Erdman, S., L. Lin, M. Malczynski, & M. Snyder (1998) Pheromone-regulated genes required for yeast mating differentiation. J. Cell Biol. 140: 461-483.
This article uses the genome-wide transposon mutagenesis approach to identify additional genes involved in cellular processes involved in mating such as agglutination, polarized growth for the formation of the mating projection, cell fusion, and nuclear fusion. Searches for mutants exhibiting defects in mating have identified a number of genes involved in these processes. However, it is likely that many genes have been missed possibly because of redundancy in the Saccharomyces genome or because alterations in these genes do not cause defects severe enough to produce a sufficiently distinct phenotype. The approach described here uses transposon mutagenesis (see Chapter 12) to produce random lacZ fusions to ORFs throughout the genome and to screen these for pheromone regulation.
1. Describe the lacZ insertional mutagenesis scheme used in this article to randomly tag genes in the Saccharomyces genome. Include:
(a) The structure of a typical library plasmid carrying a yeast DNA fragment with a Tn insert creating a lacZ fusion.
(b) The genotype of the host strain into which the library was transformed.
(c) How were transformants selected?
(d) How were transformants screened to identify fusions to pheromone-regulated genes?
2. For this study:
(a) Why did the authors use a bar 1 IS. strain?
(b) Why were both haploid MATn and homozygous MATa/MATa diploid strains used?
3. List the phenotypes used to test the novel pheromone-regulated genes identified in this study for a potential affect on mating. Describe the reasons for selecting FIG I, FIG2, FIG3, and FIG4 for further study.
4. What is a PRE sequence and what is the significance of finding PRE sequences in the promoter regions of FIG I, FIG2, FIG3, and FIG4?
5. Describe the method used to demonstrate that cell cycle arrest does not alter the expression of the FIG genes.
6. Describe the complete phenotype of the fig2 mutant in detail. Include results discussed throughout this article.
7. Describe the Fig2 protein. Include sequence motifs and the results of cellular localization studies. What do the authors propose is a likely function of Fig2p?
8. The study identified 54 pheromone-regulated genes but the authors estimate that there are between 67 and 132 such genes in the Saccharomyces genome. Describe how they derived this estimate.
9. Only nine previously known pheromone-regulated genes were identified in this screen.
(a) Name one gene that you know to be pheromone regulated that was not identified.
(b) How do the authors explain the fact that they missed many genes?
(c) How might the Tn library be improved so as to make the mutagenesis method more random?
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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