Trends in Microbiology

Trends in Microbiology

Volume 10, Issue 4, 1 April 2002, Pages 169-173
Journal home page for Trends in Microbiology

Opinion
The Schaechter–Bentzon–Maaløe experiment and the analysis of cell cycle events in eukaryotic cells

https://doi.org/10.1016/S0966-842X(02)02322-3Get rights and content

Abstract

The Schaechter–Bentzon–Maaløe (SBM) experiment, performed more than 40 years ago, provides an important lesson for the analysis of the eukaryotic cell cycle. Before this experiment, temperature shifts had been used to synchronize bacteria and determine the pattern of DNA synthesis during the bacterial division cycle. These experiments indicated that DNA replication occurred during a fraction of the division cycle with gaps before and after DNA synthesis, a pattern similar to the eukaryotic division cycle. The SBM experiment studied DNA replication during the division cycle by labeling an unperturbed culture with a short pulse of tritiated thymidine. All cells were found to be labeled, indicating that unperturbed cells synthesize DNA throughout the division cycle. Thus, the SBM experiment was a control experiment demonstrating that artifacts can be introduced by synchronization methods. The idea of an control experiment under unperturbed conditions is proposed for the analysis of data on cell-cycle-specific gene expression in yeast and mammalian cells.

Section snippets

DNA synthesis during the bacterial division cycle

More than 40 years ago, Lark and Maaløe performed a series of experiments on the timing of DNA synthesis during the bacterial division cycle. Bacterial cells were ‘synchronized’ by temperature shifts [7]. Subsequent measurements of DNA synthesis during the division cycle of these temperature-entrained cells indicated that synthesis occurred during the middle of the division cycle, with a gap at either side [8]. This result was similar to the proposed pattern of DNA replication during the

The Schaechter–Bentzon–Maaløe experiment

The experiment performed by Schaechter, Bentzon and Maaløe was extremely simple [6]. Exponentially growing bacteria were pulse-labeled for a short time with tritiated thymidine, the cells were fixed and the amount of radioactivity in individual cells was determined by autoradiography. If the results using synchronized cells were correct, then only a proportion of the cells would be expected to be labeled as cells that were in the G1 and G2 phases during the labeling period would be unlabeled.

Analysis of gene expression during the eukaryotic cell cycle

Let us now apply the lessons of the SBM experiment to the problem of the timing of gene expression during the division cycle of human and yeast cells.

One of the most striking results obtained using S. cerevisiae is that there is continuous variation in the timing of gene expression during division [1]. This implies that the cell has a timing mechanism to turn on gene expression for different genes at different points during the division cycle. The methods used to analyze the cell cycle,

Analysis of the cell cycle without synchronization

There are many ways to test the pattern of gene expression during the division cycle without synchronization. Here, I will suggest two methods to illustrate possible approaches. This list should not be taken as exhaustive or as the final list as, with technical improvements, other methods are sure to be developed that will test the proposition that one or another gene is expressed in a cell-cycle-specific pattern.

One method that would be strictly analogous to the SBM experiment would be to look

Rb phosphorylation during G1 phase

One of the most well known G1-phase events is the specific phosphorylation of the retinoblastoma protein (Rb). I will not review the data for this proposal as I have done so elsewhere [18]. However, I will point out that this classic example of a G1-phase event has an alternative explanation. It appears that much of the data supporting G1-phase Rb phosphorylation is based on a problem with how the cells are grown and analyzed. Not only is it possible to grow cells where all cells have only

The ultimate test: reproducibility and standardization

The ultimate demonstration and convincing proof that a gene is synthesized in a meaningful way during the cell cycle is that this pattern of gene synthesis is studied in more than one laboratory and can be analyzed in different laboratories in a reproducible manner. I have previously proposed that the ultimate analysis of gene expression requires a standard system allowing researchers in different laboratories to reproduce work from other laboratories [20]. Similarly, if a G1-phase

Controls and the burden of proof

It is sometimes difficult to do control experiments. One has a result, the results look satisfying, so it is difficult to be self-critical and to test the experimental results by running controls to eliminate possible artifacts. Part of the brilliance of the SBM experiment is that it was a control experiment that questioned a result that not only appeared consistent with eukaryotic data but could also be understood and explained in its own right. There was no reason to question the original

Acknowledgements

Charles Helmstetter, my friend and scientific colleague for almost 40 years, was helpful in suggesting improvements to the proposals made here. Kerby Shedden of the University of Michigan Dept of Statistics was invaluable in clarifying many ideas regarding the cell cycle. Some papers of interest can be read at http://www.umich.edu/∼cooper.

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