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The overexpression of histones in the wild-type cells induces G1 delay.

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posted on 20.05.2010, 02:43 by Macarena Morillo-Huesca, Douglas Maya, Mari Cruz Muñoz-Centeno, Rakesh Kumar Singh, Vincent Oreal, Gajjalaiahvari Ugander Reddy, Dun Liang, Vincent Géli, Akash Gunjan, Sebastián Chávez

(A) Wild-type cells were transformed with pRS316 (empty vector) or with the analogous centromeric plasmids expressing either HTA1–HTB1 or a mutant version of this locus lacking the sequence that mediates its transcriptional repression in response to the free histones (HTA1–HTB1ΔNEG). The transformants were grown exponentially in a selective medium (AS), synchronized at START by treatment with alpha-factor for two hours (ST) and released from the arrest by washing out the alpha-factor. Samples were then taken at the different time points to analyze the DNA content by flow cytometry and the proportion of unbudded cells by microscopy. (B) Aliquots from the synchronized cells used in (A) were taken to analyze the CLN3 mRNA levels in START by Northern blot. The results of a typical experiment and the average quantification of three independent experiments are shown. The CLN3 mRNA levels have been normalized to ADH1.