Question

a mutant hunt for genes involved in regulating the expression of the BAK3 gene, which is required to make yeast smell good. Rather than sniffing individual yeast colonies to find relevant mutants, use a reporter to rapidly select for colonies in which BAK3 expression is lost. put the entire BAK3 promoter upstream of the ZOM1 gene. ZOM1 expression makes yeast sensitive to zoomidine, because the Zom1 protein converts zoomidine into a deadly poison. grow mutagenized yeast carrying the [BAK3 promoter- ZOM1]reporter construct in the presence of zoomidine and are excited to see that many colonies survive! However, when clone and analyze these colonies, find that many of them are mutations in genes required for zoomidine import into the cell - boring! want to introduce a second construct into the yeast that will allow to distinguish mutations in zoomidine transport from the (much more exciting) mutations that affect regulation of BAK3. Which one of the following constructs use? An additional copy of the BAK3 promoter + ZOM1 C BAK3 promoter + Lacz C ZOM1 promoter + Lacz Half of the BAK3 promoter + a weak non-specific activator + Lacz

Answer 1

BAK3 promoter + LacZ

The LacZ reporter gene codes for the enzyme beta-galactosidase. Beta-galactosidase is involved in the breakdown of lactose into glucose and galactose. LacZ can be exploited to work as a reporter gene by providing the yeast culture plates with X-gal, which is another substrate for beta-galactosidase. X-gal, when metabolised by beta-galactosidase, results in the formation of a blue-coloured dye. So, by using the BAK3 promoter + LacZ construct, we ensure that all those colonies which have normal BAK3 expression will also have beta-galactosidase expression. Hence, all those colonies with normal BAK3 expression will appear blue. Colonies without BAK3 expression will appear white.

Hence, once we have screened colonies using the BAK3 promoter + ZOM1 reporter construct, the remaining colonies can then be screened with respect to the BAK3 promoter + LacZ construct. The use of this second construct will allow us to differentiate between those colonies which survived the zoomidine selection because of absence of BAK3 expression and those which had mutations in the genes responsible for zoomidine import. The latter colonies will appear blue, thereby making it clear that their resistance to zoomidine is not due to loss of BAK3 expression. Only those colonies which appear white will have loss of BAK3 expression, as only these colonies will show no expression of Zom1 (and will therefore be resistant to zoomidine) and also no expression of beta-galactosidase (and therefore appear white).