Neigeborn, L., P. Schwartzberg, R. Reid, & M. Carlson (1986) Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations. Mol. Cell. Biol. 6: 3569-3574.
1. The following questions relate to the construction and use of the library to isolate SNF3.
(a) What vector was used to construct the genomic library used to isolate SNF31
(b) Name the SNF3 mutant allele used in the host strain.
(c) URA3 is the nutritional selection marker on the library plasmid. The host strain used was MCY657. Describe the details of how the transformants were selected and how those containing SNF3 were identified.
2. Plasmid pLN185 (Figure 1) contains the indicated Bam HI fragment in the BamHl site of vector YIp5, a pBR322 derivative containing the yeast URA3 gene. Plasmid pLN185 was used to demonstrate that this fragment is derived from the SNF3 locus by targeted integration. Describe the targeted integration of pLN185 in detail. Include the method of integration (Hint: the BglII site in the insert fragment is unique to the plasmid), a diagram of the SNF3 locus before and after the plasmid integration, and diagram the cross showing that pLN185 integrated at the SNF3 locus and not elsewhere in the genome.
3. Two library plasmids complemented the snf3 mutation in the host strain, pPSC4 and pPSC5.
(a) How were these two plasmids helpful in localizing the position of SNF3 in this region?
(b) The fragments in plasmids pPSC4 and pRRC5 do not differ significantly in their ability to complement snf3 mutations. How do you know that from the data in Table 2?
Northern analysis was used to characterize the transcription of SNF3. (a) Briefly, what is a Northern analysis and how does it differ from a Southern analysis?
(b) Is the expression of SNF3 transcription regulated, and if so how?
(c) Why do the authors include URA3 in Figure 2?
5. Diagram the one-step gene disruption used to construct snf3-A4::HIS3.
6. Table 3 presents data indicating that a snf3 null allele differs from the mutations isolated in previous mutant hunts.
(a) Describe the different phenotypes of snf3-A4::HIS3 and snf3-217.
(b) Which allele is dominant in the following gene pairs: SNF3 and snf3-217; SNF3 and snf3-A4::HIS3; snf3-217 and snf3-A4::HIS3?
(c) Why does this information indicate that snj3 missense mutations exhibit abnormal function rather than a complete loss of function?
(d) How do the authors explain the finding that growth on sucrose and raffinose is poor despite normal derepression of secreted invertase?
7. The authors evaluated the pleiotropic effects of the snf3 mutations on other glucose-regulated genes. (Please ignore all data on snf3-39 which later turned out to carry multiple mutations.)
(a) Diagram the reporter gene used to assess the effects on GAL10 expression.
(b) What pieces of data indicate that snf3-A4::HIS3 has little effect on galactose induction and glucose repression of GAL101
8. 'We speculate that the aberrant glucose repression observed in snf3 missense mutants results from a defect in sensing or signaling the availability of glucose in the environment.' What reasons are given for this conclusion?
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