Molecular Mechanisms Of Dna Replication And Cell Division

One of the most significant contributions of Saccharomyces cerevisiae (the brewer's, baker's or budding yeast) and the fission yeast Schizosaccharomyces pombe to biology is in the understanding of eukaryotic cell cycle. The mitotic cell division is accomplished by a temporal sequence of events in which the cell first duplicates the DNA (S-phase), followed by precise separation of the sister chromatids into daughter nuclei (M-phase), leading finally to the separation of the two daughter cells. Each phase is separated from the preceding phase by an interval of time. The gap G1 is the time interval between mitosis and the initiation of new DNA synthesis; G2 is the gap between completion of DNA synthesis from the initiation of mitosis. In the late sixties, Leland H. Hartwell took a genetic approach to understand the biochemical basis behind the orderly sequence of events of the mitotic cycle. He capitalized on the genetic advantages of S. cerevisiae to generate cell division cycle (cdc) mutants (Hartwell et al., 1974). The cdc mutants were conditional temperature sensitive alleles (ts alleles), which could be stably maintained by growing the mutant cells at a permissive temperature of 23°C and exhibited the mutant phenotype of cell cycle arrest only at 37°C. The growth-arrested cdc mutants could be observed under a light microscope to determine accurately the position within the cell cycle at which they were arrested. The mapping of the cell cycle position with growth is possible in yeast by observing the ratio of the size of the bud (daughter cell) to the mother (Figure 6.2). The technique used by Hartwell for generating yeast cdc-mutants is shown in Figure 6.3.

Early genetic work with yeast accelerated the understanding of the eukaryotic cell cycle, first by forging a link between the events in the cycle with specific genes and second by establishing that the orderly sequence of events during cell cycle is a result of biochemical dependency, meaning that a prior event needed to be completed before the initiation of the

Figure 6.2 The S. cerevisiae cell cycle. The shape of a cell shows its position in its division cycle. The position START within G1 is the point at which the cell is committed to complete the cell cycle. The bud emerges at the beginning of S-phase and enlarges during G2 and M. The spindle pole bodies in yeast are embedded on the nuclear membrane. Yeast, like other fungi, has a closed mitosis—the nuclear envelope never breaks down. Reproduced with permission from Watson, J.D., Gilman, M., Witkowski, J. and Zoller, M. (1992), Recombinant DNA. © Scientific American Books.

Nucleus

Nucleus

Figure 6.2 The S. cerevisiae cell cycle. The shape of a cell shows its position in its division cycle. The position START within G1 is the point at which the cell is committed to complete the cell cycle. The bud emerges at the beginning of S-phase and enlarges during G2 and M. The spindle pole bodies in yeast are embedded on the nuclear membrane. Yeast, like other fungi, has a closed mitosis—the nuclear envelope never breaks down. Reproduced with permission from Watson, J.D., Gilman, M., Witkowski, J. and Zoller, M. (1992), Recombinant DNA. © Scientific American Books.

EMS-treated cells

EMS-treated cells

Plate cells grow at 23:C

Plate cells grow at 23:C

Make replica ,

Make replica ,

Recover is lethal mutant from master plate

Is mutant does not grow at 37 °C

Collection of is lethals

Growth at 23°C

Shift to 37°C growth ceases

Collection of is lethals

Growth at 23°C

Shift to 37°C growth ceases

View cells using microscope

Asynchronous Synchronous Asynchronous Synchronous arrest G1 arrest arrest G2 or M arrest cdc mutant cdc mutant

View cells using microscope

Asynchronous Synchronous Asynchronous Synchronous arrest G1 arrest arrest G2 or M arrest cdc mutant cdc mutant

Figure 6.3 Isolation of cdc mutants. Cells, mutagenized with the DNA-methylating agent ethyl methanesulfonate (EMS), were grown at 23°C and then spread on agar at 23°C (the permissive temperature). The colonies that were formed were replica-plated and grown at 37°C (the non-permissive temperature). Individual colonies that failed to grow at 37°C were noted as temperature sensitive (ts) mutants. The ts-mutants from the master plate were grown at 23°C, shifted to 37°C for a few hours and examined by microscopy. Cells in most cultures arrested at random points in the cell cycle (asynchronous arrest) but some cells arrested at the same point of the cycle (synchronous arrest). The latter class of cells was called cdc mutants. Reproduced with permission from Watson, J.D., Gilman, M., Witkowski, J. and Zoller, M. (1992), Recombinant DNA. © Scientific American Books.

next. Finally, the availability of the cdc mutants allowed the cloning and identification of the genes regulating cell cycle by complementation as described in the next section.

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