Part I (50 points)

Section A (20 points)

Answer ALL questions

(1) The dut- ung- E. coli double mutant is used to increase the efficiency of site-directed
mutagenesis using the primer extension technique. Briefly explain the basic
principles. (5 points)

(2) Protein-protein interaction plays an important role in many biological processes.

Suppose you have cloned gene "X" which encodes Protein X and you know that
Protein X will interact with Protein Y. Briefly explain how you could make use of
the yeast two-hybrid technique to clone gene "Y" which encodes Protein Y. (5
points)

(3) What is a retrovirus? What is the major advantage of using retroviruses as vectors for
gene transfer into animal cells? (5 points)

(4) Ti plasmids are frequently employed as vectors for plant gene transfer. However, to
improve the efficiency of gene transfer, modified version of Ti plasmids are usually
used in place of the native Ti plasmids. One of the commonly used system is called
binary vectors system. Briefly explain how this system works. (5 points)
Part I (con'd)

Section B (30 points)

Read the instructions carefully and answer ALL parts of the following question.

(1) Yeast is a prefect model to study the control mechanism of eukaryotic cell cycle.
Many cell division cycle mutant (cdc mutant) were identified in yeast. Suppose you
have a temperature sensitive yeast mutant cdcX- which will undergo normal division
at 25 °C but not at 30 °C.

Q.1 Making use of the yeast mutant mentioned above, design an experiment to get the
rat cdcX cDNA clone. (7.5 points)

(2) You want to study the function of cdcX gene in the cell division cycle of rat cell
culture. Suppose you have an alcohol inducible promoter which will express
strongly when the rat cell culture is grown in the presence of a non-toxic dose of
alcohol but remain silent in the absence of alcohol.

Q.2 Design an experiment to show that cdcX gene is essential for the cell division cycle
of rat cell culture. (7.5 points)

(3) Expression study of the cdcX gene in rat cell culture shows that the mRNA levels is
positively regulated by growth factor Z. Suppose you also obtained the promoter of
the rat cdcX gene.

Q.3 Design an experiment to locate the cis-element on rat cdcX promoter which is
responsible for the induction of growth factor Z. (7.5 points)

In the genetic model plant Arabidopsis, a mutant was found to be defective in its cdcX
gene. This mutant is temperature sensitive so that the plant can continue to grow
only under 15 °C but all the cell division will stop at 22 °C.

Q.4 Design an experiment to test whether the rat cdcX gene can also function in
Arabidopsis. (7.5 points)