You have received a prestigious summer research internship at the University of

Question

You have received a prestigious summer research internship at the University of California Berkeley studying nuclear transport in yeast. Your advisor puts you on a project to develop a screening method for identifying temperature-sensitive mutants in the nuclear transport apparatus. A. In stage 1 of the project, your advisor provides you with two plasmids - each contains a hybrid gene under the control of a regulatable promoter. The hybrid gene is a fusion between a gene whose product is normally imported into the nucleus and the gene for the restriction nuclease EcoRI. The plasmid pNL^+ contains a functional nuclear localization signal (NLS); the plasmid pNL^- does not have a NLS. The promoter, which is from the yeast Gall gene, allows transcription of the hybrid gene only when the sugar galactose is present in the growth medium. Your advisor instructs you to introduce the plasmids into yeast (in the absence of galactose) and then assay the transformed yeast under two conditions: in medium containing glucose and in medium containing galactose. You document the following results: Explain why the yeast with the pNL^+ plasmid grow in the presence of glucose but die in the presence of galactose, whereas yeast with the pNL^- plasmid grow in both media. B. Using this screening tool that you developed, in no more than three sentences outline an experiment that will allow you to identify temperature-sensitive mutants in the nuclear transport apparatus.

Explanation / Answer

1.Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources. Some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression.

In this question, The plasmid pNL+ contains a functional nuclear localization signal. The plasmid pNL- contains a nonfunctional signal. The promoter, which is from the yeast GAL1 gene, allows transcription of the hybrid gene only when the sugar galactose is present in the growth medium. EcoRI is a restriction enzyme that cleaves DNA based on a particular sequence. Introducing the restriction enzyme into a cell’s nucleus, will result in many double strand breaks of the host’s chromosomes and will ultimately kill the cell. Hence, the cells gets killed in the galactose medium.

2. Mutagenize cells containing the pNL+ plasmid at low temperature and then shift them to high temperature in the presence of galactose. At the restrictive temperature the nuclear transport mutants will not take up the killer protein encoded by pNL+ and, therefore, will not be killed. Normal cells, however, will transport the killer protein into the nucleus and die. After one or two hours at the high temperature to allow selection against normal cells, you intend to lower the temperature and put the surviving cells on nutrient agar plates containing glucose. You anticipate that the nuclear translocation mutants will recover at the lower temperature and form colonies.

Related Questions

Navigate

• Browse (All)
• Browse Y
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.