0102 Fin Mark

Part I (Dr. Lam’s section; 35 marks) Question 1 We have covered the yeast two-hybrid system in the class. However, we have not discussed the yeast one-hybrid system that is actually a simpler system allowing us to isolate DNA binding proteins. Based on your knowledge on yeast two-hybrid system and the hints given below, explain how you can use yeast one-hybrid system to identify proteins specifically binding to a target DNA segment. [Hint: many activation domains can initiate transcription so long as it is placed near the RNA polymerase; yeast one-hybrid does not require the interaction of two proteins] (8 marks) Construct a cDNA library in which random cDNA fragments (0.5) are fused in frame (1) to the activation domain (1) of a transcription factor (0.5). Place the target DNA segment in the promoter (0.5) and close to the transcriptional start (1) of marker genes (1). Two marker genes will be used for double marker (1). Introduce the marker genes into the chromosome of a yeast strain (1). Transform the yeast strain with the cDNA library constructed (1). When both marker genes are turned on, the transformed clone(s) will be candidates for DNA-binding proteins (1). Question 2 VNTR is used by FBI as evidence to prosecute a criminal suspect. Explain the principle of VNTR and why it is suitable for this purpose (5 marks). VNTR depends on the variable of number of tandem repeats between individuals (2). Minisatellites are used (0.5). A pair of specific primers flanking the tandem repeats will be used to amplify the repeated region (1) and the differences will be visualized by gel electrophoresis (0.5). Since it is a single locus method (0.5), statistical analysis (1) can be performed which is important for identification of individuals (0.5).

Question 3 A scientist decides to study genes that control the flowering mechanism of a plant. He has three graduate students. He asked one student to make DNA chips from a flower library, another student to construct genetic and physical map of the target plant, and the remaining student to generate flowering mutants. Explain the overall strategies used by this scientist (10 marks) DNA chips: to perform comprehensive gene expression profile analyses (2) to identify differentially-expressed genes (1) that may be related to the flowering mechanism (1). Genetic and physical map: to prepare for map-based cloning approach (2). Precise map location (1) and DNA markers (1) are important for this strategy. Mutants: to determine gene function by “loss-of-function” approach (2). Mutants defective in the flowering process will help to locate genes controlling flowering mechanism (2). If mutants are generated by insertional mutagenesis, strategies such as plasmid rescue can be used to clone the corresponding genes (1). If mutants are point mutations by chemical mutagenesis, the corresponding genes can be cloned by map-based cloning approach (1). Question 4 Distinguish the terms differential expression, differential display and differential hybridization (6 marks) Differential expression: the study of differences in gene expression pattern (1). Comparison can be made between different treatment conditions, cells, tissues, individuals, species, etc. (1) Differential display: a PCR assisted method used to visualize differential expression (1). The basic principle of this technique involves the use of short random primers that lead to the amplification of a subset of cDNA (from mRNA) species (2). Differential hybridization: a library screening method for cloning of differentially expressed genes (1). Total RNA extracted from different samples will be used to make probes and hybridized to duplicated filters of a cDNA library (2). Question 5 Answer either (a) or (b) of the following questions (6 marks) (a)

State any one good question asked during the BIO4320 student presentations. Explain why you think this question is good and give an answer to this question. [Questions asked in your presentation cannot be used. However, questions asked by yourself in other student’s presentation is acceptable.]

(b)

Within the topics covered in Dr. Lam’s lecturing materials, state one question you think that should have (but not) appeared in this examination. Explain why this question is so important and provide a brief answer for it. - THE END -