Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells

Waechter, Dieter E.; Avignolo, Carlo; Freund, Erwin; Riggenbach, C. Mark; Mercer, W E; McGuire, Patricia; Baserga, Renato

doi:10.1007/bf00226301

Cited by 17 publications

(10 citation statements)

References 13 publications

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“…This corresponds to early-to-mid G1 for these cells. After selection for poly(A)+ mRNA, 1.4 ,ug of double-stranded cDNA with an average size of 800 base pairs was synthesized from 5 ,ug of RNA. The cDNA was inserted into the tetracycline-resistance gene of pBR322 at the BamHI site after digestion with Sau3A1 to an average of 250-300 base pairs.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, it has been demonstrated that a G1-specific temperature-sensitive mutant, tsAF8, which arrests in G1 at the restrictive temperature (2), is a mutant of RNA polymerase II (3)(4)(5)(6). In addition, the progression of cells in culture through G1 can be inhibited by the intracellular microinjection of a-amanitin (5), a drug that at low concentrations is known to have one and only one specific site of action, the large subunit of RNA polymerase II (7).…”

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confidence: 99%

See 1 more Smart Citation

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

Hirschhorn¹,

Aller²,

Yuan³

et al. 1984

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

181

135

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A library of double-stranded cDNA was constructed from ts13 cells, a Gl-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)+ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34°C.Differential screening of the library with G -specific and Gospecific single-stranded cDNA probes prepared from the same cells identified five cDNA clones whose sequences were preferentially expressed in G1. (2), is a mutant of RNA polymerase II (3-6). In addition, the progression of cells in culture through G1 can be inhibited by the intracellular microinjection of a-amanitin (5), a drug that at low concentrations is known to have one and only one specific site of action, the large subunit of RNA polymerase II (7). These studies conclusively showed that a functional RNA polymerase II is an absolute requirement for the transition of cells from a resting to a growing state. This requirement justifies the search for genes transcribed by RNA polymerase II that control the transition from Go to G1 to S. As a first step in this direction, we have begun a search for genes whose expression is specifically increased in G1. A cDNA library was constructed from poly(A)+ mRNA isolated from serumstimulated ts13 cells that are a G1-specific temperature-sensitive mutant of the cell cycle (2,8). Genes that were preferentially expressed in G1 were identified by differential hybridization. The combination of molecular biology techniques with the use of Gl-specific temperature-sensitive mutants should ultimately allow us to select among the preferentially expressed genes those that play a major role in cell-cycle progression. This initial report describes the identification and characterization of five cDNA clones that are preferentially expressed in G1 at the permissive temperature yet are differently affected by the temperature-sensitive block at the restrictive temperature. MATERIALS AND METHODSCell Lines and Culture Conditions. The cell lines ts13 and tsAF8 are G1-specific temperature-sensitive mutants originally isolated from baby hamster kidney (BHK) cells (2, 8). Both lines were maintained under culture conditions described in detail (9, 10). The permissive temperature for both cell lines was 34WC, and the nonpermissive temperature was 39.60C for ts13 and 40'C for tsAF8. Cells were made quiescent by serum deprivation [i.e., maintained 48-50 hr in Dulbecco's minimal essential medium (DME medium) supplemented with 0.5% calf serum]. Quiescent populations were stimulated with fresh DME medium containing 15% fetal calf serum. The entry of cells into DNA synthesis was routinely monitored by continuous labeling with [3H]thymidine (6.7 Ci/mmol; 0.5 ,Ci/ml; 1 Ci = 37 GBq; New England Nuclear) followed by autoradiography.Enzymes. All restriction endonucleases were purchased from Bethesda Research Laboratories or New England Biolabs and were used according to the manufacturers' directions. Avian myeloblastosis virus reverse transcriptase was provided by...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

Hirschhorn¹,

Aller²,

Yuan³

et al. 1984

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

181

135

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“…The other example of G1 arrest caused by a mutation in the general transcriptional machinery is an RNA polymerase II mutant (Burstin et al 1974;Waechter et al 1984).…”

Section: How Does the Ccg1 Mutant Cause G1 Arrest?mentioning

confidence: 99%

Drosophila 230-kD TFIID subunit, a functional homolog of the human cell cycle gene product, negatively regulates DNA binding of the TATA box-binding subunit of TFIID.

Kokubo¹,

Gong²,

Yamashita³

et al. 1993

Genes Dev.

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“…The expression of particular genes during the GI phase of the mammalian cell cycle is required for the commitment of the cell towards division (3,4,40). Genes that possess the characteristic of enhanced expression when quiescent cells in culture are stimulated to proliferate by mitogens therefore constitute an interesting class in which functions that are essential for cell proliferation may be sought.…”

mentioning

confidence: 99%

Transcriptional regulation of two serum-induced RNAs in mouse fibroblasts: equivalence of one species to B2 repetitive elements.

Edwards¹,

Parfett²,

Denhardt³

1985

Mol. Cell. Biol.

126

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We obtained eight cDNA clones that define five genes whose expression (appearance of transcripts in the cytoplasm) is enhanced when quiescent mouse fibroblasts are stimulated with serum to divide. Two of these clones (designated 49C8 and 16C8) correspond to RNA species that are present in the cytoplasm of quiescent cells at very low levels. After serum stimulation, the level of 16C8 mRNA rose more rapidly than that of 49C8 RNA, reaching a maximum around 6 to 12 h. The data suggest that 49C8 and 16C8 RNAs are induced as a result of independent stimnuli. Either fibroblast growth factor or 12-tetradecanoylphorbol-13-acetate alone could induce 16C8 expression almost as effectively as serum; in contrast, 49C8 was not efficiently induced by epidermal growth factor, fibroblast growth factor, insulin, or 12-tetradecanoylphorbol-13-acetate. Inhibitors of transcription and translation diminished the induction of 16C8, while 49C8 expression was sensitive to actinomycin D but not cycloheximide or 5,6-dichloro"1-j--D-ribofuranosylbenzimidazole. In vitro transcription experiments with isolated nuclei revealed a peak in transcriptional activity of the 16C8 gene at around 3 h after serum stimulation. Sequence analysis of the 49C8 clDNA clone showed >90% homology of a large portion to a consensus rodent B2 repetitive elemnent.

show abstract

Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells

Cited by 17 publications

References 13 publications

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

Drosophila 230-kD TFIID subunit, a functional homolog of the human cell cycle gene product, negatively regulates DNA binding of the TATA box-binding subunit of TFIID.

Transcriptional regulation of two serum-induced RNAs in mouse fibroblasts: equivalence of one species to B2 repetitive elements.

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