Iyer, V.R., C.E. Horak, C.S. Scafe, D. Botstein, M. Snyder, & P.O. Brown (2001) Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409: 533538.
This article uses a combination of DNA microarray analysis and chromatin immunoprecipitation to identify potential binding sites of the transcription factors SBF and MBF. The method is interesting because it provides an example of how DNA microarray analysis can be used to identify sequences that are enriched in a particular sample. Here the sample is enriched for DNA fragments that are specifically bound by a particular protein. One could also use this method to identify sequences that are enriched or depleted from a genome because of duplications, deletions, or aneuploidy.
1. Describe the transcription factors SBF and MBF and their role in regulating the cell cycle in Saccharomyces.
2. Describe chromatin immunoprecipitation (often referred to as CHIP) and the specific method used in this article to isolate SBF- or MBF-binding DNA fragments.
(a) The cross-linking step is done in vivo in whole cells. Why is this important?
(b) What is the size range of the DNA fragments generated by their method? Why was this size range chosen instead of shearing the samples to produce smaller or larger fragments?
(c) CHIP was done on unsynchronized cells, a-factor treated cells, and benomyl-treated cells. The last two treatments synchronize cells at which point in the cell cycle?
3. Describe the primer pairs used for the PCR amplification of the DNA fragments and synthesis of the intergenic DNA microarray.
4. Discuss the reason for each of the control experiments (Lanes 1, 2, and 3).
5. Describe the criteria used to evaluate the microarray data in order to identify potential SBF-regulated genes.
6. Describe the criteria used to evaluate the microarray data in order to identify potential MBF-regulated genes.
7. The authors present three lines of evidence in support of their conclusion that valid in vivo SBF- and MBF-binding sites have been identified. Describe this evidence.
8. The authors ask the question, 'Why are two different transcription factors used to mediate nearly identical transcriptional programmes during the cell-division cycle in yeast?' Summarize their reasons for making this statement and their answers to this question.
9. Many of the SBF- and MBF-binding sites identify potential novel genes involved in regulating the cell cycle. You wish to explore the function of these novel genes.
(a) Give criteria that you might use to select a specific novel ORF for further study.
(b) List the biochemical, cell biology, and genetic characterizations you would carry out to explore the cell cycle role of this novel gene.
Genetic Techniques for Biological Research Corinne A. Michels Copyright © 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)
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