0001 Fin Mark

Question 1 A dicot plant has the following properties: (i) salt tolerant, and (ii) ready transformable with Agrobacterium. (a)

Using a yeast system, design one strategy to clone the genes conferring salt tolerance. (5 marks) • Identify a yeast strain that is sensitive to salt (0.5) and determine the killing dosage (0.5). • Construct a cDNA library of the target plant (0.5) using a yeast expression vector (0.5), preferably carrying an inducible promoter (0.5). • Transform the yeast strain with the cDNA library (0.5). • Screen/select on medium containing killing dose of salt (0.5); untransformed yeast cells should be included as the negative control (0.5); the survived yeast cells may contain cDNA clone of the salt tolerance gene (0.5). • Verify the results by (i) growing the yeast with or without the inducer on medium containing killing dose of salt (0.5) and (ii) re-transforming the original yeast strain with plasmids containing putative clones and looking for a high survival rate (e.g. 100%) of the transformed cells (0.5).

(b)

After you have successfully cloned the putative genes in (a), suggest two tests to verify that the cloned genes are responsible for salt tolerance in plants. (5 marks) • Engineer the cDNA clone from (a) into a plant expression vector containing appropriate selection markers (0.5) and a constitute promoter that can function in plant (1); • Drive the expression is either in sense (0.5) or antisense (0.5) orientation; • Use co-integration / binary vector system (0.5) for Agrobacterium-mediated (0.5) plant transformation; • Transform the sense construct into a salt sensitive plant to see if the cDNA can confer salt tolerance (1); • Transform the antisense construct into the original salt tolerant plant to see if the salt tolerance is reduced (1).

(c)

You are asked to test if a target gene cloned in (a) is only expressed in a particular cell type, explain what experimental approach you will employ. (5 marks) • Clone promoter of the target gene (0.5); • Construct a gene / an operon fusion (0.5) with a report gene (0.5) such as GUS (0.5); • Transform the construct into the target plant (0.5) to obtain stable (0.5) transformants; • Perform histochemical staining (0.5) using appropriate substrate (0.5) e.g. X-gluc for GUS (0.5); • Confirm the results by in situ hybridization (1). OR • Using the clone gene as the template, make labeled (0.5) antisense (1) RNA / single stranded DNA probes (1) by in vitro transcription / biased PCR techniques (0.5); • Prepare sections from tissues of the target plant (0.5) • Perform in situ hybridization (1) by hybridization the sections with probes (0.5) described above; • Detect the bound probes after washing steps (0.5).

(d)

Two different genes (Gene X and Gene Y) were cloned in (a). Despite that either of the Gene X or Gene Y can confer salt tolerance to certain extent, a strong synergistic effect was observed when both Gene X and Gene Y were expressed in the same cell. Design one experiment to test if the gene products of Gene X and Gene Y physically interact with each other. (5 marks) • A yeast two-hybrid system will be used (0.5); • Protein fusions will be constructed so that Protein X is fused with the DNA binding domain (or activation domain) and Protein Y is fused with the activation domain (or DNA-binding domain) of a transcription factor (TF) (2; diagrammatic representation is accepted). • The fusion constructs should be cloned into different yeast expression vectors containing different selectable markers (1) • A reporter strain with double reporters will be constructed (0.5). The expression of the reporter gene should be driven by the upstream activation sequence (UAS) of the corresponding TF used above (0.5); • Positive results, indicated by the successful expressions of both reporter genes (0.5), are due to the interaction between protein X and Y which brings the activation domain and DNA-binding domain of the TF to close proximity (0.5) and thus activate the RNA polymerase to start transcription (0.5).

Question 2 In the model plant Arabidopsis thaliana, random T-DNA insertional mutagenesis was performed. Independent mutant lines were collected and propagated separately. Making use of this tool, how can you study the functions of a cloned gene with known DNA sequence? (5 marks) • In the independently collected and propagated mutant lines, some may carry T-DNA inserted in the gene of interest (0.5) and thus represent knock-out mutants / loss-of-function mutants (0.5); • These knock-out / loss-of-function mutants, the functions of the target gene can then be studies by comparing to the wild type parent (1); • To identify T-DNA insertional mutants of the target genes, PCR approach will be used. One primer based on the sequence of the T-DNA (0.5) and one primer based on the sequence of the cloned gene (0.5) will be used to perform PCR reactions (0.5) on the genomic DNA prepared from randomly generated mutant lines (0.5). Positive results indicate that the T-DNA and target gene are in close proximity (0.5) suggesting that the T-DNA might have inserted in the target gene. To facilitate screening, genomic DNA from pooled lines can be used (0.5). Confirmation of successful insertions can be achieved by sequencing the PCR products (0.5). Question 3 The pharmaceutically important compound EPO is a glycoprotein. An animal specific complex sugar attached to EPO is important to prolong the clearance time (time for the kidney to remove EPO). Design a strategy to produce purified recombinant EPO glycoprotein that is ready for clinical use. (5 marks) • An EPO gene construct will be made so that the EPO is fused to a tag (0.5) such as HIS-Tag (0.5); a cleavage site (0.5) should be introduced between the EPO and His-Tag (0.5) for future separation of these two protein domains; • The construct, driven by an appropriate promoter in a suitable vector (0.5); check the host system) will be transformed into an animal cell (1; 0.5 if other eukaryotic cell lines were used; no mark if bacteria were used) such as insect cells (0.5; when baculovirus vector was used; using an animal cell line is to ensure that animal specific complex sugar will be attached to the recombinant EPO protein (0.5); • After growing and harvesting the cells (0.5), the protein extract will be purified through an affinity column (0.5).