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Problems of Cell Cycle 1. If the most basic function of the cell cycle is to duplicate accurately the DNA in the chromosomes and then distribute the copies precisely to the daughter cells, why are there gaps between S phase and M phase? 2. Many cell-cycle genes from human cells function perfectly well when expressed in yeast cells. Why do you suppose that is considered remarkable? After all, many human genes encoding enzymes for metabolic reactions also function in yeast, and no one thinks that is remarkable. 3. What determines the length of S phase? One possibility is that it depends on how much DNA the nucleus contains. As a test, you measure the length of S phase in dividing cells of a lizard, a frog, and a newt, each one of which has a different amount of DNA. As shown in Table 17-1, the length of S phase does increase with increasing DNA content. Even though these organisms are similar, they are different species. You recall that it is possible to obtain haploid embryos of frogs and repeat your measurements with haploid and diploid frog cells. Haploid frog cells have the same length S phase as diploid frog cells. Further research in the literature shows that in plants, tetraploid strains of beans and oats have the same length S phase as their diploid cousins. Propose an explanation to reconcile these apparently contradictory results. Why do you suppose the length of S phase increases with increasing DNA content in different species, but remains constant with increasing DNA content in the same species? 4. The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe provide facile genetic systems for studying a wide range of eukaryotic cell biological processes. If cell-cycle progression is essential for cell viability, as it is in these yeasts, how is it possible to isolate cells that are defective in cell-cycle genes? 5. You have isolated a new Cdc mutant of budding yeast that forms colonies at 25°C but not at 37°C. You would now like to isolate the wild-type gene that corresponds to the defective gene in your Cdc mutant. How might you isolate the wild-type gene using a plasmid-based DNA library prepared from wild-type yeast cells? 6. Fertilized eggs from the frog Xenopus, which contain 100,000 times more cytoplasm than a typical mammalian cell, are a favorite choice for studying the biochemistry of the cell cycle. Why isn't it just as easy to study these biochemical questions by growing large numbers of mammalian cells, which is a straightforward process? 7. As genes encoding cyclin-dependent kinases were cloned from fission yeast, budding yeast, and animal cells, investigators working in plant systems designed experiments to determine whether plants also possessed CDKs that functioned as the catalysts for cell cycle progression. To isolate a cDNA encoding a CDK in maize, investigators aligned the predicted amino acid sequences of human cdc2, S. pombe cdc2, and S. cerevisiae cdc28 genes (see the figure below). Degenerate oligonucleotide PCR primers corresponding to the boxed amino acids were generated and a DNA fragment of the expected size was synthesized by PCR using these primers and maize cDNA library as a template. Why were the boxed regions selected as the basis for primer design? The PCR product was then used as a probe to screen the maize cDNA library, and a full-length cDNA clone was isolated. The clone was sequenced, and the predicted amino acid sequence was 64% identical to human cdc2 and 63% identical to S. pombe cdc2 and to S. cerevisiae cdc28. What does the level of sequence similarity suggest about the evolution of cdc2 genes? A complementation experiment was performed with S. cerevisiae cells possessing a temperature-sensitive cdc28 mutation. Wild-type cells, cdc28ts cells, and cdc28ts cells transformed with the maize cdc2 cDNA under the influence of a strong promoter were grown at the permissive (25 C) or restrictive (37 C) temperatures. Cell proliferation was monitored by the growth of colonies on the culture plates (shown in the figure below). Why do the cdc28ts cells form colonies at 25 C but not 37 C? What is the significance of colony formation of the cdc28ts + maize cdc2 cells at 37 C? What does this experiment tell us about the functional homology of cyclin-dependent kinase genes among eukaryotic species? 8. Order the following events in animal cell division. A. Alignment of chromosomes at the spindle equator. B. Attachment of micro tubules to chromosomes. C. Breakdown of nuclear envelope. D. Condensation of chromosomes. E. Decondensation of chromosomes. F. Duplication of centrosome. G. Elongation of the spindle. H. Pinching of cell in two. I. Re-formation of nuclear envelope. J. Separation of centrosomes. K. Separation of sister chromatids.