Bisson, L.F., L. Neigeborn, M. Carlson, & D.G. Fraenkel (1987) The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae. J. Bacteriol. 169: 1656— 1662.
Neigeborn et al. (1986) (Article 5) describe the cloning of SNF3 using a low-copy vector library. In Article 7, they use a high-copy vector library. Fragments from six different genomic regions were isolated. They all cannot be the real SNF3\ Thus, they must be suppressors of snf3.
1. Define the terms high-affinity glucose transport and low-affinity glucose transport. You may have to read the enzyme kinetics section of a biochemistry text.
2. Discuss the genes encoding hexose kinases in Saccharomyces and compare them functionally.
3. The library used in this study was constructed using the vector YEp24.
(a) What is the yeast origin of replication of this vector and what is the average copy number per cell?
(b) What is the nutritional selection marker carried by this vector?
(c) How were suppressed transformants identified?
(d) What methods were used to distinguish the 11 different plasmids that resulted from this selection and place them into groups?
4. Strain MCY657 (snf3-72) transformants carrying plasmids pSC2 and pSC7 had a different phenotype compared with those carrying plasmids pSC8 and pLN 133-7, and the pSC2 and pSC7 transformants were more similar to transformants carrying the actual SNF3. Discuss.
5. In your own words, explain why the authors propose that 'the complementing genes carried by pSC2 and pSC7 are perhaps the prime candidates for actually specifying a glucose carrier involved in high-affinity uptake'.
6. What caveats do the authors mention with regard to the other complementing genes that increase low-affinity glucose uptake?
7. Several of the complementing genes carried by the plasmids isolated in this study were characterized by molecular genetic analysis in future studies by these authors. They encode proteins that fall into the same multigene family as the Snf3 protein but are not highly sequence homologous to SnBp. HXT1 encodes a protein which is 30% identical to Snf3p and appears to function as a high-affinity glucose transporter (Lewis & Bisson, 1991). HXT2 is another of the multicopy suppressors of snf3. HXT2 encodes a protein which is 69% identical to Hxtlp and 66% identical to Gal2p, the galactose transporter (Kruckeber & Bisson 1990). Based on these statements, would you or would you not be surprised if I told you that one of these sn/3 complementing genes is GAL21 Explain your answer.
Lewis, D.A. & L.F. Bisson (1991) The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters. Mol. Cell. Biol. 11: 3804-3813. Kruckeberg, A.L. & L.F. Bisson (1990) The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport. Mol. Cell. Biol. 10: 5903-5913.
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