Defective RNA polymerase II in the G 1 specific temperature sensitive hamster cell mutant TsAF8

Microinjection experiments demonstrated a requirement for cellular ras activity late in G1. In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the GO/GI phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G1 as in previous studies by injecting anti (17). In support of this notion, a physical association between ras-GTP and cellular raf proteins has been demonstrated with several different experimental approaches (16,20,40,41,43,44).As further evidence that ras functions to transduce signals initiated by tyrosine kinases, the action of growth factor receptors leads to an elevation of the proportion of cellular ras associated with GTP (12, 31). Phosphorylated receptor molecules bind a number of proteins (22), including adapter molecules which associate with the ras exchange factor mSOS (2,4,8,11,23,29,33). It is likely that ras proteins are activated by the exchange factors positioned at the plasma membrane by their association with activated growth factor receptors. Genetic studies of Caenorhabditis elegans and Drosophila melanogaster have independently forged a link between ras, tyrosine kinases, and exchange factors. In each organism, the action of a developmentally required tyrosine kinase has been shown to * Corresponding author. Phone: (216) 444-0633. depend on a number of separate genes, including analogs for ras, ras exchange factors, and adapter proteins (7,13).While the data presented above leave little question that ras functions to transduce the signal initiated by growth factor receptors, there is a critical inconsistency between them in relationship to cell cycle timing. When quiescent cells are stimulated with serum and injected with anti-ras antibody (or either of the dominant ras inhibitors) at various times thereafter, DNA synthesis within the recipient cells is efficiently inhibited so long as the injection occurs prior to the initiation of S phase. This indicates that ras is required late in the G, phase of the cell cycle, presumably at the G1/S-phase boundary (21). The action of growth factors, however, alters ras activity much earlier in the cell cycle. The proportion of ras-GTP increases within 5 min following serum addition to quiescent cells (12,31). In addition, many of the intermolecular associations studies described above take place early in GJ/G1. Furthermore, injection of oncogenic ras into quiescent cells has been shown to induce the rapid expression of cellular fos protein, while the fos expression induced soon after growth factor addition can be partially inhibited by previous injection of anti-ras antibody (37). Finally, studies with dominant inhibitory ras mutants make it clear that the ability of peptide growth factors to stimulate mitogen-activated protein kinase activity is depende...

Section: Resultsmentioning

confidence: 99%

Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells.

Dobrowolski¹,

Harter²,

Stacey³

1994

“…The experiments presented in this paper have two purposes: to explore the effect of a small deoxyribonucleic acid (DNA) oncogenic virus, simian virus 40 (SV40), on sodium butyrate inhibition of growth and to try to understand some of the mechanisms involved in such inhibition.…”

mentioning

confidence: 99%

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid.

Kawasaki¹,

Diamond²,

Baserga³

1981

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells. Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in G1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serumstimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the Go -GI -S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.

“…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).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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). We have interpreted these data as implying that the TS AmaK mutations (11) of CHO cell lines and the TS mutation in TsAF8 cells are mutations in the same complementation group.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Self Cite

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