The gene complementing a temperature-sensitive cell cycle mutant of BHK cells is the human homologue of the yeast RPC53 gene, which encodes a subunit of RNA polymerase C (III).

M. Ittmann, J. Ali, A. Greco, C. Basilico

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Abstract

The temperature-sensitive BN51 cell cycle mutant of BHK cells arrests in G1 at the nonpermissive temperature (39.5 degrees C). We have previously reported cloning the gene which complements this mutation. The complementing gene encodes a highly charged protein with a basic amino-terminal domain and an acidic carboxyl-terminal domain. We have recently found that the predicted BN51 protein shows significant homology to the 53 kilodalton subunit of RNA polymerase C (III) from Saccharomyces cerevisiae. Consistent with this, antibodies raised to fusion proteins containing BN51 coding sequences and antipeptide antibodies reveal that the BN51 gene encodes a 48 kilodalton protein which appears to be located primarily in the nucleus following subcellular fractionation and by immunohistochemistry. Analysis of RNA polymerase III activity in temperature-sensitive BN51 cells by nuclear runoff transcription assay reveals a marked drop in RNA polymerase III transcription after 48 h at the nonpermissive temperature (39.5 degrees C). This is correlated with a significant decrease in low molecular weight RNAs after 48 h at 39.5 degrees C. In addition, RNA polymerase III activity in S100 extracts of BN51 cells is more sensitive to heat inactivation at 39 degrees C than control extracts from BHK cells. When the yeast gene is introduced into the BN51 cells in a mammalian expression vector, it weakly complements the BN51 mutation in that it prevents cell death at 39.5 degrees C. The mechanism by which inhibition of RNA polymerase III activity leads to arrest in G1 is unclear but is not due to a marked decrease in total protein synthesis.

Original languageEnglish
Pages (from-to)503-511
Number of pages9
JournalCell Growth and Differentiation
Volume4
Issue number6
Publication statusPublished - Jun 1993

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RNA Polymerase III
Cell Cycle
Yeasts
Temperature
Genes
Proteins
Cell Extracts
Mutation
Antibodies
Saccharomyces cerevisiae
Organism Cloning
Cell Death
Hot Temperature
Molecular Weight
Immunohistochemistry
RNA

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

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title = "The gene complementing a temperature-sensitive cell cycle mutant of BHK cells is the human homologue of the yeast RPC53 gene, which encodes a subunit of RNA polymerase C (III).",
abstract = "The temperature-sensitive BN51 cell cycle mutant of BHK cells arrests in G1 at the nonpermissive temperature (39.5 degrees C). We have previously reported cloning the gene which complements this mutation. The complementing gene encodes a highly charged protein with a basic amino-terminal domain and an acidic carboxyl-terminal domain. We have recently found that the predicted BN51 protein shows significant homology to the 53 kilodalton subunit of RNA polymerase C (III) from Saccharomyces cerevisiae. Consistent with this, antibodies raised to fusion proteins containing BN51 coding sequences and antipeptide antibodies reveal that the BN51 gene encodes a 48 kilodalton protein which appears to be located primarily in the nucleus following subcellular fractionation and by immunohistochemistry. Analysis of RNA polymerase III activity in temperature-sensitive BN51 cells by nuclear runoff transcription assay reveals a marked drop in RNA polymerase III transcription after 48 h at the nonpermissive temperature (39.5 degrees C). This is correlated with a significant decrease in low molecular weight RNAs after 48 h at 39.5 degrees C. In addition, RNA polymerase III activity in S100 extracts of BN51 cells is more sensitive to heat inactivation at 39 degrees C than control extracts from BHK cells. When the yeast gene is introduced into the BN51 cells in a mammalian expression vector, it weakly complements the BN51 mutation in that it prevents cell death at 39.5 degrees C. The mechanism by which inhibition of RNA polymerase III activity leads to arrest in G1 is unclear but is not due to a marked decrease in total protein synthesis.",
author = "M. Ittmann and J. Ali and A. Greco and C. Basilico",
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T1 - The gene complementing a temperature-sensitive cell cycle mutant of BHK cells is the human homologue of the yeast RPC53 gene, which encodes a subunit of RNA polymerase C (III).

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AU - Greco, A.

AU - Basilico, C.

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