Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines.

Guialis, Apostolia; Morrison, Kelly; Ingles, C J

doi:10.1016/s0021-9258(18)50711-0

Cited by 31 publications

(2 citation statements)

References 25 publications

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“…The central role of RNAP II in the regulation of gene expression makes it an important subject for mutational analyses, particularly in a genetically tractable organism such as S. cerevisiae. RNAP subunit mutations can be used to study the regulation of polymerase biosynthesis (19) and to reveal a function for evolutionarily conserved sequence domains. Conditional lethal mutants also provide hosts for the selection of unlinked pseudorevertants; such suppressor mutations may identify functional interactions between RPO21 and other RNAP subunits or transcription factors.…”

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confidence: 99%

Isolation and Characterization of Temperature-Sensitive Rna Polymerase II Mutants of Saccharomyces cerevisiae

Himmelfarb¹,

Simpson²,

Friesen

1987

Molecular and Cellular Biology

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Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.

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confidence: 99%

Isolation and Characterization of Temperature-Sensitive Rna Polymerase II Mutants of Saccharomyces cerevisiae

Himmelfarb¹,

Simpson²,

Friesen

1987

Molecular and Cellular Biology

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“…Such semi-dominance is also seen in fusions of amanitin-resistant cultured cells (SOMERS, PEARSON and INGLES 1975). GUIALIS, MORRISON and INGLES (1979) reported that, in the presence of amanitin, hybrid CHO cells carrying both the amanitin-resistance allele and the wild type allele produce sensitive and resistant polymerases at the same rate; however, the sensitive polymerase is degraded rapidly. It apperars, therefore, that there is an increase in the amount of resistant polymerase in these cells.…”

Section: Amanitin-resistance Mutationsmentioning

confidence: 96%

Genetic analysis of Amanitin resistance in the Nematode Caenorhabditis Elegans

Bullerjahn

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Mutants resistant to oc-amanitin, a fungal toxin that interferes with messenger RNA production by binding to RNA polymerase II, make possible genetic analyses of RNA polymerase II function. The work reported here involves studies of amanitin resistance in the nematode, Caenorhabditis elegans. The frequency of the amanitin resistance mutation was 3 x 10"6 following EMS mutagenesis. An allele of ama-1, an essential gene encoding the amanitin-binding subunit of RNA polymerase II, was isolated, and the gene was mapped. A recessive-lethal mutation that confers dominant resistance to amanitin defined another gene, ama-2. The ama-2 gene has been mapped to linkage group (LG) V, and the mutant phenotypes have been characterized. Resistance to various levels of amanitin has been tested for several mutant strains carrying mutations in either ama-1 or ama-2. Growth of strains carrying one or two copies of m l 18m252, the embryonic-lethal allele of ama-1, has been characterized, and the data suggest that this allele encodes a deleterious product that interferes with normal RNA polymerase II function. A fine-structure genetic map has been constructed for ama-1. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination distances from m l 18, the resistance mutation. Intragenic recombination between the lethal site and the parental resistance mutation was detected by means of resistance to amanitin. Recombinants were detected at frequencies as low as 2 x 10 ^-6. The segregation of closely linked flanking markers, tine-17 and unc-5, revealed whether the lethal mutation was to the left or right of m ll8, To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication mDpl [unc-17(el 13) dpy-13(+) ama-l(+) ]. The heteroallelic strains were sensitive to amanitin, and recombination events between the lethal mutations were specifically selected by means of the dominant amanitin resistance encoded on the recombinant chromosome. The segregation of outside markers revealed the order of the lethal mutations. By adding the distances between the extreme left and right mutations, the ama-1 gene was estimated to be 0.011 map unit long. The position of the mutations within the gene is non-random. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.

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Human mutant cell lines with altered RNA polymerase II

Shander

Croce

Weinmann

1982

Journal Cellular Physiology

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A human fibrosarcoma cell line, HT-l080-6TC-9AM, resistant to a-amanitin at concentrations up to 10 pdml, was isolated after ethylmethanesulfonate mutagenesis and stepwise selection. The mutation is stable and dominant. RNA polymerase II purified from the mutant cells showed an altered affinity for labeled a-amanitin and the sensitivity of the enzyme to the fungal toxin was decreased 50-to 100-fold. This functional test demonstrated that the biochemical basis for the resistanceofthecellstoa-amanitin isduetoan alteration of RNApolymerase II.Our current knowledge of the transcriptional events required for the production of mRNAs derives from the purification and in vitro activity of DNA-dependent RNA polymerase I1 (EC 2.7.7.6) (Roeder, 1976). An approach to first-hand knowledge of the biological properties of the enzyme is through the use of cellular mutants that affect the RNA polymerase 11 or other elements of the transcriptional machinery. RNA polymerase I1 mutants have been used to study the role of the host enzyme in viral gene expression (Ben Ze'ev and Becker, 1977;Silver et al., 1979;Gram-Jensen et al., 1979) and in the regulation of enzyme biosynthesis (Crear and Pearson, 1977; Guialis et al., 1979). Recently, a yeast RNA polymerase I1 mutant and a Drosophila mutant were shown to have altered transcriptional properties (Ruet et al., 1980; Coulter and Greenleaf, 1982).The fungal toxin a-amanitin has been shown to selectively inhibit the activity of RNA polymerase I1 in isolated nuclei and in crude and purified preparations of the enzyme (Wieland and Faulstich, 1978). The a-amanitin, a bicyclic octapeptide, binds stoichiometrically to the 140 kd subunit of RNA polymerase TI (Brodner and Wieland, 1976). Several rodent cell mutants have been selected by their ability t o grow in high concentrations of a-amanitin (Chan et al., 1972; Amati et al., 1975; Lobban and Siminovitch, 1975; Lobban et al., 1976;Somers et al., 1975;Ingles et al., 1976). Until now, only one other human cell mutant described was derived from WI-38 cells (WI-38) with a limited lifespan and heterozygous (Buchwald and Ingles, 1976). A new stable, homo-or hemizygous mutant can be used to determine the chromosomal location of the gene, to analyze the effect of these alterations in transcriptional regulation and specificity of the enzyme, and eventually to serve as genetic markers for the purification of the DNA sequences coding for the gene itself.We describe here human fibrosarcoma-derived a-amanitin-resistant mutants obtained by stepwise selection in medium containing the fungal toxin. Biochemical analysis of the partially purified RNA polymerase I1 from the mutant cells indicates that one of them possibly presents an altered permeability, whereas the other has an altered RNA polymerase 11. The latter mutant is homo-or hemizygous and stable. MATERIALS AND METHODSCells Human fibrosarcoma cells HT-1080-6TG were mutagenized when subconfluent by adding 200 pg/ml ethylmethanesulfonate (EMS) for 18 hours. After washing, the cells were fed with E...

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Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines.

Cited by 31 publications

References 25 publications

Isolation and Characterization of Temperature-Sensitive Rna Polymerase II Mutants of Saccharomyces cerevisiae

Isolation and Characterization of Temperature-Sensitive Rna Polymerase II Mutants of Saccharomyces cerevisiae

Genetic analysis of Amanitin resistance in the Nematode Caenorhabditis Elegans

Human mutant cell lines with altered RNA polymerase II

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