1 binding sites and the lack of TATAA and CAT motifs shows similarities to the promoters of thymidylate synthetase (96) and ribonucleotide reductase (97). Folate cofactors may be involved in the regulation of these housekeeping genes as well (92) .…”

Section: Eukaryotesmentioning

confidence: 95%

Regulation of Gene Expression by Cofactors Derived from B Vitamins.

Brandsch

1994

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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SummaryVitamins represent precursor molecules for the majority of cofactors essential for various enzyme activities. Is the expression of proteins that require a specific cofactor for their function correlated to its availability? Examples are presented and discussed of the involvement of vitamin-derived cofactors in the regulatory mechanisms controlling gene expression in bacteria and eukaryotes at the transcriptional and trans lational level.

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Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Cited by 41 publications

References 36 publications

Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis

Regulation of Gene Expression by Cofactors Derived from B Vitamins.

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