Changes in RNA polymerase II in a cell cycle‐specific temperature‐sensitive mutant of hamster cells

Rossini, Mara; Baserga, Susan J.; Huang, C.L.; Cj, Ingles; Baserga, Renato

doi:10.1002/jcp.1041030114

Cited by 52 publications

(20 citation statements)

References 28 publications

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“…To test accumulated levels of each RNA after stable DNA transfection, polyclonal cell lines were generated by transfection of each construct into mouse L cells or tsAF8 cells, a line of baby hamster kidney cells that contains a temperature-sensitive RNA polymerase II (Meiss and Basilico 1972;Rossini et al 1980; ]askulski and Baserga 1988). After selection for G418 resistance, >200 colonies were pooled for each construct.…”

Section: Reduced Accumulation Of Mrnas Containing the Gm-csf A U Domamentioning

confidence: 99%

“…The baby hamster kidney cell line tsAF8 (Rossini et al 1980; obtained from J. Corden, Johns Hopkins University School of Medicine, Baltimore, MD) was maintained at 34°C in Dulbecco's modified Eagle medium (DMEM) with 1 gram/liter of glucose and supplemented with 10% fetal calf serum and 10% COz. To obtain stable polyclonal lines, 18-24 hr before transfection, -106 cells were seeded per 100-mm culture dish.…”

Section: Cell Culture and Transformationsmentioning

confidence: 99%

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Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex.

Savant-Bhonsale¹,

Cleveland²

1992

Genes Dev.

187

106

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Many short-lived mRNAs, including those encoding lymphokines, cytokines, and proto-oncogenes, contain an AU-rich sequence in their 3'-untranslated regions. These AU domains and, more specifically, AUUUA motifs within them, are widely thought to mediate the extreme instability of the corresponding mRNAs. This is most clearly true for granulocyte monocyte colony stimulating factor (GM-CSF) mRNA whose AUUUA motifs are conserved phylogenetically and whose presence in an otherwise stable 13-globin mRNA results in a 50-fold decrease in accumulated mRNA level. We show that RNA instability conferred by the GM-CSF AU motif requires the mRNA to be actively translated and the AU motif to be within the 3'-untranslated region. By analysis of the sedimentation characteristics, we identify a large (>20S), divalent cation-independent complex found only on unstable RNAs. Like instability, complex formation (1) is dependent on translation of the RNA, (2) requires intact AUUUA motifs, and (3) is blocked by ribosome translocation across the AU-rich motif. We propose that RNA instability mediated by the AU motif is achieved through translation-dependent assembly of this large mRNA-destabilizing complex.

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Section: Reduced Accumulation Of Mrnas Containing the Gm-csf A U Domamentioning

confidence: 99%

Section: Cell Culture and Transformationsmentioning

confidence: 99%

Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex.

Savant-Bhonsale¹,

Cleveland²

1992

Genes Dev.

187

106

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“…The second hypothesis has been taken into account by a number of authors to explain the relationships between r RNA synthesis and cell cycle progression [4, 19, 201. However, it has been shown, at least for mammalian cells, that the inhibition of rRNA processing is not related to the beginning of the S phase [21, 221, while the possibility cannot be excluded that a continued nucleolar transcription activity is a prerequisite for DNA synthesis [22, 231. Instead, a clear correlation has been observed between mRNA synthesis and the G1/S transition [24,25]. Specific gene products are required at this point of the cell cycle, and mutants that have a temperature-sensitive RNA polymerase activity, such as TSAF8 [24], or cells treated with inhibitors that specifically inhibit mRNA synthesis are not able to progress towards DNA synthesis and remain in G, phase [25].…”

Section: Ribosomal R N a Synthesis And Processingmentioning

confidence: 99%

Inhibition of RNA Synthesis in Neurospora crassa Hyphae Treated with Picolinic Acid

Martegani

1981

European Journal of Biochemistry

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Picolinic acid, a metal-chelating agent, blocks the nuclei of germinating conidia of Neurosporu crassa in the GI phase. The addition of picolinic acid to exponentially growing cultures of N. c r a m in glucose medium causes an immediate inhibition of growth. Concentrations higher than 50 mM completely inhibit growth of mycelia, whereas lower concentrations (20-30 mM) give only a partial inhibition. Picolinic acid, added at the concentration of 20 mM, immediately blocks the accumulation of stable RNA, while protein accumulation continues at an unchanged rate for at least 30 min and only thereafter is slightly inhibited. DNA accumulation rapidly slows down after the addition of the drug. The level of polyadenylated RNA decreases quickly after the addition of picolinic acid, and the rate of its synthesis also appears to be inhibited. Picolinic acid greatly affects the metabolism of stable RNA in N. crassa: both processing and transcription of rRNA are sequentially inhibited with a moderate accumulation of precursor rRNA. It is concluded that the effects of picolinic acid on the nuclear division cycle in Neurospora may be related to the requirement of specific gene products to enter the S phase, whose synthesis is inhibited following the reduction of polyadenylated RNA synthesis.In eukaryotic cells there is a coordination between cytoplasmic growth and the nuclear division cycle [I]. The molecular basis of such a coordination is not known; however, a number of conditions that restrict protein synthesis and/or RNA synthesis cause an arrest of the cell cycle in a restriction point usually located in the G I phase [2]. In the yeast Saccharomyces cerevisiae this restriction point has been identified as 'start', and it has been shown that the transition through 'start' needs the expression of specific genes [3].The arrest in GI phase in both mammalian cells and yeast has also been observed after treatment of the cells with some ion-chelator substances such as 8-hydroxyquinoline, o-phenanthroline and picolinic acid [4]. In a previous paper from our laboratory [5] it has been shown that picolinic acid, a chelator of ions of transition metals, inhibits DNA synthesis in germinating conidia of Neurospora crassa and in the meantime blocks Neurospora nuclei in GI phase: the block appears to be specific since when picolinic acid is removed a synchronous wave of DNA synthesis takes place [5]. The mechanism of action of picolinic acid is not known, although some experiments done on mammalian cells [6] indicate that this inhibitor interferes with RNA metabolism.In this paper the effects of picolinic acid on the macromolecular syntheses in N. crassa were studied. The results indicate that picolinic acid strongly inhibits within a few minutes both rRNA processing and transcription and poly(A)-rich RNA synthesis, while it has only a moderate inhibitory effect on protein accumulation. The cell cycle effects of picolinic acid appear to be related to the requirement of specific gene products to enter in the S phase, whose synthesis is i...

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“…The CHO cell lines are notoriously poor recipients for DNA transformation, but it is not immediately apparent why transfer to the quite competent Ltk-cell line was unsuccessful. One explanation may be that a-amanitin too rapidly inactivates RNA polymerase II activity, whereas the half-life of polymerase II of the TS mutant TsAF8 when shifted to 40°C is at least 12 to 15 h and the cells remain viable at nonpermissive temperatures for as long as 40 h (20,21). Retention of some recipient cell RNA polymerase II activity during the early stages of selection of transformants may be essential.…”

mentioning

confidence: 99%

“…Previous studies of this mutant suggested that its RNA polymerase II could be the function which has the TS mutation. Growth at 40°C results in a selective loss of the activity of RNA polymerase II (20) and a loss of polymerase II molecules as quantitated by [3H]amanitin binding (21). More importantly, this TS mutation in TsAF8 was complemented by fusion with wild-type CHO cells, but not by fusion with the CHO cell TS polymerase II mutant TsAmaR-1 (22).…”

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confidence: 99%

DNA-Mediated Transfer of an RNA Polymerase II Gene: Reversion of the Temperature-Sensitive Hamster Cell Cycle Mutant TsAF8 by Mammalian DNA

Ingles¹,

Shales²

1982

Mol. Cell. Biol.

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An understanding of the biochemical mechanisms which regulate both the activity and the selectivity of RNA polymerase (RNA nucleotidyltransferase) in mammalian cells should be facilitated by the availability of mutant cell lines containing altered RNA polymerase activities. Mutations affecting the activity of RNA polymerase II in several cultured cell lines have already been described. These mutations are of three types: resistance to the RNA polymerase II inhibitor a-amanitin (4, 12, 23), conditional lethal temperature-sensitive (TS) mutations in polymerase 11 (11), and several second-site mutations that revert the TS effects on growth of a Chinese hamster ovary (CHO) TS polymerase II mutation (22). With this limited number of mutants, it is clear that only a modest beginning has been made in genetically defining the various components of the transcription complex in animal cells; indeed, both the AmaR and the TS mutations are in the same complementation group (22). Although these mutations have been of some use in demonstrating the regulated synthesis of polymerase II subunit polypeptides (7,8,24), there exists a need for both better characterization of the existing mutations and the isolation of a wider spectrum of mutations affecting RNA synthesis in animal cells. Such goals could be more readily realized were the gene(s) for eucaryotic RNA polymerase II isolated.Gene isolation has, for the most part, been accomplished to date by using DNA cloning of abundant mRNA species, but the isolation of genes represented only infrequently in mRNA populations, such as those for RNA polymerase II polypeptides, requires different experimental approaches. DNA transfer of genes expressing selectable phenotypes to appropriate recipient cells, followed by recombinant DNA screening to rescue transforming DNA, has been shown to be a successful method for the isolation of the genes coding for less abundant enzymes, such as thymidine kinase (19) and adenine phosphoribosyltransferase (10). However, despite the codominant inheritance of a-amanitin resistance, gene transfer of the AmaR phenotype has proven difficult in several laboratories. In this study we show that with an alternative approach, involving transfer of the dominant wild-type TS' gene to the TS BHK-21 cell line TsAF8, it is possible to establish that the TS defect in TsAF8 is a polymerase II mutation and that the gene determining sensitivity to a-amanitin is the TS' polymerase II gene. MATERILS AND METHODS

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Changes in RNA polymerase II in a cell cycle‐specific temperature‐sensitive mutant of hamster cells

Cited by 52 publications

References 28 publications

Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex.

Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex.

Inhibition of RNA Synthesis in Neurospora crassa Hyphae Treated with Picolinic Acid

DNA-Mediated Transfer of an RNA Polymerase II Gene: Reversion of the Temperature-Sensitive Hamster Cell Cycle Mutant TsAF8 by Mammalian DNA

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